Methods of treating inflammatory diseases of colon

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine, in particular to gastroenterology, and deal with treatment of ulcerative colitis and Crohn's disease. Method of treatment includes introduction into organism of therapeutically efficient quantity of attaching cells from placenta, cultivated in such a way as not to differentiate into adipocytes or osteocytes. Also claimed is application of said cells for obtaining medication, intended for treatment of ulcerative colitis or Crohn's disease. Claimed produced product for treatment of ulcerative colitis or Crohn's disease includes in packing form pharmaceutically efficient quantity of said cells.

EFFECT: inventions ensure essential reduction of inflammatory process in colon in modelling of said diseases, as well as due to pathological mechanisms of treatment in addition to T-lymphocyte suppression.

31 cl, 5 ex, 12 tbl, 16 dwg

 

The technical field to which the invention relates, and the prior art

The present invention in some embodiments of implementation relates to methods of treatment of inflammatory diseases of the colon with the use of attached cells from adipose tissue or placental tissue and, more particularly, but not exclusively, to methods for treating ulcerative colitis or Crohn's disease using fixed cells.

In developing the medical world there is a growing need for large quantities of stem cells of the adult person with a view to their transplantation and tissue engineering. In addition, the stem cells of an adult human is constantly evolving in relation to therapy and treatment of various conditions, such as haematopoietic disorders, heart disease, Parkinson's disease, Alzheimer's disease, stroke, burns, muscular dystrophy, autoimmune disorders, diabetes, and arthritis.

In recent years, considerable activity focused on therapeutic potential of mesenchymal stem cells (MSCs) in terms of their various medical applications, including tissue repair damaged organs such as the brain, heart, bone and liver, and to support bone marrow transplantation (BMT). MSCs, a heterogeneous population of cells derived from, for example, bone mo is ha adipose tissue, placenta and blood, capable of differentiation into various types of Mature mesenchymal cells (for example, in the reticular endothelial cells, fibroblasts, adipocytes, osteogenic precursor cells), depending on the influences of various biologically active factors. Accordingly, MSCs are widely investigated in regenerative medicine as the basis for building new tissues, such as bone, cartilage and adipose tissue for repair of damage or replacement of pathological tissues and treatment of genetic and acquired diseases. Moreover, the ability of MSCs to multipotential, ease of selection and cultivation, as well as their high potential of ex vivo expansion makes them attractive therapeutic tool.

Inflammatory bowel disease (IBD), a group of inflammatory conditions of the colon and small intestine includes Crohn's disease and ulcerative colitis is a chronic, recurrent and constant condition of unknown origin that affects at least 1 out of 1000 people in Western countries.

Crohn's disease (also known as granulomatous colitis and regional enteritis), an autoimmune disease caused by the damaging effects of the immune system in the gastrointestinal tract and the induction of inflammation in the gastrointestinal tract, predstavljaet an inflammatory disease, which can affect any part of the gastrointestinal tract from mouth to anus, causing a wide range of symptoms. First of all, it causes pain in the abdomen, diarrhea, vomiting and weight loss, but may also cause complications outside the gastrointestinal tract such as skin rashes, arthritis and eye inflammation. Currently there is no known pharmaceutical or surgical cure for Crohn's disease, and treatment options are restricted to controlling symptoms, maintaining remission and preventing relapse (for example, compounds 5-aminosalicylic (5-ASA) acid, corticosteroids, such as prednisone and hydrocortisone, and immunomodulators, such as azathioprine and mercaptopurine).

Ulcerative colitis, a form of colitis is a disease of the intestine, specifically the large intestine or colon, that includes characteristic ulcers, or open sores in the colon. The main symptom of active disease is usually constant diarrhea mixed with blood. Modern treatment of ulcerative colitis includes anti-inflammatory drugs, immunosuppression and biological treatment aimed at specific components of the immune response. Sometimes required colectomy (partial or complete removal of the colon by surgical vmichael the CTV), and this is considered as the treatment of disease.

Okamoto et al. [Okamoto et al., above] and Matsumoto et al. [Matsumoto et al., Gastroenterology (2005) 128: 1851-1867] demonstrated that cells derived from bone marrow (BMDCs)can revive the population of the epithelium of the gastrointestinal tract of man after the disease graft-versus-host or formation of gastric ulcers after irradiation and bone marrow transplantation. Komori et al. 2005 [Komori et al., J Gastroenterol (2005) 40:591-599] also reported a temporary increase in epithelial mucosal cells originating from bone marrow, and myofibroblasts in the healing process of gastric ulcers and induced trinitrobenzenesulfonic acid (TNBS) colitis in rats. In addition, the company Osiris (www.osiris.com)specializing in clinical medicine, gives an estimate of Prochymal, the product derived from MSCs bone marrow for the treatment of Crohn's disease. Osiris is currently conducting a multicenter trial to evaluate the safety and efficacy of Prochymal for Crohn's disease.

In PCT publication no WO 2008/100498 disclosed methods of treatment of diseases associated with the immune system (e.g., inflammatory bowel disease, disease, graft-versus-host), using stem cells placenta or stem cells of the umbilical cord. Disclosed stem cells come from placental mammals, regardless of morphology, is Askerov cell surface or the number of transfers after initial cultivation, and attached to the tissue culture substrate (e.g. plastic for culturing tissue or coated with fibronectin plate for tissue culture).

In the publication U.S. No. 20080213227 disclosed methods of treatment of autoimmune diseases and inflammatory diseases (e.g. inflammatory bowel disease and Crohn's disease) through the introduction of mesenchymal stem cells in an effective amount. Disclosed mesenchymal cells can be obtained from the attached cells from bone marrow or periosteal cells, or alternatively from the blood, skin, umbilical cord blood, muscle, adipose tissue, bone or pericentre.

In PCT publication no WO 2007/108003 disclosed methods of cell expansion, which include the cultivation of attached cells from the placenta or adipose tissue under conditions of three-dimensional cultivation, which supports cellular expansion. There are also cells thus obtained, and their uses.

Summary of the invention

In accordance with an aspect of some embodiments of the present invention features a method of treating ulcerative colitis or Crohn's disease in need of this individual, and the method includes the administration to an individual a therapeutically effective amount of attached cells isplenty or adipose tissue, resulting in the treatment of ulcerative colitis or Crohn's disease.

In accordance with an aspect of some embodiments of the present invention, it is proposed the use of attached cells from the placenta or adipose tissue to obtain medicines identified as for the treatment of ulcerative colitis or Crohn's disease.

In accordance with an aspect of some embodiments of the present invention suggests a product of manufacture, comprising packaging material includes a label for use in the treatment of ulcerative colitis or Crohn's disease, and packing material Packed pharmaceutically effective amount of attached cells from the placenta or adipose tissue.

In accordance with some variations of the invention, attached cells include a positive marker expression selected from the group consisting of CD73, CD90, CD29 and CD105.

In accordance with some variations of the invention, attached cells include negative marker expression selected from the group consisting of CD3, CD4, CD45,
CD80, HLA-DR, CD11b, CD14, CD19, CD34 and CD79.

In accordance with some variations of the invention, attached cells are able to suppress an immune response.

In accordance with some variations is Tami embodiment of the invention, suppression of the immune response includes suppressing the activity of T cells.

In accordance with some variations of the invention, attached cells obtained from three-dimensional (3D) culture.

In accordance with some variations of the invention, three-dimensional (3D) culture includes 3D bioreactor.

In accordance with some variations of the invention, the cultivation of attached cells in 3D culture produced in conditions of perfusion.

In accordance with some variations of the invention, the cultivation of attached cells produce for at least three days.

In accordance with some variations of the invention, the cultivation of attached cells to produce until at least 10% of the attached cells proliferate.

In accordance with some variations of the invention, attached cells include the expression profile of genes as described in table 11.

In accordance with certain variants of the invention, the attached cells include cultured cells of placenta or adipose tissue under conditions of 2-dimensional (2D) culture.

In accordance with some variations of the invention, at least 12% of the attached cells n which appears in S and/or G2/M phase of cell proliferation.

In accordance with some variations of the invention, attached cells include the expression profile of genes as described in table 8.

In accordance with some variations of the invention, attached cells less commiteeman towards osteogenic line of differentiation compared to fixed cells derived from the bone marrow, while growing and providing opportunity to differentiate in the same conditions.

In accordance with some variations of the invention, attached cells less commiteeman towards adipogenic line of differentiation compared to fixed cells derived from the bone marrow, while growing and providing opportunity to differentiate in the same conditions.

In accordance with some variations of the invention, the product also includes an additional drug for the treatment of inflammation of the colon.

In accordance with some variations of the invention, the product further includes immunosuppressive agent.

In accordance with some variations of the invention, the product further includes an anti-inflammatory agent.

If not specified the way, all technical and/or scientific terms used in this description have the same meaning as commonly understood by a specialist in the field of technology to which the invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, illustrative of the methods and/or substance described below. In the event of a conflict should be controlled by the specification, including definitions. In addition, substances, methods, and examples are only illustrative and not intended to be binding constraints.

Brief description of figures

Herein only as an example, described some embodiments of the invention with reference to the accompanying figures. In this section, with specific reference to the figures in detail, it is emphasized that presents the details are given as example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken together with the figures, makes obvious to a person skilled in the art how embodiments of the invention can be implemented in practice.

Figa-B are figures depicting analysis of the cell cycle 2D is attached to etoc placenta, suitable for use in accordance with these instructions (figa), or fixed cells obtained in accordance with the instructions of the patent WO/2007/108003 designated as PLX (pigv). The cells were fixed in 70% EtOH'ev O.N, centrifuged and resuspendable in a solution of iodide of propecia (PI) and then analyzed using FACS.

Figure 2 is a histogram depicting the expression of markers 2D attached cells of the placenta that are suitable for use in accordance with these instructions. It is noted that the negative expression was registered for CD11b, CD34, HLA-DR, CD14, CD19 and CD45, while positive expression was observed for CD29, CD73, CD90 and CD105.

Figure 3 is a histogram depicting the reduced response of cells of the lymphocytic series under the action of the 2D clip of placental cells that are suitable for use in accordance with these instructions. Mononuclear cells (MNCs) peripheral blood (PB) stimulated with PHA (10 μg/ml). One of four different batches of 2D attached cells were added to stimulated MNCs. Three parallel from each group were sown in 96-well plates.

Figa-F is a group of photos depicting the growth of bone marrow cells and placenta in osteogenic conditions or adipogenic lines of differentiation. Cells originating from bone marrow (figa-C),or cells, originating from the placenta (fig.4D-F), were sown in the growth medium (figa and 4D), in the environment for osteogenic differentiation (figv and 4E) or in the environment for adipogenic differentiation (figs and 4F) in 24-hole tablet coated with vitronectin and collagen. The medium was changed every 3-4 days. At the end of the growth period the cells were fixed, stained and photographed as described in the following examples section.

Figa-F is a group of photos depicting the growth of bone marrow cells and placenta in the modified conditions osteogenic or adipogenic lines of differentiation. Cells originating from bone marrow (figa-C), or cells derived from placenta (fig.5D-F), were sown in the growth medium (figa and 5D), the environment for osteogenic differentiation (figv and 5E) or in the environment for adipogenic differentiation (figs and 5F) in 24-hole tablet coated with vitronectin and collagen. The medium was changed every 3-4 days. At the end of the growth period the cells were fixed, stained and photographed as described in the following examples section.

On figa-B shows the cell cycle analysis of 3D clip-on cells obtained Plurix (denoted as PLX, pigv) and Celligen (PLX-C, figa). The cells were fixed in 70% EtOH'ev O.N, centrifuged and resuspendable in a solution of iodide of propecia (PI) and then analyzed using FACS.

On figa-C shows the expression of typical DL the fibroblast markers, but the lack of expression of typical endothelial markers on PLX-C. Piga depicts the negative expression of the endothelial marker CD31; figv depicted negative expression of endothelial marker KDR; and figs shows positive expression of the marker in human fibroblasts (D7-FIB). It is noted that the red histogram for isotype IgG1 (FITZ) represent the negative control, whereas the blue histograms represent the positively stained cells.

On figa-D shows the expression stimulatory and co-stimulatory molecules on the cell PLX-C. Piga depicts the expression of CD80 on PLX-C; figv depicts the expression of CD86 on PLX-C; figs depicts the expression of CD40 on PLX-C; and fig.8D depicts the expression of HLA-A/B/C PLX-C. Negative controls were obtained with fluorescent molecules of the appropriate isotype. It is noted that the red histograms indicate the population of cells expressing the marker PLX-C, blue histograms indicate the population of cells expressing a marker of bone marrow (BM), and green histograms indicate the population of cells expressing the marker of mononuclear cells (MNC).

On figa-B shows the inhibition of proliferation of lymphocytes under the action of PLX-C. Piga depicted tests mixed reactions lymphocytes (MLR), performed with 2×105mononuclear cells (MNC, donor A), about the coming of peripheral blood (PB), stimulated equal number of irradiated (30 j/kg) MNCs originating from PB (donor), followed by the addition of increasing quantities of cells PLX-C cultures. Three parallel from each group were sown in 96-well plates. The rate of proliferation was measured by incorporation of [3H]-thymidine; figv depicted MNCs originating from peripheral blood (PB), ConA stimulated (1.5 mg/ml). The cultures were added increasing the number of cells PLX-C. Three parallel from each group were sown in 96-well plates. The rate of proliferation was measured by incorporation of [3H]-thymidine.

On figa-C depicts the regulation of PLX-C secretion of proinflammatory and anti-inflammatory cytokines after co-culture with peripheral blood cells. On figa-B shows the secretion of IFNγ (figa) and TNFα (pigv) after culturing MNCs human-derived (isolated from peripheral blood), ConA stimulated with PLX-C; figs shows the secretion of IFNγ, TNFα and IL-10 after culturing MNCs human-derived (isolated from peripheral blood), LPS-stimulated, with PLX-C. Supernatant collected and analyzed for cytokines using ELISA.

11 is a histogram depicting the macroscopic evaluation of the tissues of the colon of mice with colitis in terms of Wal is ena. TNBS (mouse modeling colitis), TNBS+5-ASA (mice with colitis who received treatment on the gold standard), TNBS+2D attached cells (part 1)/br, TNBS+3D attached cells (PLX-C, part 2)/br, TNBS+2D attached cells (part 1)/and TNBS+3D attached cells (PLX-C, part 2)/century Macroscopic evaluation was performed blindly by two investigators.

Fig is a histogram depicting microscopic evaluation of tissues of the colon of mice with colitis indicator Ameho. TNBS (mouse modeling colitis), TNBS + 5-ASA (mice with colitis who received treatment on the gold standard), TNBS+2D attached cells (part 1)/br, TNBS+3D attached cells (PLX-C, part 2)/br, TNBS+2D attached cells (part 1)/and TNBS+3D attached cells (PLX-C, part 2)/century Histological evaluation was performed blindly by two investigators.

Fig is a histogram depicting the expression level of mRNA of IL-1β in the tissues of the colon of mice with colitis. In mice caused colitis by vnutribruchinnogo the introduction of TNBS and they were introduced 2D or 3D (PLX-C) attached cells intraperitoneal or intravenous route. Total RNA was isolated from tissues of the colon different experimental groups and the levels of expression of IL-1β was assessed using RT-PCR.

Fig represents the histogram, izobretatelskuju evaluation of the tissues of the colon of rats with colitis. In rats caused colitis by TNBS injection inside the colon and they were introduced PLX-C cells intraperitoneal (br) or intravenous (IV) route.

Description of specific embodiments of the invention

The present invention in some embodiments of implementation relates to methods of treatment of inflammatory diseases of the colon with the use of attached cells from adipose tissue or placental tissue, and more specifically, but not exclusively, to methods for treating ulcerative colitis or Crohn's disease using fixed cells.

The principles and process of the present invention can be better understood with reference to the figures and accompanying description.

Before a detailed explanation of at least one variant of the invention should be understood that the invention is not necessarily limited to its application to the details set forth in the following description or illustrated in the examples. Allowed other embodiments of the invention, or enforce it in practice or perform in a variety of ways. It should be clear also that used in the present description of the language and terminology used to describe and should not be construed as limiting.

When implementing the present invention on practitionerlater of the present invention unexpectedly discovered, that attached cells from the tissues of the placenta can be effectively used for the treatment of ulcerative colitis and Crohn's disease.

As shown in the present description below and in the following examples section, the inventor of the present invention discovered through complex experiments that attached cells derived from placental tissue or adipose tissue and grown in culture in 2D conditions (example 2) or 3D (examples 1 and 3) cultivation, can be effectively used to treat inflammation of the colon, such as ulcerative colitis, as shown on murine (example 4), and rat (example 5) experimental models. The inventor of the present invention have shown that intravenous (IV) or intraperitoneal (/br) introduction 2D or 3D clip-on cells of the present invention leads to a significant improvement in the inflammatory condition of the tissue of the colon that is defined using both macroscopic and microscopic evaluation of the colon (11, 12, and 14). This anti-inflammatory effect was as effective as the gold standard treatment with 5-ASA. Taken together, the instructions of the present invention describe the value of anti-inflammatory action is attached cells of the present invention and suggest their use for the treatment of inflammatory diseases of the colon the colon, such as ulcerative colitis and Crohn's disease.

Thus, in accordance with one aspect of the present invention features a method of treating ulcerative colitis or Crohn's disease in need of this individual, and the method includes the administration to an individual a therapeutically effective amount of attached cells from placental tissue, or adipose tissue, thereby making the treatment of ulcerative colitis or Crohn's disease.

Used in the present description, the term "treatment" refers to the prevention, treatment, handling, reducing, alleviating, minimizing, suppressing or halting the harmful effects of ulcerative colitis or Crohn's disease. Experts in the art should understand that in order to assess the development of the pathology can be used a variety of methods and tests and similar various methods and tests can be used to assess depression, remission or regression of disease.

Used in the present description, the term "ulcerative colitis" refers to a pathological condition of the intestine, a form of inflammatory bowel disease (IBD), specifically the large intestine or colon, that includes characteristic ulcers, or open grooves in the colon. Disease ulcerative colitis is usually diagnosed by the following recurrent symptoms - constant dia is it mixed with blood, with the gradual emergence. Ulcerative colitis in accordance with the instructions of the present invention applies to any stage or severity of ulcerative colitis (e.g., remission of the disease or the acute form of the disease).

Used in the present description, the term "Crohn's disease" refers to an inflammatory condition that can affect any part of the gastrointestinal tract from the mouth to the anus, also known as granulomatous colitis or regional enteritis, is a form of inflammatory bowel disease (IBD). Crohn's disease is a type of autoimmune disease and is usually diagnosed by the following recurrent symptoms - abdominal pain, diarrhea (which may be mixed with blood), vomiting, weight loss, skin rashes, arthritis and eye inflammation. Crohn's disease in accordance with the instructions of the present invention applies to any stage or severity of Crohn's disease (for example, to remission of the disease, the acute form of the disease, relapse).

Used in the present description the expression "requires that the individual" refers to a mammal, preferably to the individual, which is the person, male or female of any age, who has been diagnosed with probable or exact ulcerative colitis or Crohn's disease, such as an individual the mind, who moved inflammatory disease of the colon. Diagnosis of ulcerative colitis or Crohn's disease may include any diagnostic test, such as, for example, laboratory tests, endoscopic evaluation, a biopsy of the mucosa (for ulcerative colitis), implementation of the x-ray with barium (Crohn's disease) and CT or MRI scanning (Crohn's disease).

It should be understood that the present invention also involves the treatment of other inflammatory conditions of the colon, including, but not limited to, chronic inflammatory bowel disease (Garcia Herola A. et al., Gastroenterol Hepatol. 2000 Jan 23; (1):16), gluten enteropathy (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16; 138 (2):122) and REIT, using fixed cells of the present invention.

As mentioned in the present description above, the method in accordance with this aspect of the present invention is carried out by introducing the individual a therapeutically effective amount of attached cells from the placenta or adipose tissue.

Used in the present description, the expression "fixed cells" refers to a homogeneous or heterogeneous population of cells, the growth of which depends on the attachment, i.e. for growth in vitro requires attaching to the surface.

Used in the present description the expression "fatty tissue" refers to soybeans is internai fabric, which includes fat cells (adipocytes).

Used in the present description, the term "placenta tissue" refers to any part of the female organ of mammals, which lines the uterine wall during pregnancy is a shell of the fetus, to which it is attached via the umbilical cord. After birth, the placenta is expelled (and referred to as postpartum placenta). In the illustrative embodiment, the placenta refers to a placenta.

In accordance with the instructions of the present invention is attached cells derived from placenta or adipose tissue, can be reproduced using two-dimensional (2D) or three-dimensional (3D) culturing conditions.

Used in the present description, the expression "two-dimensional culture refers to a culture in which cells are placed in conditions that are compatible with cell growth, along with the fact that the cells are allow to grow in one plane. Conditions in two-dimensional culture inventions are created in such a way as to make possible the dissemination of attached cells.

Used in the present description, the expression "three-dimensional culture refers to a culture in which cells are placed in conditions that are compatible with cell growth, along with the fact that the cells to grow in more than one layer. It is quite clear that the environment of the cells in situ in W is the same organism (or tissue) is a three-dimensional structure. Cells are surrounded by other cells. They are supported in a complex network of fibers of the extracellular matrix of nanoscale, which allows you to create different local microenvironment. Its extracellular ligands mediate not only the attachment to the basal membrane, but also access to a variety of blood and lymphatic vessels. Oxygen, hormones and nutrients are transported to cells and wastes are removed. Conditions in three-dimensional culture of the invention are designed to simulate such an environment, as will be further illustrated below.

It should be clear that the conditions in two-dimensional and three-dimensional cultivation are such as to make possible the distribution of attached cells.

Used in the present description, the terms "pervasive" and "distribution" refer to the maintenance of cells essentially in the absence of their differentiation and, ultimately, in terms of cell growth, i.e. increase in the population of cells (e.g., at least 2 times) without differentiation accompanying this increase.

Used in the present description, the terms "supported" and "maintenance" refers to the regeneration of cells essentially in the absence of their differentiation, i.e. essentially to a stable cell population without differentiation, accompanying with the giving of such stability.

As mentioned, attached cells of this aspect of the invention derived from adipose tissue or placental tissue.

Cells of the placenta can be obtained from the placenta after a full-term pregnancy or placenta after premature birth. The placenta is preferably collected immediately after the outflow of blood. The placenta preferably has perfesional over a period of time sufficient to remove residual cells. Used in the present description, the term "perfusionist" or "perfusion" refers to the act of pouring or transmission fluid across or through the organ or tissue. Placenta tissue may occur from any mammal, for example, the source of placental tissue is the man. A suitable source of placental tissue is placenta after full-term pregnancy (for example, through 1-6 hours), but the source of the tissue or cells of the placenta or method for isolating placental tissue is not critical to the invention.

Attached cells derived from the placenta, can be derived from both fetal (i.e. amnion or internal parts of the placenta, see example 1)and maternal (i.e. basal decidual membranes and lining the uterus decidual membranes) parts of the placenta. Tissue samples are washed in physiological buffer [e.g., phosphate buffered saline (PD is) or buffer Hanks]. Suspension of single cells do by processing tissue hydrolytic enzyme (see below) and/or grinding and pumping the pieces of tissue through a nylon filter, or by careful pipetting (Falcon, Becton, Dickinson, San Jose, CA) washing buffer.

Attached cells derived from adipose tissue, can be isolated using a variety of methods known to experts in this field of technology. For example, such methods are described in U.S. patent No. 6153432. Adipose tissue may occur from omental/visceral fat, the adipose tissue of the mammary gland, gonads or other sources of adipose tissue. One source of adipose tissue is fat omentum. In humans, adipose tissue is usually distinguished by liposuction.

Allocated fixed cells of placenta or adipose tissue can be extracted by processing the tissue by hydrolytic enzymes, such as collagenase, trypsin and/or dispute; and/or effective concentrations of hyaluronidase or Gnkazy; and ethylenediaminetetraacetic acid (EDTA); at a temperature of between 25-50°C for periods from 10 minutes to 3 hours. Cells can then pass through a nylon or gauze filter with cells from 20 microns to 1 mm Cells are then subjected to differential centrifugation directly in the media or through the gradient Ficoll or Percoll or another to build the molecular gradient. Cells are centrifuged at speeds from 100 to 3000 x g for periods of from 1 minute to 1 hour at a temperature of between 4-50°C (see U.S. patent No. 7078230).

In addition to the attached cells derived from placenta or adipose tissue, the invention also contemplates the use of attached cells from other tissue sources, which are characterized by the phenotype of stromal stem cells (as will be further described herein below). The tissue sources from which can be extracted is attached cells include, but are not limited to, umbilical cord blood, scalp, hair follicles [for example, as described in patent application U.S. 20060172304], testes [for example, as described in article Guan K., et al., Nature. 2006 Apr 27;440(7088): 1199-203], mucous membrane of the nasal cavity [for example, as described in Marshall, CT., et al., Histol Histopathol. 2006 Jun;21(6):633-43], embryonic yolk SAC [for example, as described in article Geijsen N, Nature. 2004 Jan 8;427(6970): 148-54] and amniotic fluid [Pieternella et al. (2004) Stem Cells 22:1338-1345], all sources, as is known, include mesenchymal stem cells. Attached cells from these tissue sources can be selected by culturing cells on the surface to attach, isolating in this way is attached cells from other cells in the initial population.

Regardless of the source of the ICA (for example, placenta or adipose tissue) removing the cells are preferably produced in a sterile environment. After receiving the extracted cells give them a chance to bind to the substance to be attached (for example, with a configuration in the form of a surface) to highlight the result of adherent cells. Cultivation then occurs in the context of 2D (as described in example 2 of the examples section), and the cells can then be moved in the 3D conditions (as described in examples 1 and 3 of the examples section).

Used in the present description, the term "substance for attaching" refers to synthetic substance, a natural substance or their combination, altocomisionado (i.e. biologically compatible), having the chemical structure (for example, charged groups exposed on the surface)that can keep cells on the surface.

Examples of substances to be attached, which can be used in accordance with this aspect of the invention, include, but are not limited to, polyester, polypropylene, polyalkylene, polyphthalamide, polyvinyl chloride, polystyrene, polysulfone, cellulose acetate, glass fiber, ceramic particle, Matrigel, a component of the extracellular matrix (e.g., fibronectin, chondroitin, laminin, collagen, poly-L-lactic acid and inert metal fiber.

SL is blowing to consider, the seeding of the cells of placenta or adipose tissue is usually made when the density in the culture 3±0,2×103cells/cm2. After sowing cell culture is usually cultivated in an incubator for tissue cultures in moisture conditions with 5% CO2at 37°C.

Further stages of purification or enrichment of stromal stem cells can be produced using methods which are well known in the art (such as by using FACS using the expression of markers of stem cells, as will be further described herein below).

Non-limiting examples of key environments, suitable for cultivation in accordance with the invention, include minimum essential medium Needle, ADC-1, LPM (not containing bovine serum albumin), F10 (HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ medium (with or without modification Fitton - Jackson), a basic environment the Needle (BME-with the addition salts of the base of the Needle), the modified Dulbecco Wednesday Needle (DMEM without serum), Yamane, full-20, a modified Glasgow Wednesday Needle (GMEM), environment Leibovitz L-15, Wednesday McCoy''s 5A medium M199 (M199E - salt of the base of the Needle), medium M199 (M199H - salt base Hanks), minimum essential medium Needle (MEM-E - salt of the base of the Needle), minimum essential medium Needle (MEM-H - salt base Hanks) and minimum essential medium Needle (MEM-NAA with interchangeable amine is a acid), among many other containing medium 199, CMRL 1415, CMRL 1969, CMRL 1066, NCTC 135, MB 75261, MAB 8713, DM 145, Williams' G, Neuman &Tytell, Higuchi, MCDB 301, MCDB 202, MCDB 501, MCDB 401, MCDB 411, MDBC 153. The preferred environment for use according to the invention is DMEM. These and other suitable environments are commercially available, among others, from GIBCO, Grand Island, N. Y., USA, and Biological Industries, Bet HaEmek, Israel. Description of some of these environments are summarized in the book Methods in Enzymology, Volume LVIII, "Cell Culture", pp. 62 72, edited by William B. Jakoby and Ira H. Pastan, published by Academic Press, Inc.

The medium may be supplemented, for example, serum, such as serum fruits calves or other types, and optional or alternative growth factors, vitamins (e.g. ascorbic acid), cytokines, salts (e.g., B-glycerol), steroids (e.g. dexamethasone) and hormones such as growth hormone, eritropoyetina, thrombopoietin, interleukin-3, interleukin-6, interleukin-7, colony stimulating factor, macrophage, the ligand of c-kit/stem cell factor, the ligand of osteoprotegerin, insulin, insulin-like growth factors, epidermal growth factor, factor growth of fibroblasts, nerve growth factor, ciliary neurotrophic factor, growth factor from platelets and morphogenic protein bone concentrations from PG/ml to milligram/ml

Additionally recognize that culture is the ed may be added as additional components. Such components may include antibiotics, antifungal agents, albumin, amino acids, and other components known in the field of technology of cultivation of cells. Additionally, there may be added components to increase during differentiation, when it is necessary (see extras below).

You should take into account that when attached cells of the invention are introduced to the individual, which is the person, the cells and culture medium (for example, with the above-described additives environment) should be essentially free of xenobiotics, i.e. devoid of any impurities of animal origin, such as Mycoplasma. For example, the culture medium may contain additives Vice serum, addition of human serum and/or factors obtained by synthetic or recombinant means.

As mentioned, once attached, the cells are obtained, they can be sown in 2D or 3D environment (see examples 1, 2 and 3 of the examples section below). It should be borne in mind that the cells can be transferred to the matrix with a 3D configuration directly after separation or, alternatively, they can be planted in a 3D environment after 2D conditions (as mentioned in the present description above).

You should take into account that in the process of cultivation in terms of 2D attached cells m is tenderly constantly to reseed. In accordance with the embodiment of the present invention, the cells can be Persiani for at least 4 passages, at least 5 passages, at least 6 passages, at least 7 passages, or at least 8 passages. It should be clear that cells usually subcultured when the culture reaches about 70-80% of confluently, usually within 3-5 days (1.5-2 udayani). Moreover, under cultivation in the conditions of 2D cells can be grown in culture medium devoid of additives antibiotics for at least 2 passages, at least 3 passages, or at least 4 passages.

Thus, in the case of 2D culture cultivation of produce for about at least 2 days, 3 days, 4 days, 5 days, 10 days, 20 days, months or even more. To increase the number of cells can also produce pereselenie. It should be clear that the culture medium may be replaced in order to extend or improve the conditions of cultivation.

2D adherent cells can be collected when proliferate, at least approximately 12% of the cells, in order to avoid uncontrolled differentiation and aging.

2D adherent cells of some embodiments of the present invention include at least about 10%, 28%, 30%, 50%, 80% or more about iterative-active cells (as may be assessed by monitoring S and/or G2/M phases by FACS).

As mentioned, attached cells can be transferred into a 3D environment.

Thus, the substance to attach to this aspect of the invention is shaped for 3D cultivation, thereby providing a matrix for growth, which significantly increases the available contact surface for attachment of the cells so that creates a simulation infrastructure tissues (e.g. placenta).

To obtain large-scale cultivation can be done in the 3d bioreactor.

Examples of such bioreactors include, but are not limited to, the bioreactor flow displacement, bioreactor with continuous stir tank, bioreactor with a porous layer, bioreactor system CelliGen Plus® (New Brunswick Scientific (NBS) or bioreactor system BIOFLO 310 (New Brunswick Scientific (NBS).

As shown in example 3 of the examples section, the Celligen bioreactor capable of 3D expansion of attached cells under controlled conditions (e.g. pH, temperature, and oxygen level) and at a constant perfusion medium for continuous cell growth. Moreover, in cell cultures, it is possible to directly monitor the concentration levels of glucose, lactate, glutamine, glutamate and ammonium. The rate of consumption of glucose and the rate of formation of lactate is attached cells makes it possible to measure the speed kletochnogo and to determine the time of collection.

Other 3D bioreactors, which can be used in the invention include, but are not limited to, bioreactor with continuous stir tank, in which the culture medium is continuously fed into the bioreactor, and the product is continuously output to maintain a constant stationary phase in the reactor. Bioreactor with a mixing tank with a drum with a fibrous substrate is commercially available, for example, New Brunswick Scientific Co., Edison, NJ, bioreactor with a stationary substrate, the bioreactor with the rise of air flow, where the air is usually fed through the bottom of the Central child pipe, rises up with the bubbles and releases the exhaust gas at the top of the column, with perfusion bioreactor inoculated cells using polyactive pen [as described in article Wendt, D. et al., Biotechnol Bioeng 84: 205-214, (2003)], the perfusion bioreactor with radial flow with a tubular porous frameworks of poly-L-lactic acid (PLLA) [as described in Kitagawa et al., Biotechnology and Bioengineering 93(5): 947-954 (2006)]. Other bioreactors, which can be used according to the invention described in U.S. patent№№ 6277151, 6197575, 6139578, 6132463, 5902741 and 5629186.

The seeding of the cells is preferably at 100000-1500000 cells/mm at the time of sowing. In the illustrative embodiment, planted a total of 150±30×106cells planted 3-5×10 6cells/g of carrier or duration of 0.015 to 0.1×106cells/ml

The cultivation is performed for at least about 2 days, 3 days, 4 days, 5 days, 10 days, 20 days, months or even more. It should be clear that cultivation in the bioreactor can extend this period. Cultivation of attached cells in 3D culture can be performed in continuous flow culture medium. To increase the number of cells may also be pereselenie. You must also take into account that the culture medium can be replaced to extend or improve the conditions of cultivation.

In accordance with the embodiment of the present invention cultivation of attached cells in 3D culture can be done in terms of perfusion of culture medium. Usually the rate of perfusion is determined by the concentration of glucose in the culture medium is attached cells. Thus, in accordance with the instructions of the present invention the culture medium may be replaced, when the glucose concentration of approximately 500 mg/l, approximately 550 mg/l, or approximately 600 mg/L.

3D adherent cells can be collected when proliferate, at least about 10% of the cells, in order to avoid uncontrolled differentiation and staren who I am.

3D adherent cells of some embodiments of the present invention include at least about 10%, 28%, 30%, 50%, 80% or more proliferative-active cells (as may be assessed by monitoring S and/or G2/M phases by FACS).

Adherent cells of some embodiments of the present invention may include at least one phenotype of stromal stem cells".

Used in the present description, the term "phenotype of stromal stem cell" refers to a structural or functional phenotype typical of stromal cells (i.e. mesenchymal) cells originating from bone marrow.

Used in the present description the expression "stem cell" refers to a cell that has not passed the final differentiation.

Thus, for example, cells may be spindle shaped. Alternative or additionally, cells can Express the marker or set of markers (for example, the marker surface), typical of stromal stem cells. Examples of surface markers of stromal stem cells (positive and negative) include, but are not limited to, CD105+, CD29+, CD44+, CD73+, CD90+, CD3-, CD4-, CD34-, CD45-, CD80-, CD19-, CD5-, CD20-, CD11B-, CD14-, CD19-, CD79-, HLA-DR - and FMC7-. Other markers of stromal stem cells include, but are not limited to, tyrosine is roxelana, nestin and H-NF.

In accordance with a specific embodiment of the present invention is attached cells do not Express Oct-4.

It should be clear that 2D is attached cells placental tissue, created in accordance with the instructions of the present invention, have the profile of gene expression essentially as described in table 8 the next section examples. At the same time, 3D is attached cells placental tissue, created in accordance with the instructions of the present invention, have the profile of gene expression essentially as described in table 11 the next section examples.

In accordance with an illustrative embodiment of 2D and 3D clip-on cells of the present invention is less commiteeman to differentiation towards osteogenic or adipogenic line compared to attached cells from the bone marrow, growing and differentiating in the same conditions.

Examples of functional phenotypes typical of stromal stem cells include, but are not limited to, suppressor activity against T-cells (they do not stimulate T-cells, and visa versa) and activity-aware of haematopoietic stem cells.

In accordance with one embodiment of the invention attached cells of the invention is capable of being in order to suppress an immune response in an individual.

Used in the present description, the expression "the suppression of the immune response in the individual" refers to the reduction or inhibition of the immune response that occurs in an individual in response to an antigen (e.g., alien cell, or part of it). Immune response, which can be suppressed attached cell, includes the humoral immune response and cellular immune responses, which include specific recognition of antigens of a pathogen using antibodies and T-cell proliferation T cells), respectively.

As shown in examples 4-5 the next section examples of 2D and 3D clip-on cells of the present invention, as detected, induce anti-inflammatory effects in inflammatory conditions of the colon. It should be further understood that this effect may be mediated by cells or secretively their factor with anti-inflammatory action even in the absence of cells. Thus, the fixed cells of the present invention preferably can be used to treat inflammation of the intestine, such as in case of ulcerative colitis and Crohn's disease.

The expression "the introduction of the individual" refers to the introduction of the cells of the invention into the target tissue. Cells can occur from the recipient or from allogeneic or xenogeneic donor. This expression is s also covers "transplantation", "replacement cells or transplantation of cells of the invention the individual.

According to a particular variant embodiment of the invention attached cells you can enter individual by any means known to a person skilled in the technical field, for example by intravenous (IV), intramuscular (I/m) or intraperitoneal (/br) introduction.

Cells that can be entered in accordance with this aspect of the invention, include the above-described fixed cells that can be grown in three-dimensional or two-dimensional environment, and their partially or completely differentiated mesenchymal and amazingingly derivatives.

Methods of obtaining lines of differentiation of specific cells from stromal stem cells of the invention are well known in the art. See, for example, U.S. patent No. 5486359, 5942225, 5736396, 5908784 and 5902741.

Cells may not be subjected to any influence or be genetically modified so as to be directed toward the desired line of differentiation (see patent application U.S. No. 20030219423).

Cells can be derived from autologous or neautrogena source (i.e., allogeneic or xenogeneic) fresh or frozen (e.g., cryogenic canned) products.

As neautrogena cells can induc is its immune response when introduced into the body, developed several approaches to reduce the likelihood of rejection neautrogena cells. They include or suppress the immune system of the recipient, or encapsulating neautrogena cells in isolation from the immune system, a semi-permeable membrane prior to transplantation.

Methods of encapsulation are usually classified as microencapsulation, including small spherical fillers, and microcapsulation, including larger sheet layered membrane and the membrane of the hollow fibers (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64).

Methods of obtaining microcapsules known in the art and include, for example, disclosed Lu MZ, et al., Cell encapsulation with alginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol Bioeng. 2000, 70: 479 - 83, Chang TM and Prakash S. Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu MZ, et al., A novel cell encapsulation method using photosensitive poly(allylamine alpha - cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.

For example, microcapsules are obtained by integration of the modified collagen triple copolymer shell-2-hydroxyethylmethacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), which leads to the thickness of the capsule 2-5 microns. Such microcapsules can be further encapsulated in an additional 2-5 µm membrane of the ternary copolymer to give the negative the positive charge of the smooth surface and to minimize the absorption of plasma proteins Chia, S.M. et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56).

Other microcapsules are created on the basis of alginate marine polysaccharide (Sambanis, A. Encapsulated islets in diabetes treatment. Diabetes Technol. Ther. 2003, 5: 665-8) or its derivatives. For example, microcapsules can be obtained by polyelectrolyte complexation of polianionov of sodium alginate and sodium sulfate pulp with polycation hydrochloride, poly(methylene-co-guanidine)and in the presence of calcium chloride.

It should be clear that the encapsulation of the cells is improved when using smaller capsules. Thus, quality control, mechanical stability, diffusion properties, and activity in vitro encapsulated cells is improved when the size of the capsule is reduced from 1 mm to 400 microns (L. Canaple et al., Improving cell encapsulation through size control. J Biomater Sci Polym Ed. 2002; 13:783-96). Moreover, biocapsule with nanopores with well-controlled pore size as small as 7 nm, with chemically tailored surfaces and exact microcephala, as found, create successful microenvironment for immunoisolation cells (Williams D. Small is beautiful: microparticle and nanoparticle technology in medical devices. Med Device Technol. 1999, 10: 6-9; Desai, T.A. Microfabrication technology for pancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).

Examples of immunosuppressive agents that may be used include, but are not limited to, methotrexate, cyclophosphamid is, cyclosporine, cyclosporine a, chloroquin, hydroxychloroquin, sulfasalazin (sulfasalazine), gold salts, D-penicillamine, Leflunomide, azathioprine, anakinra, infliximab (REMICADE), etanercept, blockers of TNF-alpha, biological agent, the target of which are inflammatory cytokines, and nonsteroidal anti-inflammatory drugs (NSAIDs). Examples of NSAIDs include, but are not limited to, acetylsalicylic acid, holinsalitsilat magnesium, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, Ketoprofen, Ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.

Depending on the pathological condition of the individual, you can enter additional chemical drugs (e.g., immunomodulatory, chemotherapeutic, anti-inflammatory and so on) or cells.

For the treatment of inflammatory conditions of the colon, including ulcerative colitis and Crohn's disease, can be used any treatment known to a person skilled in the art including, for example, aminosalicylate (for example, mesalazine, balsalazide, olsalazine), corticosteroids (e.g., cortisone, prednisone, prednisolone, cartitem, hydrocortisone, methylprednisolone, becom tazon, budesonide), immunosuppressive drugs (e.g., mercaptopurine, azathioprine, methotrexate, tacrolimus), biological treatment (e.g., infliximab, visilizumab), heparin, low molecular weight (LMWH), modification of diet (e.g., fiber) and surgery.

The individual can also enter an anti-inflammatory agent, such as, but not limited to, alclofenac; alklometazon dipropionate; algestone acetonide; alpha-amylase; antenatal; aminated; amfenac sodium salt; amiprilose hydrochloride; anakinra; aniolek; unitrazepam; Amazon; balsalazide disodium salt; bendazac; benoxaprofen; benzydamine hydrochloride; bromelain; properly; budesonide; carprofen; cicloprofen; zintzen; lipofen; clobetasol propionate; clobetasone butyrate; kopinak; claudicationa propionate; Normethadone acetate; ortodoxo; deflazacort; desonide; desoximetasone; dexamethasone dipropionate, diclofenac potassium salt; diclofenac sodium salt; diflorasone diacetate; diploidea sodium salt; diflunisal; difluprednate; deltalon; dimethylsulfoxide; tracename; andreson; enlimomab; anayama sodium salt; epirizole; etodolac; etofenamate; felbinac; panamal; fenbufen; fenclofenac; vinklarek; fendosal; tinpiple; fentiazac; flusalan; flashcard; floranova acid; flunizol; flunisolide and the Etat; flunixin; flunixin meglumin; fluocortin butyl; permetrina acetate; flucuate; flurbiprofen; Floralife; fluticasone propionate; paraprotein; furubotn; halcinonide; halobetasol propionate; halopedia acetate; ibufenac; ibuprofen; ibuprofen aluminum salt; ibuprofen of Picanol; ilonday; indomethacin; indomethacin sodium salt; indoprofen; indexa; entresol; isoflupredone acetate; isoxepac; isoxicam; Ketoprofen; levamisole hydrochloride; amoxicil; loteprednol etabonate; meclofenamate sodium salt; meclofenamic acid; Malorita dibutyrate; mefenamovaya acid; mesalamine; Meselson; methylprednisolone, sulatan; modifiant; nabumetone; naproxen; of naproxen sodium salt; naproxen; namazon; olsalazine sodium salt; orgotein; herpanacine; oxaprozin; oxyphenbutazone; phrenilin hydrochloride; pentosan polysulfate sodium salt; fenbutatin nitroglycerin; pirfenidone; piroxicam; piroxicam cinnamate; piroxicam alamin; pirprofen; prenasal; prifile; Prozorova acid; Poquoson; proxitol; proxitol citrate; rimexolone; romazarit; calculex; salnacedin; salsalate; Sanguinaria chloride; seclusion; serotonin; sudoxicam; sulindac; suprofen; tolmetin; talniflumate; talksalot; tebufelone; tenidap; tenidap sodium salt; tenoxicam; testam; taskid; tetragamy; typing;tixocortol pivalate; tolmetin; tolmetin sodium salt; triclinic; trifluralin; zidometacin; zomepirac sodium salt.

In any of those described herein methods cells can be entered either as such or, preferably, as part of pharmaceutical compositions, which optionally include pharmaceutically acceptable carrier.

Used in the present description, the term "pharmaceutical composition" refers to the drug attaches itself to the cells of the invention (i.e. attached cells from the placenta or adipose tissue, which is obtained after 2D or 3D cultivation), with other chemical components such as pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration of cells to the individual.

In the present description hereinafter, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation of the individual and does not abrogate the biological activity and properties of the input connections. Examples of media without restrictions are propylene glycol, saline solution, emulsion, or mixture of organic solvents with water.

In the present description, the term "filler" refers to an inert substance added to a pharmaceutical composition to further ease the introduction of the connection. Examples of fillers without restrictions include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

In accordance with a preferred embodiment of the invention, the pharmaceutical carrier is an aqueous physiological saline solution.

Methods of preparation and administration of drugs may be found in the "Remington''s Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated into this description by reference.

The pharmaceutical composition can be entered systematically (as described herein above). Alternatively, the pharmaceutical composition can be entered locally, for example by injection of the pharmaceutical composition directly into a tissue region of the patient.

The pharmaceutical compositions of the invention can be obtained using methods well known in the art, for example by means of conventional mixing, dissolving, granulating, creating bean, grinding into powder, emulsifying, encapsulating, on or lyophilization.

Pharmaceutical compositions for use in accordance with the invention, thus, can be prepared in the traditional way using one or more fisiologicas is acceptable carriers, including fillers and auxiliary agents which facilitate processing of the active ingredients into preparations which can be used pharmaceutically. The appropriate structure depends on the chosen route of administration.

For injection, the active ingredients of the pharmaceutical compositions can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks solution, ringer's solution, physiological saline solution or freezing medium containing cryoconserved. For insertion through the mucous in the composition of the penetrants are used, suitable for penetration through the barrier. Such penetrants are usually known in this technical field.

Determination of a therapeutically effective amount really is within the abilities of experts in this field of technology, particularly in light of the proposed in the present description detailed disclosure.

For any preparation used in the methods of the invention, therapeutically effective amount or dose can be determined initially from tests in cell culture in vitro. Preferably the dose is determined in animal models to achieve the desired concentration or titer. Such a composition can be used to more accurately determine useful doses in humans.

The toxicity of therapeutic effectiveness of the active ingredients, described herein, can be determined using standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. Data obtained from these tests in vitro and in cell cultures and animal studies can be used to determine the range of dosages for use in humans. The dosage may vary depending on the shape of the dosage and the route of administration. The exact composition, route of administration and dosage can choose the treating of the individual physician, taking into account the patient's condition (see, e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).

The number of dosed composition and interval may be adjusted individually to the levels of the active ingredient which are sufficient for the effective regulation of the synthesis of neurotransmitters implanted cells. Dosages necessary to achieve the desired effect should depend on individual characteristics and route of administration. To determine concentrations in plasma can be used methods of their detection.

Depending on the severity of the condition and the response to treatment dosing can be a single or multiple introduction during the course of treatment lasting from several days to several weeks or until it reaches the Eisenia pathological conditions.

A number of compositions for injection should, of course, depend on the individual being treated, severity of the disease, the route of administration, the decision of the doctor prescribing the treatment, and so the Dosage and timing of treatment should depend on a careful and constant monitoring of the changing state of the individual.

Models of inflammatory diseases of the colon include animal models of ulcerative colitis, such as, but not limited to, colitis, induced trinitrobenzenesulfonic acid (TNBS), in rats and mice [Komori et al., J Gastroenterol (2005) 40: 591-599; and examples 4-5 in the present description below].

Compositions comprising the product of the invention prepared with pharmaceutically compatible carrier, can also be obtained, placed in an appropriate container and labeled for treatment of the specified condition.

Compositions of the invention may be provided, if desired, in a package or the metering device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The package may, for example, comprise metal or plastic foil, such as blister packaging. Packing or dosing device can be accompanied by instructions for administration. The pack or dispenser may also be provided in the notification the receiving, associated with the container in form prescribed by a governmental Agency regulating the manufacture, use or sale of pharmaceutical agents, and this notice reflects approval by the Agency of the form of the compositions or administration to humans or animals. Such notice, for example, can be labeled with approval by management under the control over products and medicines of the USA for prescription drugs or liner approval of the product.

Attached cells of the invention can be suitably made in the form of pharmaceutical compositions, which can be properly Packed in the form of product production. This product includes markings for use in the treatment of ulcerative colitis or Crohn's disease, packaging material, which is Packed pharmaceutically effective amount of attached cells from the placenta or adipose tissue.

It should be clear that the product may optionally include an extension of the medicinal product for the treatment of inflammatory conditions of the colon, including, for example, anti-inflammatory agents, immunomodulatory agents, anti-inflammatory agents and other drugs for the treatment of inflammatory conditions of the colon (as described in more detail in esteem document above).

Used in the present description, the term "about" refers to ±10%.

The terms "includes", "include", "includes", "including", "having" and their cognates mean "including, but not limited to".

The term "comprising" means "including and limited to".

The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, stage and/or parts, but only if the additional ingredients, stage and/or parts not actually alter the basic and novel characteristics of the claimed composition, method or structure.

Used in the present description only form "a", "an" and "the" include references to the plural, unless the context clearly indicates otherwise. For example, the term "connection" or "at least one connection can include a variety of compounds, including mixtures thereof.

Throughout this application, various embodiments of the present invention can be presented in the format ranges. It should be clear that the description in the format ranges are given merely for convenience and brevity and should not be construed as a strict limitation of the scope of the invention. Accordingly, the description of the range should be viewed as a necessary disclosure of all possible subgroups of the range is a, as well as individual digital values within the range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subgroup range, such as from 1 to 3, 1 to 4, from 1 to 5, 2 to 4, from 2 to 6, from 3 to 6 and so on, as well as individual numbers within that range, for example 1, 2, 3, 4, 5 and 6. This applies regardless of the breadth of the range.

Whenever in the present description indicates the digital range, it is understood that it includes any quoted numbers (fractional or whole) within the specified range. The expression "wingspan ranges/ranges between"a first indicate number and a second indicate number and magnitude ranges/ranges from" a first indicate number of "up", the second specifies the number, they are used in the present description are interchangeable and mean the inclusion of the first and second indicated numbers and all the fractional and integer numbers between them.

Used in the present description, the term "method" refers to the manner, methods, techniques and procedures for the implementation of this task, including, but not limited to, such actions, methods, techniques and procedures either known to, or readily developed based on the known mode of action, methods, techniques and procedures by practitioners of the chemical, pharmacologists who eskay, biological, biochemical and medical fields.

It should be clear that certain features of the invention, which for clarity are described in the context of separate embodiments, may also be offered in combination in a single embodiment. Conversely, various features of the invention, which, for brevity, described in the context of a variant of implementation, can also be offered separately or in any suitable more fractional combination or as it is suitable for any other described embodiment of the invention. Certain distinctive features that are described in the context of the various embodiments should not be regarded as a necessary distinguishing features of these embodiments up until an implementation option is not effective without these elements.

Various embodiments of aspects of the present invention, as defined in the present description above and as stated in the section of the claims below, find experimental confirmation in the subsequent examples.

EXAMPLES

Currently, reference is made to the following examples, which together with the above description illustrates the invention in a non-limiting form.

In General, the COI is lizama in the present description nomenclature used in the present invention laboratory methods include molecular, biochemical, microbiological methods and techniques of recombinant DNA. Such methods are fully disclosed in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); the methods presented in U.S. patent No. 4666828; 4683202; 4801531; 5192659 and 5272057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunotest widely described in the patent and scientific literature, see, for example, U.S. patents№№ 3791932; 3839153; 3850752; 3850578; 3853987; 3867517; 3879262; 3901654; 3935074; 3984533; 3996345; 4034074; 4098876; 4879219; 5011771 and 5281521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "reduced and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. L, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996), all of which are incorporated by reference as if fully set forth in esteem description. Throughout this paper proposes other General references. Used in this description of the methods that are supposed to be well known in the art and are available for the convenience of the reader. All the information included in this description by reference.

EXAMPLE 1

Methods of creating 3D attached cells from the placenta

Attached cells were obtained as previously described (see WO/2007/108003), in a bioreactor system containing 3D media to obtain 3D-attached cells (referred to in this description as PLX).

Materials and experimental procedures

Attached cells from the placenta

The internal part of the full-term placenta (medical center, Bnei Zion, Haifa, Israel) were cut under sterile conditions, washed 3 times with buffer Hank and incubated for 3 hours at 37°C With 0.1% collagenase (1 mg/ml tissue; Sigma-Aldrich, St. Lewis, MO). By gentle pipetting suspended cells were then washed in DMEM with the addition of 10% FCS, a mixture of Pen-Strep-Nystatin (100 U/ml:100 μg/ml:of 1.25 U/ml) and 2 mm L-glutamine, were sown in vessels of 75 cm2and incubated at 37°C in incubator for cell cultures in moisture conditions with 5% CO2.

Two-dimensional (2D) growing cells

The cells were allowed to attach to the surface of plastic within 72 hours, last is what the medium was changed every 3-4 days. After 2-3 passages the cells were kept at cryogenic temperature were thawed and were sown for re-growing in containers. After reaching 60-80% of confluently cells were separated from the vessel for growing using 0.25% trypsin-EDTA and were sown in new vessels (typically every 3-5 days) with an additional 2-5 passages. Thereafter, the cultured cells were collected for analysis or for cultivation in bioreactors.

PluriX bioreactorTMPlug Flow

PluriX bioreactorTMPlug Flow (Pluristem, Haifa, Israel; see U.S. patent No. 6911201 and WO/2007/108003) downloaded 1-100 ml 3-dimensional porous media (4 mm in diameter), made of a matrix of non-woven cloth or polyester. These carriers are able to provide the reproduction of a large number of cells in a relatively small volume. Glassware was designed and manufactured Pluristem (Pluristem, Haifa, Israel). The bioreactor was placed in the incubator at 37°C at a flow rate regulated and monitored through the valve and peristaltic pump. The bioreactor contains a port for introduction of the sample and injection to ensure the consistent seeding of cells. The culture medium with a pH of 6.7 to 7.4 came out of the tank. The tank has introduced the filtered gas mixture containing air/CO2/O2in different proportions, depending on the density of cells in the bio is auctore. The share of O2ever the level of dissolved O2at the outlet of the bioreactor, which is defined by monitor. The gas mixture is directed into the tank through the silicone tube or diffuser (Degania Bet, Emek Hayarden, Israel). The culture medium was passed through the separating container for the collection circulating, unattached cells. The recirculation was provided by a peristaltic pump. The bioreactor was further provided with an additional port for introducing the sample and containers for continuous replacement of the environment.

Obtaining 3D-attached cells (PLX)

Confluently primary 2D culture attached in human cells, grown as described above were treated with trypsin, washed, resuspendable in DMEM with the addition of 10% FCS, a mixture of Pen-Strep-Nystatin (100 U/ml:100 μg/ml:of 1.25 U/ml) and 2 mm L-glutamine and were sown (103-105cells/ml) port for injection on 3D media in a sterile bioreactor Plug Flow. Before inoculation of the bioreactor was filled with PBS-Ca-Mg (Biological Industries, Beit Ha emek, Israel), autoclaved (120°C, 30 min) and washed with culture medium on Dulbecco containing 10% inactivated by heating the serum fruits of the calves and the mixture Pen-Strep-Nystatin (100 U/ml:100 μg/ml:of 1.25 U/ml). The flow rate was maintained at a level of 0.1-5 ml/min, the Process of sowing included cessation of circulation for 2-48 h, which is thus on the Valo an opportunity for the cells to settle to the media. The bioreactor was maintained under conditions of controlled temperature (37°C) and pH (pH of 6.7 to 7.4) using an incubator supplied with sterile air and CO2as needed. The culture medium was changed 2-3 times per week. The circulating medium was replaced with fresh DMEM every 4 hours to 7 days. When the density of cells 1×106-1×107cells/ml (after 12-40 days of cultivation) of the bioreactor was removed the whole environment and the bioreactor and the media were washed 3-5 times SFR. 3D-attached cells were separated from media by using trypsin-EDTA (Biological Industries, Beit Ha emek, Israel; 3-15 minutes with mild stirring, 1-5 times) and then resuspendable in DMEM and stored at cryogenic temperatures.

EXAMPLE 2

Methods of obtaining 2D-fixed cells for use in accordance with these instructions and obtain thereby 2D attached cells

Received 2D attached cells exhibiting properties different from the properties described above 3D attached cells (PLX, example 1). After that 2D is attached cells from the bone marrow or placenta were grown in conditions that stimulate differentiation of osteocytes or adipocytes.

Materials and experimental procedures

The process of obtaining 2D attached cells

Obtaining human tissues

All of the placenta were obtained in the maternity ward with about the Abrene Committee of Helsinki on medical care. Accordingly, all donors placenta was signed informed consent and were screened donors and testing of donors (IPC1). Immediately upon receipt of the placenta from the donor (during the procedure caesarean section) were placed in a sterile plastic bag and then in a foam box with bags of ice. The placenta was removed and immediately placed in quarantine zone to obtain permission for use by quality control (QC) and quality assurance (QA). All subsequent phases of obtaining produced in quarantine, adapted the clean room before entering the approved QC test results for Mycoplasma, and cells were isolated for 2D cell cultivation.

The selection and processing of attached cells

First, the placenta was cut into pieces under aseptic conditions in a fume hood with laminar flow, washed with buffer solution Hank and incubated for 3 hours at 37°C With 0.1% collagenase (1 mg collagenase/ml tissue). Added 2D cellular environment (2D environment, including DMEM with addition of 10% FBS, 0.25 microgram/ml Fungizone and 50 μg/ml gentamicin) and digested tissue is roughly filtered through a sterile metal grid, collected in a sterile beaker and centrifuged (10 min, 1200 rpm, 4°C). Using easy pipetting suspended cells were then washed 2D environment with doba the population of antibiotics, sown in 80 cm2vessels and incubated at 37°C in incubator for tissue culture in moisture conditions with a flow of 5% CO2. After 2-3 days, during which the cells are allowed to attach to the surface of the vessel, the cells were washed SFR and added a 2-dimensional environment.

Two-dimensional (2D) growing cells

Before the first passage of the samples environment for growing at 10% of the total number of vessels in quarantine were combined and passed testing Mycoplasma (IPC2). If it was found that cells negative in relation to Mycoplasma (set EZ-PCR Micoplasma, Biological Industries, Israel), cells were released from quarantine. After 1-2 additional passages of the cells was transferred into a clean room to obtain 2D culture (2DP). In the room 2DP cultivation was continued by another 3-5 passages (note that cells were grown in 2D environment with the addition of antibiotics to the passage 2, and then were grown in 2D environment without antibiotics). After passage of the 4 selected sample IPC-3 to determine the immunological phenotype. During the cultures were grown in an incubator for tissue culture in moisture conditions with 5% CO2at 37°C. After a total of 6-8 passages (9-16 doublings of cells) cells was collected and stored at cryogenic temperatures as stock 2D cells (2DCS).

The first passage is usually carried out after 10-15 bottom is. Since passage 2 until the passage of 6-8 cells were passively when the culture 70-80% of confluently, usually within 3-5 days (1,5-2 double). Cells were separated from blood vessels using 0.25% trypsin-EDTA (4 minutes at 37°C) and were sown with obtaining density culture 3±0,2×103cells/cm2. The size of the vessels for tissue culture increased in accordance with the continuation of the passages. The process of cultivation began in the vessel to tissue culture 80 cm2continued 175 cm2then 500 cm2(triple vessel) and, finally, the cells were sown in cell factory 10 trays (6320 cm2).

Before storage at cryogenic temperatures at the end of the cultivation period 2DSC collected culture medium and received a sample to send in GLP certified laboratory for testing Mycoplasma (IPC4).

Cryopreservation procedure stored product 2D cells

For cryopreservation 2DCS 2D cultured cells were collected in aseptic conditions using 0.25% trypsin-EDTA. Cells were centrifuged (1200 rpm, 10 min, 4°C)were calculated and resuspendable in a 2D environment.

For freezing the cell suspension was diluted 1:1 2D environment for freezing (final concentration was 10% DMSO, 40% STR and 50% 2D environment). From one placenta has received approximately 1.5 to 2.5×109cells. 4 ml of cells were stored at con who offered a concentration of 10×10 6/ml in 5-ml polypropylene containers for cryopreservation. The vessels were marked and transferred to the freezer with a controlled cooling rate for a process with a gradual decrease in temperature (1°C/min), and then transferred for storage in the gas phase of the freezer liquid nitrogen in the storage room for cooling. This material was designated as reserved party 2D cells (2DCS).

Analysis of the cell cycle

2D is attached cells and PLX cells were fixed with 70% EtOH'ev O.N, centrifuged and resuspendable in the solution of propecia iodide (PI)containing 2 μg/ml PI (Sigma), 0.2 mg/ml RNase A (Sigma) and 0.1% (vol./about.) Triton (Sigma) for 30 minutes. Cell cycle was analyzed using FACS.

Matrix (micrometric) for analysis of gene expression

Attached cells were obtained from full-term human placentae and were propagated using 2D cultures or in accordance with the instructions WO/2007/108003 (as described in examples 1-2). For further analysis were obtained on three different batches of cells using each of the methods of reproduction.

Cells were extracted RNA (kit Qiagen-Rneasy micro) and were applied to the matrix Affymetrix expression of the whole genome GeneChip® Human Exon 1.0 ST Array (Affymetrix, Santa Clara, California, USA).

Analysis of membrane markers using FACS

Cells were stained with monoclonal antibodies as described is about before. Briefly, 400000-600000 cells suspended in 0.1 ml of buffer for the flow cytometer in 5-ml test tube to test for 15 minutes at room temperature (RT) in the dark with each of the following monoclonal antibodies (MAbs): conjugated with FITC MAb against human CD29 (eBioscience), conjugated with PE MAb against human CD73 (Becton Dickinson), conjugated with PE MAb against human CD105 (eBioscience), conjugated with PE MAb against human CD90 (Becton Dickinson), conjugated with FITC MAb against human CD45 (IQProducts), conjugated with PE MAb against human CD19 (IQProducts), conjugated with PE MAb against human CD14 (IQProducts), conjugated with FITC MAb against HLA-DR human (IQProducts), conjugated with PE MAb against human CD34 (IQProducts), conjugated with FITC MAb against human CD31 (eBioscience), conjugated with FITC MAb against human KDR (R&D systems), MAb against marker of fibroblasts (D7-FIB) (ACRIS), conjugated with FITC MAb against human CD80 (BD), conjugated with FITC MAb against human CD86 (BD), conjugated with FITC MAb against human CD40 (BD), conjugated with FITC MAb against HLA-ABC man (BD), conjugated with FITC-isotype IgG1 (IQ Products), conjugated with PE isotype IgG1 (IQ Products).

Cells were washed twice with a buffer for the flow cytometer, resuspendable in 500 μl of buffer for the flow cytometer and analyzed using flow cytometry with the use of the flow cytometer FC-500 (BeckmanCoulter). Negative controls were obtained with fluorescent molecules of the appropriate isotype.

Analysis of immunomodulation

Mononuclear cells from human (MNCs) were isolated from peripheral blood. Suspension 200000 MNCs in 200 μl of medium (RPMI medium 1640 containing 20% SPR 96-well) stimulated with 10 μg PHA/ml (SIGMA) in the presence of 20000 2D attached cells for 5 days in moisture conditions, 5% CO2at 37°C. Used 4 different parties 2D attached cells. Three repetitions of each group were sown in 96-well plate. During the last 18 h of a 5-day cultivation, the cells were subjected to pulse 1 µci3H-thymidine and then harvested on glass fiber filter. Capture thymidine was quantitatively evaluated using a scintillation counter.

Induction of osteogenesis in 2D attached cells

Osteogenesis was performed in accordance with the set for osteogenesis Chemicon (cat No. scr028, Millipore, MA, USA).

Environment for the induction of osteogenesis

Preparing a fresh environment for the induction of osteogenesis before each new environment using the components set (see table 1 below).

Table 1
The components of the environment for osteogenesis
The components is t Source
concentration
NumberEnd
concentration
DMEM low glucose (Invitrogen, Gibco)8,7 ml87%
Serum (inactivated by heating)1 ml10%
Dexamethasone1 mm1 ál0.1 ám
A solution of ascorbic acid-2-phosphate0.1 M20 ál0.2 mm
The solution glycero-2-phosphate1 M100 µl10 mm
L-glutamineX 100100 µlX 1
Pen&StrepX 100100 µlX 1

To obtain a 1 mm solution of dexamethasone to 100 ál of 10 mm solution of dexamethasone was added to 900 ál of this is Ola. The original solution was kept together with other components of the kit at -20°C. the Vessel containing 50 ml of serum iactiveaware heating, was divided into 5-ml aliquots and kept at -20°C until use.

The coating 24-well plates for culturing tissue

Mixture for coating, comprising 12 μg/ml vitronectin and 12 μg/ml collagen (both are included in the kit), were prepared by diluting each of them 1×SFR.

After this mixture for coating was added to the wells to cover the surfaces of the hole (prepared 5 holes x 2 tablet). The plates were incubated over night at room temperature. After this mixture for coating was removed and the wells were washed SFR. The tablets were dried immediately prior to use.

Growing cells

Cells from the placenta (plc11-3-1) or cells from the bone marrow (BM108) were placed (200,000 cells per well) in 1 ml of medium for cultivation, including DMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 mm L-glutamine (Sigma-Aldrich), 45 μg/ml of gentamicin-IKA (Teva Medical) and 0.25 µg/ml Fungizone (Invitrogen, Gibco). Cells from the placenta (4 holes x 2 tablet) or cells from the bone marrow (1 hole × 2 tablet) were grown to 100% of confluentes (usually overnight) before initiation of osteogenic differentiation.

After reaching the cells 100% confluently the culture medium was aspirated and replaced with 1 ml of medium for induction of osteogenesis (day differenziali). Environment for the induction of osteogenesis was replaced with fresh medium every 2-3 days for a total of 14-17 days.

As a control, one of the two tablets (for each type of cells not incubated with medium for osteogenic differentiation, and with growing medium (described in the present description above).

On the 17th day osteocytes were fixed and stained with a solution of alizarin red as described in detail below.

The staining Protocol

Staining of osteocytes were made, starting with gentle suction of the medium from each well (careful not to suck the cells). Then the cells were fixed by incubation in chilled on ice 70% ethanol for 1 hour at room temperature. The alcohol was then carefully aspirated and cells were washed twice with water (5-10 minutes per wash). The water then was aspirated and the cells were added to a solution of alizarin red (500-1000 ml). Cells were incubated with a solution of alizarin red at room temperature for 30 minutes. Alizarin red was removed and cells were washed 4 times with 1 ml of water and drained after each wash. Finally, each well was added 1-1,5 ml of water to prevent drying of the cells. Tablets microscopically visualized using an inverted Nikon microscope.

Induction of osteogenesis in a modified environment in the products osteogenesis (2D attached cells)

Environment for the induction of osteogenesis was prepared afresh before each replacement medium using the components listed in table 2 below, along with vitamin D.

Table 2
The components of the environment for osteogenesis
ComponentSource
concentration
NumberEnd
concentration
DMEM with high glucose (Biological Industries, Bet Haemek, Israel)8,7 ml87%
L-glutamineX 100100 µlX 1
Serum (inactivated by heating)1 ml10%
Dexamethasone (Chemicon)10 mm10 ál10 µm
A solution of ascorbic acid-2-phosphate (Chemicon)0.1 M20 ál 0.2 mm
The solution glycero-2-phosphate (Chemicon)1 M100 µl10 mm
Vitamin D (Sigma)10 µm10 ál10 nm
Gentamicin (Biological Industries, Bet Haemek, Israel)X 100100 µlX 1

A vessel with 50 ml of serum iactiveaware heating, was divided into 5-ml aliquots and kept at -20°C until use.

Coated 48-well plates for culturing tissue

Mixture for coating, comprising 12 μg/ml vitronectin and 12 μg/ml collagen (both from Chemicon), were prepared by diluting each of them 1×SFR.

After this mixture for coating was added to the wells to cover the surfaces of the hole (prepared 5-hole × 2 tablet). The plates were incubated over night at room temperature. After this mixture for coating was removed and the wells were washed once SFR. The tablets were dried immediately prior to use.

Growing cells

Cells from the placenta (fetal cells PLC 8-2-1, PLC 15 3-4-2 or PLC 19-4-3-1) were placed (100,000 cells per well) in 0.5 ml of medium for cultivation, including DMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 mm L (Sigma-Aldrich), 45 µg/ml of gentamicin-IKA (Teva Medical) and 0.25 µg/ml Fungizone (Invitrogen, Gibco) (4 holes x 2 tablet). Cells from the bone marrow (BM109) were placed (150,000 cells per well) in 0.5 ml of medium for cultivation (as described above) (1 hole × 2 tablet). Cells were grown to 10% of confluentes (usually overnight) before initiation of osteogenic differentiation.

After reaching the cells 100% confluently the culture medium was aspirated and replaced with 0.5 ml of medium for induction of osteogenesis (day 1 of differentiation). Environment for the induction of osteogenesis was replaced with fresh medium every 2-3 days for a total of 26 days.

As a control, one of the two tablets (for each type of cells not incubated with medium for osteogenic differentiation, and with growing medium (described in the present description above).

On day 26 osteocytes were fixed and stained with a solution of alizarin red as described in detail below.

The staining Protocol

Staining of osteocytes were made, starting with gentle suction of the medium from each well (careful not to suck the cells). Then the cells were fixed by incubation in chilled on ice 70% ethanol for 1 hour at room temperature. The alcohol was then carefully aspirated and cells were washed twice with water (5-10 minutes per wash). The water then was aspirated and the cells doba is Lyali solution of alizarin red (500-1000 ml). Cells were incubated with a solution of alizarin red at room temperature for 30 minutes. Alizarin red was removed, and cells were washed 4 times with 1 ml of water and drained after each wash. Finally, each well was added 1-1,5 ml of water to prevent drying of the cells. Tablets microscopically visualized using an inverted Nikon microscope.

Induction adipogenesis in 2D attached cells

Adipokines conducted in accordance with the set for adipogenesis Chemicon (set to adipogenesis Chemicon, cat No. scr020, Millipore, MA, USA).

Environment for the induction of adipogenesis

Environment for the induction or maintenance of adipogenesis was prepared immediately before each replacement medium using the components described in tables 3 and 4 below.

Table 3
The components of the environment for the induction of adipogenesis
ComponentSource
concentration
NumberEnd
concentration
DMEM with low glucose (Biological Industries, Bet Haemek, Israel)4,4 ml 90%
Serum (inactivated by heating)0.5 ml10%
Dexamethasone (Sigma)10 mm0,5 ál1 micron
IBMX (Sigma)0.5 M5 ál0,5 mm
Insulin (Sigma)10 mg/ml5 ál10 mg/ml
Indomethacin (Sigma)10 mm50 µl100 mm
Pen & StrepX 10050 µlX 1

Table 4
The components of the environment to maintain adipogenesis
ComponentSource
concentration
NumberEnd
concentration
DMEM low is by cutting down glucose 4,4 ml90%
Serum (inactivated by heating)0.5 ml10%
Insulin (Sigma)10 mg/ml5 ál10 mg/ml
Pen & StrepX 10050 µlX 1

Growing cells

Cells from the placenta (plc11-3-1) or cells from the bone marrow (BM108) were placed (200,000 cells per well) in 1 ml of medium for cultivation, including DMEM (Invitrogen, Gibco), 10 % FCS (Invitrogen, Gibco), 2 mm L-glutamine (Sigma-Aldrich), 45 μg/ml of gentamicin-IKA (Teva Medical) and 0.25 µg/ml Fungizone (Invitrogen, Gibco). Cells from the placenta (4 holes x 2 tablet) or cells from the bone marrow (1 hole × 2 tablet) were grown to 10% of confluentes (usually overnight) before initiating adipogenic differentiation.

After reaching the cells 100% confluently the culture medium was aspirated and replaced with 1 ml of medium for the induction of adipogenesis (day 1 of differentiation). Environment for the induction of adipogenesis was replaced with fresh medium every 2-3 days for a total of 25 days (as detailed in table 5, the lower is in the present description). Note that the monolayers adipogenic cells were extremely fragile and could be easily removed from the plates, and therefore replacement of the medium produced through careful substitutions environment to prevent the destruction of lipid droplets.

As a control, one of the two tablets (for each type of cells not incubated with medium for adipogenic differentiation, and with growing medium (described in the present description above).

Table 5
Scheme adipogenic differentiation
DayWednesday
1Environment for the induction of adipogenesis
3Environment for the induction of adipogenesis
5Environment for the induction of adipogenesis
7Environment to maintain adipogenesis
9Environment for the induction of adipogenesis
11Environment for the induction of adipogenesis
13The environment is Nuccio of adipogenesis
15Environment to maintain adipogenesis
17Environment for the induction of adipogenesis
19Environment for the induction of adipogenesis
21Environment for the induction of adipogenesis

On the 25th day adipocytes were fixed and stained with a solution of oil red, as described in detail below.

The staining Protocol

Staining of adipocytes produced, starting with gentle suction of the medium from each well (careful not to suck the cells). Then the cells were fixed by incubation in 4% paraformaldehyde for 30-40 minutes at room temperature. The clamp was then carefully aspirated and cells were washed three times SFR (5-10 minutes per wash). SFR then was aspirated and cells washed twice with water. After that the water was aspirated and the cells were added to the solution of oil red (500-1000 ml). Cells were incubated with a solution of oil red at room temperature for 50 minutes. The solution of oil red was removed and cells were washed 4 times with 1 ml of water and drained after each wash. Finally, each well was added 1-1,5 ml of water to prevent drying of the cells. The tablets of microsc pichaske visualized using an inverted Nikon microscope.

Obtaining a solution of oil red

Used 0.25 g original oil red, which was dissolved in 50 ml isopropanol by incubation for 10-15 minutes in a bath at 37°C.

For use with 30 ml of the original dye was mixed with 20 ml DDW (left on for 10 minutes and then filtered through a paper coffee filter). The solution of oil red was prepared anew for each use.

Induction adipogenesis in a modified environment for the induction of adipogenesis (2D attached cells)

Environment for the induction of adipogenesis prepared afresh before each replacement medium using the components described in table 6 below.

Table 6
The components of the environment for the induction of adipogenesis
ComponentSource
concentration
NumberEnd
concentration
DMEM with low glucose content4,4 ml90%
Serum (inactivated by heating)0.5 ml 10%
Dexamethasone (Sigma)1 mm5 ál1 micron
IBMX (Sigma)0.5 M5 ál0,5 mm
Insulin (Sigma)10 mg/ml5 ál10 mg/ml
Indomethacin (Sigma)10 mm200 ál100 mm
Gentamicin (Biological Industries)10 ál

Growing cells

Cells from the placenta (fetal cells PLC 8-2-1, PLC 15 3-4-2 or PLC 19-4-3-1) were placed (100,000 cells per well) in 0.5 ml of medium for cultivation, including DMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 mm L-glutamine (Sigma-Aldrich), 45 μg/ml of gentamicin-IKA (Teva Medical) and 0.25 µg/ml Fungizone (Invitrogen, Gibco) (5 holes × 2 tablet).

Cells from the bone marrow (BM109) were placed (100,000 cells per well) in 0.5 ml of medium for cultivation, including DMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 mm L-glutamine (Sigma-Aldrich), 45 μg/ml of gentamicin-IKA (Teva Medical) and 0.25 µg/ml Fungizone (Invitrogen, Gibco) (4 holes x 2 tablet). Cells were grown to 100% confluent the activity (usually overnight) before initiating adipogenic differentiation.

After reaching the cells 100% confluently the culture medium was aspirated and replaced with 0.5 ml of medium for the induction of adipogenesis (day 1 of differentiation). Environment for the induction of adipogenesis was replaced with fresh medium every 2-3 days for a total of 3-4 weeks.

As a control, one of the two tablets (for each type of cells not incubated with medium for adipogenic differentiation, and with growing medium (described in the present description above).

On day 26 adipocytes were fixed and stained with a solution of oil red, as described in detail below.

The staining Protocol

Staining of adipocytes produced, starting with gentle suction of the medium from each well (careful not to suck the cells). Then the cells were fixed by incubation in 4% paraformaldehyde for 30-40 minutes at room temperature. The clamp was then carefully aspirated and cells were washed three times SFR (5-10 minutes per wash). SFR then was aspirated and cells washed twice with water. After that the water was aspirated and the cells were added to the solution of oil red (500-1000 ml). Cells were incubated with a solution of oil red at room temperature for 50 minutes. The solution of oil red was removed and cells were washed 3 times with 1 ml of double-distilled water and drained after each promicing, to each well was added 1-1,5 ml of water to prevent drying of the cells. Tablets microscopically visualized using an inverted Nikon microscope.

Obtaining a solution of oil red

Used 0.25 g original oil red, which was dissolved in 50 ml isopropanol by incubation for 10-15 minutes in a bath at 37°C.

For use with 30 ml of the original dye was mixed with 20 ml DDW (left on for 10 minutes and then filtered through a paper coffee filter). The solution of oil red was prepared anew for each use.

Results

As shown in table 7 below, the processing of 2D attached cells suitable for use in accordance with these instructions, is different from the stage 2D PLX (WO/2007/108003) in several aspects. First, a new medium for cultivation 2D attached cells antibiotics were added only during the initial stage of cultivation (up to passage 2). In addition, the new 2D attached cells were kryokonservierung only after 5-8 passages (i.e. at the end of the culture), and not during the intermediate stages of the 2D growth, as in the case of process PLX.

Table 7
Comparison of 2D attached cells, suitable for the use of the Oia in accordance with these instructions, with cells obtained for PLX in WO/2007/108003
WO/2007/1080032D attached cells these instructions
Vessel to tissue culture80 cm2and 175 cm2175 cm2, tripled vessels and Multi Tray
Environment with the addition of antibioticsAt all stages of the processPrior to passage 2 (inclusive)
Cryopreservation 2DCSAfter 2-3 passages, then kryokonservierung were thawed and were sown for the secondary cultivation in containers for 2-5 passages before planting in the bioreactorAfter 2-3 passages, then kryokonservierung and thawed before use
Container for freezing2-ml cryogenic vessels5-ml cryogenic vessels
Freeze volume1-1,5 ml4 ml
A method of freezingContainer for freezing (contains isopropyl alcohol) Freezer with controlled speed

Changes in the process of obtaining 2D attached cells resulted in changes in the properties of the obtained cells. These differences are summarized in the present description below.

Analysis of cell cycle 2D attached cells compared to 3D attached cells WO/2007/108003- 2D attached cells was compared with 3D attached cells to estimate the distribution of cells between the different phases of the cell cycle. As is clear from figa-B, 2D attached cells showed typical proliferative profile (distribution of cells between the different phases of the cell cycle). Specifically, 28% of cells were in S and G2/M phases (figa). These results showed that the cells were collected during proliferation and that the cultivation conditions supported cell growth.

On the contrary, 3D attached cells showed a lower rate of cell proliferation. Less than 8% of cells were in S and G2/M phases (pigv). These results showed that the cells were then collected, when there has been a low level of proliferation, and that the conditions in the bioreactor were suboptimal to maintain cell growth.

Comparison between 2D cells suitable for use in accordance with these instructions, and the cells obtained according to the instructions WO/2007/108003 using microarrays. Analysis of the expression of genes by using microarrays allowed us to simultaneously monitor the expression profiles of the whole genome of attached cells, obtained from human placentas with full-term pregnancy, multiplied with 2D cultures or in accordance with the instructions WO/2007/108003 (PLX, see example 1 in the present description above). These results allowed us to estimate the molecular mechanism underlying phenotypic differences between cells obtained by these different methods of cultivation (see table 8 below).

protein 1, binding SH2 domain of SHC
Table 8
Gene expression in 2D attached cells suitable for use in accordance with these instructions, compared with genes expressed PLX WO/2007/108003
Gene2D against Plurix (rate of change)P-value (brought)
induced by interferon protein with tetratricopeptide repeats21,820,0401812
argininebenefits of leukocytes14,563,88E-06
signal peptide, CUB domain, EGF-like 310,820,0255115
homolog 1dickkopf (Xenopus laevis) 6,843,06E-07
integrin, alpha 66,760,0411667
keratin 27 pseudogene 276,390,000224998
similar to keratin, type I cytoskeletal 18 (cytokeratin6,240,000304949
aldehyddehydrogenase family 1, member A1of 5.840,00145807
coupled with G protein-coupled receptor, family C, group 5, member Aof 5.753,39E-05
the coagulation factor III (thromboplastin, tissue factor)5,550,012192
inhibitor 3 cyclin-dependent kinase (associated with CDK2 double5,510,000732492
coupled with G protein-coupled receptor 1265,500,00197635
containing the domain DEP 15,410,000370513
4,960,00430878
protein of the centrosome 55 kDa4,780,0021952

induced by interferon protein with tetratricopeptide repeatsof 4.660,0139777
NUF2, a component of the NDC80 kinetochore complex, homolog (S. cerebr4.610,00276524
mal, like a squirrel differentiation of T-cellsof 4.440,00664216
induced by interferon protein with tetratricopeptide repeats4,420,00357376
the member 18 family of kinesin4,330,00134108
cholinergic receptor, muscarinic 24,070,0320078
the cycle of cell division 2, G1 to S and from G2 to M4,060,0017111
4,060,00537097
denticleless homolog (Drosophila)4,060,00141153
shugoshin-like 1 (S. pombe)4,000,00101318
open reading frame 3 chromosome 133,980,000548296
linking-bearing kinase3,970,00784983
cytosolic protein 1 lymphocyte (L-plates)3,970,0049584
WAS3,960,00178153
cyclin E23,940,000203389
cathepsin C3,930,00532262
integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-43,910,0158411
KIAA01013,900,0105909
member 20A of the family of kinesin3,900,00582352
receptor opioid growth factor like 1a 3.870,00114551

Anilin, actin binding protein3,830,010923
catenin (associated with Katherina protein), alpha-like 13,767,46E-05
homolog 20 cycle of dividing cells (S. cerevisiae)3,700,00514206
diaphanous homolog 3 (Drosophila)3,690,00107709
family with sequence similarity 111, member B3,690,000125819
the kinase Aurora A3,660,00632571
the fibroblast growth factor 7 (keratinocyte growth factor)of 3.640,0328983
maternal embryonic kinase with alnovol zipper 3,630,00908391
the inhibitor dissociation of Rho GDF (GDI) beta3,630,00200066
protein N centromere3,620,000540143
MAD2 failure stopping mitosis-like 1 (yeast)3,620,00488102
timedilation3,610,00685584
cyclin B23,600,016544
the controller 4 signaling G-protein3,590,00781061
the open reading frame 173 chromosome 6to 3.580,00222408
receptor mediated by hyaluronan motility (RHAMM)3,550,00467816
BUB1 of budding, not inhibiting the benzimidazole, homolog 1 (yeast3,540,0108258
SPC25, component ND80 kinetochore complex, homolog (S. ce3,530,00568662
the establishment of the clutch 1 homolog 2 (S. cerevisiae)3,520,000773033
cyclin A23,510,00965934

regulatory subunit 2 protein kinase CDC283,510,0128024
keratin 183,470,000514523
ribonucleotides M2 polypeptideof 3.460,00834059
arylacetamides-like 13,440,000902645
the member 11 family of kinesin3,430,00915145
protein 11A, activating GTPase Rho3,410,00834174
subunit 1 of complex GINS (homolog Psf1)3,390,00104515
disc, large homolog 7 (Drosophila)3,380,0317074
protein kinase TTK3,380,0112171
remote in lymphocytic leukemia, 23,380,0109528
the replication factor C (activator 1) 3, 38 kDa3,370,00109668
family of 7 vectors of dissolved substances (Transporter of cationic amino acids3,360,00688017
kinase with dual specificity regulated by phosphorylation of tyrosine (Y)3,340,0234606
member of the 2C family of kinesin3,340,0059888
protein 8 heat shock 22 kDa3,320,0219583
polo-like kinase 1 (Drosophila)3,300,0140309
like (avian) homolog of the viral oncogene myeloblastosis v-myb 3,280,0043878
trypsinogen C3,280,00416276
thymidine kinase 1, solublewith 3.270,00124134
NAD(p)H dehydrogenase, quinone 1with 3.270,000282423

unit 2 high-mobility group3,240,0196872
associated 2 with the cycle of cell division3,240,0122226
enzyme mRNA editing of apolipoprotein B, catalytic polypep3,230,00308692
inhibitor Sarbinowo peptidases, group B (ovalbumin), member3,220,0190218
binding protein goinnovate nucleotides (G protein), gamma 113,220,00140559
the open reading frame 23 chromosomes 153,21 0,000147331
the member 14 of the family of kinesin3,190,00947901
transmembrane protein 1543,180,0045589
glycerokinase3,162,66E-05
KIAA15243,150,0380688
the coagulation factor XIII polypeptide B3,140,0294465
Protein 2 dense contacts (zona occludence 2)3,130,00012562
nei endonuclease VIII-like 3 (E. coli)3,120,00115606
pleckstrin 23,110,0304429
the member 23 family of kinesin3,090,00790585
protein 1, activating GTPase Rac3,090,00381613
protein 1-like growth factor keratinocyte of 3.070,0300588
protein 1-like growth factor keratinocyteof 3.070,0300588
protein 1-like growth factor keratinocyteof 3.070,0300588
transcription factor 19 (SC1)of 3.070,00109627

containing OCIA domain 2of 3.070,00122147
protein associated with the metastasis of lung cancer3,060,00148024
transcription factor 19 (SC1)3,050,00124327
transcription factor 19 (SC1)3,050,00124327
protein 29, activating GTPase Rho3,050,0466211
glucosaminyl(N-acetyl)transferase 1, base 2 (beta-1,6-N-3,050,0197148
the replication factor C (activator 1) 4, 37 kDa3.04 from0,00164152
protein regulator of cytokinesis 13,010,0325664
transforming containing acid double helix protein 32,980,0014577
candidate 5 exposure cancer2,960,0330594
protein 1 associated with nucleolar and spindle2,960,00520875
cyclin B12,960,0103092
transmembrane protein 482,960,00458248
interacting with ZW102,951,88E-05
containing endonuclease domain 12,950,000429245
hypoxanthineguanine 1 (Lesch-Nyhan syndrome2,940,000634057
fucosidase, alpha-L-2, plasma2,940,00540929
ubiquitin-conjugating enzyme E2T (potential)2,930,00741886
lipase A, lysosomal acid, cholesterylester disease Wolman2,920,0167385
villin 2 (Ezrin)2,920,0131934
glycerokinase2,903,37E-06
domain repeat, WD 762,890,0023531

the CD97 molecule2,890,00994045
the open reading frame 24 chromosome 182,890,00347442
topoisomerase (DNA) II alpha 170 kDa2,890,0321109
integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA-32,87member A family with sequence similarity 292,850,00111165
member 4A family of kinesin2,850,0114203
associated with BRCA1 domain 1 RING2,850,000540414
serum2,840,0387246
the RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae)2,830,000854739
anemia Fanconi's syndrome, a group of complementaly I2,830,00464532
dihydrotetrazolo2,820,00178879
homolog claspin (Xenopus laevis)2,810,00683624
ornithindecarboxilase 12,810,00144868
antigen 5, associated with semen2,800,00906321
the histone cluster 1, H3b2,800,0304598
the collection ATPase containing domain 2 AAA2,790,00415258
protein KIAA02862,790,00130563
binding protein goinnovate nucleotides (G protein), alpha Ingawas 2.760,00184597
BUB1 of budding, not inhibiting a benzimidazole, a homolog of the beta 12,740,0166047
digidrofolatreduktazy pseudogene2,740,00141306
containing the domain brix 12,730,00471977

protein 2 associated with the cytoskeleton2,720,0030499
protein S28 ribosomes of mitochondria2,720,00298194
polymerase (directed DNA), Epsilon 2 (p59 subunit) 2,720,00479612
member A family with sequence similarity 722,720,00143248
protein 2, EBNA1 binding2,700,00296292
similar to protein SA 40S ribosomes (P40) (342,700,0385298
protein associated with adipose differentiation2,700,000331751
thioredoxins 12,700,000197486
component 5 complex save minichromosome2,690,00475504
protein 1, binding Hippel-Lindau2,690,00329061
SCL2,680,00390288
anemia Fanconi's syndrome, a group of complementaly D22,680,0281405
kinase 2, related NIMA (a gene never in mitosis) 2,680,00289469
protein 19 with PHD finger2,680,000177604
microsomal glutathione S-transferase 12,680,041701
breast cancer 2 early onset2,680,00586847
non-SMC complex condensin I, subunit Hto 2.670,0216752
the open reading frame 27 chromosome 13to 2.670,0234588
the histone cluster 1, H2bgto 2.670,000180822

non-SMC complex condensin II, subunit G22,660,0130322
protein I centromere2,640,0106816
stomatin2,640,00387095
glutathione S-transferase omega 1 2,630,000648379
contains A domain similar to proteincarbohydrate2,620,0419644
protein binding calcyclin2,620,00524566
ligand KIT2,610,00641955
ubiquitin-conjugating enzyme E2L 32,610,00343347
inhibitor Sarbinowo peptidases, group B (ovalbumin), member2,600,0030439
ATPase, Ca++transporting, plasma membrane 42,600,023011
TPX2 associated with microtubules, homolog (Xenopus laevis)2,600,0253137
interacts with thyroid hormone receptor 132,590,0118319
member of the Z family of histone H2A2,590,0129697
regulatory subunit 1B protein kinase CDC28to 2.570,0107391
associated with the cycle of cell division 3to 2.570,006289
component 8 complex save minichromosometo 2.570,000841489
transcription factor 2 E2F2,550,0496479
protein that interacts with TIMELESS2,550,00771062
component 4 complex save minichromosome2,540,00342054
polo-like kinase 4 (Drosophila)2,530,00209633

member of the C1 family of kinesin2,530,00821937
dihydrotetrazolo2,520,00307793
glycero-3-phosphatedehydrogenase 2 (mitochondrial) 2,520,00211969
induced TGF beta nuclear protein 1of 2.510,0365579
integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptorof 2.510,0210165
protein that interacts with MLF1of 2.510,0177203
protein 2 heat shock 70 kDa2,500,0215102
hairy and enhancer of splitting 1 (Drosophila)2,500,000283509
ATP-binding cassette, subfamily C (CFTR2,490,00382491
serpitine2,480,0443487
domain sema, immunoglobulin domain (Ig), short basic home2,470,008548
domain 1 with ancyranum repeat (heart muscle)2,470,00911953
the Transporter 1, ATP-St is binding cassette, subfamily B (MDR2,470,00859077
the Transporter 1, ATP-binding cassette, subfamily B (MDR2,470,00859077
the Transporter 1, ATP-binding cassette, subfamily B (MDR2,470,00859077
the histone cluster 1, H1b2,460,0470898
family with sequence similarity 72, member A2,460,00165234
associated with the membrane containing the domain O-acyltransferase 12,460,01185
the substrate 8 ways receptor epidermal growth factor2,450,0194949

homolog B ASF1 antisilence function 1 (S. cerevisae)2,450,00543408
opening cytokinesis 112,440,00697577
this is istwo with sequence similarity 72, member A2,440,00162905
protein 2, related to actin2,440,000288443
TTF-synthase2,438,80E-05
the phosphoprotein 1 M-phase2,430,0271814
regulatory subunit 1B protein kinase CDC282,430,0145263
the histone cluster 1, H2ai2,430,0161621
nucleosomal binding domain 2 high-mobility group2,420,0030536
protein 1A heat shock 70 kDa2,420,00734287
protein 1A heat shock 70 kDa2,420,00674816
carnitinelongevity 1A (liver)2,410,00170894
neurofilament, medium polypeptide 150 kDa 2,410,0190611
transmembrane protein 622,410,00761064
kinase 1, akin to cow pox2,400,0233182
geminin, inhibitor of DNA replication2,400,00167629
phosphoglucomutase 22,400,00818204
Lamin B12,400,0477748
keratin 182,400,000112551
deafness, autosomal dominant 52,390,00235481
proteasome (proteasomes, macrofauna) subunit, type beta 9 (lar2,390,0202595
proteasome (proteasomes, macrofauna) subunit, type beta 9 (lar2,390,0202595

proteasome (proteasomes, macrofauna) sub who dinica, type beta 9 (lar2,390,0202595
the open reading frame 31 chromosome 122,390,0173089
izotsitratdegidrogenazy 3 (NAD+) alpha2,390,00297129
villagelevel block M12,380,0203154
transmembrane protein 106C2,380,000214223
hypothetical protein LOC7290122,380,000446087
protein 1 PHD finger2,370,010191
protein L15 ribosomes of mitochondria2,370,0306092
part 2 elastin microfibril2,370,0192072
hypothetical protein DKFZp762E13122,370,00726778
retinoblastoma-like 1 (p107)2,36 0,00319946
the inhibitor of the tissue factor pathway (associated with lipoprotein2,360,0356227
oncogene sequence 2, transforming epithelial cells2,360,000571152
crystallin, Zeta (heneretta)2,360,0370884
domain hect and RLD42,360,00679184
nucleosomal binding domain 2 high-mobility group2,360,00384071
a homologue of A cycle of cell division 25 (S. pombe)2,360,000341692
thymopoietin2,350,0223176
induced by interferon protein with tetratricopeptide repeats2,340,0177928
syndrome bloom2,340,0209259
phosphatase 1 dual specificity what was mentioned 2,340,00211272
the elongation factor, RNA polymerase II, 22,340,0130017

polypeptide 16 kDa small nuclear ribonucleoprotein D12,340,0334665
CDC45 cycle cell division 45-like (S. cerevisiae)2,330,00735977
the exonuclease 12,330,00739393
similar to ribosomal protein L39 are effective2,330,00429384
the histone cluster 1, H2bh2,330,0377748
polypeptide M1 ribonucleotides2,330,000170076
homolog sulfiredoxin 1 (S. cerevisiae)2,325,14E-05
factor 2 multiple failure coagulation2,310,0116892
proteasome (proteasomes, macrofauna) subunit, type alpha, 32,310,0195874
ribonuclease H2, subunit A2,300,00669936
component 10 complex save minichromosometo 2.290,0037925
protein 1B heat shock 70 kDa2,280,0048959
protein 1B heat shock 70 kDa2,280,0054404
protein 1B heat shock 70 kDa2,280,0054404
ATPase, Na+2,280,000381464
hypothetical protein LOC2017252,280,000313319
cathepsin L12,270,0314419
associated 5 with the cycle of cell division2,270,01021
RAB8B, member of the N. family of RAS oncogene 2,270,00417066
SPC24, a component of the NDC80 kinetochore complex, homolog (S. ce2,270,00287227
gamma glutamylcysteine (conjugase, polypolylineof 2.260,0195219

homolog of the C cycle of cell division 25 (S. pombe)2,250,0169914
mutS homolog 2, colon cancer colon nonpolyposis type 1 (E. coli)2,250,00578953
metallothionein 1L (gene2,250,00709646
homolog controller biogenesis of ribosomes RRS1 (S. cerevisiae)2,240,0120061
associated 8 with the cycle of cell division2,240,00619878
shugoshin-like 2 (S. pombe)2,240,000852557
homolog mRNA turnover 4 (S. cerevisiae) 2,240,00373104
ST6 (alpha-N-acetylneuraminic-2,3-beta-galactosyl-1,2,240,00830766
homolog 2 oncogene v-ets virus erythroblastosis E26 (bird)2,230,0364123
the replication factor C (activator 1) 2, 40 kDa2,230,00768959
kinase 7, related NIMA (a gene never in mitosis)2,230,00159114
the main lacinova zipper and W2 domains 22,230,0190782
the histone cluster 1, H2bf2,230,0124279
factor 1A initiation of translation in eukaryotes, X-linked2,230,00330183
the Transporter 1, ATP-binding cassette, subfamily B (MDR2,220,0164234
the Transporter 1, ATP-binding cassette, subfamily B (MDR2,22 0,0164234
the Transporter 1, ATP-binding cassette, subfamily B (MDR2,220,0164234
polymerase (RNA) III (directed DNA)polypeptide G2,220,0298794

phosphatidylinositol-4-phosphate 5-kinase, type II, Alf2,220,00964099
proteasome (proteasomes, macrofauna) 26S subunit, ATPase, 62,220,024269
transforming into a pituitary tumor 1of 2.210,0485166
the histone cluster 2, H3dof 2.210,0102932
SolidColorBrush-like (yeast)of 2.210,0473641
serpitine2,200,00880325
ribosomal protein L22-like 12,200,00335381
2,200,000396285
the family of 24 vectors of solutes (sodium2,200,0328774
STAM-binding protein-like 12,200,0181743
protein 1 WD repeat and block HMG DNA binding2,200,0034833
such CSE1 segregation of chromosomes 1 (yeast)2,200,0013662
complex recognition of the origin, similar to the subunit 6 (yeast)2,200,00182466
transcription factor A, mitochondrial2,190,0110092
component 8 actsoma2,190,00132017
mitochondrial ribosomal protein L12,190,0361058
sphingomyelinase 22,19 0,0020701
deoxycytidylate2,180,00101444
family with sequence similarity 29, member A2,180,00469407
the open reading frame 167 chromosome 62,180,0011095

phosphatase 11 with dual specificity (RNA2,180,00426788
protein 45 block F2,180,00510098
related ras substrate 2 toxin botulinum C3 (rho family, sma2,170,0292466
protein 5, FK506 binding2,170,0193805
breast cancer 1, early onset2,170,0180553
nuclear factor I2,170,0010313
thioredoxin 2,170,009636
4A, containing SH2 domain2,160,0323646
induced TGF beta nuclear protein 12,160,00285964
PSMC3 interacting with protein2,160,00766442
the open reading frame 14 chromosome 32,150,0377617
ring finger 5 polycomb group2,150,000294142
protein 27 kDa centrosome2,150,00931602
family with sequence similarity 64, member A2,140,0019785
acid (enriched with leucine) the nucleolar phosphoprotein family 32, m2,140,0300263
Sterol O-acyltransferase (acyl-coenzyme A:cholesterol ACI2,140,0193637
factor associated the p protein, connecting block TATA (TBP), RNA-poly2,130,00514451
complex recognition of the origin, similar to the subunit 5 (yeast)2,130,049697
pseudogene protein 1, activating GTPase Rac2,130,000269488
LSM5 homolog, is associated with U6 small nuclear RNA (S. cerevisia2,130,00264664

component 7 complex save minichromosome2,130,0457691
protooncogene met (growth factor receptor of hepatocytes)2,130,0318147
containing a triple motif 252,130,0456344
the open reading frame 34 chromosomes 132,130,000702936
containing patatin-like phospholipases domain 42,130,0168306
component 6 complex save minichromosome2,120,0161279
homolog vnutrijeludochkovogo transport 80 (Chlamydomonas)2,120,0247286
peptidyltransferase F (cyclophilin F)2,120,00093846
UTP15, U3 small nucleolar ribonucleoprotein, homolog (S. c2,120,00482559
TAF9B RNA polymerase II binding protein block TATA (TBP)-AC2,120,0170365
TAF9B RNA polymerase II binding protein block TATA (TBP)-AC2,120,0170365
website integration 2B ecotropica virus2,120,0171408
3'-phosphoadenosine-5'-phosphosulfate 22,121,43E-05
proteasome (proteasomes, macrofauna) activator subunit 2, (PA282,120,00609885
metal is peptidase ADAM motif thrombospondin type 1 2,120,0102751
endonuclease 1, specific patterns flap2,120,006882
S100 calcium-binding protein A32,120,0324073
RAD18 homolog (S. cerevisiae)2,110,0016685

component 3 complex save minichromosome2,110,0018389
component 3 actsoma2,110,0249115
cysteinyl-tRNA-synthase 2, mitochondrial (presumably)2,110,00564558
glutamate-cysteinemia, modifier subunit2,110,00378868
containing the domain brix 12,110,00981178
the member 22 family of kinesin2,11,0192406
UTP11-like, U3 small nucleolar ribonucleoprotein (yeast)2,100,0132794
homolog B viral oncogene v-ral leukemia monkeys (related ras2,100,012225
homolog of meiotic division of the nucleus 1 (S. cerevisiae)2,100,00164447
phenylalanine-tRNA-synthase, subunit beta2,100,000245973
similar to ubiquitin-conjugating enzyme E2S (ubiqui2,100,000415822
containing the domain with the double helix 682,100,00227586
the receptor of laminin B2,100,000151784
the Niemann-pick disease, type C12,100,0108117
hydroxysteroiddehydrogenase-similar 22,093,71E-05
RMI1, mediated RcQ genome instability 1, homolog (S. cerev2,090,00294705
hyperexpression in carcinoma of the colon-12,090,015322
hypothetical protein FLJ204252,090,0174225
primase, polypeptide 1, 49 kDa2,090,00801018

the open reading frame 121 chromosome 202,090,0146323
associated with microtubules serine2,080,00536974
differentiation of the endothelium, sphingolipids coupled with G-proteins2,080,0132848
homeobox A92,080,00520942
protein L centromere2,080,000880856
homolog-associated nucleolar complex 3 (S. cerevisiae)2,07 0,000373346
the fibroblast growth factor 7 (keratinocyte growth factor)2,070,0173208
knob 1 is enriched in cysteine transmembrane BMP (such jordino)2,070,0267286
nucleoporin 155 kDa2,070,00218453
protein FLJ20105to 2.060,0127979
molecule CD44 (Indian blood group)to 2.060,000651436
polymerase (directed DNA), alpha 2 (70 kDa subunit)to 2.060,0033903
a similar 2 (avian) homolog of the viral oncogene myeloblastosis v-mybto 2.060,00989416
complex recognition of the origin, similar to the subunit 1 (yeast)to 2.060,00207753
hypothetical protein FLJ25416to 2.060,000177531
the member 22 family of kinesinto 2.060,0242075
protein 1 heat shock 60 kDa (chaperonin)to 2.060,0327412
component 2 complex save minichromosome2,050,0021347

fumarylacetoacetase (fumarylacetoacetase)2,053,88E-05
pseudogene glycerokinase 32,050,0103203
pigmentary degeneration of the retina 2 (X-linked recessive)2,050,0264185
kinase 1 motif (UHM), homologous U2AF2,050,0255167
chaperonin containing TCP1, subunit 5 (Epsilon)2,040,00125909
ATPase transporting H+, lysosomal 34 kDa, V1 subunit D2,040,031745
factor in the termination of transcription, RNA polymerase II2,040,000393489
succinate-CoA ligase, forming a GDF, beta subunit2,040,0028167
inhibitor 1B cyclin-dependent kinase (p27, Kip1)2,040,00183021
tyrosine-3-monooxygenase2,040,00021508
cofactor required for the activation of Sp1 transcription, subje2,040,00141809
contains 3 domain glycosyltransferases 82,030,022868
a homologue of ribosomal RNA processing 15 (S. cerevisiae)2,030,0274884
glycogenin 12,030,0224317
hypothetical protein FLJ408692,030,00444509
nuclear antigen of proliferating cells 2,030,0031727
contains 12 domain, a sterile alpha motif2,030,0232188
the open reading frame 59 chromosome 162,030,00185191

cofilin 2 (muscle)2,030,0459235
factor 2 initiation of translation in eukaryotes, subunit 2 Beth2,030,0139947
factor chromatin Assembly 1, subunit B (p60)2,030,0119687
homolog Zwilch associated with kinetochores (Drosophila)2,020,000725107
ATP-binding cassette, subfamily E (OABP), member 12,020,00454751
LSM3 homolog, is associated with U6 small nuclear RNA (S. cerevisia2,020,0199824
containing the IQ motif activates GTPase protein 3 2,020,0495882
tubulin, alpha 1c2,020,00862586
the DBF4 homolog (S. cerevisiae)2,010,0458795
protein 1, binding precursor protein, amyloid beta2,010,000910538
homolog 1 suppressor coloring pages 2-9 (Drosophila)2,010,00224324
homolog complex 7 THO (Drosophila)2,010,0047251
amyotrophic lateral sclerosis 2 (juvenile) chromosome re2,010,0484466
nucleoporin 37 kDa2,010,00652747
nucleolar protein 112,010,000852662
ATP synthase, transporting H+, mitochondrial F0 complex2,010,00866627
the histone cluster 1, H2ai2,0 0,0129155
phytoceramides, alkaline2,010,0157729
primase, polypeptide 2A, 58 kDa2,010,00290097
protein B1, similar to high-mobility group (Vysokomol2,000,000363158

such Manager 3 (Drosophila)-2,000,00386667
UDF-N-acetyl-alpha-D-galactosamine:polypeptide N-acetyle-2,010,0268634
protein with a ring finger 122-2,010,0236621
chromolaena helicase DNA binding protein 3-2,016,39E-05
centaurin, gamma-like family, member 10 pseudogene-2,018,70E-05
the open reading frame 10 chromosomes of 7-2,01the open reading frame 111 chromosome 6-2,010,0104492
centaurin, gamma-like family, member 10 pseudogene-2,010,000334818
area 1 chromosome syndrome Prader-Willi-2,010,0415526
KIAA1245-2,010,0186309
homolog peroxidation (Drosophila)-2,010,00219049
the melanoma antigen family D, 4-2,020,0263076
the melanoma antigen family D, 4-2,020,0263076
glucosidase, alpha, acid (illness Pump, Bo storing glycogen-2,020,000418401
receptor 1 phospholipase A2, 180 kDa-2,030,00069343
contains 2 domain glycosyltransferases 8-2,03 0,0173546
KIAA1546-2,030,000255634
protocadherin beta 9-2,030,0285124
family with TBC1 domain, member 3B-2,030,000414974
sushi, Neogene and EGF-like domains 1-2,030,00161129

factor 1, cross-linking microtubule-actin-2,040,00216
the family area containing the stop neuroblastoma-2,040,0213393
Golgi autoantigen, subfamily holdin a-like pseudogene-2,040,0182674
like transducin enhancer splitting 4 (E(sp1) homolog, Drosop-2,040,0164153
the family of 22 vector of dissolved substances (Transporter of organic cations), -2,050,0137275
neighbor of Punc E11-2,050,0184739
protein 5 linking insulin-like growth factor-2,050,011614
KIAA1245-2,060,0185376
the receptor for vitamin D (1,25-dihydroxyvitamin D3)-2,060,000192208
CLL B-cells-2,060,00343507
KIAA1305-2,060,00813727
KIAA1245-2,060,0185609
centaurin, gamma-like family, member 10 pseudogene-2,073,08E-05
family with TBC1 domain, member 3B-2,070,00141297
similar to the member 3 family with TBC1 domain (Rab GTPase--2,080,00105098
Mann is sidasa, alpha, class 2B, member 1-2,080,000353303
enriched with cysteine inhibitor of PAK1-2,080,000125336
midline 1 (Opitz-2,080,00130803
small nucleolar RNA, H-2,090,017124
urocortin 2-2,090,00172263

-2,09
family stop neuroblastoma, member 11-2,090,0138065
collagen, type VI, alpha 3-2,092,09E-06
family stop neuroblastoma, member 11-2,090,0148372
hypothetical white LOC646870-2,090,0117625
calcitonin 3-2,090,00300887
protein 2, binds cortactin2,28E-05
glycoprotein 2A synaptic vesicles-2,100,00704212
similar to dinamina-1 (D-100) (dynamin, brain (B-DIN-2,100,0190733
similar to dinamina-1 (D-100) (dynamin, brain (B-DIN-2,100,0190733
similar to the member 3 family with TBC1 domain (Rab GTPase--2,100,00108467
homolog 2 Notch (Drosophila) N-end-like-2,100,0193058
associated 5 with the restructuring of the matrix-2,110,000317637
complement component 1, s subcomponent-2,110,0395863
the decarboxylase cysteinsulfinic acid-2,110,00428211
hypothetical protein FLJ36144-2,110,00958437
hypothetical protein FLJ36144-2,110,00958437
dihydropyrimidinase-similar 3-2,120,0165203
enhancer of procollagen C-endopeptidase-2,120,0039236
Golgi autoantigen, subfamily holdin a-like pseudogene-2,120,00720508
family with TBC1 domain, member 3B-2,120,00122924

td align="justify"> 0,00139674
collagen, type VII, alpha 1 (epidermolysis a bullosa, distr-2,130,00109233
version-2,140,023885
the mannose receptor, C type 2-2,140,00012142
Golgi autoantigen, subfamily holdin a-like pseudogene-2,140,00767095
dynamin 1-2,15
family with TBC1 domain, member 3B-2,160,00130459
protein 21A with PHD finger-2,170,00980401
centaurin, gamma-like family, member 10 pseudogene-2,170,000180846
homolog 3 slit (Drosophila)-2,170,02844
transforming gene 1 neuroepithelial cells-2,180,0109689
cyclin L2-2,180,00093459
similar to dJ402H5.2 (novel protein similar to that in-2,180,00621503
phospholipase D family, member 3-2,181,17E-05
collagen, type VIII, alpha 1-2,190,00187242
cyclin L2-2,190,00109621
protocadherin beta 14 -2,200,0103892
metallopeptidase 2 matrix (gelatinase A, gelatinase 72 kDa,-2,205,59E-05
the lysyl oxidase-like 4-2,210,0120148
Golgi autoantigen, subfamily holdin a-like pseudogene-2,210,00977719
the regulator of transcription 1, containing a WW domain-2,210,0379899
containing the domain-bearing with RING finger 3-2,210,00931014

the open reading frame 37 chromosome 14-2,210,0182453
brain and acute leukemia, cytoplasmic-2,220,0476919
calcium channel potential-dependent, L type, alpha 1C sub-2,220,0189661
oncogene jun-2,23 7,21E-05
interleukin 19-2,230,0310328
centaurin, gamma-like family, member 10 pseudogene-2,230,000595086
centaurin, gamma-like family, member 10 pseudogene-2,230,000595086
----2,240,00666187
Golgi autoantigen, subfamily Goldin b, macropolicy (-2,240,0164005
the open reading frame 51 chromosome 15-2,240,0123547
similar to dinamina-1 (D100) (dynamin, brain (B-DIN-2,240,0123547
similar to dinamina-1 (D100) (dynamin, brain (B-DIN-2,240,0123547
protein 1, binding AE-2,250,000105628
Golgi autoantigen, subfamily holdin a-like dog who Dogen -2,260,00770626
transmembrane protein 16A-2,270,0481085
hypothetical LOC399844-2,270,000491694
oculometer-2,270,00778869
protein 1, related to low-density lipoprotein (alpha-2-microgel-2,284,26E-05
fibronectin enriched with leucine transmembrane protein 2-2,280,0135122
protein transport phospholipids-2,290,00999206

similar to dinamina-1 (D100) (dynamin, brain (B-DIN-2,290,0122573
SATB with homeoboxes 2-2,310,039781
similar to the member 3 family with TBC1 domain (Rab GTPase--2,32 0,000870285
homolog 1 tweet (Drosophila)-2,320,00450824
molecule CD24-2,340,0340122
chemerin (chimaerin) 1-2,350,0287031
AHA1, activator ATPase heat shock protein 90 kDa homolog-2,370,00979472
homolog 1 dwuhfaznosti C (Drosophila)-2,380,0347162
family of 6 vectors of dissolved substances (conveyor neurotransmitters, the-2,380,00729635
protein milk fat globule-EGF factor 8-2,390,000987073
the protein kinase 1 WNK deficiency of lysine-2,401,57E-05
small nucleolar RNA, H-2,410,00843141
homolog 3 songs (Drosophila)-2,42 0,000165552
2B domains SH3 and PX-2,420,0244357
containing 1 WD repeat and block SOCS-2,440,0387851
hypothetical protein PRO2012-2,450,00756704
Golgi autoantigen, subfamily holdin a-like pseudogene-2,460,00320764
protein 2 associated with microfibrillar-2,470,0152901
collagen, type XII, alpha 1-2,470,000204664
ST6 beta-galactosamide alpha-2,6-sialyltransferase 2-2,470,0216987

tr>
protein that interacts with thioredoxin-2,480,0135494
protein 2 linking of latent transforming growth factor beta-2,494,08E-05
Golgi autoantigen, subfamily holdin a-like pseudogene-2,490,00603583
like protein 1, binding Fomin-2,500,00290401
expressed in the mother 3-2,520,0112259
PTK7 proteincontaining 7-2,540,000116114
component H1 of ribonuclease P RNA-2,570,0156126
protein containing repeat sushi associated with X 2-2,580,0253856
related sortilin containing VPS10 domain receptor 2-2,580,00936311
similar to RIKEN cDNA 11100118M03-2,590,00516476
contains 2 domain paradoksaalsemaks decarboxylase-2,600,00683647
Enah-2,610,0077547
aspirin-2,620,000659873
small specific bullock Kajala RNA 17-2,630,0301336
protein that interacts with the nuclear pore complex-2,670,00988632
sushi, the factor a background of Villebranda of type A, EGF and pentraxin house-2,692,23E-05
patientinitiated, receptor type, U-2,690,0270428
collagen, type V, alpha 1-2,700,0166427
protein that interacts with the nuclear pore complex-2,730,0018339
transformer-2 alpha-2,740,012256

protein 2, related dystrophin-2,790,0137557
Golgi autoantigen, the treatment tip can astwo Goldin a, 8A-2,800,0111179
collagen, type VI, alpha 2-2,810,0149554
transforming growth factor, beta 3-2,810,0287865
Tropinin-2,820,00298044
hypothetical protein MGC24103-2,860,0346673
supervillin-2,870,0412717
metallopeptidase ADAM motif thrombospondin type 1-2,900,0113968
member 26B family of kinesin-2,910,00363199
protein that interacts with the nuclear pore complex-2,910,00160273
trichorhinophalangeal syndrome 1-2,940,00557712
protein that interacts with the nuclear pore complex -2,960,00111223
small nucleolar RNA, C-2,960,00666866
homeobox A2-2,970,0435423
homeobox 5 without distal-3,000,000640157
dachsous 1 (Drosophila)-3,000,00697244
small nucleolar RNA, C-3,060,0274043
small nucleolar RNA, C-3,060,0274043
protein that interacts with the nuclear pore complex-3,090,00583397
small nucleolar RNA, C-3,140,0104491
small nucleolar RNA, C-3,140,0104491
protein containing repeat sushi associated with X-3,160,00370941
white is 521 zinc finger -3,170,00668815

protein that interacts with the nuclear pore complex-3,170,00117582
open reading frame 3 of chromosome 9-3,180,00410177
Golgi autoantigen, subfamily Goldin a, 8B-3,180,0121417
gamecenter 1-3,210,0461603
small nucleolar RNA, C-3,240,00765575
the sequence of syndrome 1 Kallmann-3,250,000548703
tenascin C (hexabrachion)-3,268,26E-05
protein that interacts with the nuclear pore complex-3,290,00282604
protein that interacts with the nuclear pore complex-3,34 0,00263888
homeobox B2-3,360,00665994
similar to protein that interacts with the nuclear pore complex-3,410,0187322
protein that interacts with the nuclear pore complex-3,460,00354416
cholesterol-25-hydroxylase-3,510,0445558
protein 144 with zinc finger-3,520,0135334
protein that interacts with the nuclear pore complex-3,550,00316496
calbindin 2, 29 kDa (calretinin)-3,560,0290743
protein that interacts with the nuclear pore complex-3,580,00032839
protein that interacts with the nuclear pore complexof-3.600,000414309
protein that interacts with to the complex nuclear pore -3,620,00283418

protein that interacts with the nuclear pore complex-3,640,000213956
protein that interacts with the nuclear pore complex-3,660,000377834
KIAA1641-3,690,0191782
UDF-N-acetyl-alpha-D-galactosamine:polypeptide-N-acetyle-3,720,00964109
protein that interacts with the nuclear pore complex-3,730,000352007
containing 17 enriched with leucine repeat-3,750,0263961
open reading frame 3 of chromosome 9-3,800,0233723
protein that interacts with the nuclear pore complex-3,820,00368967
neurotronik3,78E-06
patientinitiated, receptor type, N-4,020,0294569
KIAA1641-4,020,00659194
----4,060,00488845
KIAA1641-4,160,0170531
integrin, alpha 11-4,160,000390317
KIAA1641-4,270,013175
odz, odd Oz-4,280,00172671
transmembrane protein 119-4,340,00801387
contains 2 domain plexin-4,440,031799
a family of genes of the ras homologue member J-4,590,00197982
homeobox B3-4,600,0354368
similar to protein KIAA0220-4,720,0302619
member 2 family rattlin-4,790,0260454

protein 1 pathway signaling induced by WNT1-5,990,000672342
clusterin-6,400,0303973
inhibitor Sarbinowo peptidases, group F (alpha-2 antiplasmin-6,470,00362941
sulfatase 2-6,585,88E-05
efectin-6,740,0123141
the adhesion molecule 2 contacts-7,330,0306758
containing 1 domain of fibronectin type III-7,460,0334696
sarcoglycan, Delta (35 kDa associated with distrofia glycoprotein-7,690,000881984
cystatin SN-8,270,0496433
protein 4 is associated with microfibrillar-8,670,00155578
biglycan-8,700,00161284
transmembrane induced by androgen in the prostate RNA-10,540,000100935
the carboxypeptidase E-12,480,00738131

Characterization of membrane markers in 2D attached cells suitable for use in accordance with these instructions- surface antigens expressed 2D attached cells was assessed using monoclonal antibodies. These cells represented a stable attachment of the cells, which were multiplied in vitro without loss of phenotype and without showing signs of changes in karyotype. Analysis of membrane markers 2D attached cells using flow cytometry showed a high proportion of cells expressing CD105, CD73, CD90 and CD29. Moreover, a high proportion of cells expressed CD45, CD34 and surface markers CD19, CD11b, CD14 and HLA-DR (figure 2).

And monomodality 2D attached cells - Then investigated the immunogenicity 2D attached cells. As shown in figure 3, four different parties 2D attached cells were able to reduce the proliferation of lymphocytes caused by mitogenic stimuli of phytohemagglutinin (PHA), according to the measurement of thymidine incorporation.

Induction of osteocytes- osteocyte differentiation of attached cells from the placenta or bone marrow environment for the induction of osteogenesis led to the differentiation of more than 50% of the cells from the bone marrow, as it was demonstrated positive staining with alizarin red (pigv). In contrast, none of the cells from the placenta did not show any signs of osteogenic differentiation (see figv and 4E and table 9 below).

Table 9
Total differentiation
BM108+ BM109PLC-11-3-1PLC-8-2-1Plc-15-3-4-2Plc 4-3-1
Osteocytes+++----
Adipocytes +++----

Then, 2D attached cells originating from the bone marrow or placenta, stimulated the differentiation of the modified osteogenic medium, including vitamin D and higher concentrations of dexamethasone, corresponding to the earlier instructions for modification of the Protocol of osteogenic differentiation [Parloni et al. (2008) Stem Cells 26(2): 300-11]. As follows from the results, more than 50% of the cells from the bone marrow were subjected to differentiation into osteocytes, as it was demonstrated positive staining with alizarin red (see figv). However, none of the cells from the placenta did not show any signs of osteogenic differentiation (see file and table 9 above in the present description).

Induction of adipocyte- differentiation into adipocytes 2D attached cells from the placenta or bone marrow environment for the induction of adipocytes led to the differentiation of more than 50% of the cells from the bone marrow (see figs), as demonstrated by positive staining with oil red and typical morphological changes (e.g., accumulation of fat droplets in the cytoplasm). In contrast, none of the cells from the placenta are not differentiated into adipocytes (see fig.4F and table 9 above in this OPI is offering).

Then, 2D attached cells originating from the bone marrow or placenta, stimulated the differentiation into adipocytes in a modified environment, including higher level indometacin corresponding to the previous instructions for modification of the Protocol adipocyte differentiation [Parloni et al. (2007), above]. As follows from the results, more than 50% of the cells from the bone marrow were subjected to differentiation into adipocytes (see figs), as it was demonstrated positive staining with oil red and typical morphological changes (e.g., accumulation of fat droplets in the cytoplasm). In contrast, none of the cells from the placenta showed no morphological changes typical of adipocytes (see fig.5F and table 9 above in the present description).

EXAMPLE 3

Methods of creating 3D attached cells suitable for use in accordance with these instructions, and created with them 3D attached cells

Were obtained 3D attached cells (PLX-C), which exhibit properties different from the properties described above 3D attached cells (PLX, example 1).

Materials and experimental procedures

Flow-through bioreactor with shutter CelligenTM

Obtaining 3D attached cells for use according to the present invention using CelligenTM(PLX-C cells) is ostalo of several main stages. The process begins with the receipt of the placenta during routine delivery by caesarean section at term.

Then from the whole placentas were isolated fixed cells were grown in containers for tissue culture (2D culture), was collected and stored in liquid nitrogen as a stock 2D cells (2DCS), an appropriate amount of 2DCS were thawed, washed and were sown on media in bioreactors for further cultivation in the form of 3D-culture. After 4-21 days of cultivation in bioreactors, the cells were collected and kryokonservierung in the gas phase of liquid nitrogen as PLX-C.

Obtaining human tissues

All the placenta came from the maternity Department with the approval of the Helsinki Committee for medical assistance. Accordingly, all donors placenta was signed informed consent and were screened donors and testing of donors (IPC1). Immediately upon receipt of the placenta from the donor (during the procedure caesarean section) were placed in a sterile plastic bag and then in a foam box with bags of ice. The placenta was removed and immediately placed in quarantine zone to obtain permission for use by quality control (QC) and quality assurance (QA). All subsequent phases of obtaining produced in quarantine, adapted the clean room before entering the approved QC test results is as Mycoplasma, and cells were isolated for 2-dimensional cultivation.

The selection and processing of attached cells

First, the fabric of the whole placenta was cut into pieces under aseptic conditions in a fume hood with laminar flow, washed with buffer solution Hank and incubated for 3 hours at 37°C With 0.1% collagenase (1 mg collagenase/ml tissue). Added 2D cellular environment (2D environment, including DMEM with addition of 10% FBS, 0.25 microgram/ml Fungizone and 50 μg/ml gentamicin), and digested tissue is roughly filtered through a sterile metal grid, collected in a sterile beaker and centrifuged (10 min, 1200 rpm, 4°C). Using easy pipetting suspended cells were then washed 2D environment with the addition of antibiotics, were sown in 80 cm2vessels and incubated at 37°C in incubator for tissue culture in moisture conditions with a flow of 5% CO2. After 2-3 days, during which the cells are allowed to attach to the surface of the vessel, the cells were washed SFR and added a 2D environment.

Two-dimensional (2D) growing cells

Before the first passage of the samples environment for growing at 10% of the total number of vessels in quarantine were combined and passed testing Mycoplasma (IPC2). If it was found that cells negative in relation to Mycoplasma (set EZ-PCR Micoplasma, Biological Industries, Israel), cell asvab what was expected from quarantine. After 1-2 additional passages of the cells was transferred into a clean room to obtain 2D (2DP). In the room 2DP cultivation was continued by another 3-5 passages (note that cells were grown in a 2D environment with the addition of antibiotics to the passage 3, after which the cells were grown in 2D environment without antibiotics). After passage of the 4 selected sample IPC-3 to determine the immunological phenotype. During the cultures were grown in an incubator for tissue culture in moisture conditions with 5% CO2at 37°C. After a total of 6-8 passages (9-16 doublings of cells) cells was collected and stored at cryogenic temperatures as stock 2D cells (2DCS).

The first passage is usually carried out after 10-15 days. Since passage 2 until the passage of 6-8 cells were passively when the culture 70-80% of confluently, usually within 3-5 days (1.5-2 doublings). Cells were separated from blood vessels using 0.25% trypsin-EDTA (4 minutes at 37°C) and were sown with obtaining density culture 3±0,2×103cells/cm2. The size of the vessels for tissue culture increased in accordance with the continuation of the passages. The process of cultivation began in the vessel to tissue culture 80 cm2continued 175 cm2then 500 cm2(triple vessel) and, finally, the cells were sown in cell factory 10 trays (6320 cm2).

Before cryopreservation in the rates of growing period 2DSC collected culture medium and received a sample to send in GLP certified laboratory for testing Mycoplasma (IPC4).

Cryopreservation procedure stored product 2D-cells

For cryopreservation 2DCS 2D-cultured cells were collected in aseptic conditions using 0.25% trypsin-EDTA. Cells were centrifuged (1200 rpm, 10 min, 4°C)were calculated and resuspendable in a 2D environment.

For freezing the cell suspension was diluted 1:1 2D environment for freezing (final concentration was 10% DMSO, 40% STR and 50% 2D environment). From one placenta has received approximately 1.5 to 2.5×109cells. 4 ml of cells were stored at a final concentration of 10×106/ml in 5-ml polypropylene containers for cryopreservation. The vessels were marked and transferred to the freezer with a controlled cooling rate for a process with a gradual decrease in temperature (1°C/min), and then transferred for storage in the gas phase of the freezer liquid nitrogen in the storage room for cooling. This material was designated as reserved party 2D cells (2DCS).

Initiation of procedures for three-dimensional (3D) culturing

To initiate 3D culture appropriate amount (150±30×106) cells from 2DCS were thawed at room 2DP and washed 3D environment (DMEM with 10% FBS and 20 mm Hepes) to remove DMSO before sowing in advance bioreactor system. The contents of each vessel 2DCS has pietravalle and diluted 1:9 pre-heated (37°C)3D-environment. Cells were centrifuged (1200 rpm, 10 min, 4°C) and again resuspendable in 50-100 ml of preheated (37°C) 3D environment in a 250 ml sterile bottles. Selected sample, and the cells were counted by means of a dye Trypanosoma blue to determine the number and viability of cells. The cell suspension was transferred to a fume hood laminar flow in a 0.5-l bottle for sowing. Out of the bottle for sowing cell suspension was transferred under the action of gravity through a sterile tube in the bioreactor.

Getting attached cells in the CelliGen bioreactor (PLX-C)

Description bioreactor

Phase growing 3D was performed using a bioreactor system CelliGen Plus® or BIOFLO 310 [(New Brunswick Scientific (NBS)]. Bioreactor system was used for culturing the cell culture conditions which were suitable for high concentrations of cells. The cultivation process was performed using a bioreactor in perfusion mode. The laboratory-scale bioreactor was collected from two main systems - control systems and the bioreactor (vessel and auxiliary equipment). The process parameters were monitored and controlled using the control console, which included connecting device for probes, motor and pumps, control loops dissolved oxygen (DO), pH, perfusion is shaking (with motor), control system for gas, water circulation system and heat for temperature control and an interface for the operator. The parameters of the controlled process (such as temperature, pH, DO, etc.) was displayed on the operator interface and tracked using the designed controller.

The procedure of growing cell cultures in bioreactors

As noted in the previous section of the present description, 150±30×106cells from cryopreserved 2DCS were thawed, washed and planted in sterile bioreactor. The bioreactor contained 30-50 g carriers (CDs FibraCel®, NBS), made of polyester and polypropylene, and 1.5±0.1 l 3D environment. The culture medium in the bioreactor was kept under the following conditions: 37°C, 70% dissolved oxygen (DO) and pH 7.3. The filtered gases (air, CO2N2and O2) was administered as determined by the control system to maintain the value of DO at the level of 70% and a pH level of 7.3. Within the first 24 hours the medium was stirred at 50 revolutions per minute (rpm) and increased the speed up to 200 rpm on day 2. During the first 2-3 days, the cells were grown in batch mode. Perfusion was started, when the concentration of glucose in the medium was decreased below 550 mg/L. the Medium was pumped from the container to feed into the bioreactor using sterile silicone tubing. All connections of the tubes produced under l is minirnum flow using sterile connectors. Perfusion brought daily to maintain the glucose concentration constant at approximately 550±50 mg/L. Every 1-2 days selected sample environment for growing to determine the concentration of glucose, lactate, glutamine, glutamate and ammonium (BioProfile analyzer 400, Nova Biomedical). The ratio of the rate of consumption of glucose and the rate of formation of lactate cell culture gave the opportunity to measure the rate of cell growth. These parameters are used to determine the time of collection based on the collected experimental data.

Collecting grown 3D cells PLX-C of the bioreactor

The process of collecting cells began at the end of the growth phase (4-10 days). Selected two sample environment for growing. One sample was prepared to send in GLP certified laboratory for testing of Mycoplasma in accordance with USP standards and Eu, and the other sample was transferred into a freezer-controlled rate freezing process of gradual reduction of temperature (1°C/min), after which he was transferred for storage in the gas phase of the freezer liquid nitrogen, which is located in the storage room during cooling, in case you need re-testing of Mycoplasma. These samples environment was considered as part of the Mycoplasma testing of the final product and the results were considered as part of the criteria on which I release product.

Grown in 3D culture was collected in a laminar region of a class-100 at room 3DP as follows:

The contents of the vessel bioreactor was removed to empty the container through the tube by gravity. The vessel was opened by removing the top cover and the media aseptically transferred with sterile forceps from a bunker at the top of the pancake grid. After that, the vessel bioreactor was closed and again was filled with 1.5 l of preheated SFR (37°C). The speed of agitation was increased to 150 rpm for 2 minutes. SFR was removed with the tube under pressure or under the influence of gravity into the empty vessel. The washing procedure was repeated twice.

To separate the cells from the media into the vessel bioreactor was added 1.5 l of pre-warmed to 37°C solution of trypsin-EDTA (trypsin 0,25%, EDTA 1 mm) and the media were shaken for 5 minutes at 150 rpm and 37°C. the Cell suspension was collected in 5-l sterile container containing 250 ml SPR. The cell suspension was divided between 4500-ml sterile tubes for centrifugation and selected the sample for Mycoplasma testing. Closed centrifuge tubes transferred through the core 3DP to migrate into the bathroom to fill a class 10000 (FR1), in which cells aseptically carried and kryokonservierung as PLX-C.

Analysis of the cell cycle

Cells PLX-C, recip is by using Celligen, and PLX cells obtained by Plurix, fixed with 70% EtOH'ev O.N, centrifuged and resuspendable in the solution of propecia iodide (PI)containing 2 μg/ml PI (Sigma), 0.2 mg/ml RNase A (Sigma) and 0.1% (vol./about.) Triton (Sigma) for 30 minutes. Cell cycle was analyzed using FACS.

Matrix (micrometric) for analysis of gene expression

Attached cells were obtained from full-term human placentae and multiplied with the Plurix or Celligen. For further analysis were obtained on three different batches of cells using each of the methods of reproduction.

Cells were extracted RNA (kit Qiagen-Rneasy micro) and were applied to the matrix Affymetrix expression of the entire genome. micrometric was a GeneChip® Human Exon 1.0 ST Array (Affymetrix, Santa Clara, California, USA).

Analysis of membrane markers using FACS

Cells were stained with monoclonal antibodies as described previously. Briefly, 400000-600000 cells suspended in 0.1 ml of buffer for the flow cytometer in 5-ml test tube to test for 15 minutes at room temperature (RT) in the dark with each of the following monoclonal antibodies (MAbs): conjugated with FITC MAb against human CD29 (eBioscience), conjugated with PE MAb against human CD73 (Becton Dickinson), conjugated with PE MAb against human CD105 (eBioscience), conjugated with PE MAb against human CD90 (Becton Dickinson), conjugated with FITC MAb against human CD45 IQProducts), conjugated with PE MAb against human CD19 (IQProducts), conjugated with PE MAb against human CD14 (IQProducts), conjugated with FITC MAb against HLA-DR human (IQProducts), conjugated with PE MAb against human CD34 (IQProducts), conjugated with FITC MAb against human CD31 (eBioscience), conjugated with FITC MAb against human KDR (R&D systems), MAb against marker of fibroblasts (D7-FIB) (ACRIS), conjugated with FITC MAb against human CD80 (BD), conjugated with FITC MAb against human CD86 (BD), conjugated with FITC MAb against human CD40 (BD), conjugated with FITC MAb against HLA-ABC man (BD), conjugated with FITC-isotype IgG1 (IQ Products), conjugated with PE isotype IgG1 (IQ Products).

Cells were washed twice with a buffer for the flow cytometer, resuspendable in 500 μl of buffer for the flow cytometer and analyzed using flow cytometry with the use of the flow cytometer FC 500 (Beckman Coulter). Negative controls were obtained with fluorescent molecules of the appropriate isotype.

Reaction mixed lymphocytes (MLR)

2×105MNC from peripheral blood (PB) (from A donor) stimulated an equal number of irradiated (3000 rad) MNCs from PB (donor B). The cultures were added PLX-Cs in increasing numbers. Three repetitions of each group were sown in 96-well plates. Cells were cultured in medium RPMI 1640 containing 20% SPR. Tablets pulsed marked 1 µci3 H-thymidine during the last 18 h of a 5-day cultivation. Cells were collected on glass fiber filter and capture thymidine was quantitatively determined using a scintillation counter.

For CFSE staining cells PB-MNC were stained for CFSE (Molecular Probes) to measure proliferation before cultivation. Cells were collected after 5 days and the intensity of CFSE staining was determined using flow cytometry.

ELISA

ELISA was performed as described previously. Briefly, MNCs (isolated from peripheral blood) were stimulated with 5 μg/ml ConA (Sigma), 0.5 μg/ml LPS (SIGMA) or 10 μg/ml PHA (SIGMA) in the presence of PLX-C in humidified atmosphere of 5% CO2 at 37°C. was Collected supernatant and subjected to analysis for cytokines using ELISA kits for IFNγ (DIACLONE), TNFα (DIACLONE) and IL-10 (DIACLONE).

Results

Changes in receiving with Celligen compared to the Plurix gave some important differences (summarized in table 10 below).

Table 10
Comparison of the Plurix system (WO/2007/108003) Celligen (specify present invention)
WO/2007/1080033D attached cells these instructionsImproved the e
Working volume (ml)2801500Zoomed-in process. A higher level of production in these indications (2-8 population doublings)
Mass media (g)1,430The increased scale of the process in these instructions
Configuration fundamentalsConical 50-ml columnCylindrical Packed baseThese guidelines is the best course of environment and nutrients. WO/2007/108003 ineffective flow due to the narrow shape of the outlet of the conical structure
The best uniformity of flow. The formation of channels in the plurix
The concentration of cells in culture (cells/g of carrier)3×106cells/g media5×106cells/g mediaThe best interaction between cells in these instructions

The concentration of cells in culture (cells/ml)0,015×106to etoc/ml 0,1×106cells/mlThe best interaction between cells in these instructions
The procedure of seedingSeeding with a small volume of medium for 24 h followed by the addition of the medium to a final working volumeThe seeding of the final working volume when mixingWO/2007/108003 heterogeneous distribution of cell culture inside the framework with the media
Insufficient volume of medium in the first 24 hours of the process. Leads to inappropriate working conditions (acidic)
The duration of the phase of the receiving14-21 day4-10 daysBest quality product.
Effective collection process.
The best way out.
Lower cost process in these instructions
The mode of operationRecurring batch replacing the medium twice a weekThe mode of perfusion - rate brought in accordance with the concentration of glucose (environment changed when the concentration of glucose 550±50 mg/lThese guidelines - and moderate changes in the conditions relating to the composition of the environment during the process
Continuous removal of toxic agents produced by the cells. When batch mode is a lower concentration of essential nutrients (limiting factors)
Smaller fragments of cells
Collection procedureGathering in 50-ml tubes
Treatment with trypsin 3 cycle
The collection inside the bioreactor
Treatment with trypsin 1 cycle
These guidelines - a more efficient process
The collection is produced in a closed system.
1 cycle of treatment with trypsin - best quality cells.

td align="justify"> Direct control online. Heat transfer through the water jacket.
ShakeThe circulation of the medium between the reservoir container and the column using a peristaltic pumpLifting cage impellerThese guidelines - environment flows through Packed the basis is the best supply of culture with nutrients and oxygen. The homogeneity of the environment improves other control loops (temp., DO, pH)
Temperature controlGetting produced inside an incubator. Indirect temperature control (camera incubator). Heat transfer across the boundary of contact with airThese guidelines provides more precise temperature measurement culture. Quick reply. Short time to achieve the set point.
Tracking temperatureManual.
Indirect water monitoring temperature.
Direct tracking onlineThese guidelines have the best tracking and control process. Quick response on failure
Tracking DONoTracking onlineThese guidelines have the best tracking and control process. Quick response on failure
Control DOIs missing.
Introduction only air
Direct control in the operational mode of the specified installation point using air, O2and N2.These guidelines is the best level control DO. The best maintenance of specified operating conditions

Control, otslezhivanie the pH Only visual tracking (phenol red as part of the environment)Monitoring and tracking onlineThese guidelines - the best control of pH.
The best maintenance of specified operating conditions
AerationOnly bubblingApplique (bubbling as option)WO/2007/108003 - aeration by bubbling creates a foam that can damage cells.

Changes in the process has led to changes in the properties of the attached cells. These differences are summarized below.

Analysis of cell cycle PLX obtained by Plurix, compared with PLX-C obtained by Celligen

Cells PLX-C obtained by Celligen, compared with PLX cells obtained by Plurix, to estimate the distribution of cells between the different phases of the cell cycle. As is clear from figa-B cells PLX-C, multiplied with Celligen showed typical proliferative profile (distribution of cells between the different phases of the cell cycle). Specifically, 28% of the cells were in the phases S and G2/M (figa). These results showed that the cells were collected during proliferation and conditions of the Celligen bioreactor supported cell is growth.

Comparison of cells obtained by Plurix and Celligen using microarrays

Analysis of gene expression using microarrays allowed us to simultaneously monitor the expression profiles of whole genome attached cells derived from human placentas with full-term pregnancy, multiplied with Plurix (PLX) or Celligen (PLX-C). These results allowed us to estimate the molecular mechanism underlying phenotypic differences between cells obtained by these different methods of cultivation (see table 11 below).

td align="justify"> 13,99
Table 11
Expression of genes in cells Plurix (WO/2007/108003) compared with cells Celligen (specify present invention)
GeneCelligen against Plurix (rate of change)P-value (brought)
induced by interferon protein with tetratricopeptide repeats17,520,0401812
the ferment aldehyddehydrogenase as a part of the family 1, member A116,760,00145807
argininebenefits of leukocytes3,88E-06
keratin 27 pseudogene 2712,250,000224998
similar to keratin, type I cytoskeletal 18 (cytokeratin11,830,000304949
coupled with G protein-coupled receptor, family C, group 5, member A10,353,39E-05
integrin, alpha 69,840,0411667
coupled with G protein-coupled receptor 1268,730,00197635
the coagulation factor III (thromboplastin, tissue factor)of 7.360,012192

the inhibitor dissociation of Rho GDF (GDI) betaof 7.360,00200066
signal peptide, CUB domain, EGF-like 37,200,0255115
induced by interferon protein with tetratricopeptide repeats7,0 0,0139777
homolog 1 dickkopf (Xenopus laevis)7,063,06E-07
NAD(p)H dehydrogenase, quinone 16,630,000282423
keratin 186,460,000514523
receptor opioid growth factor like 15,960,00114551
mal, like a squirrel differentiation of T-cells5,950,00664216
neurofilament, medium polypeptide 150 kDa5,860,0190611
containing the domain DEP 1of 5.820,000370513
cathepsin C5,720,00532262
WAS5,470,00178153
inhibitor Sarbinowo peptidases, group B (ovalbumin), member5,440,0190218
family of 7 vectors of dissolved substances (Transporter of cationic amino acids5,330,00688017
induced by interferon protein with tetratricopeptide repeat5,180,00357376
NUF2, a component of the NDC80 kinetochore complex, homolog (S. cereof 5.050,00276524
protein 1, binding SH2 domain of SHC4,950,00430878
thioredoxins 14,860,000197486
protein associated with the metastasis of lung cancer4,850,00148024
protein 29, activating GTPase Rho4,850,0466211

nei endonuclease VIII-like 3 (E. coli)
homolog 20 cycle of dividing cells (S. cerevisiae)4,800,00514206
family with sequence similarity 111, member B4,630,000125819
linking-bearing kinase4,540,00784983
the establishment of the clutch 1 homolog 2 (S. cerevisiae)4,530,000773033
guanylatzykalse protein 44,470,000215944
lipase A, lysosomal acid, cholesterylester disease Wolman4,420,0167385
member 20A of the family of kinesin4,390,00582352
KIAA01014,280,0105909
inhibitor 3 cyclin-dependent kinase (associated with CDK2 double4,250,000732492
the timidilatsintetazu4,230,00685584
open reading frame 3 chromosome 134,180,000548296
the kinase Aurora A4,160,00632571
4,140,00115606
protein of the centrosome 55 kDa4,130,0021952
receptor 1 (similar to lectins) oxidized low density lipoprotein4,110,0205198
denticleless homolog (Drosophila)4,050,00141153
Anilin, actin binding protein4,010,010923
ribonucleotides M2 polypeptide3,980,00834059
domain 1 with ancyranum repeat (heart muscle)3,930,00911953
transcription factor 19 (SC1)3,890,00109627
keratin 183,890,000112551

td align="justify"> 0,00124327
non-SMC complex condensin I, subunit G3,88 0,00537097
cyclin E2a 3.870,000203389
trypsinogen C3,860,00416276
small nucleolar RNA, C3,810,0334484
protein 2 dense contacts (zona occludence 2)3,810,00012562
member 18A family of kinesin3,780,00134108
member of the 2C family of kinesinof 3.770,0059888
shugoshin-like 1 (S. pombe)3,760,00101318
polo-like kinase 1 (Drosophila)3,750,0140309
thymidine kinase 1, solubleto 3.730,00124134
transcription factor 19 (SC1)to 3.730,00124327
transcription factor 19 (SC1)to 3.73
homolog claspin (Xenopus laevis)3,710,00683624
subunit 1 of complex GINS (homolog Psf1)3,690,00104515
microsomal glutathione S-transferase 1to 3.670,041701
arylacetamides-like 1to 3.670,000902645
SPC25, a component of the NDC80 kinetochore complex, homolog (S. cethe 3.650,00568662
integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-43,620,0158411
catenin (associated with Katherina protein), alpha-like 13,577,46E-05
discs, large homolog 7 (Drosophila)of 3.560,0317074
like (avian) homolog of the viral oncogene myeloblastosis v-myb3,550,0043878
serpitine3,540,0443487

protein N centromere3,530,000540143
cyclin A23,530,00965934
protein 8 heat shock 22 kDa3,520,0219583
domain sema, immunoglobulin domain (Ig), short basic home3,490,008548
protein 11A, activating GTPase Rho3,490,00834174
anemia Fanconi's syndrome, a group of complementaly I3,430,00464532
BUB1 of budding, not inhibiting the benzimidazole, homolog 1 (yeast3,420,0108258
specific to ovarian acidic protein3,420,00334641
cholinergic receptor, muscarinic 23,41 0,0320078
the cycle of cell division 2, G1 to S and from G2 to M3,410,0017111
protein regulator of cytokinesis 13,390,0325664
component 5 complex save minichromosome3,380,00475504
antigen 5, associated with semen3,370,00906321
maternal embryonic kinase with latinboy zipper3,340,00908391
small nucleolar RNA, C3,330,0298703
carnitinelongevity 1A (liver)3,330,00170894
similar to ubiquitin-conjugating enzyme E2S (ubiqui3,330,000415822
the member 11 family of kinesin3,330,00915145
kinase 7, related NIMA (gene a sa is in mitosis) 3,330,00159114
metallopeptidase ADAM motif thrombospondin type 13,320,0102751

transforming containing acid double helix protein 33,310,0014577
cyclin B13,290,0103092
MAD2 failure stopping mitosis-like 1 (yeast)3,280,00488102
dihydrotetrazolo3,280,00178879
contains 3 NIPA-like domainwith 3.270,00164708
associated 2 with the cycle of cell division3,260,0122226
enzyme mRNA editing of apolipoprotein B, catalytic polypep3,260,00308692
cyclin B23,25 0,016544
containing endonuclease domain 13,240,000429245
digidrofolatreduktazy pseudogene3,230,00141306
ATPase, Na+3,230,000381464
the replication factor C (activator 1) 3, 38 kDa3,230,00109668
domain repeat, WD 763,220,0023531
pleckstrin 23,170,0304429
protein 1, activating GTPase Rac3,170,00381613
protein 19 with PHD finger3,170,000177604
remote in lymphocytic leukemia, 23,150,0109528
protein I centromere3,150,0106816
associated with BRCA1 domain 1 RING 3,140,000540414
the controller 4 signaling G-protein3,130,00781061
STAM-binding protein-like 13,110,0181743
homolog sulfiredoxin 1 (S. cerevisiae)3,105,14E-05

the open reading frame 23 chromosomes 15is 3.080,000147331
protein kinase TTKis 3.080,0112171
non-SMC complex condensin II, subunit G2is 3.080,0130322
villin 2 (Ezrin)of 3.070,0131934
stomatin3,060,00387095
contains A domain similar to proteincarbohydrate3,060,0419644
inhibitor Sarbinowo peptidases, group B (ova albumin), member3,050,0030439
member 4A family of kinesin3,050,0114203
hypothetical protein DKFZp762E13123,050,00726778
ubiquitin-conjugating enzyme E2S3.04 from0,00118205
hydroxysteroiddehydrogenase-similar 23,033,71E-05
the collection ATPase containing domain 2 AAA3,010,00415258
TPX2 associated with microtubules, homolog (Xenopus laevis)3,000,0253137
the histone cluster 1, H4d3,000,030183
the member 23 family of kinesin2,990,00790585
protein 2 heat shock 70 kDa2,990,0215102
complex recognition of the origin, such su is jedinice 1 (yeast) 2,990,00207753
dihydrotetrazolo2,980,00307793
receptor mediated by hyaluronan motility (RHAMM)2,970,00467816
3'-phosphoadenosine-5'-phosphosulfate 22,971,43E-05

glycero-3-phosphatedehydrogenase 2 (mitochondrial)2,950,00211969
protein 1 associated with nucleolar and spindle2,950,00520875
diaphanous homolog 3 (Drosophila)2,950,00107709
the member 14 of the family of kinesin2,940,00947901
the histone cluster 1, H1b2,930,0470898
binding protein goinnovate nucleotides (G protein), alpha Inga2,92 0,00184597
component 8 complex save minichromosome2,920,000841489
candidate 5 exposure cancer2,920,0330594
leukotriene B4 12-hydroxydehydrogenase2,920,000685452
glutamate-cysteinemia, modifier subunit2,910,00378868
villagelevel block M12,910,0203154
protein associated with adipose differentiation2,900,000331751
associated with the membrane containing the domain O-acyltransferase 12,900,01185
ubiquitin-conjugating enzyme E2T (potential)2,900,00741886
associated 3 with the cycle of cell division2,890,006289
integrin, al is 3 (antigen CD49C, alpha 3 subunit of VLA-32,880,00574148
the coagulation factor XIII polypeptide B2,880,0294465
the RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae)2,870,000854739
ATP-binding cassette, subfamily C (CFTR2,870,00382491

family with sequence similarity 29, member A2,850,00111165
4A, containing SH2 domain2,840,0323646
membrane protein palmitoylation 1, 55 kDa2,840,000396285
regulatory subunit 1B protein kinase CDC282,840,0107391
PSMC3 interacting with protein2,840,00766442
part 2 elastin microfibril 2,840,0192072
topoisomerase (DNA) II alpha 170 kDa2,830,0321109
transmembrane protein 106C2,820,000214223
the histone cluster 1, H3b2,800,0304598
the open reading frame 24 chromosome 182,800,00347442
the substrate 8 ways receptor epidermal growth factor2,790,0194949
nucleosomal binding domain 2 high-mobility group2,780,0030536
SCL2,780,00390288
domain hect and RLD42,780,00679184
homolog B ASF1 antisilence function 1 (S. cerevisae)2,770,00543408
interacts with thyroid hormone receptor 13 was 2.760,0118319
associated 8 with the cycle of cell division2,750,00619878
member of the C1 family of kinesin2,740,00821937
nucleosomal binding domain 2 high-mobility group2,730,00384071
ornithindecarboxilase 12,730,00144868

a similar 2 (avian) homolog of the viral oncogene myeloblastosis v-myb2,710,00989416
ligand KIT2,700,00641955
kinase with dual specificity regulated by phosphorylation of tyrosine (Y)2,700,0234606
homolog vnutrijeludochkovogo transport 80 (Chlamydomonas)2,700,0247286
transmembrane protein 482,69 0,00458248
protein 2, EBNA1 binding2,690,00296292
interacting with ZW102,691,88E-05
the exonuclease 12,680,00739393
transketolase (syndrome Wernicke-Korsakov)2,681,92E-05
the somatostatin receptor 12,680,0144901
izotsitratdegidrogenazy 3 (NAD+) alphato 2.670,00297129
protein 2 associated with the cytoskeletonto 2.670,0030499
component 4 complex save minichromosometo 2.670,00342054
inhibitor 1 DNA binding, dominant negative helix-loop-sleep2,660,036485
regulatory subunit 1B protein kinase CDC282,66 0,0145263
keratin 182,668,40E-05
the CD97 molecule2,660,00994045
the open reading frame 173 chromosome 62,640,00222408
containing BTB (POZ) domain 32,620,0166824
deafness, autosomal dominant 52,620,00235481

protein KIAA02862,620,00130563
anemia Fanconi's syndrome, a group of complementaly D22,610,0281405
polo-like kinase 4 (Drosophila)2,600,00209633
ribonucleotides M1 polypeptide2,600,000170076
malic enzyme 1, NADP(+)-dependent, cytosolic2,590,0435444
non-SMC complex condensin I, subunit H2,590,0216752
S100 calcium-binding protein A32,580,0324073
ubiquitin-conjugating enzyme E2L 3to 2.570,00343347
BUB1 of budding, not inhibiting the benzimidazole, homolog beta2,560,0166047
glycerokinase2,552,66E-05
TAF9B RNA polymerase II binding protein block TATA (TBP)-AC2,540,0170365
TAF9B RNA polymerase II binding protein block TATA (TBP)-AC2,540,0170365
the histone cluster 1, H2bg2,520,000180822
unit 2 high-mobility group2,520,0196872
kinase 2, related NIMA (a gene never in mitosis)250 0,00289469
enriched with Proline 112,500,0357125
myoperation2,490,0255088
containing the domain brix 12,490,00471977
associated 5 with the cycle of cell division2,490,01021
fucosidase, alpha-L-2, plasma2,490,00540929
cyclin-dependent kinase 22,490,00250724
receptor lamina B2,490,000151784

hypoxanthineguanine 1 (Lesch-Nyhan syndrome2,490,000634057
containing a triple motif 252,470,0456344
proteasome (proteasomes, macrofauna) subunit, type beta 9 (lar 2,460,0202595
proteasome (proteasomes, macrofauna) subunit, type beta 9 (lar2,460,0202595
proteasome (proteasomes, macrofauna) subunit, type beta 9 (lar2,460,0202595
sphingomyelinase 22,460,0020701
transmembrane protein 622,450,00761064
glucose-6-phosphatedehydrogenase2,440,00278311
protein 1 PHD finger2,440,010191
retinoblastoma-like 1 (p107)2,440,00319946
KIAA15242,430,0380688
ST6 (alpha-N-acetylneuraminic-2,3-beta-galactosyl-1,2,430,00830766
cofilin 2 (muscle)2,430,059235
hypothetical protein LOC2017252,420,000313319
a homologue of A cycle of cell division 25 (S. pombe)2,420,000341692
breast cancer 1 early onset2,410,0180553
transaldolase 12,410,00199537
homolog mRNA turnover 4 (S. cerevisiae)2,410,00373104
glucosaminyl(N-acetyl)transferase 1, base 2 (beta-1,6-N-2,410,0197148
knob 1 is enriched in cysteine transmembrane BMP (such jordino)2,410,0267286

the inhibitor of the tissue factor pathway (associated with lipoprotein2,400,0356227
the open reading frame 59 chromosome 162,400,00185191/td>
glycogenin 12,390,0224317
transmembrane protein 1542,390,0045589
such 1 antigen canalave-interstitial nephritis2,390,00510812
TTF-synthase2,388,80E-05
phenylalanine-tRNA-synthase, subunit beta2,380,000245973
geminin, inhibitor of DNA replication2,380,00167629
Lamin B12,370,0477748
SPC24, a component of the NDC80 kinetochore complex, homolog (S. ce2,360,00287227
the glutathion reductase2,360,00353875
such 1 ribosomal protein L222,360,00335381
fumarase acetylhydrolase (fumarylacetoacetase) 2,363,88E-05
small nucleolar RNA, C2,350,0188991
family with sequence similarity 64, member A2,350,0019785
oncogene sequence 2, transforming epithelial cells2,350,000571152
polymerase (directed DNA), Epsilon 2 (p59 subunit)2,340,00479612
glycerokinase2,343,37E-06
glutathione-S-transferase M2 (muscle)2,330,0402076
the elongation factor, RNA polymerase II, 22,330,0130017
thioredoxin2,330,009636

polymerase (directed DNA), alpha 2 (70 kDa subunit)2,32 0,0033903
breast cancer 2 early onset2,320,00586847
CDC45 cycle cell division 45-like (S. cerevisiae)2,320,00735977
member of the Z family of histone H2A2,320,0129697
the Transporter 1, ATP-binding cassette, subfamily B (MDR2,310,0164234
the Transporter 1, ATP-binding cassette, subfamily B (MDR2,310,0164234
the Transporter 1, ATP-binding cassette, subfamily B (MDR2,310,0164234
homolog-associated nucleolar complex 3 (S. cerevisiae)2,300,000373346
ATPase, Ca++transporting, plasma membrane 42,300,023011
component 7 complex save minichromosome2,30 0,0457691
protein that interacts with TIMELESSto 2.290,00771062
protein 1, binding Hippel-Lindau2,280,00329061
related ras substrate 2 toxin botulinum C3 (rho family, sma2,280,0292466
thymopoietin2,280,0223176
peptidyltransferase F (cyclophilin F)2,280,00093846
molecule cell adhesion of activated leukocytes2,270,00242163
ring finger 5 polycomb group2,270,000294142
protein 1, activating GTPase Ran2,279,68E-05
the replication factor C (activator 1) 4, 37 kDaof 2.260,00164152

of 2.26
tubulin, beta 2C0,000346744
component 10 complex save minichromosomeof 2.260,0037925
the family of histone H2B, member S2,250,000885505
gamma glutamylcysteine (conjugase, folylpolyglutamate2,250,0195219
factor in the termination of transcription, RNA polymerase II2,250,000393489
polymerase (directed DNA), Delta 2, regulatory subunit 502,250,0123823
the Transporter 1, ATP-binding cassette, subfamily B (MDR2,250,00859077
the Transporter 1, ATP-binding cassette, subfamily B (MDR2,250,00859077
the Transporter 1, ATP-binding cassette, subfamily B (MDR2,250,00859077
the histone cluster 1, H2bf 2,250,0124279
factor 1A initiation of translation in eukaryotes, X-linked2,240,00330183
phosphoglucomutase 22,240,00818204
D3,D2-enoyl-CoA-isomerase peroxisome2,240,00148722
induced by interferon protein with tetratricopeptide repeats2,240,0177928
expressed 1 in G-2 and S-phase2,230,0241887
component 2 complex save minichromosome2,230,0021347
member A family with sequence similarity 722,230,00143248
RMI1, RecQ mediated genome instability 1, homolog (S. cerev2,230,00294705
protein FLJ201052,230,0127979

factor 2 multiple failure coagulation2,220,0116892
phytoceramides, alkaline2,220,0157729
containing the domain with the double helix 682,220,00227586
opening cytokinesis 11of 2.210,00697577
the alpha polypeptide growth factor from plateletsof 2.210,00176418
N-acetylphosphoramidothioate (nelsonmandela the Church2,200,00728536
protein 2-associated kinase S-phase (p45)2,200,00230153
polymerase (RNA) III (directed DNA)polypeptide G (32 kDa)2,200,0298794
protein 1, interacts with like 6 factor ADP-ribosylate2,200,00139745
to the Aster histone 1, H2bh2,190,0377748
complex recognition of the origin, similar to the subunit 5 (yeast)2,190,049697
regulatory subunit 2 protein kinase CDC282,190,0128024
the histone cluster 1, H4c2,190,0112695
hypothetical protein LOC7290122,190,000446087
polypeptide 39 unit DEAD (Asp-Glu-Ala-Asp)2,190,000340561
factor chromatin Assembly 1, subunit B (p60)2,180,0119687
protein that interacts with MLF12,180,0177203
associated with microtubules serine2,180,00536974
the sequence B, a related polypeptide MHC class I2,180,0165406
shugoshin-like 2 (S. pombe)2,180,000852557

subunit 6 of the COP9 constitutive photomorphogenic homolog ('Arab ' 2,180,000793512
methylentetrahydrofolate (NADP+ dependent)2,180,00119726
the open reading frame 167 chromosome 62,180,0011095
transforming pituitary tumor 12,170,0485166
ribonuclease H2, subunit A2,170,00669936
x-ray repair complementing insufficient reparation in Chinese XOM2,160,0369865
membrane protein palmitoylation 5 (MAGUK p55 subfamily of memb2,160,00211873
karyopherin alpha 2 (RAG group 1, importin alpha 1)2,16 0,000650645
containing the domain homology with pleckstrin, family A (posfai2,150,0256434
similar to ribosomal protein L39 are effective2,150,00429384
karyopherin alpha 2 (RAG group 1, importin alpha 1)2,150,000700649
binding protein precursor amyloid beta (A4), family B, m2,150,00201004
component 3 complex save minichromosome2,140,0018389
the histone cluster 1, H2ai2,140,0129155
the open reading frame 34 chromosomes 132,140,000702936
RAD18 homolog (S. cerevisiae)2,140,0016685
protein 1 WD repeat and block HMG DNA binding2,130,0034833
solidcolored chasa-like (yeast) 2,130,0473641

the open reading frame 63 chromosome 162,120,000804179
the phosphoprotein 1 M-phase2,120,0271814
component 6 complex save minichromosome2,120,0161279
homeobox A92,110,00520942
the fibroblast growth factor 9 (activating stroma factor)2,100,0475844
homolog of the C cycle of cell division 25 (S. pombe)2,100,0169914
the open reading frame 64 chromosomes 92,100,0265979
kinase 1 motif (UHM), homologous U2AF2,090,0255167
the replication factor C (activator 1) 2, 40 kDa2,090,00768959
hypothetical protein LOC4408942,090,0103358
small nucleolar ribonucleoprotein D1 polypeptide 16 kDa2,090,0334665
such CSE1 segregation of chromosomes 1 (yeast)2,090,0013662
biosynthesis phosphatidylinositol anchors, class W2,090,0151967
protein O centromere2,090,00397056
family with sequence similarity 20, member B2,090,00460031
hypothetical protein FLJ408692,090,00444509
binding protein goinnovate nucleotides (G protein), gamma 112,080,00140559
protein binding calcicoles2,080,00524566
ATP-binding cassette, subfamily E OABP), member 12,080,00454751

2,04
molecule CD44 (Indian blood group)2,080,000651436
component 8 actsoma2,080,00132017
family with sequence similarity 102, member B2,080,025743
the histone cluster 2, H3d2,070,0102932
family with sequence similarity 33, member A2,070,000318673
anemia Fanconi's syndrome, a group of complementaly B2,070,000255109
the member 22 family of kinesin2,070,0192406
the histone cluster 1, H2ai2,070,0161621
kinase 1, akin to cow poxto 2.060,0233182
subunit 7 of the complex integratorto 2.060,000841371
endonuclease 1, specific patterns flapto 2.060,006882
hypothetical protein FLJ25416to 2.060,000177531
ectopic site 2B integrating virusto 2.060,0171408
pigmentary degeneration of the retina 2 (X-linked recessive)2,050,0264185
protein centromere L2,050,000880856
cofactor required for the activation of Sp1 transcription, subje2,040,00141809
the open reading frame 121 chromosome 202,040,0146323
family with sequence similarity 72, member A2,040,00162905
family with sequence similarity 72, member A0,00165234
factor 1A initiation of translation in eukaryotes, X-linked2,040,00520549
the elongation factor, RNA polymerase II, 22,030,0458007

ATPase, Na+2,030,0189108
the histone cluster 1, H3a2,030,0244273
containing the domain brix 12,030,00981178
containing the domain sushi 12,030,0258164
actonelclopidogrel 6 (predpolagaem2,030,00423628
fructosamine-3-kinase2,030,00470972
syndrome bloom2,020,0209259
tubulin, alpha 1c2,01 0,00862586
transcription factor 2 E2F2,010,0496479
component 2 actsoma2,010,00649147
the member 22 family of kinesin2,010,0242075
homolog LTV1 (S. cerevisiae)2,010,00812652
dihydrolipoamide-S-acetyltransferase (component E2 piruw2,010,00179011
homolog B viral oncogene v-ral leukemia monkeys (related ras2,010,012225
domain 3 with the ring finger and WD repeat2,010,0013797
annexin A12,010,0173578
homolog 2 elaC (E coli)2,000,00266504
aldehyddehydrogenase family 9, member A12,000,00911609
tubulin, alpha 4a2,000,0435427
protein that interacts with the nuclear pore complex-2,000,00111223
oculometer-2,010,00778869
similar to kinase SMG-1, related PI-3-kinase-2,010,0356628

Golgi autoantigen, subfamily holdin a-like pseudogene-2,010,00770626
containing repeating spectrin, nuclear envelope 1-2,010,00438469
protein that interacts with the nuclear pore complex-2,010,00117582
sushi, nitogen and EGF-like domains 1-2,010,00161129
integrin, alpha V (vitronectin receptor, alpha polypeptide-2,020,00252702
inhib the tor 2B cyclin-dependent kinase (p15, inhibits CDK4)-2,040,0150268
similar to the lysyl oxidase 4-2,040,0120148
protein that interacts with the nuclear pore complex-2,040,000213956
calcium-2,040,00657494
calcitonin 3-2,040,00300887
the cell adhesion molecule 1-2,050,0261129
the family of 22 vector of dissolved substances (Transporter of organic cations),-2,050,0137275
containing the domain RUN and FYVE 3-2,050,00387265
glucosidase, alpha, acid (illness Pump, Bo storing glycogen-2,050,000418401
protein that interacts with the nuclear pore complex-2,050,00988632
enriched with Proline coactivator 1 nuclear receptors-2,060,0039587
membrane metalloendopeptidases-2,060,0152684
protein 21A with PHD finger-2,060,00980401
protein that activates GTPase Rho-2,060,00705186
homeobox B6-2,060,00301714

protein that interacts with the nuclear pore complex-2,070,00032839
receptor 1 phospholipase A2, 180 kDa-2,070,00069343
protein that interacts with the nuclear pore complex-2,080,000352007
homolog 3 slit (Drosophila)-2,080,02844
protein that interacts with the nuclear pore complex -2,090,000414309
cyclin-dependent kinase 6-2,090,0456892
dynamin 1-2,090,00139674
jumonji enriched AT domain interactions 1B-2,090,00861002
calcium-binding and contains the double helix of domain 1-2,090,00370041
the receptor for insulin-like growth factor 1-2,090,00114467
protein that interacts with the nuclear pore complex-2,100,000377834
the CD82 molecule-2,100,0175517
bromodomain, neighboring domain zinc finger, 2B-2,109,88E-05
----2,100,00666187
synaptotagmin XI-2,11 0,0129428
KIAAA1546-2,110,000255634
protooncogen jun B-2,120,0120169
the CXXC finger 6-2,120,0277527
protein that interacts with the nuclear pore complex-2,140,00282604
Cdon homolog (mouse)-2,150,0350357
CLL B-cells-2,150,00343507

protein that interacts with the nuclear pore complex-2,150,00263888
homolog 1 viral oncogene v-abl leukemia of Abelson mice-2,160,0136688
protein that interacts with the nuclear pore complex-2,160,00583397
homolog 1 tumor suppressor FAT (Drosophila)-2,18 0,0158766
TGF-alpha 2-2,180,012256
chemerin (chimaerin) 1-2,180,0287031
protein milk fat globule-EGF factor 8-2,180,000987073
the receptor for vitamin D (1,25-dihydroxyvitamin D3)-2,190,000192208
neuroblastoma, a suppressor of cancer 1-2,200,00090639
contains a jumonji domain 1A-2,200,0188513
the protein kinase 1 WNK deficiency of lysine-2,211,57E-05
protocadherin beta 14-2,210,0103892
protein 2, binds cortactin-2,212,28E-05
the regulator of transcription 1, containing a WW domain-2,220,037989
cyclin L1-2,220,00831474
nuclear factor of activated T-cells, cytoplasmic, kaltsin-2,220,00786451
the homologue of pellino 1 (Drosophila)-2,230,00939357
Golgi autoantigen, subfamily holdin a-like pseudogene-2,240,00603583
the open reading frame 10 chromosomes of 7-2,260,00738442
Golgi autoantigen, subfamily holdin a-like pseudogene-2,270,00320764

0,0111179
RNA 17, specific to the calf of Kajala-2,270,0301336
protein 2 linking of latent transforming growth factor beta-2,294,08E-05
Golgi autoantigen, subfamily Goldin a, 8A-2,29
inhibin, beta A (activin A, activin AB alpha polypeptide)-2,290,00877271
the family of 41 vector of dissolved substances, member 2-2,300,00453672
villagelevel block P1-2,300,0463138
metallopeptidase 14 matrix (inserted into the membrane)-2,311,93E-05
transcription factor 4-2,310,0367869
oncogene jun-2,327,21E-05
gene 1, transforming neuroepithelial cells-2,330,0109689
aspirin-2,330,000659873
homolog of the viral oncogene v-fos FBJ osteosarcoma in mice-2,350,0138624
ephrin-B2-2,360,0061147
containing 1 WD repeat and block SOCS-2,360,0387851
similar to dJ402H5,2 (novel protein similar to that in-2,360,00621503
containing serine PX domain-2,380,000927628
collagen, type VII, alpha 1 (epidermolysis a bullosa, the soap-2,380,00109233
protein 1, binding AE-2,390,000105628
homolog peroxidation (Drosophila)-2,400,00219049
calcium channel potential-dependent, L type, alpha 1C sub-2,410,0189661

area 1 chromosome syndrome Prader-Willi-2,450,0415526
midline 1 (Opitz-2,450,00130803
protein that interacts with complexo the nuclear pore -2,450,00354416
the open reading frame 54 chromosomes 1-2,470,0186089
transmembrane protein 16A-2,480,0481085
contains a basic domain helix-loop-helix, class B, 2-2,490,00270257
protein that interacts with the nuclear pore complex-2,500,00316496
transcription factor 1, runt related (acute myeloid leiko-2,500,000607387
protein 292 zinc finger-2,500,029832
fibronectin enriched with leucine transmembrane protein 2-2,510,0135122
protein that interacts with the nuclear pore complex-2,510,00283418
controlled potential potassium channel, subfamily G,member 1 -2,540,0244306
interleukin 19-2,340,0310328
transforming growth factor, beta 3-2,540,0287865
dihydropyrimidinase-similar 3-2,550,0165203
Golgi autoantigen, subfamily Goldin a, 8B-2,560,0121417
hypothetical protein PRO2012-2,570,00756704
homeobox 2 SATB-2,570,039781
t-complex 11 (mouse)-like 2-2,570,0324227
protein 122 with zinc finger-2,570,0236621

the open reading frame 57 chromosome 8-2,590,00261522
metallopeptidase ADAM with the ILO, the PTO thrombospondin type 1 -2,600,0113968
sushi, the factor a background of Villebranda of type A, EGF and pentraxin house-2,632,23E-05
ST6 beta-galactosamide alpha-2,6-sialyltransferase 2-2,640,0216987
related sortilin containing VPS10 domain receptor 2-2,650,00936311
protocadherin beta 9-2,660,0285124
the open reading frame 13 chromosome 5-2,670,00410172
Enah-2,680,0077547
contains 2 domain-independent method decarboxylase-2,690,00683647
similar to protein that interacts with the nuclear pore complex-2,700,0187322
protein that interacts with the nuclear pore complex-2,70 0,00368967
transmembrane protein 119-2,700,00801387
the open reading frame 37 chromosome 14-2,700,0182453
protein containing repeat sushi, X-linked 2-2,710,0253856
containing the domain-bearing with RING finger 3-2,710,00931014
collagen, type XII, alpha 1-2,720,000204664
associated 5 with the restructuring of the matrix-2,720,000317637
collagen, type V, alpha 1-2,720,0166427
protein 2, related dystrophin-2,720,0137557

ATP-binding cassette, subfamily A (ABC1), member 1-2,730,00131361
Tropinin -2,770,00298044
homolog 3 cornichon (Drosophila)-2,780,0261738
like protein 1, binding Fomin-2,780,00290401
brain and acute leukemia, cytoplasmic-2,780,0476919
patientinitiated, receptor type, U-2,800,0270428
hypothetical protein MGC24103-2,820,0346673
induced by interferon with domain 1 of helicase C-2,830,0024839
protein transport phospholipids-2,840,00999206
immediate early response 3-2,870,0152127
immediate early response 3-2,870,0152127
domain 12 metallopeptidase (ADAM maltrin alpha) -2,870,000870288
glycoprotein 2A synaptic vesicle-2,880,00704212
open reading frame 3 of chromosome 9-2,880,00410177
protein that interacts with thioredoxin-2,900,0135494
early growth response 1-2,930,000425035
small nucleolar RNA, C-2,940,00666866
small nucleolar RNA, C-2,950,00765575
immediate early response 3-2,990,0167309
protein 1, related to low-density lipoprotein (alpha-2-microgel-2,994,26E-05
homolog 1 dwuhfaznosti C (Drosophila)-2,990,0347162

-3,030,00665994
small nucleolar RNA, C-3,100,0274043
small nucleolar RNA, C-3,100,0274043
metallopeptidase 2 matrix (gelatinase A, gelatinase 72 kDa,-3,135,59E-05
KIAA1641-3,140,00659194
collagen, type VI, alpha 3-3,142,09E-06
homeobox A2-3,150,0435423
2B domains SH3 and PX-3,150,0244357
collagen, type VI, alpha 2-3,160,0149554
open reading frame 3 of chromosome 9-3,210,0233723
small nucleolar RNA, C-3,240,0104491
m is barking nucleolar RNA, C-3,240,0104491
----3,270,00488845
UDF-N-acetyl-alpha-D-galactosamine:polypeptide N-acetyle-3,350,00964109
cholesterol-25-hydroxylase-3,380,0445558
KIAA1641-3,400,013175
protein 144 with zinc finger-3,400,0135334
version-3,410,023885
such angiopoietin 2-3,420,0245161
KIAA1641-3,440,0170531
homolog B viral oncogene osteosarcoma mouse FBJ-3,540,00025573
similar to RIKEN cDNA 1110018M03-3,590,00516476
early growth of the vet 2 (homolog of Krox-20, Drosophila)-3,620,00821813

dashcous 1 (Drosophila)-3,630,00697244
member 26B family of kinesin-3,640,00363199
homeobox 5 without distal-3,660,000640157
similar to protein KIAA0220-3,690,0302619
the receptor for insulin-like growth factor 1-3,713,42E-05
patientinitiated, receptor type, N-3,770,0294569
KIAA1641-3,850,0191782
protein containing repeat sushi, coupled with X-3,850,00370941
protein 2 associated with microfibrillar-3,910,0152901
the components is t 1 complement, subcomponent s-3,970,0395863
molecule CD24-3,990,0340122
homeobox B3-4,020,0354368
trichorhinophalangeal syndrome I-4,020,00557712
the sequence of syndrome Kallmann 1-4,040,000548703
contains enriched with leucine repeat 17-4,090,0263961
contains 2 domain plexin-4,320,031799
PTK7 proteincontaining 7-4,420,000116114
supervillin-4,430,0412717
protein 521 zinc finger-4,580,00668815
calbindin 2, 29 kDa (calretinin)-4,7700290743
a family of genes of the ras homologue member J-4,790,00197982
integrin, alpha 11-4,800,000390317

odz, odd Oz-5,050,00172671
protein 32 c block F-5,520,0212957
member 2 family rattlin-5,720,0260454
clusterin-5,740,0303973
neurotronik-5,793,78E-06
protein 1 pathway signaling induced by WNT1-5,860,000672342
protein 5 linking insulin-like growth factor-6,340,011614
sulfatase 2-6,345,88E-05
protein 4 is associated with mi is afibrillar -6,930,00155578
the adhesion molecule 2 contacts-7,070,0306758
containing 1 domain of fibronectin type III-7,290,0334696
sarcoglycan, Delta (35 kDa associated with distrofia glycoprotein-7,370,000881984
refasten-7,530,0123141
inhibitor Sarbinowo peptidases, group F (alpha-2 antiplasmin-7,660,00362941
cystatin SN-7,960,0496433
gamecenter 1-8,180,0461603
tenascin C (hexabrachion)-8,328,26E-05
biglycan-8,620,00161284
transmembrane induced by androgen in the prostate RNA-1,20 0,000100935
the carboxypeptidase E-11,220,00738131

Expression of cellular markers on the cells of PLX-C

Surface antigens expressed PLX-C, was tested with monoclonal antibodies. The results showed that the cells of PLX-C was characterized by a positive markers: CD73, CD29 and CD105 and negative markers: CD34, CD45, CD19, CD14 and HLA-DR (data not shown). Description of the test on the immune phenotype were set as: ≥90% for all positive markers and ≤3% for all negative markers.

Moreover, as shown in figa-B, culture PLX-C is not expressed endothelial markers, as demonstrated by negative staining of two endothelial marker CD31 and KDR. However, the expression on PLX-C marker typical for fibroblasts, was evident (expression D7-fib, figs).

Immunogenicity and immunomodulatory properties of cells PLX-C

As PLX-C consist of attached cells derived from the placenta, it is assumed that they Express HLA class I, which is expressed by all cells of the body, and is known to induce alloreactive immune response. HLA class II and other co-stimulatory molecules normally expressed only on the surface of antigen presenting cells (APCs)

In order to assess the immunogenicity of the resulting cells PLX-C, assessing the expression of costimulatory molecules on the surface of their cell membranes. FACS analysis showed the absence of CD80, CD86 and CD40 on the cell membranes PLX-C (figa-C). Moreover, PLX-C expressed low levels of HLA class I when using staining for HLA A/B/C (fig.8D). Expression stimulatory and co-stimulatory molecules was similar to MSCs derived from bone marrow (BM) (as shown in figa-D).

For further study of the immunogenicity and immunomodulatory properties of cells PLX-C was carried out tests on the mixed lymphocyte reaction (MLR). As shown in figa-B cells PLX-C and avoid allowsave, and reduce the response of T-cells when measured by thymidine incorporation. Moreover, decreased lymphocyte proliferation (assessed by measurement of the pulse/min) was greater, the greater the number of cells PLX-C (dose-dependent). PLX-C decreased lymphocyte proliferation after mitogenic stimuli, such as concavalin A (Con A, pigv) and phytohemagglutinin (PHA), and after nonspecific stimulation with anti-CD3, anti-CD28 (data not shown).

To study the mechanism of action by which PLX-C are immune modulation of lymphocyte proliferation, and to consider whether it is mediated by the action of extracellular wsimages the quality or secretion of cytokines, mononuclear cells (MNCs)derived from PB, stimulated with PHA method milonga transfer (which prevents contact between the cells, but allows the cytokines to diffuse between the two compartments). The results showed that the inhibition of proliferation was maintained, even when the contact between cells inhibited (data not shown).

Secretion of cytokines

As described herein above, LX-C reduces the rate of proliferation of lymphocytes, obviously, through soluble factors. Further study of cytokines secreted by lymphocytes in response to PLX-C, was carried out to clarify the mechanism of action of PLX-C. As shown in figa-B, culturing mononuclear cells with PLX-C slightly reduced secretion of proinflammatory cytokines INFγ and reduced the secretion of TNFα (even in the presence of low amounts of PLX-C). Furthermore, after stimulation by lipopolysaccharide (LPS) secretion of IL-10 MNCs originating from PB was higher in the presence of PLX-C, whereas the level of TNFα secretion was decreased dose-dependent manner (figs).

You should take into account that the cells of PLX-C on the steps of the present invention were also capable of homing in ischemic tissues after intramuscular or intravenous administration to mice (data not shown).

EXAMPLE 4

Protivovesa the positive effect of PLX cells-C in vivo in a murine model of acute colitis

Materials and experimental methods

TNBS model of inflammation of intestine

Colitis was induced in susceptible lines rodents by vnutribruchinnogo backfilling kaptenisillal substances TNBS (trinitrobenzenesulfonic acid) in ethanol. The use of TNBS in ethanol based on previous reports that ethanol is required for the destruction of the mucosal barrier, whereas TNBS heptanethiol autologous or microbial proteins colon, giving them immunogenicity in relation to the immune system of the host [Wirtz et al., Nature Protocols (2007) 2(3): 541-546].

Briefly, for the induction of colitis mice were anestesiologi within 90-120 minutes and they were introduced vnutriuretrale TNBS (40 μl, 150 mg/kg)dissolved in a 1:1 mixture of 0.9% NaCl in 100% ethanol. Control mice received 1:1 mixture of 0.9% NaCl in 100% ethanol or saline using the same method.

Mice were scored 5 days after TNBS injection for evaluation of anti-inflammatory action of therapeutic cells (PLX-C cells) of the present invention. The introduction of PLX-C was evaluated by intravenous (IV) the introduction or intraperitoneal (/br) the introduction of cells 1 day after induction of colitis.

Animals

In these experiments used C57b16 mice. Total used 90 mice, which were divided into 9 the following groups:

1) 10 control the mouse is th (not receiving any treatment)

2) 10 control mice + party 1 PLX-C-I/br (2*106cells)

3) 10 control mice + party 1 PLX-C-I/(1*106cells)

4) 10 TNBS mice (mice with a model of colitis)

5) 10 mice TNBS + 5-aminosalicylic acid (5-ASA)

6) 10 mice TNBS + party 1 PLX-C-I/(1*106cells)

7) 10 mice TNBS + party 1 PLX-C-I/br (2*106cells)

8) 10 mice TNBS + party 2 PLX-C-I/(1*106cells)

9) 10 mice TNBS + party 2 PLX-C-I/(2*106cells)

Obtaining 2D attached cells derived from placenta

As described in detail in example 2, in the present description above.

Obtaining a 3D clip of cells derived from the placenta (cells PLX-C)

As described in detail in example 3, in the present description above.

Media

As a control media used PlasmaLyte containing 5% albumin.

Tests and assessment

Was carried out by macroscopic and histological assessment of colitis in samples of colon, collected from mice of different experimental groups after 5 days after administration of TNBS. Macroscopic and histological evaluations were performed blindly by two investigators.

Macroscopic analysis

The colon of each mouse was investigated under preprofile loupe (magnification × 5) for the evaluation of macroscopic damage in accordance with the criteria Wallace. The indicator is llesa were evaluated by macroscopic damage on a scale from 0 to 10 based on signs, reflecting inflammation, such as redness, thickening of the bowel and distribution of ulceration.

Table 12
Rate Wallace
IndexCriteria macroscopic evaluation
0Without inflammation
1Hyperemia without ulceration
2Hyperemia, thickening of the mucosa without ulceration
31 pitting without thickening the walls of the colon
42 or more places ulceration or inflammation
52 or more places ulceration or inflammation with the distribution of more than 1 cm
61st place ulceration or inflammation > 2 cm
71st place ulceration or inflammation of more than 3 cm
81st place ulceration or vocal is of more than 4 cm
91st place ulceration or inflammation of more than 5 cm
101st place ulceration or inflammation of more than 6 cm

Histological analysis

For histological evaluation, in accordance with the Ameho criteria used sample of the colon, located just 2 cm above the anal opening. This classification (on a scale from 0 to 6) takes into account the degree of inflammatory infiltration, erosion, ulceration, or necrosis, and the depth and surface damage spreading.

Table 13
Ameho criteria
IndexCriteria for histological evaluation
0Unchanged
1Average mucosal and/or submucous inflammatory infiltrates with swelling. Small erosion of the mucous. The integrity of the muscular mucosa.
2The same criteria as for scale 1, but more than 50% cutoff
3 A large inflammatory infiltrate with ulceration throughout the colon
4The same criteria as for scale 3, but more than 50% cutoff
5Extensive ulceration with cellular necrosis
6Extensive ulceration with cellular necrosis, but more than 50% cutoff

Molecular analysis colitis

Quantification of mRNA expression of IL-1 beta

Total RNA was isolated from whole tissue of the colon of mice using the Rneasy kit (Macherey Nagel, Hoerdt, France) according to the manufacturer's instructions. Quantification of RNA was performed using spectrophotometry. After treatment at 37°C for 30 minutes 20-50 units of Gnkazy I-free RNase (Roche Diagnostics Corporation, Indianapolis, IN, USA)was used oligo-dT primers (Roche Diagnostics Corporation, Indianapolis, IN, USA) to synthesize single-stranded cDNA. mRNA was quantitatively assessed using SYBR green Master Mix (Applera, Courtaboeuf, France) with mouse-specific oligonucleotide for IL-1β:S: 5'-gATCCACACTCTCCAgCTgCA-3' (SEQ ID NO: 1) and AS: 5'-CAACCAACAAgTgATATTCTCCATg-S' (SEQ ID NO: 2) in a GeneAmp Abiprism 7000 (Applera, Courtaboeuf, France). Calibration was performed during each test, and included controls without matrix. Each sample was applied in three repeat is. The intensity of the dye SYBR green were analyzed using the computer program Abiprism 7000 SDS (Applera, Courtaboeuf, France). All results were normalized on the unaffected gene housekeeping β-actin (oligonucleotides for β-actin: S: 5'-gggTCAgAAggATTCCTATg-3' SEQ ID NO: 3; AS: 5' ggTCTCAAACATgATCTggg-3' SEQ ID NO: 4).

Results

As described herein above, the mice were scored on day 5 after induction of colitis with vnutribruchinnogo the introduction of TNBS on day 0 and attached cells were injected on day 1. Mice were administered either 2D attached cells (in the present description next batch 1) or PLX-C attached cells (in the present description next batch 2), obtained from the placenta 1 or placenta 2, respectively. After slaughter mice spent macroscopic and microscopic evaluation of the colon.

As shown figure 11, mice treated with/in injection 2D or PLX-C cells (party 1 or 2, respectively), showed a significant improvement in the inflammatory condition of the tissues of the colon, as shown in figure Wallace. This anti-inflammatory effect was just as effective as the gold standard of treatment 5-ASA). It should be clear that/br introduction 2D attached cells (part 1) also led to a satisfactory improvement Wallace on a murine model of colitis.

Mi is roscopically evaluation of the colon showed introduction cells PLX-C (part 2) or/br or/in paths substantially decreased the inflammation of the colon at the histological level compared with TNBS mice (as shown in figure Ameho, Fig). The improvement in this treatment was significantly better compared with the TNBS groups treated with 5-ASA.

Next, total RNA was isolated from tissues of the colon and evaluated the levels of expression of IL-1β using RT-PCR (as described herein above). As is evident from the results (Fig)/introduction cells PLX-C (part 2) significantly reduced the expression level of IL-1β in the tissues of the colon. It should be clear that, although the levels of RNA expression of IL-1β was decreased insignificantly in the/br introduction (2D or 3D attached cells, party 1 and 2, respectively) and on/in the introduction (2D attached cells, part 1), introduction as PLX-C and 2D cells, however, resulted in a significant reduction of inflammation-based assessments at the macroscopic and microscopic level in mice with colitis.

Taken together, these results demonstrated that the introduction of fixed cells of the placenta of the present invention (2D and 3D PLX-C cells) leads to a significant improvement in the inflammatory condition of the colon in a mouse model of acute colitis.

EXAMPLE 5

Anti-inflammatory cells PL-C in vivo in a rat model of acute colitis

Materials and experimental methods

TNBS model of inflammation of intestine

In rats caused colitis by oral administration of the colon 22 mg of TNBS dissolved in 1:1 mixture of EtOH and water. 24 hours after induction of colitis rats were injected drugs in accordance with this type of treatment.

Rats used in this study were divided into three experimental groups (as described below). Eleven days after the induction of colitis all rats were killed and the damage of the colon was evaluated both microscopically and macroscopically.

Day of the introduction of TNBS was designated as day 0, cells PLX-C was administered on day 1 and the rats were scored on day 11.

Animals

In these experiments used 12 female Lewis rats (100-120 g). Total used 12 rats, which were divided into the following 4 groups:

1) 4 rats were injected/br 5×106cells PLX-C-I

2) 4 rats were injected in the/5×106cells PLX-C-I

3) 2 rats received/br PlasmaLyte (control group)

4) 2 rats received/PlasmaLyte (control group)

Getting attached cells derived from placenta (cells PLX-C)

The cells were obtained as described in example 3, in the present description above.

Macroscopic damage assessment of the colon

Macroscopic assessment of the damage to the colon kick is conducted in accordance with the following criteria:

0 - no damage

1 - hyperemia, but no ulcers

2 - fibrosis, but without ulcers

3 - ulceration/necrosis of less than 1 cm

4 - ulceration/necrosis of less than 2 cm

5 - ulceration/necrosis of more than 2 cm

Microscopic (histological) damage assessment of the colon

Microscopic evaluation of the damage of the colon was performed in accordance with all of the following criteria (A+B+C+D):

A. Distribution of ulceration:

0 - without ulcers

1-2 small ulcers (less than 3 mm)

3-5 large ulcers (>3 mm)

B. Submucous infiltration:

0 - none

1 - weak

2-3 - moderate

4-5 - heavy

C. Abscesses crypt:

0 - none

1-2 - rare

3-5 - diffuse

D. wall Thickness (µm):

0 - less than 470

1 - less than 600

2 - less than 700

3 - 800

4 is less than 900

5 - more than 900

Results

As is evident from Fig, the introduction of 3D clip-on cells of the present invention (cell PLX-C) leads to a significant improvement of the microscopic parameters (for histological evaluation) acute colitis in rats.

Although the invention is described in connection with specific variants of its implementation, there is no doubt that varied alternatives, modifications and variations should be obvious to experts in the given field of technology. Accordingly, assumes coverage of all such alternativeviagra, modifications and variations, which correspond to the spirit and fall into the broad scope of the attached claims.

All publications, patents and patent applications mentioned in this description are incorporated herein in full in the description, to the same extent as if each individual publication, patent or patent application were specifically and individually indicated to be included in the present description by reference. In addition, citation or reference any reference in this application shall not be construed as an admission that such reference is suitable as the level of the previous technique for the present invention. To the extent that uses section headings, they should not be construed as necessarily limiting.

1. A method of treating ulcerative colitis or Crohn's disease in need of this individual, where the method includes the introduction of a specified individual a therapeutically effective amount of attached cells derived from the placenta, which do not differentiate into adipocytes or osteocytes, resulting in the treatment of ulcerative colitis or Crohn's disease.

2. The method of claim 1, where these attached cells Express one or more of CD73, CD90, CD29, or CD105.

3. The method of claim 1, where these attached cells do not Express CD3, CD4, CD45 CD80, HLA-DR, CD11b, CD14, CD19, CD34 or CD79.

4. The method of claim 1, where these attached cells suppress the immune response by suppressing the activity of T-cells.

5. The method of claim 1, where these attached cells obtained from three-dimensional (3D) culture.

6. The method according to claim 5, where the specified three-dimensional (3D) culture includes 3D bioreactor.

7. The method according to claim 5, where the cultivation of these attached cells in a specified 3D culture performed using perfusion.

8. The method according to claim 5, where the cultivation of these attached cells is carried out for at least 3 days.

9. The method according to claim 1, where these attached cells include cultured cells from the placenta in terms of 2-dimensional (2D) culture.

10. The method according to claim 9, where at least 12% of these attached cells are in S and/or G2/M phase of cell proliferation.

11. The use of attached cells derived from the placenta, which do not differentiate into adipocytes or osteocytes, to obtain medicines for treating ulcerative colitis or Crohn's disease.

12. The use of claim 11, where these attached cells Express one or more of CD73, CD90, CD29, or CD105.

13. The use of claim 11, where these attached cells do not Express CD3, CD4, CD45, CD80, HLA-DR, CD11b, CD14, CD19, CD34 or CD79.

14. The use of claim 11, where these attached TC the TCI suppress the immune response by suppressing the activity of T-cells.

15. The use of claim 11, where these attached cells obtained from three-dimensional (3D) culture.

16. The application of clause 15 where the specified three-dimensional (3D) culture includes 3D bioreactor.

17. The application indicated in paragraph 15, where the cultivation of these attached cells in a specified 3D culture performed using perfusion.

18. The application indicated in paragraph 15, where the cultivation of these attached cells is carried out for at least 3 days.

19. The application of claim 11, where these attached cells include cultured cells from the placenta in terms of 2-dimensional (2D) culture.

20. The application of claim 19, where at least 12% of these attached cells are in S and/or G2/M phase of cell proliferation.

21. The product of manufacture, comprising packaging material includes a label for use in the treatment of ulcerative colitis or Crohn's disease, and packing material Packed pharmaceutically effective amount of attached cells derived from the placenta, which do not differentiate into adipocytes or osteocytes.

22. Product item 21, where these attached cells Express one or more of CD73, CD90, CD29, or CD105.

23. Product item 21, where these attached cells do not Express CD3, CD4, CD45, CD80, HLA-DR, CD11b, CD14, CD19, CD34 or CD79.

24. The product p is izvodstva item 21, where these attached cells suppress the immune response by suppressing the activity of T-cells.

25. Product item 21, where these attached cells obtained from three-dimensional (3D) culture.

26. Product production A.25 where the specified three-dimensional (3D) culture includes 3D bioreactor.

27. Product production A.25, where the cultivation of these attached cells in a specified 3D culture performed using perfusion.

28. Product production A.25, where the cultivation of these attached cells is carried out for at least 3 days.

29. The product manufacture in item 21, where these attached cells include cultured cells from the placenta in terms of 2-dimensional (2D) kultivirovanija.

30. The product manufacture in clause 29, where at least 12% of these attached cells are in S and/or G2/M phase of cell proliferation.

31. The product manufacture in item 21, further comprising immunosuppressive agent or an anti-inflammatory agent.



 

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FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely a method for preparing an agent possessing immunomodulatory activity. The method for preparing an agent possessing immunomodulatory activity characterized by the fact that herbal composition containing common motherwort herb, knotgrass herb, pot marigold blossom, licorice rhizome and root, Alexandrian laurel rhizome and root, rosehip, schizandra fruit, linseeds, sequentially twice extracted in 60-70% ethanol, twice in 40-50% ethanol, and once in purified water under specific conditions; then the aqueous-alcoholic extracts are concentrated in vacuum; the aqueous still residues are combined with the aqueous extract, then filtered, boiled out, purified by separation, boiled out additionally, dried in a vacuum dryer and further milled.

EFFECT: method enables preparing the agent possessing manifested immunomodulatory activity with the high content of active substances.

13 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pyridine derivatives of formula (I) wherein A, R1, R2, R3, R4, R5, R6 and R7 are presented in the description, preparing and using them as pharmaceutically active compounds possessing SP1/EDG1 receptor agonist activity.

EFFECT: using the declared compounds or pharmaceutically acceptable salts thereof for preparing a pharmaceutical composition for preventing or treating the diseases or disorders associated with the activated immune system.

13 cl, 76 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: hemorrhagic pancreonecrosis is preliminarily modelled on animals. After that, tamerit in dose 2 mg/kg is introduced intramuscularly. After that, histological analysis of pancreas tissue after the first, third and seventh day is performed and its results are used to make conclusion about sufficiency and efficiency of performed treatment.

EFFECT: method ensures correction of the course of acute phase of systemic inflammatory reaction syndrome, which leads to stimulation of migration of immunocompetent cells into necrosis foci, acceleration of formation of granulation tissue with proliferation of cells of fibroblast line without phenomena of purulent inflammation, stimulation of proliferation of pancreas duct structures.

1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to piperidine compounds of formula and their pharmaceutically acceptable salts, based on them pharmaceutical composition, treatment method with therein application and therein application for treatment of gastrointestinal diseases. In formula (I) m represents integer number 1 or 2; n represents integer number from 0 to 2, A is selected from phenyl group and benzimidazole group, where phenyl group is substituted with one or more groups, independently selected from C1-C6 linear or branched alkyl group, C1-C6 linear or branched alkoxygroup, aminogroup and halogen, and benzimidazole group is substituted with one or more groups, independently selected from C1-C6 linear or branched alkyl group, C1-C6 linear or branched alkoxygroup, C3-C7 cyclic alkyl group, aminogroup, halogen and oxogroup; X represents hydroxyl or OCONR1R2, where R1 and R2 are independently selected from hydrogen and C1-C6 linear or branched alkyl group, or R1 and R2 form 5-7-membered heterocyclic ring or 3,5-dimethylpiperidine ring, together with nitrogen atom, to which they are attached, and B is selected from phenyl group, phenoxygroup, thienyl group and naphthyl group, where phenyl group, phenoxygroup, thienyl group or naphthyl group is substituted with one or more groups, independently selected from hydrogen, halogen, nitro, cyano, trifluoromethyl, trifluoromethoxy, difluoromethoxy, phenyl, C1-C6 linear or branched alkyl group and C1-C6 linear or branched alkoxygroup.

EFFECT: obtaining novel compounds.

25 cl, 3 tbl, 163 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry, namely to enterosorbent and method of its obtaining. Enterosorbent based on peat, free from lipids, additionally contains cranberry cake, prebiotic with specified component ratio. Method of obtaining enterosorbent from preliminarily dried sphagnum peat includes extraction of lipids with solvents, mixing with cranberry cake, crushing, mixing with solution of prebiotics - lactulose or lactose and drying.

EFFECT: method makes it possible to extend assortment of natural enterosorbents with improved properties: high enterosorbing capability and prebiotic activity.

2 cl, 1 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention provides crystalline solid form of (S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalin-2-ylamino)-2-cyclohexylmethyl-butyric acid or crystalline hydrochloride of (S)-4-((2S,3S)-7-carbamoyl-1,1-diethyl-3-methoxy-1,2,3,4-tetrahydronaphthalin-2-ylamino)-2-cyclohexylmethyl-butyric acid. Invention relates to pharmaceutical composition, possessing antagonistic activity with respect to mu-opioid receptor, which contains therapeutically effective quality of crystalline solid form.

EFFECT: invention provides methods of applying claimed crystalline solid forms in treatment of diseases associated with activity of mu-opioid receptors and methods of obtaining claimed crystalline solid forms.

27 cl, 9 dwg, 17 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to organic chemistry and specifically to substituted imidazo[1,2-a]pyridines of formula I

,

where R is -CH2COOH or -COOH. The invention also relates to a method of producing a compound of formula I and use of the compound of formula I.

EFFECT: obtaining novel substituted imidazo[1,2-a]pyridines, which inhibit exogenically or endogenically stimulated secretion of gastric acid and which can be used in preventing or treating diseases associated with gastric acid, and inflammatory gastrointestinal diseases.

6 cl, 3 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to a novel prostaglandin I2 derivative or a pharmaceutically acceptable salt thereof, specifically a 7,7-difluoro-PCl2

(formula (1)) derivative, where R1 and R2 each independently denote a hydrogen atom or an alkyl group with a straight chain, having 1-3 carbon atoms, R3 is a hydrogen atom, an alkyl group, having 1-4 carbon atoms, as well as a medicinal agent based on compounds of formula 1 for treating and preventing various diseases of the gastrointestinal system.

EFFECT: obtaining a novel prostaglandin I2 derivative or a pharmaceutically acceptable salt thereof.

22 cl, 23 ex, 4 tbl, 12 dwg

FIELD: food industry.

SUBSTANCE: invention relates to compositions intended for babies with low body weight at birth. The probiotic composition intended for complete enteral nutrition of babies with body weight at birth being no more than 1500 g contains probiotic strains chosen from Lactobacillus rhamnosus, Lactobacillus reuteri, Bifidobacterium longum species or their mixtures.

EFFECT: invention allows to ensure changeover of babies with body weight no more than 1500 g to complete enteral nutrition within a period of less than 50 days after birth.

15 cl, 2 dwg, 1 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and specifically to production of human pancreatic polypeptide analogues and can be used in medicine. The disclosed analogues differ from native human pancreatic polypeptide by replacement of amino acids on one or more residues, wherein position 0 is provided with an additional amino acid which is Gly.

EFFECT: obtained analogues provide effective treatment and prevention of obesity or diabetes in a patient, and can also be effective in reducing appetite, reducing food intake or reducing calorie intake in a patient.

26 cl, 2 dwg, 2 tbl,1206 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, in particular, to surgery and can be applied for prolonged anesthetisation in early post operational period of patients with haemorrhoids of III-IV stage. For this purpose 1% solution of morphine in dose 0.1 ml per 10 kg of weight is introduced one time per day in peridural space between vertebras L2-L3 , or L3-L4 through catheter. Said quantity is diluted with 6 ml of physiological solution. Such volume of narcotic medicine ensures effective anesthetisation within 18-20 hours. After that, after said time expiry, 6.0 ml of 2% solution of lidocaine are additionally introduced, which ensures anesthetisation effect within 4 hours. Claimed procedure is repeated on 2 and 3 day in the same succession.

EFFECT: invention ensures prolonged anesthetisation in early post operational period within 3 days, due to reduction of introduced narcotic analgesic (morphine) dose, which makes it possible to reduce probability of development of addiction and development of side effects in patients.

3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel 4-aminocyclohexane derivatives, having affinity for the µ- opioid receptor and ORL1 receptor. In formula

Y1, Y1', Y2, Y2', Y3, Y3', Y4 and Y4' denote -H; Q denotes -R0, -C(O)-R0 or -C(=NH)-R0; R0 and R3 in each case independently denote -C1-8-aliphatic, -aryl, -heteroaryl, -C1-8-aliphatic-C5-cycloaliphatic, -C1-8-aliphatic-aryl; R1 and R2 independently denote unsubstituted -C1-8-aliphatic; -C1-8-aliphatic-C5-cycloaliphatic, -C1-8-aliphatic-aryl; n denotes 0; X denotes -NRa-; Ra denotes unsubstituted -C1-8-aliphatic; Rb denotes unsubstituted -C1-8-aliphatic; "aliphatic" represents a straight saturated hydrocarbon residue which is unsubstituted or mono- or multi-substituted with F atoms; "cycloaliphatic" represents a saturated, unsubstituted monocyclic hydrocarbon residue with 5 carbon atoms in the ring; "aryl" represents phenyl, which can be substituted with -F, -R0 and -OR0; "heteroaryl" represents a 5-member cyclic aromatic moiety containing 1 heteroatom. The heteroatom is N or S, and the heterocyclic ring can be substituted with -F, -R0 and -OR0; the heterocyclic ring can be part of a bicyclic system including phenyl.

EFFECT: invention also relates to a drug containing said compounds and use of the compounds to produce a drug for treating pain, stress, epilepsy, learning and memory disorders, drug dependence, cardiovascular diseases, eating disorders and locomotory impairments.

9 cl, 10 tbl, 164 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely to a method for producing a therapeutic biologically active preparation for treating dyspepsia in newborn calves. The method for producing a therapeutic biologically active preparation for treating dyspepsia in newborn calves consisting in the fact that the multiseptate gastric content and amniotic placental fluid are sampled from female foetus; the multiseptate gastric tissue is homogenised, combined with the amniotic placental fluid, settled, filtered, centrifuged; a produced liquid fraction is combined with the multiseptate gastric content in the certain environment.

EFFECT: preparation produced by the method described above possesses the improved therapeutic action in the newborn calves suffering dyspepsia.

1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely to a method for producing a therapeutic biologically active preparation for treating dyspepsia in newborn calves. The method for producing a therapeutic biologically active preparation for treating dyspepsia in newborn calves consisting in the fact that the multiseptate gastric content and amniotic placental fluid are sampled from female foetus; the multiseptate gastric tissue is homogenised, combined with the amniotic placental fluid, settled, filtered, centrifuged; a produced liquid fraction is combined with the multiseptate gastric content in the certain environment.

EFFECT: preparation produced by the method described above possesses the improved therapeutic action in the newborn calves suffering dyspepsia.

1 tbl

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