Cells as precursors of human liver
FIELD: cellular biology, medicine.
SUBSTANCE: invention relates to isolating and cryopreserving precursor-cells. Methods involve treatment of human liver tissue for preparing the essentially monocellular suspension containing precursor-cells and cells that are not precursor-cells, a single or more lines of cellular differentiation presenting in the human liver. Invention describes methods involving stage for separating cellular population resulting to reducing amount of cells that are not precursor-cells and providing preparing the separated suspension enriched with precursor-cells expressing one or more markers and associated with a single or more lines of the cellular differentiation. Also, invention describes a method for selection cells from the separated suspension wherein these cells or their progeny, or their more matured forms express one or more markers associated with lines of the cellular differentiation. These markers involve: CD14, CD34, CD38, CD45 and ICAM. Hepatic precursor-cells have diameter size 6-16 mc, they are diploid and show indices: glycoforin A-, CD45-, AFP+++, ALB+, ICAM+ and they comprise subpopulations varying with respect to expression of CD14+, CD34++, CD38++ and CD117++. These cells are useful for carrying out cellular and genetic therapy in liver treatment and for preparing artificial organs also.
EFFECT: valuable biological and medicinal properties of cells.
41 cl, 7 tbl, 13 dwg, 15 ex
The technical field to which the invention relates.
The present invention relates to hepatic stem cells, pluripotential cells give rise to hepatocytes and biliary cells, as well as to other subpopulations of progenitor cells of the liver, with the ability to multiply and to differentiate into one or more lines of differentiation of liver cells, including lines of hematopoietic (blood-forming), and hepatic mesenchymal cells. In particular, the invention relates to markers and properties used to identify precursor cells human liver, to methods for their purification and cryopreservation, to new approaches that distinguish subpopulations of hepatic cells from a subpopulation of hematopoietic cells, as well as evidence that hepatic precursor cells exist in the liver from the liver of the fetus to the adult human liver. The invention provides a Foundation for cellular and gene therapy and to obtain bio-artificial organs.
The level of technology
The primary structural and functional unit of the Mature liver is the acinus, which in cross section has the form of a wheel, located around two separate vascular lodges: 3-7 sets of portal triads (each with a gate and door venules, hepatic arterioles and bile duct) n the periphery and the Central vein in the center. Liver cells arranged in the form of cell plates, both sides of which are covered with mesh endothelium, and form a series of sinusoids adjacent to the portal and Central vascular network. Recent data show that haringay channels, small ducts located around each of the portal triads, form tiny tubules that run in the hepatic plates through zone 1 and form patterns, reminiscent of twisted wire for cleaning bottles (Theise, N. 1999, Hepatology, 30:1425-1433).
Narrow space, the space of Diss, separates the endothelium from hepatocytes along the sine wave. In consequence of this hepatocytes have two basal area, each of which is converted to a sine wave, and the apical region, which is determined by the area of contact between adjacent hepatocytes. Basal area in contact with blood and are involved in absorption and secretion of plasma components, and the apical region form bile canaliculi, specializing in the secretion of bile salts and connected by means of a connecting network of bile ducts. Blood flows from the portal venules and hepatic arterioles through the sinusoids to the terminal hepatic venules and Central Vienna.
From the point of view of the form of microcirculation acinus is divided into three zones: zone 1 - okolopostelnoy zone; zone 2 - sredneaziatskaja zone; and zone 3 - colostral the subzone. Proliferative potential, morphological features, ploidy and the majority of liver-specific genes associated with the zonal location (Gebhardt, R., et al. 1988. FEBS Lett. 244:89-93; Gumucio, J.J., 1989, Vol.19. Springer International, Madrid; Traber, P. et al., 1988. Gastroenterology, 95:1130-43). The gradients of the concentration of blood components, including oxygen, through the acinus and in the direction of blood flow from the portal triads to the Central Vienna, is responsible for some aspects of this zoning, for example for the arrangement of zones of glycolysis and gluconeogenesis. However kolovorot zoning gap junction protein of connexin 26 and ocelottrainee zoning glutamylcysteine (representing just two examples) is more intense in the direction of these gradients, and it is more typical for most tissue-specific genes, and apparently, determined by factors intrinsic to the cells or variables other than the blood flow in the micro-environment.
In addition to hepatocytes, epithelial cells of the bile duct (cholangiocytes) and endothelial cells, the area between the gate and the Central path contains other cell types, such as ito cells and Kupfer cells. They play an important role in pathogenic conditions of the liver, especially in inflammation and fibrosis, but their direct contribution to the basic homeostatic functions of normal body obviously small.
Petersville in the convergence of the diverticulum, formed from the caudal anterior intestine and transverse partitions, part vnutrennostej the mesenchyme. The formation of liver cells begins after interaction endodermal epithelium with cardiogenic mesodermal, apparently, with the help of growth factors fibroblast. Then these liver cells proliferate and penetrate mesenchyme transverse partitions, in the form of a cord, forming the primordium of the liver. Direct interaction between the epithelium and mesenchyme at these early stages the liver is crucial in determining which of the cells will become hepatocytes or cholangiocytes, or cellular epithelium, respectively. Mutations in mesenchyme-specific genes hlx and jumonji block the development of the liver, which illustrates the importance of the contribution of these tissues to the specified process. In the early phase of its development, the liver consists of a cluster of primitive hepatocytes associated continuous epithelium, without basal membrane, and of the numerous hematopoietic cells. As the endothelium becomes discontinuous honeycomb structure, the vascular system, especially the portal vascular system becomes more developed, with the formation of basement membranes. The gate of the interstitium can give impetus to the development of the bile ducts, and when he surrounds the portal venules, are formed hepatic arterioles, elonie ducts and portal triad. Immature hepatocytes rapidly proliferate and formed parenchymal plates, apparently in response to changes in the quantity and distribution of such chineorganizer molecules, as HIMSELF, 105, Agp110, E-cadherin and connexin, simultaneously with the movement of most, but not all, hematopoietic cells in the bone marrow. Recent studies confirm that some of hematopoietic precursor cells are stored in the liver of adult rodents, are at rest, and hematopoietic stem cells isolated from the liver as an adult human and mouse liver (Crosbie, O. et al. 1999. Hepatology, 29:1193-8). Mature physical organization comes in rodents in the first weeks after birth, and people - in the first few years. Metabolic zonation established in accordance with several different schemes for different enzymes, but becomes evident in the period after birth.
Stem cells and commitirovannah precursor cells
Stem cells are defined as primitive cells that have the property or infect other programs and are pluripotent, i.e. form daughter cells with more than one potential development paths, which are extensively proliferate and can restore the fabric or tissue. Most of the literature on stem cells refers to either the literature on embryos or to the literature on blood, epidermal and intestinal tissues.
Recently, the designations have been changed so that it was possible to distinguish different classes of stem cells. Cells that can participate in the development of cells of all types, including germ cells are called totipotent stem cells, and they include the zygote and normal embryonic cells until stage 8 cells (the morula stage). Embryonic stem cells, also referred to as “ES”cells, consist of permanent cell populations derived from totipotent normal cells in the blastocyst, which was first reported in the literature in the early 1980's. Line ES cells can be grown in vitro, supporting their totipotency. ES cells are tumorigenic, if they are administered to the host with impaired immunity from any site other than in utero (in the uterus), causing the formation of teratocarcinoma. However, when they are again injected into normal plasticity, they are able to resume embryonic development and to participate in the formation of normal, but chimeric mouse. Although the line of ES cells obtained from many species (mouse, rat, pig and others), but to obtain animals with new genotypes (knockouts (knockouts), transgenic animals) is usually used only with mice, by immersing the modified ES cells from the culture in blastocyte the subsequent implanting these blastocytes pseudoriemannian owners. Lines of embryonic germ (EG) cells, which exhibit many of the characteristics of ES cells, can be identified directly in vitro from a population of primordial germ cells. As in the case of ES cells, EG cells from teratocarcinoma being injected into mice with compromised immunity, participate in the formation of chimeras, including the germ line, while introducing them to blastocyte.
Deterministic stem cells are pluripotent cells, the genetic potential of which is limited to a certain number of cell types, and which have high growth potential. Increases the amount of data, for example, in the study area telomerase, suggesting that the deterministic stem cells do not possess property or infect other programs, i.e. that their offspring may have less growth potential than the parent cells. Deterministic stem cells give rise to daughter cells that lose the property pluripotency in the limits of their genetic potential to one path of development, for example, with the formation of hepatocytes and are called commitirovannah cells predecessors. In hepatic lines have commitirovannah hepatocyte precursor cells and commitirovannah biliary precursor cells.
In recent time is Ekati there are many experimental data about what culture the ES cells can be obtained from human embryos. Assume that these culture the ES cells can be injected into the tissue, in the hope that they will be able to repair damaged organs and tissues. Based on the fact that the ES and EG cells form tumors when introducing them to the sites, other than in utero (see above)suggest that the introduction of ES cells to patients is unrealistic, because there is a great danger of creating tumors in patients. To overcome this impasse, some groups of researchers are exploring the possibility of differentiation of ES cells in defined conditions microenvironment with obtaining deterministic stem cells, which then can be safely inoculate patients. For example, there is some success in obtaining hematopoietic precursor cells. However, it remains a concern that residual ES cells in culture can cause oncogenesis, if these cultures to inoculate patients. Summarizing, we can say that up until research in the field of developmental biology will reveal a myriad of governors of events that define the development of cells during embryogenesis, ES cells will remain only an experimental tool, with little hope for the possibility of clinical programs for cell or gene therapy. The only the m realistic option clinical programs for cell and gene therapy is the use of deterministic stem cells, in which the genetic potential is limited to a certain number of cell types. B contrast, ES cells can be challenging to create bioisosteric bodies for those types of tissues (e.g., hematopoietic cells), which are produced by ES cells in known conditions.
The controversy surrounding the stem cells of the liver
The presence of stem cells in normal adult liver is a subject of great controversy in the field of biology of liver cells. Below summarizes some of the dominant models that exist in this field of technology and polemiziruya each other. Text in bold indicates key idea of the different models.
Some experts in the art believe thathepatic stem cells exist only in embryonic tissuesthat in the adult liver no stem cells and that all Mature liver cells equally involved in the processes of regeneration (Farber (Farber, E. 1992), in the collection "The Role of Cell Types in Hepatocarcinogenesis" S.A.E., editor. Academic Press, New York.). Model Farber considers all Mature parenchymal cells as being phenotypically equal and believes that the well-known heterogeneity of the growth potential and gene expression in the liver is caused only by the conditions of the microenvironment. Farber suggests that korennykh conditions adult parenchymal cells to undergo reverse differentiation and become tumor cells. This model has dominated the field of carcinogenesis of the liver for decades and still has an impact on research in the field of regeneration of the liver.
Other experts believe thatall liver cells are stem cells(Kennedy, S. et al. 1995. Hepatology, 22:160-8; Michalopoulos, G.K, et al., 1997. Science, 276:60-6.). These researchers believe that all parenchymal cells are equal and high plasticity, and gene expression is determined only by the conditions of the microenvironment. Assume that in the relevant oncogenic conditions Mature parenchymal cells become stem cells, which can then turn into a tumor cell.
The model is silent stem cellsbased on the research of Wilson and Lidya (Wilson J.W. et al., 1958. J. Pathol. Bacteriol. 76:411-449). As in the field of hematopoietic cells, this concept causes the greatest confidence through intensive studies of carcinogenesis of the liver (Marceau, N. 1994. Gut. 35:294-6.). These researchers believe that precursor cells, including bipotential precursor cells can persist in adult tissues, but I think they are rare remnants of cell populations obtained from embryonic development. They believe that precursor cells play no role in ordinary, or regenerative operation pecan is, and play a role only in disease States (Overturf; K. et al. 1999. American Journal of Pathology, 155:2135-2143.). Thus, they are considered “silent”, like satellite cells in muscles. These cells are described as “oval cells”, based on the characteristic shape of the cell nucleus. These cells are of small size (~9 μm) and Express characteristic antigenic profile on the cell surface. All Mature liver cells are considered equal among themselves in respect of growth and gene expression, and believe that all aspects of the heterogeneity of gene expression is due only to the cellular microenvironment. Supporters of the model silent stem cells categorically deny any possibility of movement of parenchymal cells from kolovorot in allocentrism area. I believe that stem cells and other liver precursor cells are important only for disease States, particularly cancer. Therefore, these researchers have focused their efforts on the study of precursor cells in animals subjected to various oncogenic effects. These studies show that “oval cells” do not form a recognizable body of rapidly proliferating cells in regenerative conditions or under conditions of mild to moderate damage. A significant number of populations of proliferating oval cells observed and only after a very serious liver damage (Grisham, J.W. et al. 1997. In the book "Stem Cells", C.S. Potter, editor. Academic Press, London. 233-282).
A model based onthe flow of liver cells(Arber, N. et al. 1988. Liver, 8:80-7; Zajicek, G. et al. 1991. Liver, L 11:347-51.) was harshly criticized and most researchers are not recognized (Jirtle, R.L. 1995. Liver Regeneration and Carcinogenesis: Molecular and Cellular Mechanisms, Academic Press, New York.). This model postulates that the compartment of stem cells in each portal triad produces adult parenchymal cells, which “flow” towards the Central vein. The process flow results in daughter cells in contact with certain conditions of the microenvironment, which leads to changes in the phenotype of these cells. Again, there is the hypothesis that the conditions of the microenvironment are crucial for determining the phenotype. Most researchers argue against this model, believing that it is not consistent with studies that have not found any movement, marked with a marker of the donor cells, the newly introduced into the liver (Kennedy, S. et al. 1995. Hepatology, 22:160-8.). However, even in studies that provide the most compelling evidence in favor of the model of overflow, is still unknown what exactly influences the expression of markers used in the donor cells, the microenvironment or the position of the line cell differentiation. In addition, the hypothesis about the evolution of liver cells, probably bude is revised after recently received Taysom and his assistants (Tiese, N. Hepatology, 30:1425-1433) data, suggesting that haringay channels, which for a long time believed associated with liver cells-precursors, spread their tubules through the hepatic plate at least in zone 1.
Reid (Reid) and his assistants protect the hypothesis that the liver is a system of stem cells and differentiation of cells on maturation (Sigal, S.H. et al. 1992. Am. J. Physiol. 263:G139-48.). They suggest that the tissues are organized into lines of differentiation of cells on maturation, fueled by a population of stem cells or early progenitor cells (Brill, S. Et al. 1993. Proceedings of the Society for Experimental Biology Medicine. 204:261-9.). This fabric is defined as developing from young cells to the cells of the middle age and old cells. The maturation process is accompanied dependent on the line of differentiation and position changes in the size of cells, their morphology, antigenic profiles, Rostov potential and gene expression. It is hypothesized that these changes occur due to the combination of Autonomous cellular changes that are independent of the conditions of the microenvironment, and the changes induced by the microenvironment; the microenvironment includes nutrients, gas exchange (oxygen, CO2), pH, hormones, cellular interaction and the chemical composition of the extracellular matrix.
|Ploidy||A diploid cell||Tetraploid cells||A mixture of tetraploid and octaploid cells|
|The average size||7-20 microns||20-30 microns||30-50 microns|
|Extracellular matrix||The gradient of the chemical composition of the matrix located in the space of Diss and consisting of type IV collagen, mixed with laminin and heparansulfate in kolovorot the field and turning into fibrillar collagens, fibronectin and hearingprotection in allocentrically area.|
The authors of this hypothesis suggest that the maximum increase is observed in stem cells and early cell precursors and is gradually reduced in line cell differentiation. This model takes into account that the majority of cells in the tissue of the adult liver are polyploid, mainly tetraploid or octaploid, and less than a third cleto who are diploid. Recent data support the concept, announcing that the regenerative capacity of the tissue is determined mainly by the population of diploid cells and that older cells contribute to regeneration by increasing cell mass as a result, hypertrophic reactions associated with polyploidy (Sigal, S.H. et al. 1999. American Journal of discrimination. 276:G1260-72.). Therefore, these researchers advocate the hypothesis that the greatest hope for cell growth, by using the method as in cell or gene therapy, and for bioisosteric organs, associated with a population of diploid cells in the tissue.
The system model of stem cells and differentiation of cells on maturation inconsistent with other models for the development of liver cells in that it suggests that malignant degeneration of the liver is most frequently the result of an indirect and not a direct oncogenic effect. Suggest that oncogenic lesions kill most of the cells of the liver, particularly of Mature cells in the line of development, leading to a dramatic induction of regenerative reaction. As a consequence, the increase in the number of progenitor cells increases the risk of secondary mutational events in the fast-growing cells, cells-the precursors that may lead to malignant development. Thus, the older the hypothesis about that cancer is blokirovku differentiation or that cancer arises as a result of oncogenic lesions that affect stem cells, are accepted as true, but with the above modification.
The increasing recognition of the model lines differentiation of cells on the maturation of now is based on the fact that the liver is full of signs pointing to the process of apoptosis (loss of cells) or the process of final differentiation (Sigal, S.H. 1995. The Differentiation. 59:35-42.) and on the received data that only certain subpopulations of liver cells present in adult liver, capable of intense cell divisions (Overturf K., et al. 1999. American Journal of Pathology. 155:2135-2143; Tateno, S. et al. 2000. Hepatology. 31:65-74.). In this model, precursor cells and a subpopulation of adult cells (considered as the diploid subpopulation) is able to restore the liver tissue after their return in conditions in vivo, and capable of intensive growth, including clonal growth.
In U.S. patent No. 5559022 issued to Notone (Naughton), describes the selection of the cells from the liver and subsequent purification using gradient centrifugation. However, the selected cell population represents the population of acidophilic parenchymal cells”are cells-the precursors of liver indicated in this image the shadow.
Preclinical and clinical applicability of progenitor cells of the liver
There is a great clinical and commercial interest in the isolation and identification of immature precursor cells of the liver due to the impact that this cell population may have on the treatment of liver diseases. Each year in the United States, about 250,000 people are admitted about liver failure. Liver transplants have a therapeutic effect on some forms of liver failure, and in the United States in the year do about 4100 direct. One of the limiting factors in liver transplant is the availability of donor livers, especially considering the fact that the donor liver for transplant must come from patients who have suffered brain death, but not cardiac arrest. The use of a liver from a cadaveric donor did not lead to successful results, although recent attempts to use such donors talk about the possibility of their use in case the liver receives not later than one hour after the death of the donor.
Transplantation of cells into the liver is an attractive alternative therapy for most diseases of the liver. Surgical procedures required for the transplantation of cells, very small in comparison required for the transplant of a whole organ and poet of the mu can be used for patients with different surgical risk such as age or poor health status of the patient. Using cells of human liver preferable to use the liver cells of other mammals as potential pathogens, if any, will be of human origin and are better tolerated by patients, and, in addition, they are easier to weed out before using.
When attempting to carry out transplants of liver cells used nefrackzionirovannam Mature liver cells, and when this has reached a certain degree of efficiency (Fox, I.J. et al. 1998. New England Journal of Medicine. 338:1422-1426.). But to succeed, you must enter a large number of cells (10-20 billion), because these cells do not grow in vivo. In addition, the introduction of substantial quantities of large Mature liver cells (average cell diameter of 30-50 μm) is complicated by their tendency to form large aggregates after the introduction, which can lead to a fatal embolism. And in addition, these cells cause a noticeable reaction immunological rejection, forcing to what patients have to keep on immunosuppressants for the sake of preserving their lives. And finally achieved success in cryopreservation of Mature liver cells, and therefore it is very difficult to coordinate the possibility of obtaining a suitable liver tissue, obtaining a cell suspension, immediate delivery of cells for clinical those who FIPA.
Advances in the selection of precursor cells of the liver
It is known that the selection of precursor cells from the liver is an extremely difficult task due to the lack of markers for positive selection of cells of the liver. The only available antibodies for candidates in the hepatic precursor cells are monoclonal antibodies that receive against subpopulations of hepatic progenitor cells (oval cells), encourage proliferation after they were subjected to carcinogenic damage. However, these antibodies cross-react with antigens present in hematopoietic cells.
In the past attempted to obtain a population of hepatic progenitor cells, considering them the most appropriate population for cell and gene therapy of the liver. In U.S. patent No. 5576207 and 5789246 (Reid et. al.) used cell surface markers and flow cytometry with lateral scattering to obtain a specific subpopulation of liver cells. Subpopulations of hepatic cells of rats were isolated by removing the linear-commiteeman cells with subsequent selection of immature hepatic precursor cells, which were defined as cells having a shape other than granular bearing O-positive (antigenic marker oval cells), AFP-positive, albuminoidal and ck-19 negative (cytokeratin 19) cell markers. The above subpopulations of cells rat liver demonstrate specific characteristics that are important for the selection and identification of enriched populations of hepatic progenitor cells from the liver of rodents.
The selection of precursor cells of the liver from the liver of an adult person described here is novel and unexpected, particularly because of the controversy concerning itself only with the presence of progenitor cells of the liver in an adult, because it is believed that hepatic precursor cells in the adult human liver either not present or are physiologically silent cells, the remainder of embryogenesis. Therefore, no attempt was made their selection or study, with the exception of the study of their disease.
In contrast, the presence of cytoplasmic proteins alpha-fetoprotein (AFP) and albumin in the developing liver recognize a strong positive indicator of progenitor cells. At the earliest stages of development of the liver, these cells are able to produce offspring that come in biliary and hepatic line cell differentiation. If these daughter cells commitlimit to the lines of differentiation of biliary cells, the expression of alpha-fetoprotein is terminated. However, in hepatocytic lines differentsirovki cells expressible-fetoprotein is maintained until the perinatal period, when it is suppressed, and as one of the major characteristics of Mature hepatocytes remains the expression of the albumin.
However, because alpha-fetoprotein is an intracellular protein and becomes visible only after fixing permeabilization cells (giving the cell properties permeability), it is not suitable for use as a marker to identify viable hepatic precursor cells.
The invention relates to a method for producing a composition comprising a mixture of cells derived from the liver tissue of a person, and this mixture comprises an enriched population of human liver progenitor cells, the method includes: obtaining essentially unicellular suspension of liver cells of a person, comprising a mixture of cells of different sizes, including immature cells and Mature cells; and separating the suspension under conditions that allow to remove Mature cells and the cells of relatively large size, leaving immature cells and the cells of relatively small size, to obtain a mixture of cells comprising an enriched population of human liver progenitor cells, and these cells their progeny, or more Mature forms have one or more markers indicating the expression of alpha-fetoprotein, albumin, or the both. Alpha-fetoprotein and albumin can be full size or variant. The process of separating the suspension may include separation by size of cells, the floating density or both. The separation of the suspension can also be based on the rate of sedimentation, hydrodynamic radius and sedimentation to equilibrium density. Alternatively, the separation can be carried out on the basis of different degrees of adhesion of surface markers, glue components, such as antibodies or lectins. The selected precursor cells can be diploid and may have a size less than 15 microns in diameter. In addition, precursor cells or their progeny can synthesize macromolecules, characteristic of progenitor cells, including, but not limited to, alpha-fetoprotein and albumin. Preferably, the alpha-fetoprotein peptide includes the sequence encoded by exon 1 (aFP). Thus, alpha-fetoprotein mRNA transcribed from a larger than 2 TPN, i.e. full-aFP mRNA. Similarly, albumin preferably includes encoded by exon 1 (ALB) peptide sequence. Thus, albumin transcribed from a full-size mRNA.
In another embodiment, the present invention relates to a method of allocation, cryocans the activate your product and use of precursor cells human liver, includes treatment of liver tissue of a person to Obtain essentially unicellular suspension, including precursor cells and cells that are not precursors, one or more lines of differentiation of cells present in human liver; implementation phase separation of this suspension, which significantly reduces suspension number of cells, not cells-the precursors to obtain the separated suspension enriched cells-precursors expressing one or more markers associated at least with one of these lines cell differentiation; optional selection of the split suspension those cells, which themselves, or their descendants, or more Mature forms Express at least one marker associated at least with one line of cell differentiation; not necessarily the suspension of the cells in optimal conditions for cryopreservation; and the optional use of growth factors for the treatment of patients. Preferably selected precursor cells of the liver, expressing cytoplasmic proteins, such as alpha-fetoprotein. Processing or phase separation of the suspension according to the present invention preferably include centrifugation in density gradient or centrifuge elutriation (when the) suspension of liver cells to separate cells according to their surface density and/or size, associated with the one or more gradient fractions having a lower buoyant density and/or smaller size. The method of density gradient may include zonal centrifugation and centrifugation in a continuous stream.
One of the variants of the present invention is a negative selection of cells, which cells predecessors, including Mature hepatic, hematopoietic (blood-forming) and mesenchymal cells, using markers associated with Mature hepatic cells, such as connexin, markers associated with Mature hematopoietic cells, such as glycophorin and CD45, and/or markers associated with Mature mesenchymal cells, such as retinoids, and the factor a background of Villebranda.
The authors of the present invention have found that through the use of hepatic progenitor cells can overcome many of the disadvantages associated with the use of Mature liver cells, and make them the ideal cells for use in gene and cell therapy and to obtain bioisosteric bodies. These cells are small (7-15 μm), and therefore minimizes the possibility of formation of large emboli. In addition, these cells have high growth potential, which means that fewer cells are required to restore the fabric pecheni patient. Finally, precursor cells have minimal antigenic markers that could cause immunological rejection, which gives grounds to hope, that you only need a small amount of immunosuppressants, or they would not be necessary. Therapy with liver cells includes either extracorporeal treatment or transplantation of liver cells. Cells, preferably including precursor cells, impose any possible means, including parenteral and intraperitoneal route. You must use an effective amount of cells, preferably comprising between 103and 1010cells. More preferably transplanted between 105and 108cells, and preferably about 106cells.
In another embodiment the invention, the precursor cells of the liver are very useful for obtaining growth factors and other proteins. These factors are associated with their own growth or the growth of other precursor cells in the liver (e.g., hematopoietic or mesenchymal precursor cells) and factors associated with early stages of defining the purpose of hepatic precursor cells for specific lines of cell differentiation. These new growth factors can be used to treat diseases of the liver or against those kinds RA is a, which are transformed hepatic precursor cells. In addition, hepatic precursor cells are an important target for gene therapy, in which introduced genetically transformed or normal hepatic precursor cells improve the health of the individual such transplanted liver precursor cells.
Another aspect of the present invention is the determination of a unique antigenic profiles on the surface of cells, which correlate with the expression of alpha-fetoprotein in the cell. The definition in this way characteristics containing alpha-fetoprotein cells allows enrichment using methods flow cytometry viable liver cells-the precursors of the single-cell suspensions of living cells obtained from whole liver or lobe of the liver. In addition, the isolation and identification of hepatic precursor cells of person described here was carried out using a combination of unique ways, markers and parameters that the authors of the present invention used for the first time, to get a unique cell population of the present invention.
Another aspect of the present invention relates to precursor cells of the liver hepatic, hematopoietic or mesenchymal about the convergence. These lines of differentiation of cells, their progeny, or more Mature forms are selected using antigenic markers selected from the group consisting of CD14, CD34, CD38, CD45, CD117, ICAM, glycophorin and/or cytoplasmic markers, such as alpha-fetoprotein immunoreactivity, albuminurophobia immunoreactivity or both of these markers. Alpha-fetoprotein may occur from a full-sized mRNA (more than 2 TPN - form, usually expressed in hepatic cells predecessors) or variant forms (less than 2 TPN, i.e. approximately 0.5, and 0.8, 1, 1.5, or 2 TPN - form, usually expressed in hematopoietic cells predecessors). Precursor cells of the liver according to the present invention can be isolated from the liver of the fetus, newborn, baby, child, teenager, or adult.
In accordance with another aspect of this invention, the isolated precursor cells human liver is isolated in a highly enriched form to essentially pure form. Such precursor cells of the liver contain hepatic, hematopoietic and mesenchymal precursor cells. Hepatic precursor cells have the ability to grow in hepatocytes, biliary cells, or a combination of these cells; hematopoietic precursor cells have the ability to grow in macrophage, neutrophils, granulocytes, lymphocytes, platelets, polymorphonuclear eosinophils, basophils, or a combination of these elements. Mesenchymal precursor cells have the ability to grow in endothelial cells, stromal cells, hepatic stellar cells (ito cells), chondrocytes, bone cells, or a combination of these cells. The method according to the present invention can be used to identify mesenchymal precursor cells, expressionwhich alpha fetoprotein immunoreactivity, CD-45, albuminoidal immunoreactivity, CD34, osteopontin, bone sialoprotein, collagen (types I, II, III and IV) or a combination of both.
Another aspect of the present invention relates to precursor cells of the liver, which contain exogenous nucleic acid. Such exogenous nucleic acid may encode at least one polypeptide of the present invention, or may contribute to the expression of at least one polypeptide according to the present invention.
In accordance with another aspect of the present invention proposes a method of mitigating the negative impacts of one or more disorders or dysfunctions in humans by introducing individual suffering from such negative influences, the effective amount of the isolated precursor cells PE the new person. Precursor cells can enter either intraperitoneally or parenterally, through the vascular vessel, or to enter directly into the liver. Direct introduction can be done surgically, through the portal vein, mesenteric vein, hepatic artery, hepatic bile duct, or using combinations of these methods. In weaving alternatives hepatic precursor cells can be entered in the ectopic site of the patient's body, such as the spleen or the peritoneum.
Disorders or dysfunction in humans, which can be mitigated by using the method according to the present invention, include hepathology, softening the liver, gepatomegalia, cirrhosis, fibrosis, hepatitis, acute liver failure, chronic liver failure, or congenital metabolic disorders, and cancer of the liver, such as hepatocarcinoma or hepatoblastoma. Liver cancer can be a primary localization of cancer or cancer metastatically in the liver. Metastatic tumor can occur from any number of places primary containment, including the intestines, prostate, mammary gland, kidney, pancreas, skin, brain, lung, or a combination of these places primary forms of cancer.
In accordance with the following aspect of the present invention is proposed bioreactor, which contains biological Mat is real, including isolated precursor cells from human liver, their offspring, their Mature or differentiated descendants, or combinations thereof; and a culture medium, such as basal (minimum) environment; one or more compartments containing biological material or components, including biological material; and optionally one or more connecting holes. In addition, the bioreactor may optionally also include: extracellular matrix; hormones; growth factors, nutrients, or a combination of the above; as well as biological fluid environment, such as serum, plasma or lymph.
The bioreactor is adapted to maintain these precursor cells in viable, functional status and can support precursor cells of the liver during the period lasting from about one week to about 55 weeks. Specifically, the bioreactor adapted for use as an artificial liver, for the manufacture of products for Toxicological research or for metabolic studies, including studies of the activity of cytochrome P450 or other types of drug metabolism.
In accordance with another aspect of the present invention features a composition of isolated precursor cells human liver or susp NSIA, enriched cells-precursors derived from human liver. The cell suspension is offered in a pharmaceutically acceptable carrier or diluent and administered to a subject in need of treatment. The composition according to the present invention includes precursor cells of the liver, expressing one or more markers associated at least with one or more lines of differentiation of cells present in human liver, and essentially does not contain Mature cells. More specifically, isolated precursor cells of the liver originate from one or more lines of cellular differentiation, including lines of hepatic, hematopoietic or mesenchymal cells, and precursor cells, their progeny, or more Mature forms Express at least one or more antigenic markers CD14, CD34, CD38, CD90 or CD117, CD45, glycophorin, as well As cytoplasmic markers alpha-fetoprotein immunoreactivity, aluminumalloy therapy, or both of these markers. In another embodiment of the invention immature cells, their progeny, or more Mature forms Express osteopontin, bone sialoprotein, collagen I, collagen III, collagen IV, or a combination of both.
In accordance with another embodiment of the present invention, a system of culture cells were preceded by the tinnikov liver, including isolated precursor cells from human liver, their offspring, their more Mature or differentiated descendants, or their combination. Culture system of cells further includes extracellular matrix that includes one or more collagens, one or more adhesive proteins (laminin fibronectin) and other components such as proteoglycans, such as heparin-sulfate-proteoglycans); or individual matrix component. The matrix component includes fragments of matrix components, simulators matrix, which can be synthetic or biodegradable materials (i.e. microspheres)coated with one or more of the one or more classes of extracellular matrices. System cell culture can additionally include basal or enriched environment and other nutrients; hormones, growth factors, and not necessarily biological fluid environment, such as serum, plasma or lymph. In addition, the system of culture cells may have one or more compartments containing biological materials such as culture Cup, tablet, flask, roller bottle, transwell or other similar container.
Culture or bioreactor of the present invention can be used in one or more studies of metabolism, including the study of AK is Yunosti systems biotransformation enzymes of phase I or phase II, in one or more transport research, including the study of gene expression, regulation and activity of hepatic sinusoidal and canalave transport systems, aspects of drug metabolism as well as in the research activity of cytochrome P450, among others.
In the following embodiment, the present invention proposes a method of cryopreservation is attached to the substrate cells. Method of cryopreservation is attached to the substrate of the cell includes (a) providing attached to the substrate of the cell and matrix, or amplifier viscosity; (b) the suspension of the cells in the mixture for cryopreservation, including culture medium, an inhibitor of ice crystal formation, the regulation factor of carbohydrates, a good source of iron, lipoprotein and lipid; and (C) cooling the suspension to a temperature below the freezing temperature of the cells. The freezing temperature refers to the temperature at which cells turn into a solid mass, which can be a supercooled liquid or glassy or microcrystalline microcrystalline mass. Furthermore, it describes a mixture for cryopreservation, including culture medium, an inhibitor of ice crystal formation, the regulation factor of carbohydrates, a good source of iron, lipoprotein and lipid. The mixture for cryopreservation may also include the antioxidant, such as ascorbic acid, glycerol (10% vol.) or dimethylsulfoxide (DMSO, 10% vol.), the latter two agent can act as inhibitors of the formation of ice crystals. Factor in the regulation of carbohydrate can be an insulin or insulin-like growth factor. The source of iron, lipoprotein and lipid can be a transferrin, high-density lipoprotein and free fatty acids, respectively. Free fatty acids are not necessarily located in a complex with albumin. The mixture for cryopreservation may include collagen, collagenopathies substance, agarose, methylcellulose or gelatin, and collagen can be a collagen I, collagen III or IV collagen. The components of the environment for cryopreservation can be prepared in solution Viaspan, i.e. in the solution for cryopreservation of the University of Wisconsin.
The following variant of the invention is a collection, cell Bank, directory, or biological repository with many cryopreserved liver precursor cells and/or their offspring. Precursor cells can be isolated as described above, and may represent a hepatic precursor cells, isolated in any suitable manner that provides receipt of the hepatic cells predestin is of IKI, expressing full-sized alpha-fetoprotein, albumin, or both. Similarly, precursor cells can Express the markers, indicating the presence of a full-sized alpha-fetoprotein, albumin, or both. The repository can include a system for indexing cell markers. After thawing the cells from the repository can be used for inoculation of bioreactors for the initiation of cell cultures or for the treatment of patients.
Another variant implementation of the invention includes variant alpha-fetoprotein, which represents a gene product of a gene or mRNA without exon 1, which is described below. As described in this invention, the variant alpha - fetoprotein is often associated with hematopoietic cell precursors and their progeny and is not associated with liver cells-precursors. And another variant implementation of the invention includes a peptide comprising from three to ten amino acids taken from the sequence encoded by exon 1 of alpha-fetoprotein.
Another variant implementation of the invention includes a conjugate of a macromolecule and a peptide comprising from three to ten amino acids from the sequence encoded by exon 1 of alpha-fetoprotein, and suitable for use as antigen. The macromolecule can be a albumin, hemocyanin, asain, ovalbumin, polylysin, for example poly-L-lysine or poly-D-lysine, or any other suitable macromolecule, known to the art. You can use the antigen, generating antibodies specific for alpha-fetoprotein, the expression of which indicates the presence of hepatic progenitor cells, and does not indicate the presence of hematopoietic progenitor cells or their progeny. Antibodies can be developed by immunization of an animal with the antigen, in the absence or in the presence of adjuvant, or by treatment of spleen cells by antigen, followed by fusion of spleen cells to obtain hybridomas, as is known in the art.
In the following embodiment, the invention describes the isolation of progenitor cells from human liver, including the treatment of liver tissue of a person, providing essentially unicellular suspension, including precursor cells cells that are not predecessors of one or more lines of differentiation of cells present in human liver; division of suspension, which significantly reduces suspension number of cells, not cells predecessors, with getting sorted suspension enriched cells-precursors expressing one or more markers, wired the x for at least one or more lines of cellular differentiation, and selection of sorted suspension of those precursor cells, their descendants or their more Mature forms that Express one or more markers associated with at least one or more lines of differentiation of cells.
Brief description of figures
Figure 1. PCR analysis of mRNA for alpha-fetoprotein.
Figure 2. PCR analysis of mRNA albumin.
Figure 3. The effect of cryopreservation on the viability of cells derived from fetal liver.
Figure 4. Left panel: Histogram immunofluorescence assay of alphafetoprotein obtained by cell sorting device with excitation fluorescence (FACS). Right panel: Histogram immunofluorescence assay of albumin obtained by FACS.
Figure 5. The percentage of cells expressing surface markers CD14, CD34, CD38, CD45 and glycophorin A (GA) in nefrackzionirovannam suspensions of liver cells.
Figure 6. Coexpressed markers cell surface and alpha-fetoprotein cells of the fetal liver.
Figure 7. Top left: the Percentage of alpha-fetoprotein-positive cells.
Top right: the Percentage of albumin-positive cells.
Bottom: the Effect of fractionation in percoll on coexpression alpha-fetoprotein and albumin.
Figure 8. FACS-analysis of suspension cells derived from fetal liver to coexpression CD14, CD38 and alpha-fetoprotein.
Figure 9. The yield of alpha-fetoprotein-positive cle is OK in the selection with CD14 and/or CD38.
Figure 10. Four representative obtained using immunofluorescence assay images of fetal liver progenitor cells, stained for alpha-fetoprotein.
Figure 11. Effect of selection for CD14 (right): Image obtained using an interference microscope (top) and immunofluorescence assay (bottom).
Figure 12A. The image of a group of liver cells, obtained using phase-contrast microscopy.
Figure 12B. The image of the same group of liver cells obtained by immunofluorescence assay with antibody to alpha-fetoprotein.
Figure 12C. The imposition of a and B.
Figure 13A. Liver cells stained with callainos.
Figure 13B. Liver cells stained with alpha-fetoprotein, the same view as in panel A.
DETAILED DESCRIPTION of PREFERRED embodiments of the INVENTION
In the following descriptions of some terms often used to describe the invention. In order to provide a clear and correct understanding of the description and claims, including the amount invested in such terms, provides the following definitions.
Alpha fetoprotein immunoreactivity: Any immune reactions caused by alpha-fetoprotein. Alpha-fetoprotein can be full-length or truncated, including isomers and splice variants of al the a-fetoprotein.
Commitirovannah precursor cells: Immature cells, with one possible path of development, such as hepatocyte commitirovannah precursor cells (giving rise to hepatocytes or biliary precursor cells (giving rise to the bile ducts). The process of commitirovannah at the molecular level is not clear. It see only empirically, when the number of ways of development of the cells is reduced compared with previous generations.
Liver cells: a Subpopulation of liver cells, including hepatocytes and biliary cells.
Liver cells: As used in the present description, the term “liver cells” refers to all types of cells present in normal liver, regardless of their origin or development.
Stem cells: As used in the present description, the term “stem cells” refers to immature cells that can give rise to daughter cells with more than one possible path of development, which are pluripotent. Totipotent stem cells such as embryonic stem cells (ES cells) or embryonic cells up to 8-cell stage embryo of a mammal, have the ability of self-renewing (self-sustaining), namely, that a stem cell produces identical to itself a subsidiary of the tile is in. In contrast, deterministic stem cells, such as hematopoietic, neural, skin or liver stem cells are pluripotent and have the ability to intensive growth, but their ability to self-renewal is under question. In the case of a totipotent stem cells, some daughter cells identical to the parent cells, and some “change” them (“commitlimit”), turning on specific paths of development, which limits their genetic potential to a value less than the genetic potential parents. In the case of deterministic stem cells, some daughter cells retain pluripotency, and some lose it, commitiee to only a specific development path.
Hepatic precursor cells: These cells give rise to hepatocytes and biliary cells. Hepatic precursor cells include three subpopulations: “hepatic stem cells”, “commitirovannah hepatocyte precursor cells” and “commitirovannah biliary precursor cells”, the latter two subpopulations are immature cells that are descendants of hepatic stem cells and have one possible path of development, i.e. they can be either hepatocytes or biliary cells, but not any of them.<> Liver stem cells: a Subpopulation of hepatic precursor cells.
Precursor cells of the liver: a Population of cells from the liver, including hepatic precursor cells, hematopoietic precursor cells and mesenchymal precursor cells.
Haematopoiesis (blood): the Formation of blood cells with the ways of development of lymphocytes (b and T), platelets, macrophages, neutrophils and granulocytes.
Mesogenes: Education mesenchymal derivatives with the ways of development of endothelial cells, fat cells, stromal cells, cartilage and even bone (the last two development paths are found in the liver only disease).
Cellular therapy: As used here, the term “cell therapy” refers to the transfer of in vivo or ex vivo of specific cell populations to be used as autologous or allogenic material and transplanted in particular target cells of a patient or in the vicinity of these cells. Cells can be transplanted in any suitable medium, carrier or diluent, or with any type of system for drug delivery, including microneedle, granules, microsome assay, beads, bubbles, etc.
Gene therapy: As used here, the term “gene therapy” refers to the transfer of in vivo or ex vivo specific genetic material to specific clidamycin patient, thus changing the genotype in most cases, changing the phenotype of these cells-targets the ultimate goal of which is to prevent or modify a particular disease condition. Gene therapy may involve the modification of target cells ex vivo and the introduction of these cells to the patient. As an alternative to the cells-the precursors of the liver may be in vivo directed vector to deliver to them the exogenous genetic material and transferout these precursor cells. In addition, the bioreactor can be used obtained using genetic engineering techniques precursor cells for the treatment of patients, or as a source of biological products. In accordance with this definition, the basic premise is that these therapeutic genetic procedures designed to the maximum extent possible to prevent, cure or modify explicit or implicit pathological condition. In most cases, the ultimate goal of gene therapy is to modify the phenotype of a population of specific target cells.
CD: Cluster of differentiation” or “common determinants” in this description refers to molecules the surface of cells, recognized by monoclonal antibodies. The expression of some CD-specific cells of a certain line of differentiation or path of maturation, and ek is major depression other CD varies depending on the activation state, position or differentiation of the same cell.
When in this description uses the terms “one” he means “at least one” or “one or more”unless specified otherwise.
II. Alpha-fetoprotein and albumin as diagnostic markers for the lines of liver cells.
Alpha-fetoprotein (AFP) and albumin, both of which are cytoplasmic proteins, represent a particularly reliable markers for lines of liver cells. The expression of these proteins underlies the identification of subpopulations of liver cells, allowing to distinguish them from other types of liver cells.
Cell lines human leukemia and normal T-lymphocytes after stimulation in vitro can also Express the AFP. However, in the literature it is not reported whether AFP mRNA in cell lines of leukemia and in activated T-lymphocytes in the form, identical to the authentic AFP mRNA in the liver cells. It was necessary to determine whether it is possible to measure the expression mnrk AFP or albumin using conventional methods of analysis of proteins, such as immunofluorescence, Western blotting, etc. because the method of RT-PCR (PCR with reverse restriction enzyme) is the most sensitive of the known methods for the identification of specific matrices RNA.
To the described studies no detail was not studied forms of mRNA AFP or albumin in hemopoetic the mini-human cells. The present invention demonstrates the expression of variant forms of AFP and albumin in hematopoietic cells.
Figure 1 illustrates the analysis of liver cells and cells that are not liver cells using polymerase chain reaction (PCR), with primers to multiple exons mRNA for alpha-fetoprotein.
PCR analysis reveals truncated AFP in hematopoietic cells. Carried out analysis of RT-PCR using combinations of primers hAFPl, hAFP2, hAFP3 and hAFP4. M denotes molecular weight markers, lanes 1-3 represent NERV; tracks 10-12 - STO fibroblasts; lanes 13-15 - without RNA. Please note that on tracks 2, 4 and 8 have a common band - truncated form AFP. On tracks 1 and 4 are marked variant form of AFP unique to liver cells. Full view of AFP was observed in lanes 3 and 6. The inventors have designed nine primers for PCR to describe variant forms hAFP mRNA as described in example 1. The sequence encoding the AFP goes from exon 1 to exon 14. All combinations of primers, in addition to combinations of exon 1 mRNA AFP, amplified part of AFP mRNA in cell lines of erythroleucus person, C, and at the same time, all combinations detect AFP mRNA in the lines of liver cells human HepG2 and NERV. This shows that variant forms of AFP mRNA containing exons from exon 2 to exon 14, as it is expressed in K, but not HC is primarily the entire sequence encoding the AFP. This result confirms that the only suitable primers for the identification of liver cells are those which detect a portion of exon 1 of the AFP, the expression of which is likely to be limited tissue-specific manner. The fact that exon 1 is unique for subpopulations of hepatic progenitor cells, can be used as a probe to identify the hepatic precursor cells and to distinguish them from hematopoietic precursor cells.
Since truncated forms of AFP found in some subpopulations of hematopoietic cells, albumin also analyze in hepatic, and hematopoietic cells. The primers for albumin constructed in the same way as for the AFP (see above) and used them to analyze the expression of albumin in the lines of liver cells no comparison with lines of hematopoietic cells. As for the AFP, its truncated form was found in K, lines, hematopoietic cells, and the transcript was detected using primers for exon 12-14.
This invention describes the design and preparation of specific primers in RT-PCR to determine the type of expression of variant forms of AFP mRNA and albumin in populations of liver cells compared with populations of hematopoietic cells. As described here, the invention demonstrates that variations the fair forms of mRNA as AFP, and albumin can be found in hematopoietic cells predecessors. This means that in the case of the use of such sensitive methods of analysis it is necessary to use additional criteria, such as the probe exon 1 for AFP to distinguish populations of liver cells from populations of hematopoietic cells.
Figure 2 shows the PCR analysis of liver cells and cells that are not liver cells, for several exons of albumin. Because the truncated form of AFP mRNA was found in some subpopulations of hematopoietic cells, albumin also analyze in hepatic, and hematopoietic cells. The primers for albumin constructed in a similar manner as for the AFP (see above) and used them to analyze the expression of albumin in the lines of liver cells compared with lines of hematopoietic cells. As for the AFP, its truncated form was found in K, lines, hematopoietic cells, and the transcript was detected using primers for exon 12-14.
Studies of the development of the liver show that the liver of the fetus during prenatal development represents both hematopoiesis and hematopoietic organ. During the various stages of the development of the liver, fetal liver contains large amounts of hematopoietic cells, especially erythroid lines cell differentiation. Also what about the, there is increasing evidence that hematopoietically and hematopoietic system are closely connected and, perhaps, this relationship includes joint expression of AFP and albumin or maybe isotypes of this protein. The fact that the exon 1 AFP is unique for subpopulations of hepatic progenitor cells, allows you to identify specific subpopulations of progenitor cells of the liver according to the present invention.
Although PCR analysis shows that hematopoietic precursor cells can Express the mRNA as AFP and albumin, but the levels of expression of this mRNA is very low. Indeed, if AFP and albumin was measured by flow cytometry, in K not find detectable amounts of either AFP or albumin. Although both AFP and albumin are crucial for the identification of hepatic precursor cells, but AFP is especially valuable for the diagnosis of hepatic precursor cells after treatment by flow cytometry, thanks to his intense expression in hepatic cells predecessors. AFP is also suitable for assessing the purity of hepatic progenitor cells after any of the procedures for fractionation.
III. Processing of precursor cells human liver
The authors of the present invention have developed ways to provide the optimum yield of dissociated precursor cells of the liver of the fetus and the adult. Isolation of Mature liver cells typically includes enzymatic and mechanical dissociation of the tissue obtaining unicellular suspension, followed by fractionation using methods centrifugation in a density gradient, centrifuge elutriate (otmuchivanie), differential enzymatic degradation (namely hepatic stellate cells), and/or selection using cell culture (review in the publication Freshney, "Culture of Animal Cells, A Manual of Basic Technique", 1983, Alan R Liss. Inc. NY). Centrifugation in density gradient most researchers typically use to remove those items that they consider fragments of cells and dead cells, Olbrachtova all factions and leaving only the last residue in the test tube after centrifugation.
While all other researchers use the last residue in the test tube after fractionation in the density gradient, the method described here is unique in that it involves the use of the upper fractions of the gradient of the density and eliminates the use of sediment after centrifugation. Describe here a new variant of the method of centrifugation in density gradient is that the sediment in the tube after centrifugation was removed, and cells that have a lower buoyant density (i.e. cells that collect the I at the top or near the top of the gradient), leave. The inventors have found that younger (i.e. diploid) cells and better tolerate cryopreservation of cells are collected in the upper part of percoll used as the density gradient, but not in the sediment in the tube.
IV. Division of population
The population division is the process of enriching its cells-the precursors of the liver. Precursor cells can relate to any of the multiple lines of differentiation of cells, including hepatic, hematopoietic and mesenchymal cell lines. Because the liver has various Mature cells that can be tetraploid or polyploid, it is useful to remove some or all of the Mature cells to obtain an enriched population of progenitor cells. Preferably, but not necessarily, to carry out phase separation at 4°C.
After receiving a unicellular suspension of liver cells, the cells are divided into several fractions according to the size of cells, floating density or a combination of both these options. In accordance with the present invention, the precursor cells of the liver are smaller than 315 microns in diameter. Suitable is any way to split, to separate such small cells from larger cells and fragments of cells, including methods of separation by velocity sedimentation in the cultural panorama of the de (which can be basal or enriched environment), gradient sedimentation, chromatography using granules with larger pores for fractionation of cells, among others. Gradient material can be a covered polyvinylpyrrolidone silica (percoll), crosslinked cellulose (ficoll), dextran, or any other material known in the art and prepared in the form of isotonic to prevent lysis of the cells, for example, in phosphate-buffered saline or in basal medium Needle (Eagle) (TOGETHER). A suspension of dissociated cells is usually applied to the surface layer of the gradient material and is exposed to a centrifugal field, maintaining it at a temperature of 4°C. alternatively, the cell suspension can be applied to a device for apheresis, such as used for separation of blood components, i.e. for plasmapheresis. Large cells, including Mature parenchymal cells and tetraploid cells, settle faster than small precursor cells and diploid cells, after which the precipitated cells are removed. The centrifugation technique takes into account the sensitivity of cells to low pressure; oxygen and minimizes the time required for the enrichment of cell suspension. Using these methods, the cell suspension can be enhanced hepatic cells predecessors. In addition, the phase p is selenia may include otmuchivanie by centrifugation (elutriation), panning based on proteins adhering to the surface of cells, affinity chromatography, or the processing in periodic mode, the tagging with fluorescent tags, zonal centrifugation, centrifugation in a continuous flow magnetic separation after incubation with magnetic granules, for example with magnetic granules in combination with the antibodies, or a combination of these methods. When centrifugation in density gradient can be used intermittent or continuous gradient. Merkalova fraction suitable for immediate use, for cryopreservation, for domestication or for further enrichment. Further enrichment can be achieved by panning, affinity selection using cell sorting device with excitation fluorescence (FACS) or by any other means known in the art and described above. Negative selection is carried out by removal of cells expressing the markers CD45, glycophorin And or other markers that are mentioned below. Positive selection is performed by selection of cells expressing CD14, CD34, CD38, ICAM and other markers indicating the expression of a full-sized alpha-fetoprotein, albumin, or both.
In another embodiment, separation of populations of cells, not cells predecessors, is selectively removed using selecti the aqueous lysis. Erythrocytes are lysed by briefly placing the cell suspension in isotonic solution of ammonium chloride, followed by dilution of the culture medium and centrifugation to remove the “shadow” of erythrocytes and free hemoglobin. Similarly, cells that are not cells-precursors selectively and hydrolytically are lysed by freezing using a mixture for cryopreservation described below. Various methods of separation of suspensions allow you to delete polyploid cells; cells expressing markers associated with Mature hematopoietic cells; cells expressing markers associated with Mature hepatic cells; cells expressing markers associated with Mature mesenchymal cells, as well as the combination of these cells.
IV. Cryopreservation of progenitor cells human liver and progeny of these cells
Methodology cryopreservation in accordance with the present invention is unique and differs from the methods used in prior art. The main differences consist in the use of other buffers in the cryopreservation of a population of progenitor cells of the liver, which has a low density and, thus, is floating in a gradient centrifugation. Hepatic precursor cells are small and are di is loadname.
Successful cryopreservation of Mature cells human liver is very desirable, but it never reached in the art. As a rule, successful cryopreservation is defined as the ability to freeze cells at temperatures of liquid nitrogen (-160 -180...° (C) their subsequent thawing and then save vitality in the amount of >75%) and the ability to attach to the culture vessels. When using older methods Mature hepatocytes of rodents and humans have the vitality of 30-40%, and is not able to attach to the culture vessels after freezing under the above conditions (see Toledo-Pereya et al., U.S. patent No. 4242883; Fahy et al., U.S. patent No. 5217860; Mullon et al., U.S. patent No. 5795711; and Fahy et al., U.S. patent No. 5472876). In these patents has been reported very low viability (<50%) of the cells, and they relate mainly to the cell cultures (rather than individual cells in cell suspension) and require prolonged stay of cells in the buffer before freezing.
Figure 3 shows excellent cell viability liver after cryogenic storage in accordance with the method of the present invention. Data are expressed as the percentage change in viability, measured during processing and during thawing. These data show that cryopreservation is not provided with the significant negative influence on cell viability. Observed no significant changes in viability during the period up to 550 days of storage. Special methodology of cryopreservation of the present invention includes the use of a new buffer, a new cell population, and not necessarily the immersion of the cell in the form of extracellular matrices. Using this methodology for the first time reached viability after thawing, which did not differ from viability, measured before freezing, immediately after dispersion of the cells. The actual values of viability varies depending on the state of the tissue after delivery and from the impacts preparation of cell suspension by enzymatic and mechanical dissociation, and the average of 77% for cells derived from fetal liver. Applied methods of cryopreservation has not resulted in significant loss of viability in the freeze and the loss of the ability of cells to attach to the culture vessels to proliferate ex vivo after thawing.
V. Immunoselected precursor cells human liver
The invention provides a method of isolating precursor cells from human liver, including the provision of essentially unicellular suspension cells tissue of a human liver, with subsequent positive or negative immunoselection. The way immunos the lectures may include selection of suspension those cells, which themselves, their progeny, or more Mature forms Express at least one marker associated with at least one line of cell differentiation. These lines of differentiation of cells may represent a line of hematopoietic, mesenchymal, liver cells, or any combination of these lines of differentiation of cells. Stage selection (selection) of cells may include removal of cells expressing glycophorin A, CD45, a marker that is specific for cells of the adult liver, connexin 32 or a combination of the above. Furthermore, the method of selection may include removal of polyploid cells; cells expressing markers associated with Mature hematopoietic cells; cells expressing markers associated with Mature hepatic cells; cells expressing markers associated with Mature mesenchymal cells, or a combination of the above. The selection of cells may include the selection of cells expressing CD14, CD34, CD38, ICAM or a combination of both. In addition, using this method, you can identify and select Mature hematopoietic cells, which expertsyou glycophorin And, CD45, or a combination of both. And in addition, using this type of selection you can select Mature mesenchymal cells expressing retinoids, factor von Willebrand's disease, factor VIII, or combinations thereof.
The way they are is unselective can be implemented in conjunction with the division of a population of cells, based on the size of the cells, their buoyant density or a combination of these parameters. Using this method it is possible to select cells expressing at least one marker associated at least with one line of cell differentiation, which may be a line hematopoietic, hepatic, or mesenchymal cells. The selection of cells, their progeny, or more Mature forms may be based at least on the same token, the associated at least with one line of differentiation of liver cells. This line of differentiation can be a parenchymal cells or hepatocytes or biliary cells. Thus, the markers expressed by the cells, may represent a CD14, CD34, CD38, CD117, ICAM, or combinations thereof.
VI. Cellular markers and flow cytometry
Using your definition of progenitor cells of the liver as populations of immature cells expressing alpha-fetoprotein, along with the expression of albumin or without it, the authors of the present invention analyzed the markers, which can be selected specifically for these types of cells, using technology immunoselection. A striking discovery was that many of the markers (e.g., CD34), a classical marker for hematopoietic progenitor cells, can also in order to identify subpopulations of hepatic progenitor cells. So, solid color sorting for CD34 selection resulted in a significant (at least 9-fold) enrichment of populations of cells expressing AFP. However, not all of these F-positive cells can be confirmed that they are liver cells predecessors. Based on the percentage of albumin-positive cells, the inventors have found that 80-90% of these cells are liver cells-the precursors, and the rest are either hepatic cells predecessors, too immature to Express albumin, or perhaps subpopulations of hematopoietic cells expressing alpha-fetoprotein.
This invention uses a unique strategy of sorting using flow cytometry. Using a combination of expression of AFP and albumin, as two unique defining characteristics of hepatic precursor cells, the authors of the present invention identified antigenic markers and other flow-cytometrical the parameters that define the hepatic precursor cells. The known strategy of sorting include a selection of small cells (<15 μm, when measured with a forward scattering), which are diploid (which is determined using fluorescence staining dye Hoechst 33342), granular look is at the lateral dispersion and are negative with respect to certain hematopoietic; antigens (i.e. glycophorin And antigen of red blood cells and CD45), followed by separation using positive markers for subpopulations of hepatic and hematopoietic cells (i.e. CD14 and/or CD38).
In the described experiments, the authors present invention identify hepatic precursor cells using a selection of those cells that strongly Express alpha-fetoprotein, weakly Express albumin, and also Express CD14, CD34, CD38, CD117, or a combination of both. In addition, it describes the evidence that hematopoietic cells also Express and AFP, albeit in truncated form. The authors of the present invention describe a new cell population, methods of isolation, identification, cultivation, and how the use of such cell populations. Success in the isolation, identification and cultivation of specific cell populations of the present invention is provided, in particular, with improved methods of isolation, affinity separation of populations, high-speed sorting of cells with fluorescence excitation, increased speed and accuracy of the method, as well as using modified methods of cryopreservation and cultivation.
The inventors demonstrate strategies flowing-cytometrical of sorting and methods of isolation of precursor cells from svezhesorvannyh cell suspensions and/or thawed cryoconservation liver cells. These methods include: 1) staining of cells in several fluorous tagged probe antibodies to specific markers on the cell surface and 2) using a combination of strategies negative and positive sorting in multiparameter technologies flow cytometry. How the selection of specific stages of the lines cell differentiation of populations of hepatic kletok person can apply for liver obtained from a donor of any age, because these markers are specific for cell differentiation and its provisions.
Improved methods of labeling cells and dramatically improved the flow cytometer (flow cytometer "MoFlo" firm Cytomation, sorting cells with a speed of 40,000 cells per second and performs 8-color sorting), compared with the flow cytometer used in the past (FACSTAR PLUS, firm Becton Dickenson, which sorts cells with the speed of 2000-6000 cells per second and performs 2-4-color sorting) allow for the successful isolation and identification of this new cell population.
Figure 4 shows Univeristy sorting with excitation fluorescence (FACS-sorting). The cell suspension is prepared for immunofluorescent analysis of alpha-fetoprotein (AFP) using antibodies conjugated with red dye So, and for albumin using antibodies conjugated with the him dye (AMC). Conducted screening thirty thousand cells for the presence of red (AFP) and blue (albumin) fluorescence. The results show the presence of distinct groups of cells positive for each of these proteins. Further analysis shows that about 80% of the population positive for each of the proteins represented by the same cells (i.e. shows coexpressed these two proteins). The expression of AFP-like and aluminumalloy immunoreactivity well determined in cell suspensions, there is a strong group of cells that show a clear contrast to the background signal. Alpha-fetoprotein is expressed at 6,9±0,86% of the cells in nefrackzionirovannam cell suspensions, and the albumin is present in the 7.7±1.1% of cells. Among AFP-positive cells 75,6±4.9% of the cells coexpressing albumin, and 80±5.5% of albumin-positive cells also Express and AFP. Thus, approximately 25% of cells expressing alpha-fetoprotein, does not Express albumin, and 20% of cells expressing albumin, does not Express alpha-fetoprotein.
The proportional content of cells that carry the main surface markers used in this work are shown for the full cell suspensions (i.e. including erythrocytes) in table 2 (GA = glycophorin And surface marker on the red blood cells).
|The percentage of CD-positive cells in the original suspension of liver cells and the percentage of AFP-positive among these cells|
Figure 5 illustrates the percentage of cells expressing surface markers CD14, CD34, CD38, CD45 and glycophorin And (GA) in nefrackzionirovannam suspensions of liver cells. Note that the data on GA posted on the right axis, to keep the scale. Figure 6 illustrates the percentage of cells in the original cell suspension, expressing alpha-fetoprotein and other antigenic markers. Shown are mean values ± standard error of the mean for percentage of cells positive with respect to alpha-fetoprotein (AFP) and to specific surface markers (CD14, 34, 38, 45 and glycophorin). It is clear that glycophorin And (GA)-positive cells (i.e. erythroid cells) represent a major component in cell mass, IP the connection is negligible fraction of AFP-positive cells.
Figure 7 (top) shows coexpression alpha-fetoprotein and albumin. Shows the expression of alpha-fetoprotein (left panel) and albumin (right panel) in cell suspensions of fetal liver with selective reduction or without selective decrease the number of red blood cells by fractionation in percoll. The percentage of AFP-positive cells, coexpression albumin, also increased to 80,5±8.2%, and proportional to the content of albumin-positive cells, coexpression AFP increased to 89±3,1%, although none of these changes is not statistically significant.
Figure 7 (below) illustrates the effects of the separation of cell populations using fractionation in percoll on coexpression alpha-fetoprotein and albumin. The proportional content of cells expressing both alpha-fetoprotein and albumin, expressed as percent of AFP or albumin-positive cells. Data for cells with reduced or without reducing the number of red blood cells obtained by the use of fractionation in percoll. It is seen that when in cell suspensions by fraktsionirovanija in percoll reduced number of erythrocytes, the proportional content of cells expressing AFP, was significantly increased to 12.9±1.9%, and cells expressing albumin - to 12.1±2.3 per cent.
The impact of this PR the procedures of the proportional content of the cells, bearing surface markers, are shown in table 3, together with a proportional content of each subgroup, showing positive staining for AFP.
|The percentage of CD-positive populations in suspensions of liver cells after reducing them in number of red blood cells and the percentage of AFP-positive among these cells|
red blood cells
|% AFP-positive||by 89.8±1,3||77,1±2,9||53,5±7,2||32,5±1,3||1,8±0,9|
Figure 8 illustrates FACS analysis of suspension cells derived from fetal liver to coexpression CD14, CD38 and AFP. Bivariate scatterplot shows the distribution of staining flag on CD14 (ordinate) compared with staining fluoresceinisothiocyanate (FITZ) CD38 (abscissa). Created Windows fluorescence excitation to select specific groups of cells in accordance with the signals CD14 and CD38. They were used to demonstrate the intensity of AFP-occasianally each of these radgroupreply by AFP shows the high enrichment level of AFP was obtained by selection of cells positive for both CD38 and CD14. AFP-the signal generated by the whole cell suspension (30,000 cells), shown at the bottom left. In most cases, the presence of AFP in subgroups, selected using the cell surface marker has a continuous distribution with a clear predominance of cells, demonstrating the intensity of staining in the positive range. However, the distribution of CD38-positive cells by co-expression of AFP was unique. The CD38-positive cells was apparent bimodal distribution of co-expression of AFP, which clearly shows two distinct groups of cells, one of which is the AFP-positive and the other negative.
The results show that alpha-fetoprotein (AFP) is present in 7% of the cells in one cell suspensions of fetal tissue of the liver (i.e. in the original cell suspension). It is established that the antibody to glycophorin And (antigen on red blood cells, erythrocytes) identifies a subpopulation of cells expressing AFP. Thus, cells expressing this antigen (i.e. erythroid cells) exclude from cells, characterized as hepatic precursor cells. The CD38 antigen identifies a population of cells, demonstrating a significant increase in the proportional content AFP-the good cells (namely, their proportional content; more than 7 times higher than in nefrackzionirovannam samples). Both antigen demonstrate the existence of several isoforms, depending on the presence or not there are parts of molecules encoded splicing variants. There are antibodies that identify different isoforms.
It is established that the classical marker for hematopoietic progenitor cells, CD34 is present on many cells, which also Express and AFP. Selection of CD34-positive cells leads to enrichment of AFP-positive cells in at least 9 times compared to the original cell suspension (67% in CD-positive cells compared with 7% in the original cell suspension). However, the most effective single antibody to enrich the content of AFP-positive cells is CD14, which leads to a more than 11-fold increase in the proportional content of these cells compared to the source population (81% vs. 7%).
Suggested that the release of AFP-positive cells can be increased by using a combination of surface markers. Therefore, determined the size of the co-expression of AFP with the selected combinations of surface markers to identify to what extent it is possible to increase the selection of intracellular markers. Data are expressed as proportional containing the Oia AFP-positive cells, expressing surface markers (labeled “exit” AFP-positive cells) and the proportional content of all F-positive cells in the population, defined by surface marker (indicated as a factor of “enrichment” to AFP-positive cells). The results for combinations of CD14, CD34 and CD38 are shown in table 4 together with the results obtained for individual markers, for comparison.
The enrichment. The percentage of cells expressing either (or both) of surface markers, which are also AFP-positive.
Output. The percentage of all AFP-positive cells that also Express one (or both) combinations of surface markers.
Figure 9 shows how the selection for CD14 and CD38 enriches the content of AFP-positive cells. The proportional content of AFP-positive cells in cell suspensions, recip is the R from fetal liver, increases dramatically in the selection of cells having on their surface a positive label indicates the markers CD38 and CD14. The combination of these two markers provide a much better enrichment AFP-containing cells than the use of any of these markers individually.
Figure 10 illustrates the results of fluorescence microscopy of hepatic precursor cells of human rights. Representative liver precursor cells from fetal liver were stained for determination of AFP. The size of the cells indicate that there is an earlier precursor cells, and more advanced hepatic precursor cells. The morphology of the cells, giving a positive staining for AFP, different and covers a full range of sizes and shapes of cells in suspension, obtained from the liver of the fetus, but not from adult human liver. The largest of AFP-positive cells, approximately 12-15 µm, much smaller than Mature hepatocytes, having a size in the range from 20 to 50 microns.
Figure 11 illustrates a representative cells selected on the basis of expression of AFP. Cells with positive staining for CD14 (right) are typical for hepatoblastoma. Cells with negative staining for surface markers are smaller in size and morphology similar to the early pechanec the suspended cells predecessors. In all cases, a certain part of the AFP-positive cells expresses no surface antibodies used in this study. The appearance of such AFP-positive “zero” cells are shown in figure 11, where they can be compared with the appearance D14-positive/F-positive cells selected from the same suspension. It is clear that although both of these types of cells are AFP-positive cells, giving negative staining for the presence of surface antigens, much smaller and less complex than CD14-positive cells.
Thus, a possible marker for selection of the hepatic precursor cells are glycophorin A-, CD45-, ICAM+one or more of CD14+, CD34, CD38+, CD117, diploid, agranular (lateral diffusion), less than 15 μm (when the forward scattering). The phenotype of these selected cells are small (<15 μm) cells with little cytoplasm (with a high ratio of nucleus to cytoplasm), albumin+and/or AFP+++.
VII. Confocal description expressing alpha-fetoprotein cells in the liver of the fetus and in the adult human liver
Confocal microscopy was used to obtain images of the liver cells of fetal and adult expressing alpha-fetoprotein. This methodology the Oia allows us to observe the morphology and size of these cells, and directly shows the location of intracellular proteins, such as AFP and ALB, and proteins membrane surface markers such as CD34 and CD38.
Figure 12 illustrates confocal microscope expressing alpha-fetoprotein cells, i.e. hepatic precursor cells in the adult human liver. The figure shows three views of the same field, and shows that in this field there are two AFP-positive cells. Panel (C) shows the overlay panel (A) and panel (C) show the morphology of AFP-positive cells (red in the original) in the group of liver cells.
Figure 13 illustrates cells labeled by kalayna (A)to show all types of cells. Figure 13 (B) consists of the same cells, coexpression AFP, and shows that only two cells are F-positive. The size of cells is not a factor involved in AFP-positive status.
Expressing AFP cells detected in the liver of the fetus and in the adult human liver. The liver of the fetus, as expected, has the highest percentage (6-7%) of these cells, while the liver of an adult human has a low percentage (<1%), and the number of these cells decreases with age of the donor. Available in adult human liver small amount of hepatic progenitor cells can greatly enrich with what omashu method of fractionation in percale, receiving the output of the cells to 2% in parcelling fractions 1 and 2 obtained from adult human liver (table 5). No one expressing AFP cells are not found in liver donors at the age of 71 years.
Table 5 shows the size and viability of cells from selected using percoll fractions of cells of the adult human liver. Smaller cells (fractions 1-3) have a higher viability than larger cells (fraction 4), after cryopreservation, carried out in the same conditions.
|Fraction of percoll||Viability (%)||The size of the cells (µm)||% Of AFP-positive cells|
These results confirm that the bodies of donors suitable for use in cell therapy of liver and transplantation of organs include the liver from young donors (aged about 45 years). In addition, the liver from elderly donors (age is m ore than 65 years) is not suitable as donor material for cell therapy, and possibly for transplanting whole organs, especially for children, because the liver is very little or no high regenerative capacity of liver precursor cells, it is well known that Mature cells have regenerative ability, which is characterized only as a moderate to minimal.
VIII. The line of differentiation of cells on maturation
Thus, the Mature liver contains a population of hepatic progenitor cells capable of growth and differentiation of hepatocytes and biliary cells, both in normal and in disease. The present invention protects the assumption that each position in the line of differentiation of liver cells represents a separate stage of maturation, and that in the liver, there are numerous populations of stem cells.
It was found that embryonic liver in accordance with the present invention provides an output of precursor cells for three separate lines of differentiation of cells on maturation: line hematopoesis, in which cells can develop into hepatocytes and biliary cells (cells of the bile ducts); line hematopoiesis, in which cells can develop into lymphocytes (b and T), platelets, macrophages, neutrophils and granulocytes; and line mutagenesis, in which cells can razvivat is camping in the endothelium, fat cells, stromal cells, cartilage, and even bone cells (the latter two types of cells found in the liver only disease).
Typically, stem cells are immature cells that can give rise to daughter cells with more than one possible path of development. Stem cells produce daughter cells, some of which are identical to the parent cells, and some “commitlimit” (go) on a special path of development. The process of commitirovannah at the molecular level is unclear. Its existence is recognized only at the empirical level, when you see that the possibilities of development of cells are narrowed in comparison with the possible development paths of the preceding cells. “Commitirovannah precursor cells” is defined as immature cells with only one possible path of development, such as hepatocyte commitirovannah precursor cells (giving rise to hepatocytes or biliary commitirovannah precursor cells (giving rise to the bile ducts).
The transition from stem cells to adult cells occur in the form of a stepped process that leads to the formation of lines of differentiation of cells on maturation, in which the size of the cells, their morphology, growth potential and expression of genes associated with a particular line. For definition wide-angle is this process good metaphor “aging”. “Young” cells have early gene expression and the highest growth potential; later the cells in this line of differentiation have “late” gene expression and usually limited growth, or not grow at all. Late in the cells can be considered as “old”, or using biological term, apoptotic, and eventually disappearing from the population. The development of the line of differentiation of cells on maturation leads to the natural circulation cells in the tissue and allows regeneration after damage. Tissues differ in what happens in them kinetic processes and the processes of maturation. The line of differentiation to Mature cells of the intestine develops very quickly, coming full cycle of development in less than a week, and the line of differentiation to Mature cells of the liver develops slowly, and for cells of rat liver full development cycle requires about a year.
Forms of liver cells in rats, developing approximately 10-th day of embryonic life, called “embryonic cells of 10-day or E-10, are itacimirim cardiac mesenchyme by endodermal located in the area of mid-gut of the embryo (Zaret, K. 1998. Current Opinion in Genetics & Development. 8:526-31). Most early recognition of liver cells in the embryos was obtained in studies of mRNA that code is it alpha-fetoprotein (AFP), where used in situ hybridization ((Zaret, K. 1998. Current Opinion in Genetics & Development. 8:526-31); Zaret, K. 1999. Developmental Biology (Orlando). 209:1-10). Expressing AFP cells were observed in the area of mid-gut of the embryo around the mesenchyme that gives rise to heart 9-10 day, all of the studied samples the liver of rats and mice. The liver becomes macroscopically visible on the 12th day of embryo development (E-12) and reaches values of about 1 mm on the 13th day (E-13).
Parallel to this is haematopoiesis, when first identified hematopoietic cells 15-16 days (E-15 E - 16 (in rodents) and on the 3-4th month of embryonic development in humans, with a peak of erythropoiesis (formation of erythroid cells or red blood cells), celebrated on the day E-18 (in rodents) and on the 5-6th month people. During the peak of erythropoiesis red blood cells predominate in the liver, and their number is more than 70% of the total number of cells in the liver. The end of the period of pregnancy in rodents falls on the 21st day, and people for 9 months. During the first hours after birth the number of hematopoietic cells is dramatically reduced, so on the second day of postnatal life (in rodents) and in the first week or two (people) the majority of hematopoietic cells disappear, migrating to the bone marrow. Nobody knows the cause of this migration of hematopoietic cells. However, there are two snowmageddon.
The first is that hematopoietic cells prefer relatively anaerobic conditions and go in the bone marrow (which is relatively anaerobic), as in the liver oxygen levels increased upon activation light; and the second assumption is that the cause of migration is the loss of the pregnancy hormones. After birth, the loss of hematopoietic precursor cells in the liver is associated with a sharp decrease in the amounts of hepatic progenitor cells and a parallel increase in the number and maturity of hepatocytes and biliary cells. The full maturation of the liver fails to 2-3 weeks of postnatal life (in rodents) and in a few months (in humans). By this time the remaining hepatic precursor cells are localized in areas of the portal triads at the periphery of each of the liver acinus.
After this is the classic structure of acinus liver, each acinus in its periphery defined by six sets of portal triads, each of which has a bile duct, hepatic artery and hepatic vein, and in the center there is a Central vein, which connects to the Vena cava. Plates of liver cells, as spokes in the wheel, are arranged from the periphery to the center. Conventionally, these plates can be divided into three zones: zone 1 is located on the olo portal triads; zone 2 is srednetirazhnoy, and zone 3 is located around the Central vein. Diploid cells of the liver are only in zone 1; tetraploid cells are located in zone 2; and tetraploid, octaploid and multinucleated cells are located in zone 3. This structure is very likely to line differentiation of cells on maturation, which completes the process of apoptosis (Sigal, S.H. et al. 1995, Differentiation, 59:35-42);
IX. Using the concept of lines of differentiation of cells in preclinical and clinical studies of the biology of the liver
Characteristics of growth and differentiation in vitro and in vivo cell population of the present invention are consistent with the concept and applications of the model line differentiation of liver cells, based on the position of the line of differentiation. For example, in the culture of parenchyma in vitro the ability of parenchymal cell division and cell number of cell divisions is strictly predetermined by the dependence of the position of the line cell differentiation. So kolovorot parenchyma cells will have a higher potential divisions than allocentrically cells. This allows you to answer the question, he had not had a response, which is why primary cultures of liver cells, a body known as the most regenerative throughout the body, demonstrate such low ability to divide in culture.
For STV the new cells and their transformed copies Hepatol predicts the expression of early manifested genes, such as genes of alpha-fetoprotein and insulin-like factor II, but not genes, later expressed in the line of differentiation of cells. In the model lines differentiation of cells on maturation none hepatoma not late expresses genes, because the full passage along the entire line of cell differentiation requires undisturbed control the differentiation, growth and cell cycle. And this is indeed observed in the cell population of the present invention. In studies on the molecular biology that compares specific liver gene expression in embryonic and adult tissues, define several classes of genes: genes with which you can diagnose compartments (stem cells, amplification, differentiation); genes expressed zonal able to cross the boundaries of the compartments; and genes expressed at early, middle or late stage in the line of differentiation of cells, but discretely in a small number of cells.
Different morphological types of gene expression in primary tumors of the liver can be identified by examining the cell population of the present invention. If tumors are the proliferation of transformed stem cells with different abilities to di is ferentiable, the total expresia alpha-fetoprotein in hepatoma is not a token, induced by the tumor, is an indicator of the presence of increased populations of immature cells normally expressing alpha-fetoprotein.
The population of isolated cells of the present invention is of great importance for the success of cell and/or gene therapy of the liver. The present invention, as described in the examples, defines key terms, which can be successfully crioconservated liver cells of human and non-human primates.
Due to its ability to increase significantly in vitro, the cell population of the present invention, like the cells in hematopoietic lines of differentiation, can be used as a material for the performs for the source of cells for reproduction of their ex vivo. This eliminates the need for more invasive surgical interventions on the patient's liver.
After the obtained culture of hepatic progenitor cells, transfer of genes. This can be accomplished using a number of different systems of vectors for gene delivery. It is important to keep in mind that successful gene transfer requires a rapidly growing culture, and because of hepatic precursor cells of human of the present invention successfully share the normal physiological conditions, these cells are ideally suited for gene transfer to the liver. In addition, the growth characteristics of the cell population of the present invention can be used for gene transfer ex vivo with specific vectors for gene delivery (i.e. retroviral vectors), for efficient insertion and expression of genes requires cell proliferation.
An alternative approach to gene therapy is the design vector, the target of which is specific precursor cells, with the subsequent introduction of this vector associated with the desired gene, directly to the patient. Vectors are sent to the endogenous population of progenitor cells and modify them.
The population of progenitor cells of the present invention can be used in autologous and allogeneic cell or gene therapy. It is clear that the use of autologous liver precursor cells eliminates serious fears associated with rejection of transplanted cells. The cell population of the present invention particularly attractive for allogeneic transplantation of cells, because of their antigenic profile requires a minimum of immunological rejection reaction. In addition, other cellular elements, such as blood cells, endothelial cells, cells of Kupffer, known is haunted as highly immunogenic, essentially eliminated during the cleaning process.
After autologous or allogeneic liver cells-predshestvenniki selected, cleaned and placed in culture, they can genetically modify or leave intact, breeding in vitro clerks, and then give them to the owner. If desired genetic modification, then after it and before the transplantation of genetically modified cells can be propagated and/or to select on the basis of the introduction and ekspressirovali dominant breeding marker. The graft can be returned in the hepatic compartment or ectopic or heterotopic site. For transplantation in the liver compartment, you can use the infusion into the portal vein or vnutricletocny injection. Vnutristenocna injection can be used in weaving ways, because hepatic precursor cells transplanted by vnutrikletochnoi injections are moved in the liver compartment.
Additional medical procedures may enhance the efficiency of engraftment in the liver of transplanted hepatic precursor cells. Models in animals have demonstrated that partial hepatectomy introduction angiogenesis factors and other growth factors promotes engraftment of transplan the ATA and increase the viability of transplanted hepatocytes. An alternative approach consists in the transplantation of genetically modified hepatocytes in an ectopic site.
To date, attempts to use cell therapy for liver proved ineffective. This may be due to the fact that used donor cells were mainly represented adult liver cells, life after separation and re-injection is small. In addition, the use of adult cells leads to a strong immunological rejection. Hepatic precursor cells of the present invention provide higher efficiency, due to their reduced tendency to cause immunological rejection reaction, and due to their high regenerative potential.
As for gene therapy, it continues to use “aimed injectable vectors, which represent the most popular of the developed approach for clinical therapy. However, these attempts have met with limited success due to immunological problems, and because of the unstable expression vectors. The only approach to gene therapy that have proven promising, was gene therapy ex vivo, and these studies have been conducted almost exclusively in hematopoietic cells predecessors. The authors izobreteny is sure that gene therapy ex vivo using precursor cells (or using injectable vectors, in any way aimed at populations of these precursor cells) will be more effective, because these vectors can be entered ex vivo purified subpopulations of progenitor cells; modified cells are selected and re-injected in vivo. The advantage of progenitor cells is their enormous reproductive potential, and that they cause only minimal rejection reaction or doesn't provoke such a reaction, and in their capacity to differentiate to the formation of a line of differentiation of Mature cells.
X. Common or interdependent lines of differentiation of cells.
Improved methodology allowed the authors of the present invention to further study and characterize the hepatic precursor cells. These studies revealed a particularly close relationship between the liver and hematopoietic cells predecessors, which confirms the close relation between these two lines cell differentiation. Indeed, these studies show that precursor cells of the hepatic and hematopoietic lines of differentiation have many of the same antigenic markers (CD14, CCD334, CCD38, CD117 or ckit, antigens oval cells),have common biochemical properties (i.e. have the same transferrin, glutathione-S-transferase and the truncated isoform of alpha-fetoprotein), and also have a great similarity in the conditions of cultivation (form extracellular matrix and specific requirements hormones) for propagation ex vivo. Precursor cells of both lines of differentiation are located in the same sites acinosa liver. And finally, in the cells of these two lines of differentiation of cells on maturation there is a paracrine signaling, namely the signals produced by each of these lines of differentiation, control cells other of these two lines. Thus, we can conclude that, apparently, there is a General line or at the very least interdependent lines of differentiation of hepatic and hematopoietic cells.
The described cell populations purified and used to obtain derivatives or malogovoryaschih, or liver cells, depending on the conditions in which these cells are isolated and cultured. Therefore, bioreactors inoculated cell populations selected on the set of antigens defines as hepatic, and hematopoietic precursor cells (e.g., CD38+, ckit+, CD45+) can provide cell populations, with many potential paths of development. The development way depends on the fact, as the cells are again injected in vivo, or in what conditions the cultivation of place cells.
Another important aspect of the cell population of the present invention lies in the fact that these cells Express specific for hematopoietic stem cell surface antigen CD34. CD34-positive bone marrow cells were used as a convenient marker for positive selection of hematopoietic stem cells. However, this increases the number of messages which Express doubts about the specificity of the antigenic marker for CD34 hematopoietic stem cells (Nakauchi H., Nature Medicine, 4:1009-1010 (1998)). Experimental evidence suggests that CD34-negative cell population of human bone marrow and umbilical cord blood are cells that can re-colonize the bone marrow immunoregulatory mice.
The present invention, as described here, offers ways to purify populations of both hematopoietic and hepatic precursor cells, which are then used in the clinical and preclinical programs, on the basis of the established close relationships between hepatic and hematopoietic cells.
The use of hepatic progenitor cells is wide and diverse. It includes: 1) research on human cells; 2) received the e vaccines and antiviral drugs; 3) Toxicological studies; 4) drug development; 5) obtaining the protein (using cells as hosts for the production of various specific for human factors); 6) cell therapy of liver; 7) gene therapy of liver; 8) receiving bioisosteres liver, which can be used in scientific research, Toxicological and antimicrobial studies to obtain proteins, or use for medicinal purposes as a system using the liver. Given the possibility for the existence of a common hepatic and hematopoietic precursor cells lines of differentiation shown by the authors of the present invention, the same cells can be used for hepatic, and hematopoietic development paths, depending on the conditions in which they are placed.
The availability of hepatic precursor cells of a person with a high degree of purification will allow for a much more intensive research on human cells, which will accelerate the development of successful forms of cell and gene therapy of the liver, and will also allow you to get bioisostere human liver for use in research and clinical devices. Currently, limited availability of healthy donor human tissue interferes with the implementation of clinical programs cleto therapy of the liver and obtaining bioisosteres liver. Population of progenitor cells have significant potential breeding that will help to overcome or at least significantly reduce the specified limited availability of donor material.
The following examples make illustrative and do not limit the scope of the present invention.
Analysis of variant forms and albumin expressed in hepatic cells compared with other cell types.
Cell lines: Two lines of human hepatoma, NERV and HepG2 support in the medium Needle MEM with the addition of 1 mm sodium pyruvate, 2mm L-glutamine, 50 u/ml penicillin, 50 μg/ml streptomycin, 0.1 mm solution of non-essential amino acids medium MEM, 5 μg/ml insulin and 10% fetal bovine serum (FBS). The cell line erythroleucus person To 562 and cell line embryonic fibroblast mice STO support in DMEM/F12 with the addition of 2 mm L-glutamine, 50 u/ml penicillin, 50 μg/ml streptomycin, 5×10-5M 2-ME and 10% fetal bovine serum.
Analysis of RT-PCR: Total RNA extracted from NERV, HepG2 and STO method Chomcznski and Sacchi, N., Anal. Biochem. 162:156-159 (1987). cDNA synthesized using oligo-DT-premirovany and subjected to PCR amplification using sets of primers designed by the authors of the present invention for human AFP or albumin. The sequence is the following primers:
The PCR reaction is carried out in a total volume of 50 μl consisting of 1 µm of each primer, 200 μm of each dNTP, 50 mm KCl, 1.5 mm MgCl2, 10 mm Tris HCl, pH 8,3, and 1.25 E Amplitaq polymerase (Cetus Corp.). Samples heated to 94°C for 3 minutes, followed by amplification for 30 cycles of 2 min at 94°C, 2 min at 62°C and 3 minutes at 72°C. After the last cycle performed by the final stage of elongation at 72°C for 7 minutes. Then 5 µl of the reaction solution in each PCR reaction was poured into 2%agarose gel containing 5 μg/ml of ethidiumbromid in Tris-acetate-EDTA buffer.
RT-PCR for AFP: AFP Gene of a person consists of 15 exons (Gibbs et al., Biochemistry, 26:1323-1343). To distinguish truncated transcripts from mRNA functional full-AFP, two different parts of the sequence cDNA AFP selected as target molecules reaction RT-CR. The combination of primers hAFP1 and hAFP2 used for amplification of exon 1 containing the initiating MET (methionine), exon 3, while hAFP3 and hAFP4 amplified exon 12 to exon 14, containing the termination codon. The PCR results are shown in figure 1. Both combinations of primers result in a clearly visible bands of amplification of RNA from NERV and HepG2 (tracks 1, 2, 4 and 5). In contrast, only one specific band With terminalno part is detected using the primer set hAFP3 and hAFP4 in mRNA from C (lanes 7 and 8). This result confirms that the cell line erythroleucus, K, expresses only a truncated form of AFP without N-end. To confirm this hypothesis held a PCR reaction for the entire region encoding AFP using primers hAFP1 and hAFP4. As expected, PCR cDNA NERV and HepG2 shows only one visible band size 1.8 TPN (lanes 3 and 6), in the absence of the band in K (track 9). The controls were the samples without RNA, and the sample derived from cell lines of human embryonic fibroblast mice (STO). None of them had any visible streaks.
Next, have designed a series of primers 5’ of exon 2 to exon 6, to see the differences between authentic and variant forms of hAFP mRNA. Presented at the figure 1 the result shows that all the region coding, except for exon 1, variant forms hAFP in C the same with the authentic form (tracks 1, 3, 5, 7, 9 and 11).
RT-PCR assay for albumin: albumin Gene of a person consists of 15 Konov (Minghetti et al., J. Biol. Chem., 261:6747-7657). As for AFP, the combination of primers hALB1 and hALB2 used for amplification of exon 1 containing the initiating MET (methionine), exon 4, whereas the combination hL3 and hALB4 amplificare exon 12 to exon 14, containing the termination codon. The PCR results are shown in figure 1. Both combinations of primers lead to good:notable bands of amplification in R Is To from NERV and HepG2 (tracks 1, 2, 4 and 5). In contrast, only one specific band of the C-terminal part is detected using the primer set hALB3 and hALB4 in RNA from C (lanes 7 and 8). PCR for the entire coding region of albumin conducted using primers hALBl and hALB4, did not show any bands in K (track 9). The controls were the samples without RNA, and the sample derived from cell lines of human embryonic fibroblast mice (STO). None of them had any visible streaks.
Suppliers of reagents include:
Sigma Chemical Companyi (St. Louis, Mo)
Gibco BRL Products (Gaxthersburgh, MD)
Worthinghton Biochemical Corporation (Frehold, New Jersey)
Dupont Pharmaceuticals (Wilmington, Delaware)
Falcon-a subsidiary of Becton Dickinson Labware (Franklin Lakes, New Jersey)
Suppliers of fabrics include:
Anatomical Gift Foundation (Atlanta, Georgia)
Advanced Biosciencies Research, ABR (San Francisco, Cal)
The local Department of transplantation surgery at the UNC hospital
Treatment of human liver
Fetal liver: Fetal liver comes from many clinics associated with Advanced Biosciencies Research (ABR), all of which are located in California, or from Anatomical Gift Foundation (AGF), with clinics in the Southern United States (specifically in the States of California, Virginia), North East (Pennsylvania) or the Midwest (Kansas, Colorado). The fruits were obtained from clinics; tissue was isolated from the fruit and put in medium RPMI 1640 (Gibco) with the addition of insulin (Sigma, 5 μg/ml), transferrin (Sigma, 5 μg/ml), the Elena (10 -9M) and 5% fetal bovine serum (Gibco). Then the samples were placed on ice and sent to the laboratory of the authors of the invention, this process could take 10-16 hours. Thus, the authors received samples of approximately 24 hours after surgery. Then the samples were assigned a number with the prefix REN, in chronological order of receipt of samples (REN 1, 2, 3, etc), where REN is an abbreviation from the word Renaissance ("Rebirth").
The liver of an adult: Samples of adult human liver came from Anatomical Gift Foundation or local surgical clinics (UNC) and represented cut out the liver tissue, explants from recipients of transplants or liver destined for transplant, but not used for these purposes for reasons other than pathogens. Patients, donor tissue Explant or rejected donor tissue were subjected to study a number of diseases, and only those samples which on the basis of these studies were deemed safe to be used for further processing of the cells. After retrieval from the patient's body, the liver samples were placed in a solution of Wisconsin University (also known as Viaspan) and ice were sent to the laboratory of the authors of the invention. The time between removing the body from the patient brain-dead (in EMA commit”) and its arrival at the laboratory were very different. The samples arrived less than 24 hours from the “time lock”, i.e. from the time when the liver is removed from the body of the donor.
Cadaveric liver: the Liver, taken after death, at least 30 hours after death, received from the local associations for the supply of organs (for example, of the Karolinska Association for the supply of organs, or SORA). The liver was treated in the same way as the liver of an adult.
The list of items reviewed for security for the researcher: HIV I and II, the virus human T-cell leukemia I and II, hepatitis B and C, tuberculosis. Checklist for clinical use include: HIV I and II, the virus human T-cell leukemia I and II, hepatitis a, b, C, and G; EBV, CMV, tuberculosis, syphilis and Mycoplasma.
The liver of the fetus and an adult were treated with a combination of enzymatic digestion and mechanical dissociation, and liver of the fetus was prepared mainly by mechanical dissociation, and liver adult - primarily through enzymatic cleavage. A description of each of these methods is given below. The liver of a fetus and an adult were digested for various periods of time in enzyme buffer, which serves to dissolve the extracellular matrix, linking the cells in the tissue. The mixture collagenase enzymes, COI is lowanna to highlight the cells of the liver, is an enzyme preparation with high purity "Liberase"manufactured by Boehringer-Mannheim, consisting of a mixture of purified collagenase and elastase. This enzyme mixture can be used at much lower concentrations and with a reduced number of side effects.
Enzyme solution: a solution of collagenase - 60-70 mg/100 ml buffer (collagenase company Sigma, type IV, No. S, or firm Worthington, type, catalog No. LS005273; both are bacterial preparations enriched with collagenase, but with the large number of enzyme impurities) or liberty (Liberase) (purified product collagenase/elastase, manufactured by Boehringer-Mannheim, catalog No. 1814184)prepared in buffer P2 (see below) and used at a concentration of 0.23 mg/ml
Solution for washing cells: a solution of RPMI 1640 (Gibco) with the addition of insulin (5 μg/ml), transferrin (5 μg/ml), a mixture of free fatty acids (see below), linked in a molar ratio of 1:1 purified bovine albumin or human serum.
The mixture of free fatty acids: the population of immature cells, and damaged older liver cells for their maintenance and synthesis of their membranes require lipids. Although fully Mature hepatocytes can synthesize their membrane from one fatty acid (linoleic acid), but the younger parthenia cells to this is we are not able, and to meet the needs of the lipids they require a mixture of many different fatty acids. The authors propose a complex mixture, which is then connected in a molar ratio of 1:1 albumin high degree of purification. Detailed description of the method of obtaining the mixture of fatty acids is given below.
Royal solutions prepared as described below, to obtain a mixture of free fatty acids (100 mm):
|Palmitic acid||31.0 mm|
|Palmitoleic acid||2.8 mm|
|Stearic acid||11.6 mm|
|Oleic acid||13,4 mm|
|Linoleic acid||35,6 mm|
|Linolenic acid||5.6 mm|
To obtain a final concentration of 7.6 μm/l, add 76 μl mixture Pitre culture medium. [Link: Chessebauf and Padieu, In vitro 20 (10):780; 1984. In accordance with this reference mixture of free fatty acids used in final concentration of 7.6 of mkaku/l (=7,6 mm) in culture medium for cells].
Getting the individual components of a mixture of fatty acids:
Each individual component is dissolved in 100% EtOH, as follows:
Palmitic acid - 1 M mother solution, soluble in hot EtOH.>
Palmitoleic acid - 1 M mother solution, soluble in EtOH.
Stearic acid - 151 mm mother solution, soluble in hot tOH in a concentration of 1 g/ 21 ml.
Oleic acid - 1 M mother solution, soluble in tOH.
Linoleic acid - 1 M mother solution, soluble in EtH.
Linolenic acid - 1 M mother solution, soluble in EtOH.
These separate uterine solutions are then mixed to obtain 100 mm of a mixture of free fatty acids. Aliquots of these separate solutions of free fatty acids and mixtures of free fatty acids is prepared, passing through them, the bubbles of nitrogen to reduce oxidation and improve stability. Royal solutions frozen at -20°C.
Perfusion buffer (the buffer to fill) P1 - not containing Alicia and magnesium perfusion buffer (pH 7,2) with final Centralia below for each of the following components: 118 mm NaCl, of 4.7 mm KCl, 1.2 mm KN2RHO4, pH 7.4, 2.5 mm NaHCO3, 0.5 mm EDTA, 5.5 mm glucose, 0.5% albumin bovine or human serum (BSA), ascorbic acid (50 μg/ml), insulin (4 mg/ml), dexamethasone (1 μm).
Perfusion buffer (the buffer to fill) P2 - modified Dulbecco medium Eagle or medium RPMI 1640, with the addition of 0.5% BSA, ascorbic acid (50 μg/ml), insulin (4 mg/ml), dexamethasone (1 μm).
DMEM - modi the data Dulbecco medium Eagle (Gibco), with glucose, sodium pyruvate and L-glutamine, which then add 5% fetal bovine serum, insulin (4 mg/ml) and dexamethasone (1 μm).
Wednesday Chee, with the addition of cultural supplements ITS (5 ml/500 ml) and dexamethasone (0.1 µm).
Percoll (Pharmacia, catalog No. 17089102), diluted in a ratio of 9:1 10-fold buffered saline phosphate buffer solution Dulbecco.
The study of the tissues of fetal liver
The fetal liver is delivered in transport buffer (described above) and on ice. The resulting material is then rinsed "buffer for washing cells”consisting of RPMI medium 1640 (Gibco) with the addition of insulin (Sigma, 5 μg/ml), transferrin (Sigma, 5 μg/ml), selenium (mass spectroscopy, Johnson Mattey; 10-9M) and a mixture of free fatty acids associated with bovine serum albumin in a molar ratio of 1:1. Then the fetal liver is placed in collagenase buffer for 15-20 minutes, then carefully push through the “collector” (Sigma) with a cell size of 800, resulting in a small cell aggregates; to facilitate dissociation of use “buffer for washing cells. The resulting cell aggregates completely dissociated, Prodanova them through the filter with cells 70 microns (cell filter Falcon, nylon cells 70 μm, catalog No. 2350), using the buffer to flush to etok, to speed up this process. Cells that have passed through the filter with holes 70 microns, are kept separately from cells that have not passed through this filter. Both samples are subjected to cryopreservation and check on the percentage of viability by staining Trifanova blue.
The study of the tissues of the adult human liver
Liver kateteriziruyut using the portal vein, Vena cava, or both veins, fill buffers to remove blood, and then with buffer containing collagenase/protease for enzymatic dissociation of cells. After splitting, which requires usually 15-30 minutes, depending on the size of the liver tissue pushes through cheesecloth or a nylon filter, or soskrebajut scraper for mechanical dissociation of cells. Dissociatively cells opolaskivayut buffer containing serum to inactivate; collagenase and other enzymes used in the fill.
Perfusion buffers P1 and P2 are placed in a water bath with a temperature of 37°C. Perfusion is carried out in a vessel Miller to fill the sample, in which during the entire process maintain a temperature of 37°C. During perfusion in the buffer serves oxygen. All tubes in the vessel rinsed with 70%ethanol, then with distilled water and then with buffer P1 to ensure that the system deleted all such. Liver kanyoro Teflon cannula, needle, 16-gauge, attached to the syringe 60 ml to pass through the liver buffer P1 with ice temperature using various blood vessels on the surface of the slice large pieces of liver (100-300 grams). In rare cases, when you get a whole lobe of the liver, you can Coulibaly remains Vena cava. Studied which of the various blood vessels present in the pieces of liver, best allow perfusion of the tissue. Using this procedure also removed from the liver of all excess blood. Selected blood vessel kanyoro and sealed in place using medical “superglue”. All other major blood vessels and holes on the surface of the liver sealed with medical glue or, if required, using Q-tips with medical glue to better seal the openings. After the glue dries, the liver sample was placed on a nylon sieve inside the glass Cup is the right size. In a Cup add buffer P1, immersing him in the liver. The Cup with the liver is placed inside the vessel perfusion and attach the tube protruding from the cannula. Buffer P1 recycle within 15 minutes, starting with a low speed of about 24 ml/min, and then slowly increase the speed to 58 and 90 ml/min to optimize spending the liquid with a suitable pressure. You must check that there are no unnecessary leakage of the perfusion solution from the liver. After 15 minutes buffer P1 is removed from the Cup and replace it with a buffer P2 containing collagenase. Buffer P2 to recycle until the liver will not be sufficiently split (which is determined by the color change of the liver from dark reddish brown to pale brown, as well as on acquisition liver mushy consistency). Buffer P2 recycle for no more than 20-25 minutes. At the end of the perfusion buffer P2 poured from a Cup, and the liver is transferred into the Cup in the biological case.
In a Cup add a medium for cell culture (DMEM), and the cannula and the glue is removed, along with the undigested parts of the liver. The fibrous capsule of the liver (pisanova capsule) are ground with tweezers and scissors. This frees split fabric in the environment by removing the connective tissue and undigested material. Split the material is placed in DMEM, and then filtered through a series of filters of different sizes. In order to facilitate the filtering, the filters are placed inside a large crater. Split the material is filtered first through a layer of gauze, and then through a nylon filter with pores of 400 μm, and finally through a Teflon filter with a pore 70 μm. The filtrate is equally distributed between the centrifuge tubes and centrifuge the shape at 70g for 4 minutes.
The supernatant, obtained after centrifugation, before adding percoll, referred to as fraction 1 (F1). To the obtained after centrifugation the precipitate cells in test tubes add DMEM and isotonic solution of percoll, so as to obtain a final ratio of 3:1, respectively. For example, a small amount of cell mass with a volume of 5 ml of sediment in the tubes are suspended in 30 ml DMEM and 10 ml of isotonic solution of percoll. The sample is centrifuged at 100g for 5 minutes. Get a supernatant liquid; the upper layer is referred to as fraction 2 (F2). The middle layer of percoll referred to as fraction 3 (F3). Remaining in the tubes sediment cells called fraction 4 (F4). Cells of different fractions of suspended and examined for viability by staining Trifanova blue. The viability of these different fractions are presented in table 3, along with their viability after cryopreservation.
Cells that remained associated with vascular or ulcerosa system liver tissue after perfusion, retain. These cells are found in the original cell suspension obtained after enzymatic perfusion, and usually remain on the screens (e.g., gauze) after filtration of cell suspensions. These residues vascular and ulcerosa systems again treated with enzymes, and the resulting cell joint is complementary with other cells.
Fractionation in percoll typically used by most researchers to remove the elements that they consider fragments of cells and dead cells; retain only the final precipitate. A new variant of normal perfusion, described here, is that the sediment rejected, and retain and use for further research only cells with the lowest surface density (i.e. the cells are gathered at the top of the gradient). These cells represent a more young parenchymal cells and much easier cryopreservation (see the section On cryopreservation).
Experiments on cryopreservation. The liver samples used for the development of methods of cryopreservation, received from donors aged from the age of the fetus (gestational age from 12 to 25 weeks) and until the age of 77 years.
“A new buffer for cryopreservation”
- Viaspan (catalogue Dupont No. 1000-46-06) with the addition of 2% human serum (Gibco) or fetal bovine serum (Biowhittaker);
- 10%cryoconserved [dimethyl sulfoxide (Sigma catalog No. D5879 or D8779)used exclusively for Mature parenchymal cells, or dimethyl sulfoxide or glycerol (Sigma catalog No. 66279)used for precursor cells];
- then add to the buffer antibiotics (penicillin, 200 u/ml); streptomycin, 100 μg/ml);
- then to the buffer DOB is make hormones and growth factors: insulin (5 µg/l), transferrin (5 μg/ml), epidermal growth factor (50 µg/ml), FGF (10 ng/ml), IGF II (10 ng/ml);
- then add to the buffer lipids: free fatty acids (7,6 m/l)associated with bovine serum albumin (BSA) or human serum (HSA)and high-density lipoprotein (10 μg/ml);
in addition, the buffer add trace elements (selenium (10-9), copper (10-9M), zinc (5×10-11M)), and antioxidant (for example, perforin representing simulator superoxide dismutase, used at a concentration of 10 μg/ml ascorbic acid, used at a concentration of about 0.1 mg/ml, or any other antioxidants known in the art).
The described version of the song lies in the blending of essential nutrients, lipids, hormones and growth factors, identified as part containing no serum medium specific hormonal composition, adapted for liver cells. Use new buffer yields a measure of the viability of liver cell fractions F4 from a low of about 50% (very poor samples), to high component of 80% (for good examples). The viability of fractions F1-F3 are consistently above 80%, which, according to the authors, due to the fact that these fractions contain more than young cells, with the ploidy level and metabolizes the second activity, more suitable for the synthesis of components of the extracellular matrix and/or other cellular factors necessary for maintaining the viability and growth; therefore, we should expect that these cells will be easier to endure freezing. The use of buffer simulator superoxide dismutase resulted in increased cell viability by 5-10%.
Alternative to the above:
- the use of the modified buffer that does not Viaspan, and to the basal medium (such as RPMI 1640) was added insulin (5 μg/ml), transferrin (5 μg/ml), free fatty acids (7,6 m/l)associated with BSA (bovine serum albumin), high-density lipoprotein (10 μg/ml), trace elements (10-9M), copper (10-7M),zinc (5×10-11M) and antioxidant;
- floor cells of one of the forms of the extracellular matrix, such as collagen type IV, mixed with laminin, or collagen type I or III, mixed with fibronectin.
The cells of fetal liver, processed as described above, suspended in the buffer for cryopreservation (described above), transferred to a separate aliquot into tubes for freezing 3 ml, 5-10×106cells/ml, and kept in these conditions for 1 to 2 hours. The cells are then frozen to liquid nitrogen temperature from -100°C to -180°C, preferably -160°using computer control for the MSE of the awn freezing (Forma Cryomed), and then store in a large steam phase in the storage tank containing liquid nitrogen (-160°). Cells tolerate this procedure, while not observed any significant loss of viability during storage periods from 50 to 270 days (see figure 3).
It is established that the fraction of cells in the adult liver (F1-F4) contain differing from other populations of cells: F1 contains debris, red blood cells, hepatic stellate cells and small liver cells (less than 10 microns), which, apparently, are the populations of progenitor cells (either hepatic or hematopoietic lines cell differentiation); fraction F2, the upper part of the solution percoll, contains a larger liver cells (10-15 μm), which is a small diploid parenchymal cells; fraction F3, the bottom part of the solution percoll has a larger parenchymal cells (15-25 μm)consisting of a mixture of diploid and tetraploid cells; and fraction F4 (used by all other researchers), consists of the largest parenchymal cells (25-50 microns), all of which are polyploid (tetraploid or octaploid). As a rule, parenchymal cell fractions F1-F3 are viable after a freeze, which is 85-95%, and parenchymal cell fraction F4 have viability after Zam is Rivonia, component 50-80% (depending on the state of the liver after its receipt). Variables that affect the viability of parenchymal cell fractions F4, the following: 1) the age of the donor (the older the donor, the worse the prognosis for cells); 2) the period of time between the “record time” and delivery to the laboratory (the shorter, the better); 3) health status of the liver tissue before removing it from the body of the donor (for example, a state of severe ischemia suggests a poor prognosis). These factors interact with each other, so fast shipping fabric from an elderly patient may give better results than the fabric taken from a young patient, but too long stationed in the path.
|The average viability and efficiency of attachment of cells to the liver of the fetus and an adult when using cryopreservation and % of hepatic progenitor cells (AFP+-cells in cell suspension|
|Cell Population||Cryoconserved||Viability after processing||Zhiznesposobnosti the SSPE defrosting||The average size of cell (µm)||Growth in culture||% F+cells|
|The liver of the fetus||76%||77% (i.e survived 100%)||7-15||Good||6-7%|
|The liver of an adult human, F1||Glycerol/DMSO||80%||82-85%||>12||Good||0,5-1%|
|The liver of an adult human, F2||Glycerol/DMSO||85%||84%||12-15||Good||2%|
|The liver of an adult human, F3||DMSO||85%||85%||15-25||Good||0,2%|
|The liver of an adult human, F4||DMSO||50-75%||56%||25-50||Poor||0,01%|
A very large range of viability fractions F4 after treatment, and after thawing because of the different length of the time interval between the “record time” and getting samples in the laboratory, as well as various state of the liver (fibrosis, ischemic and others) As a rule, the fraction F4 is most sensitive to the negative impacts associated with the processing of the liver and with the General condition Zdorovya tissue. It is noteworthy that the fractions F2 and F3 were, as a rule, viable and well maintained after cryopreservation, even the EU is and has been bad in liver samples. Fraction F1 were more variable, contained a large number of fragments of cells, fat droplets, as well as numerous small cells, which included both small parenchymal cells (containing, considered to be the hepatic precursor cells), and various subpopulations of hematopoietic cells (i.e. red blood cells).
Cells pass in one row through the flow cell, where they are subjected to laser irradiation. The approximate volume of one cell is determined using the “forward scattering”, or the amount of light reflected when crossing the beam. The scattered light or side scattering from internal structures of the cell such as the nucleus, Taurus Golgi to the endoplasmic reticulum, bubbles and the like, are used to determine the values of internal complexity (i.e. the active cells and more Mature cells that contain more internal components than resting or younger cells). More selective information about the characteristics of the cells get through high-affinity binding characteristic antigens with protein complexes on the cell surface. These antibodies can be covalently linked to a fluorescent molecule, such as fluoresceinisothiocyanate (FITZ), ficoi the trine (RE) and tandem conjugates of D and cytochrome, that fire laser beams, generating emitted light with a specific wavelength for each fluorophore. By a selection panel with a characteristic chromophores, conjugated with a specific antibody, selected interest cell population.
Cells are analyzed on the basis of the input signals of their parameters. To collect the desired cells use a number of devices for collecting cells, including test tubes and conical Eppendorf tubes and mesh tablets of any size, at a speed of up to 40,000 operations per second and above.
|Antibodies and reagents used for staining procedures|
|Antibody||Provider, no catalog, no party|
|Antibody goat to AFP person||Chemicon, AB635, SR|
|Monoclonal antibody mouse to Thy person||hemicon, MOV, 293CCD|
|Conjugate PE monoclonal antibody mouse to AFP person||hromaprobe, P41020, AR|
|Biotinylated antibody rabbit for the antibody goat||Vector Laboratories, BA-5000, J0313|
|Biotinylated antibody. rabbit for the antibody goat||Jackson Immunochemicals, 200-152-096, 25985|
|Conjugate streptavidin/AMC||Jackson Immnochemicals, 016-150-084, 40001|
|The conjugate of the AMC with the antibody ass for the antibody sheep||Jackson Immunochemicals, 713-156-4732202|
|Conjugate CY-5 antibody ass for the antibody goat||Jackson Immunochemicals, 705-156-147, 38756|
|Immunoglobulin G (IgG) goat||Jackson Immunochemicals, 005-000-002, 38837|
|Immunoglobulin G (IgG) sheep||Jackson Immunochemicals, 013-000-002, 39945|
|Antibody sheep to human albumin||Serotec, AVR, 210498|
The main solutions used to prepare cells for flow cytometry
BSA: bovine serum albumin (Pentex V);
PBS: physiological solution with phosphate buffer;
FBS: fetal bovine serum;
Modified Dulbecco medium Eagle with hormones: _DMEM
500 ml DMEM, high glucose, without phenol red
25 ml fetal bovine serum (FBS)
20 ml of 5 mm EGTA (etilenditiodiuksusnoi acid)
Insulin (5 μg/ml), transferrin (5 µg/ml)
Trace elements [(selenium (10-9M), copper (10-7M), zinc (5×10-11M)]
Antibiotics (penicillin - 100 μg/ml, streptomycin 100 µg/ml)
500 mg of bovine serum albumin (BSA) 30 mg dnaase
38 μl of a mixture of free fatty acids associated with BSA
Sterile solution, filtered through filtrivahetusest Nalgene with a pore size of 0.2 μm.
Buffered saline Hanks, modified version: HBSS-mod
50 ml of 10X HBSS (balanced salt solution Hanks)
10 ml of 1 M Hepes
Penicillin - 100 μg/ml/ streptomycin 100 µg/ml
500 mg/l BSA
30 mg/l dnaase
Bring to 400 ml
pH to 7.3
Bring to 500 ml
Filtration through a sterile filter with a pore size of 0.2 μm.
Blocking buffer for immunochemistry
100 ml HBSS_mod
2.2 ml of 45%fish Gel (teleostean) and
0.8 g BSA
0.5 ml of 1% saponin in HBSS
Casting medium for immunofluorescence microscopy
0.5 ml of 2X PBS
0.25 g n-propylgallate
5.7 g of glycerin
Methods of obtaining frozen liver tissue for flow cytometry
The frozen liver tissue is subjected to rapid thawing at 37°C. Each vessel for cryopreservation (containing about 3 ml of buffer with 5-10×106cells/ml) add environment HC-DMEM to 10 ml, with a speed of 1 ml/min, on ice. The sample was then centrifuged at 1200 rpm for 5 minutes at 4°C. the Supernatant was removed and the residue cells resuspended in 5 ml of NA-DMEM. Washing of the cells is repeated up until the supernatant is clear. The cells are then counted and determine their viability using hemocytometer using the method of staining Trifanova blue. The cells divide into factions in the CE is based on the technique of the experiment. To obtain the control data preparing standard tubes containing from 1 to 2×106cells/ml, which is usually done by adding to each tube 200 μl of cell suspension containing 5-10×106cells/ml, the following standard tube:
1) OSC. The original cell suspension consisting of unstained control cells.
2) FITZ to adjust compensation. To 200 μl of cell suspension, add 5 ál labeled by FITZ of antiglycation A. Alternative is a mixture of labelled on FITZ CD34, CD38 and D45 added in an amount of 7 µl of each component to 200 μl of cells.
3) RE to adjust compensation. Use glycophorin-RE (2 µl per 1 ml HC_DMEM by adding 30 ml of the resulting solution to 200 μl of cells.
4) 7AAD for compensation. A good signal is obtained by fixing 200 ál of cell suspension with 2%paraformaldehyde, followed by adding 5 ál of 100 ám 7AAD and 5 μl of detergent (1% saponin) to 1 ml of the suspension of these cells in HBSS-mod. Permeabilization (which permeability property) cells are intensely stained with 7AAD.
5) So for compensation. 200 ál fixed with 2%paraformaldehyde cells incubated for 40 minutes in 2%goat serum, to label cell surface immunoglobulin G (IgG) sheep. The cells are then incubated with A, conjugated with the antibodies is ω ass to goat IgG (1:800) for 40 minutes.
6) of the AMC for compensation. As for 7AAD, generated artificial intensive signal to adjust the compensation. 200 microns fixed with 2%paraformaldehyde cells incubated for 40 minutes in 2%sheep serum, to label cell surface immunoglobulin G (IgG) sheep. The cells are then incubated with the AMC, conjugated with the antibody ass to sheep IgG (1:800) for 90 minutes.
7) Controls the AMC/Su. Incubated fixed (2%paraformaldehyde) and permeabilization (0,05%saponin) cells with the antibody ass to sheep IgG, conjugated with the AMC and with the antibody ass to goat IgG, conjugated with So, within 90 minutes.
8) Controls for monoclonal isotypes. Cells incubated with the conjugate PE with IgG1 mouse and conjugate FITZ with IgG2 mouse. Concentrations should correspond to the concentrations used for labelling tubes for analysis and sorting.
9) Controls for intracellular isotypes. Incubated fixed (2%paraformaldehyde) and permeabilization (0,05%saponin) cells with non-immune IgG sheep and goat IgG, within 90 minutes, as controls for antibody used to identify albumin and alpha-fetoprotein. Continue incubation with the antibody ass to goat IgG, conjugated with Su, and antibody ass to sheep IgG, conjugated to AMCA, within 90 minutes.
Goth is let arterie tubes to obtain populations of selected cells, ekspressiruyushchikh a specific combination of markers CD. Typically, these tubes contain 50-70×106cells. Cells resuspended in 1 ml of dye buffer containing HC_DMEM+1% BSA+500 PM 7AAD (5 µl of the stock solution with a concentration of 100 μm). To kasashima buffer add 15-25 μl CD34 FITZ and CD38 PE, or CD45 PE, in accordance with the number of cells (usually use 3 ál of antibody firm Pharmingen 10×106cells). Antibody to c-Kit added at a dilution of 1:60, glycophorin And used at a dilution of 1:500. Paint for 40 minutes on ice in the dark. After staining, the cells washed twice with medium HBSS-mod and fixed with 2%formaldehyde in S for 30 minutes on ice.
Intracellular staining for sorting cells
For intracellular staining cells with the aim of testing for alpha-fetoprotein (AFP) using flow cytometry cell suspension permeabilizing mixture of saponin (Sigma S4521) 0.05% in HBSS_mod for 10 minutes on ice. The cells are then blocked in a mix environment HBSS_mod containing 1%fish (teleostean) gel, 0.8% of BS and 0.005% saponin for 20 minutes, followed by incubation with the antibody goat to AFP person and antibody sheep to human albumin (both antibodies used in the dilution 1:800 in blocking buffer) for 90 minutes at room temperature in the dark. Cells double-industrial is up environment HBSS-mod, containing 0.01% saponin and then incubated with the antibody ass to goat IgG, conjugated with Su, and antibody ass to sheep IgG, conjugated with the AMC, in the course of 90 minutes.
Alternatively, after the primary antibody, the cells are incubated with biotinylated antibody rabbit to goat IgG (1:500 in blocking buffer containing 2% human serum and 0.01% saponin, for 90 minutes at room temperature in the dark). After that, the cells washed twice with medium HBSS-mod containing 0.01% saponin and then incubated with 9 μg/ml conjugate streptavidin/SW environment HBSS-mod with 0.01% saponin for 90 minutes at room temperature in the dark. And finally, the cells washed twice with medium HBSS-mod and resuspended in HBSS-mod, then filtered through a sieve with a pore size of 50 microns to remove clumps of cells for further analysis and sorting on a flow cytometer.
If you plan to carry out the selection (selection) hepatic precursor cells, immunoselected involves removal of polyploid cells and/or cells expressing markers associated with Mature hematopoietic cells from the liver, such as glycophorin And red blood cells. In addition, remove cells expressing CD45, which is expressed on all Mature hematopoietic cells; cells expressing markers associated with Mature liver and cells, such as connexin 32, which is found in all hepatocytes and biliary cells; and cells expressing markers associated with Mature mesenchymal cells, such as retinoids in hepatic stellate cells or factor von Willebrand's disease, or a factor of 8 in the endothelium.
Immunohistochemical staining of sorted cell populations.
Cells stained for alpha-fetoprotein after analysis and sorting on a flow cytometer. Sorted cell fractions collected in 0.3% environment HBSS-mod containing 1% BSA. After returning to the laboratory the volume of collected samples adjusted so that they contained 0,5×106cells/ml, and aliquots of 200 µl put circles on the slide using the device Shandon Cytospin. The preparation air-dried and retained for future staining for alpha-fetoprotein and/or albumin. Attached to subject the glass cell in the disc is surrounded by a rubber roller to get the “hole” for the application of immunohistochemical reagents. Glass is soaked in Tris-buffer (“nitrosonium” 10 mm Tris with 0,09% NaCl with pH 7.4)containing 0.3% Triton X for 10 minutes, then 10 minutes in one only nitrosonium Tris-buffer.
The cells are then blocked in 10%rabbit serum contained in the blocking solution described above fish Geel is m (teleostaen), for more than 90 minutes at room temperature. After twice washing in nitrosonium Tris buffer cells incubated until the next morning at 4°With antibody goat to AFP person, diluted in the ratio 1:100 in blocking buffer containing 2% rabbit serum. After twice washing in Tris-buffer cells incubated for 90 minutes with biotinylated antibody rabbit to goat IgG (1:200) in blocking buffer at room temperature. The final incubation with streptavidin complex/AMC (9 μg/ml in nitrosonium Tris-buffer) is used for localization of AFP-like immunoreactivity by binding fluorochrome of AMS with biotinylated antibody rabbit. After two washes with Tris-buffer cell preparations dried to a state close to dry before you cover them with a covering glass layer antifatigue (antifade) of potting medium (0.25 g n-propylgallate 5.7 g of glycerin with 1 ml PBS). When necessary, cells doubly stained for albumin by incorporating conjugated with Texas red rabbit antibodies to human antibody against albumin, with a primary antibody against fetoprotein.
Control preparations prepared without primary and secondary antibodies to demonstrate the absence of labeling of cells by the AMC in the absence of any antibodies against alpha protein, is because of the biotinylated secondary antibody. The drugs examined using EPI-fluorescence microscopy using excitation by ultraviolet dye of the AMC, which emits light in the blue part of the spectrum (450 nm).
Cell and/or gene therapy
Since plasminogen activator human urokinase (uPA) can activate plasminogen in the form of a recombinant adenoviral vector that expresses human urokinase under the control of the promoter, RSV-LTR, constructed Ad-RSV-uPA, with the aim to induce liver regeneration. For design and production of recombinant adenoviral vectors to obtain cDNA for uPA person in the following way. The Hindill fragment/Asp718 value 1,326 TPN that contains protein-coding sequence is inserted into the Hindill sites/Asp718 pXCJL.1 under the transcriptional control of the promoter sarcoma virus Rosa LTR (RSV)above plot, generating the polyadenylation signal of bovine growth hormone. The virus get after co-transfection pJMI7 and the vector was designated as Ad-RSV-uPA. Selection of Ad-RSV-uPA carried out by amplification of individual zones of hemolysis in 293 cells. Three days after infetsirovaniya the supernatant examined for immunological reactive Ira using ELISA analysis and determination of fibrinolytic activity using Cup test to determine hemolysis, demons Ryuusei catalytic activity of the Ira, produced after infetsirovaniya Ad-RSV-uPA. The purified virus is stored in aliquot at -80°and immediately before injecting dilute environment HGDMEM. Viral titers determined by measuring optical density using the standard method of determination of hemolysis. Construction of vectors essentially carried out as described in U.S. patent No. 5980886. Viruses titrated on 208F cells.
Female C57BL/6 aged 5 to 6 weeks old (Jackson Laboratories, Bar Harbor, ME) kept in an environment that is free from specific pathogen. Samples of ischemic liver at different time periods collected from dead mice and isolated precursor cells of the liver, as described above. For kanalirovaniya portal vein mouse-recipient anaesthetize by intraperitoneal administration of 0.5 ml of a solution containing 20 mg/ml of 2,2,2-tribromoethanol. Perform laparotomy on the middle line and separate the skin from the abdomen to create a subcutaneous pocket. Open the peritoneum and expose the portal vein. In the portal vein insert a silicone tube with an inner diameter of 0.02 inches, an outer diameter 0,037 inch, S/P Medical Grade, Baxter, 111) and fill in with heparinised saline. After that, through the peritoneum hold the cannula and fix its seam, made of silk thread to 4.0. Cannula length 3 cm loose at the distal end and is placed on the skin in a pre-formed pocket. Mice injected infecting virus precursor cells not earlier than 24 hours later. Some mice kanalirovanie portal vein carry out together with the removal of 2/3 of the liver. Then carry out a partial removal of the liver. To fill the portal vein, the anaesthetize mice, reveal the skin on the proximal side of the existing incision of the peritoneum. Expose the cannula and combine it with a syringe pump. For infusion of the virus preparations of adenovirus in DMEM administered for 5 to 10 minutes into the portal vein through the cannula.
All biochemical and histological analyses performed after the introduction of the living adenovirus hepatic precursor cells into the portal vein through the cannula. ELISA analysis on the Ira is based on two different monoclonal antibodies directed against the catalytic and binding to the receptor region of the Ira. One of the monoclonal antibodies have been labelled with peroxidase. Total serum protein and albumin analyze using conventional automated methods in clinical pathology laboratories. It is known that infusion of adenovirus in the portal vein of C57BL/6 leads to the transduction of 100% of hepatocytes more than one copy of adenoviral DNA in the cell. The same dose of Ad-RSV-uPA leads to a 90% mortality rate which is at least partially associated with hemorrhage. If you use a lower Ad-RSV-uPA, the mortality is less than 5%, and the dose chosen for the experiments on the regeneration of the liver. Infusion of Ad-RSV-uPA leads to transient increases in levels of urokinase in serum, peak values up to about 350 ng/ml (70-100 times higher than endogenous levels) four days later, and then fall to the background concentrations to the 12 th day. Rebound Ira is also related to the increased concentrations of SGPT in serum. In different periods of time after infusion of adenovirus animals injected 3H-thymidine and determine the amount of radioactivity incorporated into the DNA of the liver, as a way of quantifying cell proliferation. Animals treated with Ad-RSV-uPA, had an increased period of uptake of thymidine, which began on day 3 and continued for 8 days.
Thus, the period of hepatic uptake of 3H-thymidine in the introduction of Ad-RSV-uPA/oval cells is much larger than obtained by partial removal of the liver. Recipients treated with negative control adenovirus showed peak hepatic uptake of 3H-thymidine on day 4, and after 24 hours, these parameters returned to baseline levels, and minimum rise of absorption of 3H-thymidine was observed on the 11th day. Summarizing, we can say that the liver damage, as measured by levels SPGT and high rates of uptake of 3H-thymidine, obyasnyau the Xia production in the liver urokinase, that indicates that the liver is significant biosynthetic regeneration. The use of hepatic precursor cells, pour without Ira, better than using adenovirus without inserting the Ira.
The results of microscopic histological studies of animals treated with recombinant adenovirus/precursor cells, derived from cadaveric donors with a stopped heart, indicate that to the 3 rd day of receiving said treatment mice had moderate inflammatory infiltrate, which contained macrophages and neutrophils. Degenerative changes in hepatocytes included vacuolization, microtecnica kernel and a small number of mitotic nuclei. After 8-10 days after injection of Ad-RSV-uPA/oval cells showing signs of recovery of the liver, including the presence of multifocal regeneration, different size cores and a much reduced inflammatory response in a small number of degenerating hepatocytes. To the third-fourth week infiltration was resolved and liver looked normal.
In General, these studies demonstrate that the expression of urokinase in combination with cells-precursors induces significant regeneration of parenchymal cells of the liver.
Separation of cell populations by centrifugation in percoll
this example describes how to enrich the population of cells-the precursors of liver, including the enrichment of stem cells, committeename cells predecessors and commitirovannah cells predecessors. Variations of these methods known to experts in the art and are equally suitable if they meet their goal, namely to obtain an enriched population of progenitor cells.
Essentially single cell suspension of liver cells in culture medium, i.e. in basal medium Eagle (BME), is applied over a layer of 15%solution of percoll in BME. With the help of a centrifuge Sorvall RT7 and 14 cm rotor or equivalent combination of rotor and centrifuge the gradients are centrifuged at 600-1200 rpm, preferably at 750-1000 rpm, for 10 minutes. Collect the supernatant and re-centrifuged her, but when 1200-2000 rpm, preferably at 1500 rpm and the supernatant Fraction enriched cells predecessors, and the residue in the test tube (fraction F3) contains cells capable of at least one cell cycle. The cell supernatant is collected separately, and again centrifuged at 2000-3000 rpm, preferably at 2500 rpm When this last centrifugation cell precursors are often collected in the upper layers of percoll, leaving the wreckage of the cells in the lower levels, and the residue in the test tube contains cells capable passer the structure of several mitotic cycles. Ercolina fraction suitable for immediate use, cryopreservation, obtaining a culture of cells or for further enrichment. Further enrichment can be achieved by panning, affinity selection, FACS sorting, or any other method known in the art and described above. Negative selection is carried out by removal of cells expressing the markers CD45, glycophorin And other markers mentioned below. Positive selection is performed by selection of cells expressing markers CD14, CD34, CD38, ICAM or other markers indicating the expression of a full-sized alpha-fetoprotein, albumin, or both.
Obtaining precursor cells using elutriate
This example shows the stage of selection commiteeman and decommisioning precursor cells of the liver. Although in the art there are many known ways, in this embodiment of the invention described one of the preferred ways, but it is understood that other ways of obtaining information can be equally suitable, provided that they meet the objectives. Preferred examples of methods of preparation, do not limit the present invention are described in U.S. patents№№5807686, 5916743, 5672346, 5681559, 5665557, 5672346 and 5663051, which are included in the present description, the image quality is as references.
Pluripotent or commitirovannah small liver cells may first be isolated using any percoll, or other suitable density gradients, such as Histopaque, a after centrifugation, the cells washed twice with medium and resuspended in 10 ml elutriation environment. For countercurrent elutriation washed with small mononuclear cells injected through a branch on the side of the sampling in the input stream centrifuge Beckman J6M/E, equipped with a rotor JE-5 and a standard camera. However, you can use any other of the many commercially available centrifuges continuous action or devices to otmuchivanie, which are preferably used disposable plastic liners, including the camera for improved separation based on density, namely devices such as Fenwal Models CS 3000" and "Autopheresis C", sold by Baxter International Inc. from Deerfield, IL., or Spectra Apherisis v 7/6 sold by Kobe manufacturing of Lakewood, CO. The selection of devices remains for specialists in this field of technology. The peristaltic pump (Cole Palmer Instruments, Chicago, IL) provides a constant flow elutriation environment, which is a standard of 0.9% saline solution with 100 mg/DL D-glucose, 0.3 mm dinitroanthraquinone acid (EDTA) and 50 mg/DL albumin bovine serum, with pH adjusted to 7.2. Before and the use of the medium is sterilized. Cells served with the General speed of 15 ml/min, with a rotor speed of 900 g and at room temperature. Once collected 100 ml of the eluate, the flow rate (feed rate) is increased to 25 ml/min At a constant rotor speed feed rate consistently increased to 29 ml/min, 33 ml/min and 37 ml/min, collecting 200 ml for each increase in speed. Cells remaining in the chamber, collecting, stopping the rotor and rinsing the camera 100 ml elutriation environment. Each cell fraction was washed and centrifuged at 300 g for 10 minutes. Collect the desired fraction, determine cell viability by staining Trifanova blue and determine the yield of cells with counter cells (Coulter Electronics, Hialeah, FL).
Alternatively, cells do not divide by separation in a density gradient, and suspended in physiological solution with phosphate buffer (PBS) with pH 7.4, containing 5% fetal calf serum, 0.01% by vol. EDTA and 1.0 g/l D-glucose, and enter into countercurrent centrifuge elutriate Beckman at 10°s, when the rotor speed of 1950 rpm using a rotor JA-17 and standard chamber for separation (Beckman Instruments), and the samples elute at flow rates between 12 and 14 ml/min
The resulting cell in suitable fractions typically have diameters of from 5 to 15 microns, preferably from 8.0 to 9.4 microns; most of the cells had a diameter of about the 8.3 to 9.2 microns. These diameters are measured by methods known in the art. If necessary, take further positive or negative selection based on cellular markers.
Specialists in the art there are known various antibodies that can be used alone or in combination with markers of progenitor cells. Their choice depends on what type of cells you wish to isolate or enrich, and includes, but is not limited to the following, antibodies specific for hematopoietic and lymphoid agents, such as anti-CD2, anti-D2R, anti-CD3, anti-CD4, anti-D5 and anti-CD8-specific T cells; anti-D6 specific subpopulations of T cells and subpopulations of b-cells; anti-BD7, specific to the main subpopulations of T cells; anti-D12, anti-D19 and anti-D20, anti-CD72, anti-CDw78 specific for b-cells; anti-D13 and anti-CD14-specific for monocytes; anti-D16 and anti-D56 specific for natural killer cells; anti-D41 for platelets; anti-CD1a, anti-CD1b and anti-CD1c-specific cortical thymocytes and Langerhans cells; anti-D9 specific to pre-b cells, monocytes, and platelets; anti-CD10-specific lymphoid precursor cells, S-A11-cells and granulocytes; anti-CD11a-specific cells; anti-CD11b, specific for granulocytes, monocytes cells and natural-killer is in; anti-CD11c, specific for monocytes, granulocytes, natural killer cells and hairy cell leukemia; anti-CD15, specific for granulocytes; anti-CDw17 specific for granulocytes, monocytes and platelets; anti-D18-specific cells; anti-D21 specific for Mature b-cells; anti-D22 specific to the cytoplasm of b-cells and Mature b-cells; anti-CD23-specific activated b-cells; anti-D24-specific b cells and granulocytes; anti-D25 and anti-D26 specific for activated T - and b-cells and activated macrophages; anti-D27 and anti-D28 specific to the main population of T cells; anti-D30 specific for activated T - and b-cells and cells of Sternberg-reed; anti-D31 specific to platelets, monocytes/macrophages, granulocytes and b cells; anti-CDw32 specific for macrophages, granulocytes, b cells and eosinophils; anti-D33 specific for monocytes, myeloid precursor cells and for myeloid leukemia; anti-CD34-specific hematopoietic precursor cells; anti-CD35-specific for granulocytes, monocytes, b cells, some natural killer cells and red blood cells; anti-D36 specific for monocytes/macrophages and platelets; anti-D37 specific for Mature b-cells; anti-D38 specific plasmacytoma of thymocytes and activated the cells; anti-D39 specific for Mature b-cells; anti-CD40-specific b-cells and carcinoma; anti-D42 and anti-D42b specific to platelets and megakaryocytes; anti-D43 specific for leukocytes, with the exception of circulating b-cells; anti-D44 specific for leukocytes and erythrocytes; anti-D45-specific cells; anti-D45R-specific T cells, a subpopulation of b-cells, monocytes and macrophages; anti-45R specific for b-cells, monocytes and a subpopulation of T-cells; anti-D45R specific for b-cells, subpopulations of T cells, monocytes, macrophages and granulocytes; anti-D46, CD55, CD58 and CD59, specific for hematopoietic and megamarketinc cells; anti-CD47 specific to all types of cells; anti-D48 specific for leukocytes and nitroflow anti-Dw49b specific to platelets, activated and long-term cultured T-cells; anti-CDw49d specific for monocytes, T-cells and b-cells; anti-CDw49f specific for platelets and megakaryocytes; anti-CDw50 and anti-Dw52-specific cells; anti-CD51, specific for platelets; anti-D53 specific for leukocytes, including normal and neoplastic plasma cells; anti-D54 specific for endothelial cells; anti-CDw60, specific subpopulations of T cells and platelets; anti-D61 specific to platelets and megakaryocytes; anti-CD62-specific AK is verovnik platelets; anti-D63 specific for activated platelets, monocytes/macrophages; anti-D64 specific for monocytes (activated by interferon gamma); anti-CDw65, specific for granulocytes and having different reactivity towards monocytes; anti-D66 and 67 that are specific for granulocytes; anti-CD68, specific for monocytes and macrophages; anti-D69 specific for activated b - and T-cells, activated macrophages and natural killer cells; anti-CDw70, specificy for activated T - and b-cells, cells of Sternberg-Reed and both anaplastic lymphoma; anti-D71 specific for activated T and b cells, macrophages, proliferating cells; anti-D73 specific to subpopulations of b-cells and a subpopulation of T-cells; anti-D74 specific for b-cells and monocytes/macrophages; anti-Dw/75 specific for Mature b-cells; anti-D76 specific for Mature b-cells and a subpopulation of T-cells; anti-D77-specific b cells with follicular center; antibodies to the cytokines and growth factors (e.g., IL1-IL13, EGF, IGF I and II, TGF-alpha. and beta., TNF-alpha. and beta., FGF, NGF, CIF, IFN--alpha. and beta., CSF's); viral antigens, such as membrane proteins of hepatitis b virus or protein shell HIV), hormones, cellular or associated with tumor antigens or markers, adhesion molecules, hemostatic molecules and endothelial to EDI. Other markers and procedures enrichment equally suitable, such as described in U.S. patent No. 5840502 included in the present description by reference.
High-performance bioreactor (HPBR) used for the cultivation of human cells-precursors of hepatocytes and their offspring. This process ensures the production of large quantities of cells, suitable for further use for medical purposes, or bioreactor itself serves as a device for the production of biologically useful secreted by cells proteins and factors, which may include, but are not limited to the following, a growth factor for hepatocytes (HGF), insulin-like growth factor-I and II (IGF-I)and II) growth factor epidermal cells (EGF), factor transforming growth type a and type b (TGF-a and TGF-β), nerve growth factor (NGF), factor growth of fibroblast (EGF), growth factor trombotsitnoy origin (PDGF), growth factor sarcoma (SGF), the factor stimulating the growth of colonies of granulocytes and macrophages (GM-CSF), vascular endothelial growth factor (VEGF), a factor in the release of prolactin and growth hormone (GHRF), as well as various hematopoietic growth factors such as interleukins (IL) IL-1, IL-2, IL-3, IL-4, IL-5, EL-6, IL-7, IL-8, IL-10, IL-11, and others, the differentiating factor of erythroid (EDF) or the protein, releasing hormone, which is uliuli follicles (FRP), inhibin, a factor in the proliferation of stem cells (SCPF), as well as active fragments, subunits, derivatives and combinations of these proteins, among many others known in the art. Typically, in the present description, these cellular factors indicate the secretory protein, which is selected from the group consisting of a cytokine, lymphokine, interleukin, colony stimulating factors, hormones, chemotactic factors, antigemorragicescoe factor, coagulation factor, thrombolytic protein, the complement protein, enzyme, immunoglobulin and antigen. Among these biologically active proteins specialists in the art can choose the factor VIII, factor IX, factor VII, erythropoietin, alpha-1-antitripsin, calcitonin, growth hormone, insulin, low-density lipoprotein, apoliprotein E, the receptor for IL-2 and its antagonist, superoxide dismutase, immune modulators, parathyroid hormone, interferons (IFN alpha, beta or gamma), nerve growth factors, glucocerebrosidase, factor, colony stimulating, interleukins (IL1 to IL15), factor, colony stimulating granulocyte (G-CSF), factor stimulating colonies of macrophages and granulocytes (GM-CSF), factor, colony stimulating macrophages (M-CSF), growth factor fibroblast (FGF), growth factor trombotsitnoy origin (PDGF), aminoindoles, insulin ro the domestic factors (IGF-1 and IGF-2), stimulate megakaryocytes ligand (MPL), thrombopoietin or combinations thereof.
Cultivation of the cells in the bioreactor is not limited to this particular method; for this purpose is suitable and can be easily adapted and others well known in the art methods, for example, published U.S. patent№№6001585, 5998184, 5846817, 5622857, 5571720, 5563068, 5512474, 5443985, 5342781, 5330915, 5320963, 5202254, 4833083 and 4760028 included in this description as a reference.
This device contains fibers from cellulose 450 10 kD, fiber polypropylene 540, and the details on the other options can be found, for example, in U.S. patent No. 5622857, which is included in the present description by reference. Cells isolated as described above. All necessary materials obtained from the company Sigma Chemical Co. or Life Technologies. Environment for the attachment of cells for long-term cultivation as follows: RPMI 1640 (500 ml); 50 ml (10%) FBS), 4 mm L-glutamine; 1 × penicillin/streptomycin; gentamicin; 15 mm HEPES; 10 IU/ml insulin; 10 IU/ml transferrin. System HPBR washed with medium for one day before inclusion in the bioreactor environment for attaching cells. In the inner annular chamber of the bioreactor HPBR placed 500 mg previously subjected to swelling microsetella Cytodex 3. Fiber oxygenator envelop micronesica, not allowing them to be distributed throughout ECS (control system in which iania environment). In the inner annular chamber of the bioreactor is placed also viable human liver precursor cells, and device shake and rotate by hand to achieve uniform mixing of the cells with Mironosetsky. Given that the precursor cells and their progeny have a size of about 10-20 microns in diameter, the ratio of cells and microsites in the AMF inoculum is about 500. The apparent viscosity of cells and microsetella increases rapidly, indicating that the attachment of cells to microsites and to each other is quickly and properly. After about 2-3 minutes of this mixing in the inner annular chamber is formed discrete gel cell and microsetella. After cultivation until the next morning at 37°From the environment to attach (stationary) environment replace environment for long-term cultivation (2 l). These volumes are in any case not limit scope of the present invention, since the specialist in the art can easily adjust the performance of the bioreactor to the desired level. Hepatocytes cultured for 5 weeks, with weekly introducing into the system a fresh environment. The metabolic function of cells tracked by daily sampling. After 5 weeks, achieve output >90% of viable cells and Micronase the oil using the following procedure: a 0.1% collagenase in PBS, mixed from 0.44 ml (0,23 M) EDTA, used to wash the ECS and HPBR incubated for 10 minutes; the contents of the ECS displace sterile air from the cylinder of the syringe; this procedure is repeated with the medium to long-term cultivation, and the collected material is washed and separated.
Bioreactor HPBR equally suitable for cultivation and genetic transformation of cells (e.g., for gene expression HGF). The following describes the genetic non-viral methods for the “anchors” of cells (for example, SW 480 P3; # CCL228 ATS (American collection tissue cultures), which experts in the art can suitably modify and optimize using culture cells and cups. For HPBR is preferable to use fiber for environment, having the properties of 10 kD. Bioreactor operates in much the same way as described above. For cultivation of anchored cells are widely used Micronesian Cytodex 1 (Pharmacia, sold by Sigma Chemical Co). In the ECS of the bioreactor HPBR you can inoculate cells at various densities, I ranges from 1×104up to 1×1015cells or higher, if desired. The recommended ratio of cells to Micronesia in the AMF inoculum is about 10, although the specialists in this field of technology can change this ratio, as desired. During the experiment condition is the device gently rotate at a speed of about 10 movements per minute (or more). After culturing the cells for about one day (or more, depending on the specific cells) is achieved fusion, optimal for efficient transfection. The ratio of cells to Micronesia inoculation can be adjusted to improve the use of this time interval to increase therapeutic non-economic efficiency. On the day of transfection prepare a solution of plasmid DNA (e.g., pCMV) and the solution of cationic lipid (e.g., LIPOFECTIN Reagent, Life Technologies). These reagents must be free of serum, even if the full process requires the presence of serum. Mix the required number of solutions of DNA and lipid, and then inject this mixture into the ECS device. After a few hours of transferowania start using the serum, if necessary, and continue to cultivate cells, as before, within a few days. Longer time periods can be used when reproduction is continuously transformed cells. Cells are harvested as described above.
As an extension of the above example, specialists in the art can easily adapt the bioreactor, turning it into a system of extracorporeal support functions of the liver. Xenotransplantation (transpla is the level of organs between different species) can help to overcome the shortage of donor liver due to the use of animal organs. However, the potential danger of transplantation of organs from animals is that viruses infecting the donor animals can infect and recipients. As the recipient of the transplant will be forced to take medications that suppress the immune system and prevent rejection of organs, they may not be able to fight off viral infection, obtained from the animal. In an even more terrible scenario, the animal virus can mutate in the infected host into a form that can infiniroute in contacting people with a normal immune system. The result may be a new pathogenic virus of the person. Most often for organ transplantation man use organs pigs and primates.
However, it is clear that if there is available an artificial liver, based on human cells, it will be preferable than the liver of animals.
After the desired time cultivating get Mature hepatocytes and/or biliary cells, originating from populations enriched cells-the precursors of the liver. Usually get 2 to 5 billion; cells with high (above 80%) viability. Typically, the cultural environment represents the environment Waymouth with the addition of hormones. To accommodate from 2 to 5 billion is in the cells bioreactor extend to two sealing vessels, each of which has an inner diameter of 40 mm and height 100 mm In this particular situation using glass beads with a diameter of approximately 2 mm and a total volume of 250 ml for sealing the vessel. Wednesday served with the degree of recirculation component 360 ml/min. On high viability of hepatocytes indicates a steady rate of oxygen consumption. Then the reactor is connected to the deprived liver recipient and liver was surgically removed because of its complete destruction. In the same way bioreactor connected to a person with a dysfunctional liver. Specialists in the art will know how to connect the bioreactor system of extracorporeal support, perform the function of the liver, or known alternative methods existing in the art, such as described in U.S. patents№№6008049, 5981211, 5976870, 5891713, 5827729, 5643794, 5622857, 5605835 and 5270192 included in this description as a reference. From these references it is obvious that the donor cells artificial liver not necessarily human cells, and now perhaps the use of such cells from other species. For example, liver cells pigs or primates equally suitable for use for humans. Clear is also the methods and compositions of the present invention allow to obtain cells human liver for use in cell therapy or extracorporeal therapy liver, with all the ensuing advantages.
Blood from the left femoral artery was sent to hemoconcentrator Minntech. In the femoral artery insert the 12-lane elecath cannula and connected to the pipeline from PVC with a diameter of1/4inches going to hemoconcentrator. Hemoconcentrator separates the blood into the cell-free fraction ultrafiltrate and the fraction of blood cells. The fraction of blood cells are returned to the femoral vein through the same pipeline. Ultrafiltrate exits hemoconcentrator through the PVC pipe with a diameter of inch and logs hepatocytes bioreactor, with a speed governed to 40 ml/min, using a roller pump. After perfusion through the bioreactor ultrafiltrate returned to the patient through the left jugular vein. To demonstrate the provision of in vitro hepatic metabolism in infiltration at the inlet of the bioreactor introduce two chemical for which it is known that they are metabolized by the liver: 7-ethoxycoumarin and lidocaine. The number of relevant metabolites, 7-Oh-coumarin and monoethylglycinexylidide (MEGX), measured at the outputs of bioreactors to return infiltrate patient. Observed and significant metabolism as 7-ethoxycoumarin, and lidocaine. Thus, the results obtained demonstrate the possibility of using a bioreactor as a support system, providing in vitro hepatic metabolism. Separation of blood cells from plasma to minimize immunological response of the recipient on alien hepatocytes. Thus, hepatic precursor cells and their progeny can be used in the bioreactor to provide extracorporeal support, providing hepatic metabolism.
Encoded by exon 1 peptides and their use as antigens
Short peptides corresponding to exon 1 of alpha-fetoprotein, are used to accurately detect alpha-fetoprotein in various lines of differentiation of cells by evaluating the expression using specific antibodies. The sequence encoded by exon 1 of the peptide is as follows:
The PLACENTA. No. 14 MKWVESIFLIFLLNFTESRTLHRNEYGI
These amino acids can also be presented in alphabetical order, in the form of ABCDEFGHIJKLMNOPRSTUVWXYZ, so that the letter a of this chain starts at position M, K, W, V, E, S, I, F, L, I, F, L, a ,L, or N peptide. Peptides encoded by exon sequences ranging in length from four to twelve amino acid residues kongugiruut with macromolecule, resulting in a gain antigen. The peptide was not necessarily associated with the macromolecule with OSU of spacer length from two to eight carbon atoms. The macromolecule is an albumin, hemocyanin, casein, ovalbumin or polylysine. Suitable peptides include the peptides listed in the table, and
their counterparts, at least 80%homology, or standard substituents of the amino acids. Below is an example of how a specialist in the art can implement the design in order to obtain the desired sequence and length of the peptide in accordance with specific needs:
where any element of A-B-C-D-E-F-G-H-I-J-K-L-M or N may be a non-polar amino acids (hydrophobic), such as glycine Gly G
alanine is Ala And
valine Val V
leucine Leu L
isoleucine Ile I
methionine Met M
phenylalanine Phe F
tryptophan Trp W
Proline Pro P
or polar (hydrophilic) amino acids
serine Ser S
threonine Thr T
tyrosine Tight Y
asparagine Asn N
glutamine is Gin Q
or electrically charged (negatively)
aspartic acid Asp D
glutamic acid Glu E
or electrically charged (positive)
lysine Lys K
arginine Arg R
histidine His H
or may be absent. The chain may consist of acceptable substituents of amino acids or of their salts. The most frequent substitutions AMI is ocelot are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly, and Vice versa.
1. A method of obtaining a composition comprising a mixture of cells derived from the liver tissue of a person, the mixture is enriched population of progenitor cells human liver, and the method includes:
(a) receiving, in essence, a single cell suspension of cells of the liver tissue of a person, comprising a mixture of cells of different sizes, including immature cells and Mature cells;
(b) separation of the suspension under conditions that allow to remove Mature cells and the cells are relatively large in size, leaving immature cells and the cells of relatively small size;
(c) selecting cells, which themselves, their progeny, or more Mature forms demonstrate the presence of one or more markers indicating the expression of alpha-fetoprotein, albumin, or both,
to obtain a mixture of cells comprising an enriched population of progenitor cells human liver.
2. The method according to claim 1, in which the liver tissue get from fetal, newborn, baby, child, teenager, or adult.
3. The method according to claim 1, in which immature cells have a diameter of less than about 15 microns.
4. The method according to claim 3, in which immature cells have a diameter of more than about 5 microns.
5. The method according to claim 3, in which oterom immature cells have a diameter, comprising more than about 8 microns.
6. The method according to claim 1, wherein the enriched population includes diploid cells of human liver.
7. The method according to claim 1, in which the precursor cells of the liver are liver precursor cells, hematopoietic precursor cells, mesenchymal precursor cells, or a mixture thereof.
8. The method according to claim 1, in which the alpha-fetoprotein is a full-sized alpha-fetoprotein.
9. The method according to claim 1, in which the separation of suspensions involves dividing the size of the cells, floating density or a combination of these parameters.
10. The method according to claim 1, in which phase separation of the suspension includes centrifuge elutriation, centrifugation in density gradient, panning, affinity chromatography, staining with fluorescent tags, countercurrent flow cytometry, continuous centrifugation, zonal centrifugation, magnetic granules or a combination of these methods.
11. The method according to claim 1 which further includes selective lysis of Mature cells.
12. Isolation of progenitor cells human liver, including
(a) receiving, in essence, a single cell suspension of cells of the liver tissue of a person, comprising a mixture of cells of different sizes, including immature cells and Mature cells;
(b) razdelennymi in terms allowing you to remove the Mature cells and the cells are relatively large in size, leaving immature cells and the cells of relatively small size;
(c) selecting cells, which themselves, their progeny, or more Mature forms demonstrate the presence of one or more markers indicating the expression of alpha-fetoprotein, albumin, or both.
13. A method of obtaining a composition comprising an enriched population of progenitor cells human liver, including
(a) receiving, in essence, a single cell suspension of cells of the liver tissue of man,
(b) implementation of a positive or negative immunoselection in suspension with a mixture of cells comprising an enriched population of progenitor cells human liver, which themselves, their progeny, or more Mature forms demonstrate the presence of one or more markers indicating the expression of alpha-fetoprotein, albumin, or both.
14. The method according to item 13, in which the precursor cells of the liver are liver precursor cells, hematopoietic precursor cells, mesenchymal precursor cells, or combinations thereof.
15. The method according to item 13, in which immunoselected includes the selection of cells expressing markers associated with hematopoietic cells; cells expressing markers, linked to the s with liver cells; cells expressing markers associated with mesenchymal cells, or combinations thereof.
16. The method according to item 13, in which immunoselected includes selection of suspension of such cells, which themselves, their progeny, or more Mature forms demonstrate the presence of one or more markers indicating the expression of alpha-fetoprotein, albumin, or both.
17. The method according to item 16, further comprising selecting cells, which themselves, their progeny, or more Mature forms produce mRNA full-sized alpha-fetoprotein.
18. The method according to item 13, in which immunoselected includes the selection of the suspensions of these cells, which Express a marker that is specific for cells of the adult liver.
19. The method according to item 13, in which immunoselected includes the selection of such cells, which themselves, their progeny, or more Mature forms Express CD14, CD34, CD38, ICAM, CD45, CD117, glycophorin And, connexin 32, osteopontin, bone sialoprotein, collagen I, collagen II, collagen III, collagen IV, or combinations thereof.
20. The method according to item 13, in which immunoselected includes the selection of such cells, which themselves, their progeny, or more Mature forms additionally Express alpha-fetoprotein immunoreactivity, albuminoidal immunoreactivity or a combination of these characteristics.
21. The way to select cells before the of estevanico human liver, including:
(a) receiving, in essence, a single cell suspension of cells of a tissue of a human liver, and
(b) implementation of a positive or negative immunoselection in suspension, which includes the selection of the suspension of such cells, which themselves, their progeny, or more Mature forms demonstrate the presence of one or more markers indicating the expression of alpha-fetoprotein, albumin, or both.
22. A composition comprising an enriched population of progenitor cells human liver, their progeny, or more Mature forms, and these cells human liver demonstrate the presence of one or more markers indicating the expression of alpha-fetoprotein, albumin, or both.
23. The composition according to item 22, in which precursor cells include liver precursor cells, hematopoietic precursor cells, mesenchymal precursor cells, or combinations thereof.
24. The composition according to item 22, in which precursor cells, their progeny, or more Mature forms Express CD14, CD34, CD38, CD117, ICAM, or combinations thereof.
25. The composition according to item 22, in which precursor cells contain an exogenous nucleic acid.
26. The composition according A.25, in which the exogenous nucleic acid encodes at least one of interest polypeptide.
27. The composition according to claim 2, in which exogenous nucleic acid stimulates the expression of at least one interest of the polypeptide.
28. A method of treating liver dysfunction or disease treatable cells-the precursors of the liver, the subject in need thereof, comprising an introduction to this subject an effective amount of progenitor cells human liver, their offspring, their more Mature forms or combinations of these cells, in a pharmaceutically acceptable carrier, and treatment of liver dysfunction or disease.
29. The method according to p, in which precursor cells human liver include hepatic precursor cells, hematopoietic precursor cells, mesenchymal precursor cells, or combinations thereof.
30. The method according to p, further comprising introducing at the same time or sequentially in any order effective number of precursor cells of the adult human liver, their offspring, their more Mature forms or combinations of these cells.
31. The method according to p, in which precursor cells human liver is administered parenterally.
32. The method according to p in which diseases, disorders or dysfunctions of the liver include hepathology, softening the liver, gepatomegalia, cirrhosis, fibrosis, hepatitis, acute liver failure, chronic liver failure, cancer, heme is ideological violations hematologic dysfunction or congenital metabolic disorders.
33. The method according to p, in which the cancer includes hepatocarcinoma, hepatoblastoma or both these types of cancer.
34. The method according to p, in which the cancer includes metastatic tumor in the liver originating from a place of primary containment, selected from the group consisting of colon, prostate, breast, kidney, pancreas, skin, brain and lung.
35. The method according to p in which hematological abnormalities or dysfunction include anemia, leukemia or impairment or dysfunction caused by chemotherapy, radiation, drugs, viruses, trauma, or a combination thereof.
36. A method of treating a disease in a subject in need thereof, comprising introducing an effective amount of hepatic precursor cells, their progeny, or more Mature forms, in which the hepatic precursor cells, their progeny, or more Mature forms contain exogenous nucleic acid.
37. Cell culture, comprising the composition according to item 22, extracellular matrix, and cultural environment.
38. Pharmaceutical composition comprising the composition according to item 22 and a pharmaceutically acceptable carrier.
39. The population of progenitor cells human liver, their progeny, or more Mature forms that demonstrate the presence of odgovori more markers, indicating on the expression of alpha-fetoprotein, albumin, or both.
40. The population of progenitor cells human liver, their progeny, or more Mature forms § 39, which show the phenotype of glycophorin A-, CD45-alpha-fetoprotein+++, albumin+and ICAM+.
41. The population of progenitor cells human liver according to § 39, which additionally Express CD14+, CD34++, CD38++, CD117+or combinations thereof.
FIELD: biotechnology, molecular biology, medicine, genetic engineering, pharmacy.
SUBSTANCE: the hemopoietic protein comprises the amino acid sequence of the formula: R1-L1-R1, R2-L1-R1, R1-R2 or R2-R1 wherein R1 represents the modified ligand flt-3; R2 represents the modified human IL-3, the modified or unmodified colony-stimulating factor. Modification of R1 is carried out by addition of N-end with C-end directly or through linker (L2) that is able to join N-end with C-end to form new C- and N-ends. The modified human IL-3 is prepared by replacing amino acids at positions 17-123. The human G-CSF is modified by exchange of amino acids. The hemopoietic protein is prepared by culturing cells transformed with vector comprising DNA that encodes the hemopoietic protein. The hemopoietic protein stimulates producing hemopoietic cells and this protein is used as a component of pharmaceutical composition used in treatment of humans suffering with tumor, infectious or autoimmune disease. Invention provides preparing multifunctional hemopoietic proteins eliciting the enhanced activity with respect to stimulation of hemopoietic cells and eliciting the improved physical indices. Invention can be used for preparing chimeric multifunctional hemopoietic proteins.
EFFECT: improved preparing and producing method, valuable medicinal properties of protein.
22 cl, 19 dwg, 18 tbl, 117 ex
FIELD: genetic engineering, immunology, medicine.
SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.
EFFECT: improved preparing methods, valuable medicinal properties of antibody.
33 cl, 5 dwg, 1 ex
FIELD: organic chemistry, natural compounds, medicine, oncology.
SUBSTANCE: invention represents new saponin mixtures used for inhibition of initiation and activation of mammalian epithelial cell in pre-malignant or malignant state, for stimulation of apoptosis of mammalian malignant cell, prophylaxis of anomalous proliferation of mammalian epithelial cell, for treatment of inflammatory and regulation of angiogenesis in mammal. These mixtures are isolated form plants of species Acacia victoriae. Also, invention relates to methods for their applying. These compounds can comprise triterpene component, such as acacic or oleanolic acid to which oligosaccharides and monoterpenoid components are joined. Mixtures and compounds elicit properties associated with regulation of apoptosis and cytotoxicity of cells and strong anti-tumor effect with respect to different tumor cells.
EFFECT: valuable medicinal properties of compositions.
43 cl, 53 tbl, 50 dwg, 44 ex
FIELD: biology, genetic engineering.
SUBSTANCE: invention relates to preparing immortalized cellular lines from health human skin tissues and can be used in immunological, pharmacological, photo- and chemical-toxicological analysis of cutaneous response, for expression of heterologous genes and for construction of artificial skin. Keratinocytes are immortalized by infection of keratinocytes of health human. The human skin sample is isolated and prepared its for culturing in vitro. Keratinocytes are prepared from this prepared human skin sample and plated in serum-free medium for growing keratinocytes in cultural plates with cover alleviating attachment and growth of cells. In the process for culturing keratinocytes the serum-free medium is replaced to provide preparing the optimal confluent growth of cells in culture with continuous maintenance of cup cover. Keratinocytes are transferred in selective serum-free medium in cultural cups with cover and infected with vectors pLXSHD + SV40(#328) and pLXSHD + E6/E7. Then prepared immortalized keratinocytes are transferred in cultural cups with cover to useful medium for proliferation. Then prepared proliferated keratinocytes are transferred in medium with high calcium content for differentiation in cultural chambers with cover. Invention provides preparing the human keratinocyte cellular line that has no oncogenic property and retains capacity for differentiation and expression of proteins and enzymes expressing by normal differentiated keratinocytes being even after increased number of passages in culture. Also, this cellular line forms lamellar and polarized epithelium with keratinized layer (stratum corneum) consisting of ortho-keratinocytes in the process for culturing in organotypical culture in serum-free medium and without layer of feeding cells.
EFFECT: improved immortalizing method, valuable biological properties of cellular line.
7 cl, 2 dwg, 4 ex
SUBSTANCE: method involves applying a composition comprising liposomes having gene structures encoding growth factors. The composition is administered for making injections into wound and impregnating materials for covering or closing wounds with the materials. Advanced bandage has coverage material and liposomes. Introducing liposome gene structures directly into wounds contributes to better healing results.
EFFECT: wide range of functional applications; enhanced treatment effectiveness and safety.
31 cl, 13 dwg, 4 tbl
FIELD: molecular biology.
SUBSTANCE: the suggested innovation deals with the fact that nucleic acids should be isolated directly out of the sample without pipetting stage but with the help of interconnected reservoirs being prepared beforehand. The above-mentioned vessels should be applied either separately or being interconnected according to standard microtitrating format. The sample should be mixed with a lyzing buffer and nucleic acids are bound with matrix in closed system including, at least, two interconnected reservoirs. Forced movement of sample's mixture and buffer back and forth from one reservoir into another one for several times through narrow passage provides their thorough intermixing. The method provides quick and safe isolation of nucleic acids.
EFFECT: higher efficiency.
44 cl, 4 dwg, 1 ex
FIELD: biotechnology, veterinary science.
SUBSTANCE: invention relates to therapeutic vector used in therapy of infectious diseases in cats that comprises at least one foreign nucleic acid each of that (a) encodes protein taken among the group consisting of feline protein CD28 represented in SEQ ID NO:8 or its immunogenic moiety; feline protein CD80 represented in SEQ ID NO:2 or 3, or its immunogenic moiety; feline protein CD86 represented in SEQ ID NO:6 or its immunogenic moiety, or feline protein CTLA-4 represented in SEQ ID NO:10 or its immunogenic moiety; and (b) nucleic acid that is able to be expressed in insertion of vector in the corresponding host. Indicated therapeutic vector is used in effective dose as component of vaccine against infectious diseases in cats for their immunization and in methods for enhancement or inhibition of immune response in cats and reducing or eradication of tumor in cats. Invention provides stimulating the activation and proliferation of T cells and to enhance effectiveness of control of infectious diseases in cats.
EFFECT: valuable biological properties of recombinant virus.
41 cl, 13 dwg