Method of cultivation pluripotential stem cells
SUBSTANCE: method of cultivation of pluripotential stem cells herewith conserving undifferentiated condition and pluripotency thereof without using feeder cells. The method applies fluid medium and culture bottle on the surface of which there are immobilised molecule of fused protein containing outer domain of E-cadherin and Fc-region of immunoglobulin.
EFFECT: invention can be used in regenerative medicine.
9 cl, 11 dwg, 7 ex
The technical field to which the invention relates.
This invention relates to a method of growing and method of gene transfer for pluripotent stem cells such as ES cells, and pluripotent stem cells produced using these methods.
The level of technology
To continue living organisms have the ability to quickly replace and repairbot lost or damaged cells and tissue, and this ability is known as "regenerative ability. Examples of "regeneration" of the higher animals include the well-known phenomena of healing skin wounds and blood vessels, but it is known that even parenchymal organs such as the liver and kidneys, are subjected to cell growth and tissue reconstruction for rapid restoration of tissue homeostasis in response to tissue damage. In recent years there were attempts to use this natural regenerative ability of biological organisms to achieve cure or ameliorate various diseases and wounds, and such new medical methods became known as "regenerative medicine".
Stem cells play a Central role in the practice of "regenerative medicine". "Stem cells" can be generally defined as undifferentiated cells that can differentiate the specialized cells or polyfunctional cells, as well as replicates, making possible the generation of cells that are identical to themselves. Unique stem cells are found in each type of tissue and cells, and, for example, blood cells such as erythrocytes, lymphocytes and megakaryocytes are produced through precursor cells produced from stem cells, known as hematopoietic stem cells, whereas cells of skeletal muscle are produced from stem cells/precursor cells, known as satellite cells and cultured myoblasts". Additional types that have been identified so far include neural stem cells, which are found in nervous tissue, such as brain and spinal cord, and produce neurons and glial cells, epidermal stem cells, which produce epidermal cells and cells of the hair follicles, oval cells (hepatic stem cells)that produce hepatocytes and cells of the bile ducts, and cardiac stem cells that produce cardiomyocytes.
Some methods of treatment regenerative medicine using stem cells or precursor cells derived from such cells have already been presented, and infusion transplant methods with the use of hematopoietic stem cells or hematopoietic glue is OK precursors are well known in the treatment of conditions caused by absence or functional deficiency of blood cells, such as leukemia and plastic anemia. However, stem cells are present in parenchymal organs such as the brain, heart or liver, are technically difficult to obtain from living tissues and/or for in vitro cultivation, and these stem cells also typically have a low potential for proliferation. Stem cells can also be extracted from the tissues of corpses, but the medical use of the thus obtained cells is associated with ethical problems. Thus, the regenerative treatment of neuropathy, cardiopathy, etc. will require the development of methods to generate the desired cell types using cells other than stem cells present in these target tissues.
First of all, how to use "pluripotent stem cells" can be mentioned as strategies based on this approach. “Pluripotent stem cells” are defined as cells capable of continuous or virtually unlimited proliferation in vitro while maintaining their undifferentiated state, manifestations of normal karyotype (chromosome) and having the ability to differentiate into all cell types of the three embryonic leaves (ectoderm, mesoderm and endoderm) when K is favorable terms. Currently, the most well-known pluripotent stem cells are embryonic stem cells (ES-cells)isolated from early embryos, and similar embryonic germ cells (EG cells)isolated from fetal primordial embryonic cells, where both are objects of the present invention.
ES cells can be isolated in the form of a population of undifferentiated stem cells by transferring the inner cell mass of the blastocyst stage embryo in vitro culture and repetition of the process of separation and passage of this cell mass. Cells have a suitable density of cells on the cell-feeders derived from primary cultured mouse embryonic fibroblasts (hereinafter referred to as MEF cells)derived from mouse fetal tissue, or murine stromal cells such as STO cells, and repeat the passage with frequent replacement of the culture medium can lead to the establishment of a cell line that retains the property of undifferentiated stem cells. Another symptom of ES cells is the presence of the enzyme telomerase, which is active in maintaining the length of chromosomal telomeres and the enzyme attaches ES cells the ability of virtually unlimited cell division in vitro.
Line ES-cell derived thisway, are "pluripotent"because they can be grown and repeatable to passivates almost indefinitely while maintaining a normal karyotype, and they are able to differentiate into diverse different types of cells. For example, the transplantation of ES cells into the body of the animal subcutaneously, intraabdominal or intratesticular they form tumors called "teratoma", but these tumors contain a mixture of different cells and tissues, including neurons, osteocytes, chondrocytes, intestinal cells, muscle cells, etc. In mice, intrauterine transplantation in pseudoriemannian mouse aggregate embryo generated infusion transplant ES-cells into the blastocyst stage embryo or aggregation (grouping) with embryo stage eight cells leads to the generation of "chimeric mouse", which is the offspring with differentiated cells derived from these ES cells in the whole body or parts of organs and tissues. This method is often used as the primary way to generate mice with a knockout,” in which certain genes are artificially destroyed or modified.
It is well known that ES cells also induced the differentiation into different types of cells cultivated in vitro. Although the exact method varies depending on t the PA cell, usually use the method of induction of differentiation through education “embroidere body” (hereinafter referred to as “EB”), which is the cell mass of the embryo-like state, formed by aggregation of ES cells by culturing in suspension culture. This method can produce cells having characteristics of endoderm, ectoderm and mesoderm fetal stage, as well as differentiated cells such as blood cells, vascular endothelial cells, chondrocytes, skeletal muscle cells, smooth muscle cells, cardiomyocytes, glial cells, neurons, epithelial cells, melanocytes, keratinocytes, adipocytes and other Differentiated cells obtained by culturing in vitro in this way, have essentially the same structural and functional characteristics of cells present in organs and tissues, and experiments with grafts using experimental animals have demonstrated that from happening ES cells cells are attached to organs and tissues and normal function.
In relation to reviews of the properties of ES cells and methods of their cultivation and their capacity for differentiation in vivo and in vitro, the authors refer to the following literature: Guide to Techniques in Mouse Development (Wasserman et al., Academic Press, 1993); Embryonic Stem Cell Differentiation in vitro (M.V. Wiles, Meth. Enzymol. 225: 900, 1993); Manipulation of the Mouse Embryo: A Laboratory Manal (Hogan et al., Cold Spring Harbor Laboratory Press, 1994) (non-Patent document 1); Embryonic Stem Cells (Turksen, ed., Humana Press, 2002) (non-Patent document 2).
EG cells can be obtained by stimulation of fetal germ cells, known as primordial germ cells, on the cell-feeders, such as MEF cells or cells of STO, in the same way as ES cells, using a factor inhibiting leukemia (hereinafter called LIF) and basic fibroblast growth factor (hereinafter referred to as bFGF/FGF-2), or chemical agents, such as Forskolin (Matsui et al., Cell 70:841, 1992; Koshimizu et al., Development 122: 1235, 1996). It was confirmed that EG cells have properties that are very close to the properties of ES cells have pluripotency (Thomson & Odorico, Trends Biotechnol. 18:53, 2000). Thus, throughout this description, the term "ES cells" can include "EG cells".
After Thomson et al. first established ES cells from Primate (rhesus macaque) in 1995, the concept of regenerative medicine using pluripotent stem cells begin to move to the field realities (U.S. Patent No. 5843780; Proc. Natl. Acad. Sci. USA 92: 7844, 1995). Later, these researchers used similar methods for the successful selection and establishment of lines of ES cells from early human embryos (Science 282: 114, 1998). Later research groups in Australia and Singapore presented a similar message (Reubinoff et al., Nat. Biotech. 18: 399, 2000; international public, the tion of patent No. WO00/27995), and now were registered 20 different lines of ES cells of human rights in the National institutes of health (NIH) (http://stemcells.nih.gov/registry/index). Gearhart et al. also managed to create a line EG-human cells from primordial germ cells (Shamblott et al., Proc. Natl. Acad. Sci. USA 95: 13726, 1998; U.S. Patent No. 6090622).
When using these pluripotent stem cells to obtain material for research or products of regenerative medicine, it is important that methods used passage maintained the undifferentiated state and high efficiency (potential) proliferation of these cells. The MEF cells or similar cells (such as cells STO) is used usually as a cell-feeders (feeder cells for ES/EG cells to maintain an undifferentiated state and high efficiency proliferation of these cells. Also important is the addition of fetal calf serum (hereinafter referred to here FBS) to the culture medium and is decisive choice FBS-product, which is suitable for the cultivation of ES/EG cells, usually with the addition of FBS in the amount of approximately 10-20%. LIF was also identified as a factor that maintains the undifferentiated state of ES/EG cells derived from mouse embryo (Smith & Hooper, Dev. Biol. 121:1, 1987; Smith et al., Nature 336:688, 1988; Rathjen et al., Genes Dev. 4:2308, 1990), and adding LIF the culture can more effectively maintain the undifferentiated state (see the following literature: Manipulating the Mouse Embryo: A Laboratory Manual (Hogan et al., Cold Spring Harbor Laboratory Press, 1994) (non-Patent document 1) and Embryonic Stem Cells (Turksen, ed., Humana Press, 2002) (non-Patent document 2)).
However, the methods of cultivation used for these classic ES/EG cells are unusable when the ES (or EG-) cells used for regenerative medicine or for other practical purposes. One reason for this is that ES cells are insensitive to the absence of LIF and cell-feeder causes the death of these cells or the loss of the undifferentiated state and differentiation in various cell types (Thomson et al., Science 282:1145, 1998). The use of cell-feeders in itself is another problem, as they both systems cocultivation increase production costs and unsuitable for large-scale cultivation, while the separation and purification of ES cells from cell-feeders is necessary, when in fact should be used in these ES cells. In the future, the application of ES cells and other pluripotent stem cells as sources of cells for regenerative medicine and, in particular, for cell transplantation therapy, the use of products of cells derived from animals (not humans), such as MEF cells, and FBS will be undesirable due to risks, including the potential infection of these ES cells by viruses other animals and pollution antigenic molecules, which can be well-known as heteranthera (Martin et al., Nature Med. 11:228, 2005).
Thus, to improve methods of cultivation of ES/EG cells and modifying them so that they were suitable for future use, must be taken active steps to develop substitutes FBS (international patent publication No. WO98/30679) and for use as cell-feeders human cells instead of MEF cells (Richards et al., Nature Biotech. 20:933, 2002; Cheng et al., Stem Cells 21:131, 2003; Hovatta et al., Human Reprod. 18:1404, 2003; Amit et al., Biol. Reprod. 68:2150, 2003). Another attractive prospect is the development of methods of cultivation without the use of feeder cells (feeders). Carpenter et al. reported that seeding ES cells coated with Matrigel or Laminin culture Cup and adding air-conditioned cells MEF medium to the culture medium makes it possible prolonged culturing of ES cells, which retain their undifferentiated state and pluripotency (Xu et al., Nature Biotech. 19:971, 2001 (non-Patent document 3); international patent publication No. WO01/51616 (Patent document 1)). The same group has also managed to create a more effective system for culturing ES cells to develop serum-free medium containing added bFGF/FGF-2 or stem cell factor (hereinafter referred to here SCF) (international publicat what I patent No. WO03/020920 (Patent document 2)). The system of cultivation of ES cells using the same serum-free medium that does not require feeder cells (feeder), it was also reported Israeli research team (Amit et al., Biol. Reprod. 70:837, 2004 (non-Patent document 4)). Recently it was also reported about the way to maintain the undifferentiated state of ES cells by adding bFGF/FGF-2 and an antagonist of bone morphogenetic protein Noggin (Xu et al., Nature Methods 2:185, 2005). Separately it was shown that the simple addition of an inhibitor glikogensintetazy-kinase (GSK)-3 to culture medium can effectively maintain the undifferentiated state of ES cells of mouse and man without the addition of growth factors or the like and without the use of cell-feeders (Sato et al., Nature Med. 10:55, 2004 (Reputedly document 5)).
Thus, while offering new ways for culturing pluripotent stem cells without the use of cell-feeders, the actual implementation and industrial application of these cells will require even greater efficiency of cultivation and methods of culturing pluripotent stem cells.
One well-known factor that maintains the undifferentiated state of mouse ES/EG cells and increases their efficiency proliferation is above LIF, and although LIF-related family of IL-6 falls under this category of elite gadgets is Yu (Yoshida et al., Mech. Dev. 45:163, 1994; Koshimizu et al., Development 122:1235, 1996), very few other examples reported in the literature. Recently, it was reported Bassington environment containing added bFGF/FGF-2 or SCF to significantly enhance the effectiveness of the proliferation of ES cells (international patent publication No. WO03/020920 (Patent document 2)).
Due to the active, i.e. proliferating nature of ES cells compared to other cell types, little attempt was made to actual studies of their effectiveness proliferation; however, the needs of regenerative medicine will require increased proliferation of these cells.
One of the problems encountered at present in cultivation of pluripotent stem cells is that these cells commonly form tight colonies, and, consequently, are difficult to manipulate for passage, etc. Undifferentiated ES/EG cells are usually detected in the state in which these cells are firmly bonded to each other, forming colonies, i.e. cell mass with indistinct borders between cells. To ensure ES/EG cells for passage or experiments on the induction of differentiation is necessary, therefore, the dispersion of these colonies within the possible shortest time period by treatment with a solution of a protease, such as trypsin or the like, But the donkey perform this dispersion of colonies of ES/EG cells individual cells requires the processing of relatively high concentrations of proteases and/or intensive mechanical mixing, such procedures significantly reduce viability and ability to adhesion of these ES/EG cells.
In addition, since ES/EG cells undergo spontaneous differentiation during continuous cultivation in a clustered state, they must be dispersed in separate cells during the passage, and the passage should be performed before the colony will grow to excessive size. For example, mouse ES cells usually require each passage within 2-3 days, and if the passage does not teach the appropriate way, cells that have deviated from their undifferentiated state, may appear in the form of a cluster, which makes these cells unsuitable for use. This cannot be overcome simply by adding a sufficient amount of factor supports undifferentiated state of ES/EG cells, such as the aforementioned LIF or inhibitors of GSK-3, and is induced by excessive growth of colonies and cells with a differentiated phenotype. Thus, it is expected that the method of cultivation of ES/EG cells without the formation of colonies, i.e. with individual dispergirovannykh cells, should be very useful for ensuring ES/EG cells for industrial applications. However, such attempts or successes to date was not.
The authors of this invention prior is entrusted sown cells F9 line of embryonal carcinoma cells, which, as you know, normally proliferate by forming colonies on the culture Cup, covered with E-cadherine (Nagaoka et al., Biotechnol. Lett. 24:1857, 2002 (non-Patent document 6)), and found that it prevents the formation of cell colonies (International Symposium on Biomaterials and Drug Delivery Systems, 2002.04.14-16, Taipei, Taiwan; 1stMeeting of the Japanese Society for Regenerative Medicine, 2002.4.18-19, Kyoto, Japan). Specifically, F9 cells showed the morphology of dispersed cells on the culture plate, with E-cadherin (epithelial cadherin, integral membrane protein), which is a known adhesion molecule for F9 cells immobilized on untreated polystyrene culture Cup (hereinafter referred to here as the "E-cad-Cup".
The F9 cells exhibit a phenotype, somewhat similar to ES cells, expressive alkaline phosphatase (hereinafter referred to here ALP) or SSEA-1 and Oct-3/4, which are known as specific markers of ES/EG cells (Lehtonen et al., Int. J. Dev. Biol. 33:105, 1989, Alonso et al., Int. J. Dev. Biol. 35:389, 1991). However, F9 cells do not require cell-feeders or LIF to maintain the undifferentiated state of these cells and, therefore, are different in mechanism of maintaining their undifferentiated state. In addition, while ES cells have triploblasty (triple) the potential of differentiation into all three sarod the cheap leaf differentiation of F9 cells is restricted to the endodermal cells, and they are not able to form chimeras. In other words, although F9 cells used as a model system ES/EG cells in some experiments, they differ from ES/EG cells in many aspects, including the method of cultivation and the cultivation conditions.
Thus, it is impossible to predict, on the basis of scientific data, whether the above-mentioned E-cad-Cup be used in the methods of culturing ES cells, which do not require cell-feeders, can ES cells cultivated in ways to passivates while maintaining their undifferentiated state and pluripotency and can be increased efficiency proliferation of these ES cells. Indeed, the efficiency of cell proliferation F9 cultured on E-cad-Cup is approximately equivalent to the efficiency of cell proliferation F9 cultured on plate for culturing normal cells, and not the obtained data that would suggest that through this can be increased efficiency proliferation of ES cells.
It is known that E-cadherin is expressed undifferentiated mouse ES cells, and is also well known that intercellular adhesion markedly inhibited ES cells that lack the gene expression of E-cadherin due to mod the classifying gene (Larue et al., Development 122:3185, 1996). Yet no attempt has been made use of E-cadherin as substrate adhesion in the way of the cultivation of ES/EG cells.
In addition to effective ways of cultivation described above, when the alleged use of pluripotent stem cells such as ES cells, as laboratory material or for manufacturing products for regenerative medicine, is also needed to develop methods for effective implementation of the selected exogenous genes in these cells and their expression. In particular, one strategy for the application of ES cells in regenerative medicine for the treatment of various diseases is a modification of the properties of these cells, such as the effectiveness of proliferation and differentiation or sensitivity to drugs, and this may be satisfactorily accomplished by the introduction of suitable expression of exogenous genes in these cells. For mouse ES cells it is well known that genes can be artificially modified to obtain transgenic mice or mice with knockout, which are particularly useful are effective methods of gene transfer.
Regular transfer of exogenous genes into cells is often performed with the use of such agents as calcium phosphate, DEAE-dextran and preparations of cationic lipids. However, it is known that the application of such methods is ES cells leads to a lower efficiency, than the efficiency in the case of other types of cells (Lakshmipathy et al., Stem Cells 22:531, 2004 (non-Patent document 8)). It was also reported about ways using different viral vectors for the transfer of exogenous genes. For example, are well-known retroviral vectors (Cherry et al., Mol. Cell Biol., 20:7419, 2000), adenoviral vectors (Smith-Arica et al., Cloning Stem Cells 5:51, 2003), lentiviral transfer vectors (Amaguchi et al., J. Virol. 74:10778, 2000; Asano et al., Mol. Ther. 6:162, 2002; international patent publication No. WO02/101057) and vectors, Sendai virus (Sasaki et al., Gene Ther. 12:203, 2005; Japanese Unexamined Patent Publication No. 2004-344001). However, designing and obtaining viral vectors are relatively complex and require time, while biological safety is also an issue, depending on the virus, and therefore, such methods are not convenient or universally applicable.
Thus, the transfer of the exogenous gene in ES cells most often carried out by a method known as electroporation. This method involves applying an electric pulse to the cells for the temporary opening of pores in cell membranes for introducing an exogenous gene into the cells, and this method is extremely flexible way. Was recently created an advanced method called nucleophile through which the exogenous gene is transferred directly in the cell is the nucleus to achieve significantly higher efficiency expression (Lorenz et al., Biotech. Lett. 26:1589, 2004; Lakshmipathy et al., Stem Cells 22:531, 2004 (non-Patent document 8)). However, this method requires special generating a pulse device, and obtaining optimal conditions is not easy. In addition, you need to process these cells by protease, such as trypsin, for dispersion of individual cells, and, consequently, the toxicity of the cells is relatively high.
Thus, the most applicable methods of gene transfer for pluripotent stem cells such as ES cells, could be ways to use agents of gene transfer, which are inexpensive and easy to obtain and could enable effective transfer of exogenous genes in cells cultured in thermostat.
Non-patent document 1: Manipulating the Mouse Embryo: A Laboratory Manual (Hogan et al., Cold Spring Harbor Laboratory Press, 1994).
Non-patent document 2: Embryonic Stem Cells (Turksen, ed., Humana Press, 2002).
Non-patent document 3: Xu et al., Nature Biotech. 19:971, 2001.
Non-patent document 4: Amit et al., Biol. Reprod. 70:837, 2004.
Non-patent document 5: Sato et al., Nature Med. 10:55, 2004.
Non-patent document 6: Nagaoka et al., Biotechnol. Lett. 24:1857, 2002.
Non-patent document 7: Nagaoka et al., Protein Eng. 16:243, 2003.
Non-patent document 8: Lakshmipathy et al., Stem Cells 22:531, 2004.
Patent document 1: international patent publication No. WO01/51616.
Patent document 2: international publication p is an awning No. WO03/020920.
Description of the invention
In light of these circumstances, an objective of this invention is the provision of a method of culturing pluripotent stem cells such as ES cells, without the use of cell-feeders, which increases the efficiency of cell proliferation and increases the efficiency of gene transfer.
To solve the above problems, the authors of this invention have investigated the possibility of increasing the efficiency of cell proliferation and increase the efficiency of gene transfer to ES cells by culturing the cells in a state without the formation of colonies or, in other words, in dispergirovannom condition.
As mentioned above, the authors of this invention have managed to cultivate cells F9 cell line embryonic carcinoma, without the formation of colonies, i.e. in dispergirovannom condition. When cooking Cup for cell culture, which had E-cadherin immobilized or applied in the form of a coating on the solid surface, (E-cad-Cup), and seeding of cells F9 on this E-cad-Cup, F9 cells showed the morphology of dispersed cells without the formation of colonies. The efficiency of cell proliferation was essentially the same for F9 cells, cultured on E-cad-Cup, and F9 cells, cultivated on a normal Cup.
When you try seeding ES cells on E-cad-Cup virtually all cells con is playlis to this Cup, and they showed the morphology of dispersed cells without the formation of cell-like morphology of F9 cells. Most notably, the effectiveness of the proliferation of ES cells, seeded on E-cad-Cup, under these culture conditions was significantly higher than the efficiency of the proliferation of ES cells cultured on a normal Cup. The efficiency of transfer of exogenous genes and the levels of expression were also significantly higher.
Additionally, it was confirmed that the ES cells passaged many times on E-cad-Cup, are still undifferentiated and retain their pluripotency when added to this liquid environment factor supports undifferentiated state. In addition, it was demonstrated that ES cells obtained with the use of the method described above, can be induced to differentiation in functional differentiated cells such as neurons and cardiomyocytes, using known methods and which can be generated chimeric mice by transplantation of these cells in the early mouse embryos, the present invention was completed.
Thus, in accordance with the first embodiment of the present invention is provided a new method of growing pluripotent stem cells such as ES cells, which does not require cell-feeder the century The method according to the invention is characterized by the fact that used for culturing vessel is a molecule that attaches pluripotent stem cells, immobilized or deposited on a solid substrate surface with a pre-defined density, whereby these cells may be cultured in dispergirovannom able to increase the ability of proliferation. Pluripotent stem cells thus obtained retain the undifferentiated state and pluripotency.
In accordance with the second embodiment of the method according to the invention is characterized by the fact that used for culturing vessel is a molecule that attaches pluripotent stem cells, immobilized or deposited on a solid substrate surface with a pre-defined density, whereby the cultivation of these cells in dispergirovannom condition can increase the efficiency of gene transfer in these cells.
As another variant implementation of the invention provides pluripotent stem cells with undifferentiated state and pluripotency, which is obtained by the method described in this invention. For the purposes of this description, “undifferentiated” state of pluripotent stem cells may is be confirmed by the expression of at least one marker of the absence of differentiation.
In accordance with another embodiment this invention provides a differentiated cell obtained by means of a suitable processing, inducing differentiation of pluripotent stem cells obtained by the method described in this invention. Differentiated cells are not particularly limited, as they are the type of cells, whose differentiation can usually be induced from pluripotent stem cells. Specifically, there may be mentioned ectodermal cells or derived from ectoderm cells, mesoderm cells or derived from mesoderm cells, endodermal cells, or produced from endoderm cells, etc.
According to another variant implementation of the invention relates to a method for generating chimeric embryos or chimeric animal with the use of pluripotent stem cells obtained by the method described in this invention, and generated chimeric embryo or chimeric animal.
First of all, this invention relates to the following aspects.
(1) a Method of growing pluripotent stem cells, characterized by the cultivation of pluripotent stem cells in dispergirovannom condition while maintaining their undifferentiated state and pluripotency, with the use of the liquid medium and the vessel for culturing, having immobilized or supported on solid-phase surface of the substrate molecule, which is adhesive in respect of these pluripotent stem cells, without using cell-feeders.
(2) Method of gene transfer for pluripotent stem cells, characterized by efficient gene transfer into pluripotent stem cells and the expression of it, using a liquid medium and a vessel for culturing having immobilized or supported on solid-phase surface of the substrate molecule, which is adhesive in respect of pluripotent stem cells.
(3) the Method according to (1) or (2) above, where the molecule that is adhesive with respect to those pluripotent stem cells, is any molecule that is expressed by these pluripotent stem cell, or a molecule that is structurally homologous with this molecule and has homophilous binding capacity with pluripotent stem cells.
(4) the Method according to (3) above, where the molecule that is adhesive with respect to those pluripotent stem cells is a molecule belonging to the family of catherinew.
(5) the Method according to (4) above, where a molecule belonging to the family of catherinew is E-cadherin or molecule that has structural homology with this mod is ecoloy, which contains the EC1 domain and one or more domains of the EC2 domain, domain IS, domain ES and domain AS E-cadherin and which has homophilous binding capacity with pluripotent stem cells.
(6) the Method according to (5) above, where E-cadherin obtained from a mammal.
(7) the Method according to (6) above, where E-cadherin derived from human or mouse.
(8) the Method according to any of (1)to(7) above, where a molecule, which is adhesive in respect of pluripotent stem cells, fused with the Fc region of immunoglobulin and immobilized on a solid substrate surface by means of this Fc-region.
(9) the Method according to any of (1)to(8) above, where these pluripotent stem cells are embryonic stem cells (ES cells) or embryonic germ cells (EG cells) of the mammal.
(10) Pluripotent stem cells obtained by the method according to any of (1)to(9) above.
Brief description of the graphical material
Figure 1 is a pair of diagrams showing the adhesion of ES cells (cell lines (R1 and EV) c E-CAD-Fc deposited on the polystyrene Cup. The degree of adhesion represents a relative value, which is 100% taken number of ES cells stuck to the Cup, covered with gelatin (0.1 per cent). BSA: the group with ES cells stuck to the Cup, covered with 0.1% bovine serum albumin. *: with regard to the group with the gelatin is,
Figure 2 is a set of photographs showing the morphology of ES cells seeded on E-cad-Fc-Cup. These images of cells were obtained two days after seeding ES cells in a Cup, covered with gelatin, collagen type I, fibronectin or E-cad-Fc.
Figa is a chart showing the effect of proliferation of ES cells, seeded on E-cad-Fc-Cup. ES cells were sown on a bowl of gelatin (specified as a Control) or a Cup of E-cad-Fc and the number of cells was compared on the third day. *: relative to the Control group, p<0,01.
FIGU is a graph showing the uptake of BrdU ES cells, seeded on E-cad-Fc-Cup. ES cells were sown on a bowl of gelatin (specified as a Control) or a Cup of E-cad-Fc and were labeled BrdU. BrdU penetrated in these cells, were detected after three days of staining using antibodies using a fluorescent dye. *: relative to the Control group, p<0,01.
Figa is a set of photographs showing the expression of the marker for the undifferentiated state of ES cells, seeded on E-cad-Fc-Cup. ES cells (cell line EV) were sown in the bowl of gelatin (specified as a Control) or a Cup of E-cad-Fc and ALP activity were detected on day 14 of cultivation. In this figure, LIF(+) and (-), respectively, indicate the addition/no addition of LIF to the culture medium.
Figv is the tsya next photos showing the expression of markers for undifferentiated ES cells, seeded on E-cad-Fc-Cup. ES cells (cell line EV) were sown in the bowl of gelatin (specified as a Control) or a Cup of E-cad-Fc and protein Oct-3/4 was detected on day 14 of cultivation. DAPI nuclear staining using DAPI. Overlay: the Overlay of DAPI and dye antibodies Oct-3/4.
Figure 5 is a set of photographs showing the expression of markers for undifferentiated ES cells, seeded on E-cad-Fc-Cup. ES cells (cell line EV) were sown in the bowl of gelatin (specified as a Control) or a Cup of E-cad-Fc and gene expression of Oct-3/4 gene and Rex-1 were investigated using RT-PCR on day 14. The symbols + and - on the form, respectively, represent the addition and no addition of LIF to the culture medium.
6 is a graph showing the LIF-reactivity ES cells seeded on E-cad-Fc-Cup. ES cells (line R1) were sown in the bowl of gelatin (specified as a Control) or a Cup of E-cad-Fc and were cultured in different concentrations of LIF for the formation of colonies and ALP activity were detected to measure the share of "undifferentiated" colonies. *relatively Cont/LIF 1000 u/ml, p<0,05.
Fig.7 is a set of photographs showing the induced pluripotent ES cells passaged on E-cad-Fc-Cup. ES cells (line R1), seeded and roasted on the Cup of desire is another (specified as a Control) or a Cup of E-cad-Fc, were regenerated and EB were formed in serum-free medium for the induction of differentiation. Samples extracted at day 16 after the formation of S (the"S" in the figure)was used to study the gene expression of various differentiation markers using RT-PCR. As control groups used ES cells before the above passirovannym and 16-day EB formed with the use of these cells (1st and 2nd from the left in the figure, respectively). T/Bra: T/Brachyury, βH1: hemoglobin, AFP: α-fetoprotein, TTR: transthyretin, GDPDH: glyceraldehyde-3-phosphatedehydrogenase.
Fig is a pair of photographs showing the pluripotency of ES cells passaged on E-cad-Fc-Cup. ES cells (line R1), sown on E-cad-Fc-Cup, induced for differentiation into neurons (top photo) and cardiomyocytes (bottom photo). These photos show images of cells fixed on the 12th day after the induction of differentiation and stained with antibodies against markers specific for neurons and cardiomyocytes.
Fig.9 is a pair of photographs showing the transmission through the germ line of germ cells ES cells cultured on E-cad-Fc-Cup. Chimeric mice generated from ES cells cultured on E-cad-Fc-Cup, was crossed with a mouse wild-type C57BL/6 mice and the offspring were subjected to PCR analysis used the eat two different microsatellite markers. M: marker DNA size, ES: ES cells (line IV), B6: mouse C57BL/6, #1-#4: individuals who, according to the base color of the coat, do not contain ES-cells, #5-#8: individuals who, according to the base color coat containing ES cells. The numbers on the vertical axis represent the DNA size (BP).
Figure 10 is a graph showing the gene transfer efficiency expression for ES cells, seeded on E-cad-Fc-Cup. Cells (EV) were sown in a bowl of gelatin (referred to as a Control) or E-cad-Fc-Cup and they were subjected to gene transfer using expressing GFP vector. GFP in these cells after one day were detected by the antibody-dye, using fluorescent dye and measuring the fluorescence intensity. *: relative to the Control group, p<0,01.
11 is a set of photographs showing the morphology of ES cells seeded on E-cad-Fc-Cup. These images of cells were obtained two days after seeding ES cells on plates coated with gelatin (Control) or E-cad-Fc.
The best option of carrying out the invention
The term "pluripotent stem cells" throughout this description refers to cells capable of prolonged and virtually unlimited proliferation in vitro while maintaining their undifferentiated state, exhibiting a normal karyotype (chromosome) and having the ability to diff rezervatsiya all three embryonic leaf (the ectoderm, the mesoderm and endoderm) under suitable conditions. The term "pluripotent stem cells" includes, but is not limited to ES cells isolated from early embryos, and their similar EG-cells derived from primordial germ cells of the fetal stage. Throughout this description, the term "ES cells" will be used as including “EG cells”.
The term “undifferentiated state throughout the present description refers to a pluripotent nature of stem cells, showing the lack of differentiation, which can be confirmed on the basis of one or more markers of undifferentiated ES cells, such as ALP activity or expression of the gene (product) Oct-3/4, or on the basis of the expression of different antigenic molecules. No differenziali pluripotent stem cells means that pluripotent stem cells are capable of prolonged or virtually unlimited proliferation and exhibit a normal karyotype (chromosomes) in the presence of the ability to differentiate into all three embryonic leaf in suitable conditions.
The term “pluripotency” in this description refers to the ability to differentiate into various cell types. Differentiated cells are not specifically limited, as they are the type of cells, where diff is retireve can usually be induced from pluripotent stem cells. Specifically, there may be mentioned ectodermal cells or derived from ectoderm cells, mesoderm cells or derived from mesoderm cells, endodermal cells, or produced from endoderm cells, etc.
The term “fluid” in the context of all descriptions shall include any fluid that can be used for conventional ways passage pluripotent stem cells.
The term “adhesion molecule pluripotent stem cells in this description may refer to a molecule that binds and sticks affine with pluripotent stem cells, and it can be a molecule of any of various types of compounds, such as protein, peptide, sahariana chain, low-molecular compound (drug) or the like as the molecules stick together with pluripotent stem cells, preferred are molecules that are expressed in these cells and have homophilous binding capacity, and as an example can be mentioned the family of molecules catherinew. It is known that E-cadherin is expressed undifferentiated ES cells and is, therefore, preferred for use, but there are no special restrictions with this protein. When sticky molecule (adhesion molecule) is a protein molecule or pepti is a, can be used peptide fragment, while it has the same activity of adhesion (adhesion), and that this molecule is a protein or peptide.
Sticking together with pluripotent stem cell molecule (molecule adhesion pluripotent stem cells) can be used for a method of cultivation of this invention by immobilization or applying it on a solid surface vessel for the cultivation or culture of the substrate (hereinafter called together "the culture substrate"). As culture substrates for the present invention can be used any substrates that are commonly used for culturing cells, such as a Cup or beaker. These culture substrates can be prepared from inorganic materials such as glass, or organic materials such as polystyrene or polypropylene, but they are preferably sterilized materials with high heat resistance and moisture resistance.
The method used for immobilization or coating adhesion molecules pluripotent stem cells on a solid phase surface of the culture substrate may be a physical way, such as adsorption, or chemical method, such as the formation of covalent bonds, but the adsorption method is preferred SLE is due to the ease of operation. Artificial antigenic molecule may be added to the adhesion molecule or merged with adhesive molecule in advance for use of the specific binding in respect of such antigenic molecules are antibodies. In this case, these specific antibodies must be immobilized on a solid phase or deposited on a solid phase culture substrate in advance by physical methods such as adsorption, or chemical means, such as the formation of covalent bonds.
The culture substrate (the substrate), thus prepared, can be used directly for routine culturing pluripotent stem cells. That is, a suitable number of pluripotent stem cells can be suspended in the commonly used liquid medium or medium for cell culture, and this mixture is applied to this culture substrate. Subsequent replacement of the liquid medium and the passage may also be carried out in the same way as they are in conventional ways.
The term "homophile binding" refers in this description to bind cell-cell or cell-substrate via adhesion molecules, which includes the binding or Association between the same type adhesion molecules.
The term "cell-feeders" in the description refers to a single tile is am, also known as the cell substrate, which are cultivated in advance and perform the role of supplying nutrients and cell growth factors, which may not be present in the medium used for culturing cells, which were not able to survive and grow by themselves. "Cell-feeders" include, but are not limited to, MEF cells and stromal cells, such as cells of STO.
The term "dispersed state" refers in this context to the state of growing cells attached to the surface of the culture substrate, where there are formed individual colonies and individual cells or are not in contact with other cells or if they are in partial contact, have a very small contact area.
The term "gene" in the description refers to genetic material and refers to a nucleic acid comprising units of transcription. The genome can be RNA or DNA, and may be naturally occurring or artificially designed sequence. Gene does not have to encode a protein, and, for example, it may encode functional RNA, such as a ribozyme or siRNA (short/small interfering RNA).
Other advantages and characteristics of the present invention, in addition to the above effect will be explained in the detailed description of the secured below p is impactfully options for the implementation.
If there are no other indications for carrying out the present invention can be used in the methods of genetic engineering used in molecular biology and recombinant DNA technology, as well as the usual protocols of cell biology and conventional methods, with reference to the standard literature in this area. They include, for example, Molecular Cloning: A Laboratory Manual, 3rdEdition (Sambrook &Russel, Cold Spring Harbor Laboratory Press, 2001; Current Protocols in Molecular Biology (Ausubel et al. ed., John Wiley & Sons, 1987); Methods in Enzymology Series (Academic Press); PCR Protocols: Methods in Molecular Biology (Bartlett & Striling, eds., Humana Press, 2003); Animal Cell Culture: A Practical Approach, 3rdEdition (Masters ed., Oxford University Press, 2000); and Antibodies: A Laboratory Manual (Harlow et al. & Lane, ed., Cold Spring Harbor Laboratory Press, 1987). Reagents and kits used for cell culture and experiments for cell biology, cited throughout this specification are available from commercial sources such as Sigma Aldrich, Invitrogen/GIBCO, Clontech and Stratagene.
Can also be the ordinary methods of cultivation and experiments on the development and biology of cells using pluripotent stem cells with reference to the standard literature in this area. This literature includes Guide to Techniques in Mouse Development (Wasserman et al. ed., Academic Press, 1993); Embryonic Stem Cells, Differentiation in vitro (M.V. Wiles, Meth. Enzymol. 225:900, 1993); Manipulating the Mouse Embryo: A Laboratory Manual (Hogan et al. ed., Cold Spring Harbor Laboratory Press, 1994; and Embryonic Stem Cells (Turksen ed., Humana Press, 2002). eagency and sets, used for culturing these cells and experiments on development and cell biology, cited throughout this specification are available from commercial sources such as Invitrogen/GIBCO and Sigma.
Standard protocols have been created for the generation, transfer and preservation of pluripotent stem cells in mouse and human, and they can be carried out using pluripotent stem cells with reference to the above literature, as well as a plethora of other literature (Matsui et al., Cell 70:841, 1992; Thomson et al., U.S. Patent No. 5,843,780; Thomson et al., Science 282:114, 1998; Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998; Shamblott et al., U.S. Patent No. 6,090, 622; Reubinoff et al., Nat. Biotech. 18:399, 2000; International Patent Publication No. WO00/27995). Well-known ways of establishing cell lines ES cells or ES-like cells for other species, such as, for example, monkey (Thomson et al., U.S. Patent No. 5,843,780; Proc. Natl. Acad. Sci. USA, 92, 7844, 1966), rats (Iannaccone et al., Dev. Biol. 163:288, 1994; Loring et al., International Patent Publication No. WO99/27076), chickens (Pain et al., Development 122:2339, 1996; U.S. Patent No. 5,340,740; U.S. Patent No. 5,656,479), pigs (Wheeler et al., Reprod. Fertil. Dev. 6:563, 1994; Shim et al., Biol. Reprod. 57:1089, 1997) and the like, and the ES cells used for the present invention can be obtained in accordance with methods described for each of these types.
ES cells are pluripotent stem cells, identified as unit adifferential the data stem cell extraction cell mass in the inner space of the embryo to the blastocyst stage, known as the inner cell mass, and the transfer of its in vitro, with repeated separation and passirovannym this cell mass. As murine ES cells, there are various lines, including E14, D3, CCE, R1, J1, EB3, etc., some of which may be obtained from the American type culture Collection, Cell & Molecular Technologies or Thromb-x currently 50 lines ES-cells were installed (laid) worldwide, and 20 different lines registered in the National Institute of health USA (NIH) (http://stemcells.nih.gov/registry/index.asp). Some of them can be obtained from ES Cell Interntional or from the Wisconsin Alumni Research Foundation.
Line ES cells usually lay the cultivation of early embryos, but the ES cells can be derived from early embryos obtained by nuclear transfer of somatic cells (Munsie et al., Curr. Biol. 10:989, 2000; Wakayama et al., Science 292:740, 2001; Hwang et al., Science 303:1669, 2004). Were also suggested ways to generate ES cells from the embryo-like structures the blastocyst stage, obtained by transfer of nuclei of cells of the desired animals to other types of oocytes or denuclearising oocytes divided into several parts (known as the centrosome or oblastey) (International patent publication numbers WO 99/45100; WO01/46401; WO01/96532; U.S. Pregrant Publication Nos. 02/90722; 02/194637). It was also reported in an attempt to obtain ES cells from artherogenesis embryo developed to the same stage and blastocyst stage (U.S. Pregrant Publication No. 02/168763; Vrana K, et al., Proc. Natl. Acad. Sci. USA 100:11911-6) and a way to merge ES cells from somatic cells to obtain ES cells with genetic information in the nucleus of somatic cells (international patent publication No. WO00/49137; Tada et al., Curr. Biol. 11:1553, 2001). The ES cells used for the present invention include ES cells obtained by such methods, and ES cells, chromosomal DNA which has been modified by means of genetic engineering.
EG-cells used for this invention, is produced by stimulation of the fetal germ cells, known as primordial germ cells, on the cell-feeders, such as MEF cells, STO cells or cells Sl/Sl4-m220 chemical agent such as LIF, bFGF/FGF-2 or Forskolin, in the same way as in the case of ES cells (Matsui et al., Cell 70:841, 1992; Koshimizu et al., Development 122:1235, 1996), and their properties are very similar to the properties of ES cells (Thomson & Odorico, Trends Biotechnol. 18:53, 2000). As in the case of ES cells, EG cells obtained by fusion of EG cells from somatic cells (Tada et al., EMBO J. 16:6510, 1997; Andrew et al.), and EG cells, chromosomal DNA which has been modified by means of genetic engineering, can also be used for the method of this invention.
In addition, pluripotent stem cells to be used for the production method Yes the tion of the invention, not limited to ES cells or EG cells, and include all pluripotent stem cells derived from an embryo or fetus of a mammal, the umbilical cord or tissue or blood of an adult individual, for example, organs or bone marrow of adults, and with ES/EG-cell-like characteristics. For example, ES-like cells obtained by culturing gametes under special culture conditions, to exhibit symptoms similar to the symptoms of ES/EG cells (Kanatsu-Shinohara et al., Cell 119:1001, 2004), and can be used as pluripotent stem cells. As another example, there may be mentioned multipotential adults (continuing in adult individuals) precursor cells/stem cells (MAPS), secreted from cells in the bone marrow and have the potential to differentiate into all three embryonic leaf. In addition, pluripotent stem cells obtained by culturing epithelial cells of the vagina hair roots or keratinocytes (international patent publication No. WO02/51980), intestinal epithelial cells (international patent publication No. WO02/57430) or cells of the inner ear (Li et al., Nature Med. 9:1293, 2003) under special culture conditions, and pluripotent stem cells obtained by the processing of mononuclear blood cells (or stem cells contained in the cell is rakli) using M-CSF (macrophage colony stimulating factor) + PMA (phorbol-12-myristate-13-acetate) (Zhao et al., Proc. Natl. Acad. Sci. USA 100:2426, 2003) or antibody CR3/43 (Abuljadayel, Curr. Med. Res. Opinion 19:355, 2003), all included as well as their characteristics are similar to the symptoms of ES/EG cells. In this case, the characteristics similar to the characteristics of ES/EG cells can be defined as properties of cell biology that are unique to ES/EG cells, such as the presence of the surface (antigen) markers specific for these cells, and expression of genes specific to these cells, and the potential of education teratoid tumors and the potential formation of chimeric mouse.
This invention relates to a method of culturing pluripotent stem cells, including ES cells, and is characterized by the use of molecules sticking to pluripotent stem cells (hereinafter referred to here as “clinging to pluripotent stem cells molecules”). Stick to pluripotent stem cells molecules used for carrying out the present invention are used as a method of cultivation of this invention being immobilized or supported on solid-phase surface of the vessel for the cultivation or culture of the substrate (hereinafter called together the culture substrate). Any culture substrate can be used as a culture substrate of the present invention, while it allows you to cultivate plurimae the local stem cells, but preferably it is a culture substrate used for cultivation of cells of the previous prior art. As examples of culture substrates for cell culture can be referred Cup, tablet, bottle, slide, camera, tube, cell factory, roller bottle, rotating the flask, hollow fiber, micronesica, granules, etc. These culture substrates can be made of inorganic materials such as glass, or organic materials such as polystyrene, but it is preferable to use materials such as polystyrene, which have high adsorption properties for proteins and peptides, or materials that have been processed, for example, hydrophilic or hydrophobic treatment, to increase the adsorption properties. Preferred are also sterilized materials with high heat resistance and high moisture resistance. As an example of such a preferred substrate can be mentioned polystyrene Cup and/or tablet without special treatment for culturing cells (hereinafter referred to here as the “raw polystyrene tablet”), most commonly used for culturing E. coli and the like, and such a culture substrate is commercially available.
Stick to pluripotent the Noah stem cell molecule (i.e. the adhesion molecule pluripotent stem cells) is a molecule that is associated with affine pluripotent stem cells or sticks affine to pluripotent stem cells, and it can be of any type, such as a protein, peptide, sahariana chain, low-molecular compound or a molecule composed of two or more of these components. About a few adhesion molecules was reported for undifferentiated ES/EG cells, but it is known that these cells Express, for example, ICAM-1, VCAM-1, NCAM, belonging to the immunoglobulin superfamily (Tian et al., Biol. Reprod. 57:561, 1997). The adhesion molecule pluripotent stem cell is preferably a molecule that is expressed on the surfaces of cell membranes used pluripotent stem cells, and more preferably it is the molecule with homophiles binding ability. Homophile binding to adhesion of cells means linking cell-cell or cell-substrate via an adhesion molecule. Known adhesion molecules, having such properties include NCAM, L1, plexin and cadherin, members katerinovka family molecules are preferably used from the viewpoint of the durability of adhesion. It was also reported that E-cadherin specifically expressed undifferentiated ES cells (Larueet al., Development 122:3185, 1996), and therefore this molecule is preferred for use. However, due to the use of adhesion molecules are not limited to E-cadherine, and can be used any of the molecules of the family of catherinew or adhesion molecules homophiles binding, expressed pluripotent stem cells. Can also be carried out modification of genes in ES cells by way of genetic engineering, leading to the expression of full-length or partial gene encoding a molecule with homophiles binding ability, even if it is normal is not expressed in ES cells, for use of this molecule in the method of this invention.
Catherine are adhesion molecules involved in the CA2+dependent intercellular adhesion and binding, known as adhesive binding or adhesively connecting link, and a well-known three types, E-(epithelial)-cadherin, N(neural)-cadherin and P(placental)-cadherin. These cadherin molecules are membrane-bound glycoproteins, consisting of 700 to 750 amino acid residues, and the extracellular region contains five repeating structures, known as extracellular kuderinova (EC) domains, consisting of approximately 110 amino acid residues. For example, the domains of E-cadherin person (the amino acids follow etelnost SEQ ID NO:1 in the list of sequences) are EC1, EC2, ES, ES and ES corresponding to amino acid residues 157-262, 265-375, 378-486, 487-595 and 596-700, respectively (where these numbers provisions are remnants of the amino acid sequence of SEQ ID NO:1). The domain of mouse E-cadherin (amino acid sequence SEQ ID NO:2 in the list of sequences) are EC1, EC2, ES, ES and ES corresponding to amino acid residues 159-264, 267-377, 380-488, 489-597 and 598-702, respectively (where these numbers provisions are remnants of the amino acid sequence of SEQ ID NO:2). These EU-domains are homologous among different molecules catherinew, with particularly high homology is between domains that are located near the N-Terminus (EC1, EC2). It is now known that more than 50 cateringowych molecules exhibit such similar structure, and they are grouped together in a collection catherinew. Reviews on cadherins can be found in Takeichi, Curr. Opin. Cell Biol. 7:619, 1995; Marrs & Nelson, Int. Rev. Cytol. 165:159, 1996; Yap et al., Annu. Rev. Cell Dev. Biol. 13:119, 1997; Yagi & Takeichi, Genes Dev. 14:1169, 2000; Gumbiner, J. Cell Biol. 148:399, 2000, and elsewhere.
E-cadherin (also called Katherina-1) is widely expressed in epithelial cells, such as parenchymal cells of internal organs such as the liver, kidneys and lungs, and keratinocytes, and know that he is an important adhesion molecule for intercellular adhesion (see reviews in Mareel et al., Int. J. Dev. Biol. 37227, 1993; Mays et al., Cold Spring Harb. Symp. Quant. Biol. 60:763, 1995; El-Bahrawy & Pignatelli, Microsc. Res. Tech. 43:224, 1998; Nollet et al., Mol. Cell. Biol. Res. Commun. 2:77, 1999). E-cadherin is expressed also in abundance in undifferentiated mouse ES cells, and it is known that ES cells lacking expression of E-cadherin due to the use of genetic engineering, have markedly inhibited intercellular adhesion (Larue et al., Development 122:3185, 1996). In addition, it can be confirmed that the genes E-cadherin is expressed in the lines of ES cells, on the basis of information stored in a public database of gene expression at the U.S. National Center for Biotechnology Information (NCBI).
The method of obtaining cateringowych molecules such as E-cadherin or other adhesion molecules, for carrying out the present invention, if the molecule is a protein or peptide, preferably provides for the receipt, treatment and use of recombinant protein using molecular biological methods, although this is not limited. Can be used other methods with comparable results, and, for example, the adhesion molecule pluripotent stem cells can be used after extraction and purification from living tissue or cells, or the peptide may be chemically synthesized for use.
Standard protocols have been established for methods of production of recombinant proteins and receive the deposits of genes coding for proteins such as adhesion molecules pluripotent stem cells, and you can reference cited above literature, although there is no limitation of this literature. For example, E-cadherin, gene E-cadherin has already been isolated and identified for the animals, including human (SEQ ID NO:1), mouse (SEQ ID NO:2) and rat, and the corresponding nucleotide sequences available from public databases of DNA, such as NCBI (access number: (man) NM_004360; (mouse) NM_009864; (rat) NM_031334). Thus, the person skilled in the art can construct a primer or probe specific for the gene of interest E-cadherin, and use it in ordinary molecular biological methods for the production and application of cDNA for the gene E-cadherin. Alternatively, the cDNA for the gene E-cadherin can be obtained from RIKEN Gene Bank (Tsukuba, Japan) or the American type culture Collection (ATSS) or Invitrogen/ResGen. The gene encoding used adhesion molecule, preferably derived from the same species of animal from which pluripotent stem cells, and, for example, when carrying out the present invention using mouse ES cells, it is preferable to use cDNA of mouse E-cadherin. However, it can be used cDNA of E-cadherin from different species such as human, monkey, cow, horse, pig, sheep, poultry (e.g. the, chicken or amphibian (e.g., Xenopus laevis).
An example of a suitable method of obtaining recombinant protein adhesion molecules for use in this invention is characterized by the transfer of the gene encoding this molecule in mammalian cells such as COS cells, 293 cells or cells of Cho, and his expression. Preferably, the gene associated with the sequence of nucleic acids that make possible the transcription and expression of this gene in a wide range of mammalian cells, i.e. the sequence of the promoter so that transcription and expression are under the control of this promoter. Transcribed and expressed gene is also preferably connected with the signal attaching the poly. As a preferred promoters can be mentioned promoters from viruses such as SV 40 (simian virus), cytomegalovirus (CMV) or rous sarcoma virus, or the promoter of β-actin promoter, EF-1α (elongation factor) or the like
Gene used to obtain the recombinant protein should not be required to contain a full-sized region of the gene encoding this molecule, as it can be a partial sequence of a gene, while the molecule is a protein or peptide encoded by this partial sequence has an activity of adhesion equivalent activity or exceeding AK is Yunosti the original molecule. For example, E-cadherin, suitable for use in accordance with this invention may be a recombinant protein designed from the partial sequence comprising amino acid residues 690-710 from the N-Terminus encoding the extracellular region, i.e. a protein containing the EC1-EC domains. Because the domain that is closest to the N-end (EC1) molecule cadherin usually determines the binding specificity or the property homophiles binding of this molecule (Nose et al., Cell 61:147, 1990), can be constructed and used protein molecule containing at least EC1 and devoid of one or more other domains. Can also be used a protein having at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homology at the amino acid level with the above protein molecule and exhibiting adhesion activity.
The above-mentioned recombinant protein can also be obtained in the form of protein, fused with another protein or peptide. For example, it may be obtained in the form of protein, fused with the Fc region of immunoglobulin or protein GST (glutathione-S-transferase)protein MBP (mannosylation protein), protein Avidya, His-tagged (oligoglycines), (Hemagglutinin), Mec-label, label VSV-G glycoprotein of the virus vesicular dental is one) or the like, and Protein a/G column or a column with a specific antibody can be used for convenient and efficient purification of this recombinant protein. Fc-fused protein is particularly preferred for carrying out the present invention, as it has a higher ability to adsorb onto culture substrates made of such material as polystyrene.
Numerous genes encoding the Fc regions of immunoglobulin, have already been isolated and identified in mammals, including humans. Many of their nucleotide sequences reported, and, for example, information on nucleotide sequences containing Fc regions of IgG1, IgG2, IgG3 and IgG4, is available from public DNA databases, such as NCBI, and these sequences are registered, respectively, as access numbers: AJ294730, AJ294731, AJ294732 and AJ294733. Thus, the person skilled in the art can construct a primer or probe specific for the Fc region, and use it in ordinary molecular biological methods for the production and application of cDNA that encodes this Fc-region IgG1 or IgG2 person or IgG2 or IgG2b mouse that have a strong binding affinity in relation to Protein A/G. there are ways to enhance binding affinity in relation to Protein And the introduction of mutations in the Fc-regions (Nagaoka et al., Protein Eng. 16:243, 2003 (Repatent the initial document 7)), and can also be used Fc-proteins with genetic modifications in these methods.
Examples of methods for obtaining recombinant proteins for E-cadherin, which are preferred for carrying out the present invention, have been published in the literature by the authors of the present invention (Nagaoka et al., Biotechnol. Lett. 24:1857, 2002 (non-Patent document 6); Protein Eng. 16:243, 2003 (non-Patent document 7)).
Commercially available is also purified recombinant protein obtained by introducing in mouse cells fused gene obtained by linking cDNA having a sequence encoding a Fc-region of IgG human and sequence His-tag, with cDNA encoding the extracellular region of E-cadherin mouse or human, and the expression of this recombinant protein (Recombinant Human/Mouse E-cadherin-Fc Chimera; R&D Systems, Genzyme Techne), which can be used as protein E-cadherin mouse and human.
Method for immobilization or coating adhesion molecules pluripotent stem cells on a solid phase surface of the culture substrate for carrying out the method described by this invention can be a physical way, such as adsorption, or chemical method, such as the formation of covalent bonds, but the adsorption method is preferred due to ease of operation. When the adhesion molecule is a mole who ula protein or peptide, or when it is a high-molecular compound, containing sacharine chain, this molecule can be easily adsorbed by the contacting of the solution of this molecule with a solid-phase surface of the culture substrate, such as a Cup, and removal of solvent after the prescribed period of time. More specifically, a solution of adhesion molecules prepared using a solvent such as distilled water or PBS, can be filtered and sterilized and then contacted with the culture substrate, such as a Cup, and let it stand for a period of several hours - full day/night to get the culture substrate for cells with immobilized or supported on its adhesion molecule. It is preferably used after washing several times with distilled water or PBS and replace a balanced salt solution such as PBS.
Artificial antigenic molecule preferably connect or merge with this adhesion molecule in advance, as this will allow the use of binding with antibodies specific for this antigen molecules, and effectively attach these adhesion molecules on the surface of the substrate. In this case, these specific antibodies must be immobilized or supported on the surface of the culture substrate in advance by physical methods, such as absorption or chemical methods for the Ohm, such as the formation of covalent bonds. For example, for recombinant protein obtained by the fusion of an Fc-region of IgG with adhesion molecule, an antibody that is attached in advance to the culture substrate may be an antibody that specifically recognizes this Fc-region of IgG. For recombinant protein obtained by fusion protein - or peptide-tag adhesion molecule, antibody, specific for this merged molecules, can be attached in advance to the culture substrate for use.
The adhesion molecule, immobilized or supported on solid-phase surface of the substrate for cell culture, for the purposes of this invention may be a molecule of the same type, or can be used in combination of two or more different adhesion molecules. In such cases, solutions of each of the adhesion molecules can be mixed, and the mixed solution may be applied as described above.
The concentration of the solution of the adhesion molecules must be properly accounted for on the basis of adsorption and/or affinity of this molecule and the physical properties of this molecule, but for recombinant protein obtained by the fusion of an Fc-region with the extracellular region of E-cadherin, this concentration of approximately 0.01 to 1000 μg/ml, preferably about 0.1-200 μg/ml, even more preferably 1-50 μg/ml and Naib is more preferably 5-10 mg/ml
Pluripotent stem cells are used for carrying out the present invention, plated on a culture substrate, prepared as described above. The method of cultivation and the cultivation conditions for pluripotent stem cells can be conventional method of cultivation and conventional cultivation conditions for pluripotent stem cells, with the exception of using the above-described culture substrate. Ordinary methods of cultivation and the cultivation conditions for pluripotent stem cells described in the above literature and, specifically, in Guide to Techniques in Mouse Development (Wasserman et al., Academic Press, 1993); Embryonic Stem Cell Differentiation in vitro (M.V. Wiles, Meth. Enzymol. 225: 900, 1993); Manipulation of the Mouse Embryo: A Laboratory Manual (Hogan et al., Cold Spring Harbor Laboratory Press, 1994); Embryonic Stem Cells (Turksen, ed., Humana Press, 2002); and other sources (Matsui et al., Cell 70:841, 1992; Thomson et al., U.S. Patent No. 5,843,780; Thomson et al., Science 282:114, 1998; Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998; Shamblott et al., U.S. Patent No. 6,090, 622; Reubinoff et al., Nat. Biotech. 18:399, 2000, and International Patent Publication No. WO00/27995), although there are no specific restrictions these links.
The liquid medium used for the cultivation of AscI, can be any medium that can be used in traditional ways passage pluripotent stem cells. As specific examples may be mentioned modified Eagle medium Dulb the KCO (DMEM), minimum essential medium Glasgow (GMEM), medium RPMI 1640 and so on, usually with the addition of approximately 2 mm glutamine and/or approximately 100 μm 2-mercaptoethanol. Can also be used Wednesday KnockOut DMEM (Invitrogen), defined for ES-cells DMEM (Cell & Molecular Technology) and TX-WES (Thrpomb-X), which are commercially available as media for culturing ES cells. Such environment preferably contain FBS added to approximately 5-25%, but they can also be serum-free media, replaced by, for example, 15-20% KnockOut Serum Replacement (Invitrogen). Can also be used in the culture supernatant of cells EF or environment containing added bFGF/FGF-2, SCF, or the like, and detailed procedures for this are well known (Xu et al., Nature Biotech. 19:971, 2001; international patent publication WO01/51616; international patent publication WO03/020920; Amit et al., Biol. Reprod. 70:837, 2004).
Liquid medium for culturing pluripotent stem cells has also preferable substances and the factors added to it, which contribute to the maintenance of the undifferentiated state of pluripotent stem cells. Specific substances and factors are not particularly limited, but LIF is preferred for murine ES/EG cells. LIF is a protein factor that is well known from the published literature (Smith & Hooper, Dev. Biol. 11:1, 1987; Smith et al., Nature 336:688, 1988; Rathjen et al., Genes Dev. 4:2308, 1990), as well as access numbers X13967 (LIF person), H (murine LIF) and NM_022196 (rat LIF), and recombinant proteins can be obtained, for example, under the trade name ESGRO (Chemicon). Addition of an inhibitor of GSK-3 to the culture medium can effectively maintain the undifferentiated state of ES cells of mouse and man without the addition of other growth factors or bioactive factors (Sato et al., Nature Med. 10:55, 2004). In this case, any substance having the activity of inhibiting the activity of GSK-3 can be used, and may be mentioned, for example, Wnt family of molecules (Manoukian & Woodgett, Adv. Cancer Res. 84:203, 2002; Doble & Woodgett, J. Cell Sci. 116:1175, 2003).
By planting pluripotent stem cells that were supported by passirovannym common ways on the culture substrate prepared by the above method, and the cultivation with the above culture conditions, and method for carrying out this invention it is possible to perform the passage of these cells in dispergirovannom state while maintaining the original undifferentiated state of these cells. Because pluripotent stem cells, cultivated in this state, not physically inhibited during cell division and/or the mechanisms of inhibition of cell growth, 's mediated the intercellular contact is not operated, and/or survival of cells increases and the number of dead cells is reduced, there is a significant proliferation and growth of cells. In the case of culturing mouse ES cells according to the method of the present invention, as one example, it is possible to increase the degree of proliferation of at least 1.25 times, preferably at least 1.5 times and more preferably at least 2 times in comparison with conventional cultivation method. The passage to approximately 4 generations under these conditions makes it possible to obtain at least 3 times and preferably at least 10 times more cells than the number of cells obtained by conventional methods. The degree of proliferation can be expressed by such factors as the degree of increasing the number of cells or the rate of doubling per unit of time, and used the methods of measurement and calculation can be any well-known methods used for conventional experiments with cells.
As explained above, the lack of differeciate pluripotent stem cells means that pluripotent stem cells are capable of prolonged or virtually unlimited proliferation and exhibit a normal karyotype (chromosome) with the capacity for differentiation into all three germ is istka under suitable conditions. Preferably they are also one of the other properties of pluripotent stem cells, such as the preservation of telomerase activity, education teratoid tumors or the ability of the formation of chimeras. Methods of studying the nature and properties of cells can be easily carried out using established standard protocols with reference to the above cited literature, for example, Guide to Techniques in Mouse Development (Wasserman et al., Academic Press, 1993); Embryonic Stem Cell Differentiation in vitro (M.V. Wiles, Meth. Enzymol. 225: 900, 1993); Manipulation of the Mouse Embryo: A Laboratory Manual (Hogan et al., Cold Spring Harbor Laboratory Press, 1994); or Embryonic Stem Cells (Turksen, ed., Humana Press, 2002), but no specific limitations to these methods.
Pluripotent stem cells in an undifferentiated state can be defined as cells for which at least one and preferably more marker molecules can be confirmed using at least one and preferably more than one of the methods described below. The expression of different markers that are specific against an undifferentiated pluripotent stem cells, detects common biochemical or immunochemical methods. While there are no specific restrictions on method used, preferred are immunochemical methods, such as immunohistological staining or immunoblot-anal is Z. In such methods can be used in marker-specific polyclonal antibodies or monoclonal antibodies that are associated with undifferentiated pluripotent stem cells. Antibodies that target individual specific markers are commercially available and can be appropriately used. Specific markers for undifferentiated pluripotent stem cells include the ALP activity and the expression product of the gene Oct-3/4 gene Rex-1/Zfp42. Can also be used in a variety of antigenic molecules that include markers of lack of differentiation SSEA-1 for mouse ES cells, SSEA-3 to ES-human cells, or SSEA-4, TRA-1-60, TRA-1-81, gCTM-2, etc. Their expression is reduced or eliminated after differentiation of ES cells.
Alternatively, the expression of markers of undifferentiated pluripotent stem cells can be confirmed by molecular biological methods, often used in previous state of the art for the desired marker proteins, such as using reverse transcriptase polymerase chain reaction (RT-PCR) or hybridization analysis, regardless of the specific method. Known nucleic acid sequences for genes encoding marker proteins specific for undifferentiated, pluripotent the stem cells (e.g., Oct-3/4, Rex-1/Zfp42 or Nanog), and the marker-specific sequences useful as primers or probes can be easily identified from public databases such as NCBI.
The application of the method of gene transfer into pluripotent stem cells
According to another variant implementation of the invention, the method described by this invention can be used as a way to effectively transfer the desired exogenous gene in the pluripotent stem cells. There is no specific limitation for portable exogenous genes, and, for example, we can talk about the natural protein such as a growth factor or a receptor, enzyme, transcription factor, or the like, or synthetic protein, generated by the modification using the method of genetic engineering. Portable gene may be a gene functional RNA, such as a ribozyme or siRNA. The exogenous gene may be a gene marker for evaluating the efficiency of gene transfer or stability of expression, such as the gene encoding GFP (green fluorescent protein) or β-galactosidase, luciferase, or the like
In one preferred embodiment, subject to the transfer of the exogenous gene is associated with a sequence of nucleic acid, which makes possible the transcription and expression of this gene, i.e. a sequence promoto is a, under control of this promoter, in a form that makes possible its transcription and expression. In such cases, the gene is preferably associated with the signal sequence of the poly. As promoters, which make possible the transcription and expression of exogenous genes in pluripotent stem cells, can be mentioned promoters from viruses such as virus SV40, CMV or the rous sarcoma virus, or promoter, β-actin, EF1α promoter or the like depending on the purpose, can also be used the sequence of nucleic acids that make possible the transcription or expression of a particular gene in specific cell types/tissues or in cells with a specific stage of differentiation, i.e. the sequence of cell/tissue-specific promoter or specific in relation to the stage of differentiation of the promoter or promoter Pol. III for the expression of RNA. Sequences of these promoters can be used from public DNA databases, such as NCBI, and can be used conventional molecular biological methods for constructing vectors of genes containing the desired sequence of genes. The vectors for these promoters can be obtained from Invitrogen, Promega, Ambion and other sources.
The method of introducing the gene (vector) is not specifically limited and can be mentioned, for example, how Tr is speccie using calcium phosphate or DEAE-dextran. Methods for transfection of target cells gene transfer can also be used with the use of lipid drugs that can be absorbed by these cells and have low cytotoxicity, such as LipofectAMIN (Invitrogen), Superfect (Qiagen) or DOTMA (Roche), with the formation of complexes liposome-nucleic acid containing the gene target. Alternatively, the gene of interest can be included in a viral vector such as retrovirus or adenovirus, and this recombinant virus can be used to infect cells. In this case, this viral vector is a reconstruction of the nucleic acid sequence full-length or partially deficient or mutated viral DNA or RNA gene of interest included in such a way that it can be expressed.
The use of pluripotent stem cells grown by the method of this invention
Pluripotent stem cells, which were grown according to the production method according to this invention, can then be obtained efficiently and in large quantities in the form of pluripotent stem cells, supporting their undifferentiated state, using well-known methods of obtaining cells. The gene transfer method of the present invention enables efficient and vysokoproizvoditel the second obtaining pluripotent stem cells having a desired gene transferred and expressed in them. Thus obtained pluripotent stem cells will hereinafter be called “pluripotent stem kletke obtained according to this invention.
As ways of deriving pluripotent stem cells can be mentioned methods, using well-known enzymatic processing, which is used in the usual way for passage of pluripotent stem cells. As a concrete example can be mentioned a method in which the medium is removed from the culture vessel, in which were cultured pluripotent stem cells, use PBS to wash several times, preferably 2-3 times, add a solution containing appropriate protease (for example, a solution containing the same protease, such as trypsin or dispute), carry out the cultivation at 37°C for a suitable period of time, preferably about 1-20 minutes, and more preferably 3-10 minutes, and then this mixture is suspended in a suitable solution such as the above medium for culturing ES cells, with a separate cells. Can also be used non-enzymatic methods, and, for example, may be mentioned a method in which the medium is removed from the culture vessel, in which cultivated pluripotency the stem cells, PBS used to wash several times, preferably 2-3 times, add a solution of ethylenediaminetetraacetate (EDTA) to a final concentration of 0.01-100 mm, preferably 0.1 to 50 mm and more preferably 1-10 mm, for processing at 37°C for a suitable time, preferably about 1-60 minutes, and more preferably 10-30 minutes, to separate these cells and then this mixture is suspended in a suitable solution such as the above medium for culturing ES cells, to obtain individual cells. The same method can be carried out using ethylene glycol-bis(2-aminoethylamide ether)leads to compounds, which (EGTA) instead of EDTA.
This invention also provides differentiated cells derived a suitable inducing differentiation processing of the pluripotent stem cells obtained according to this invention. These differentiated cells are not specifically limited, as they are the type of cells, whose differentiation can usually be induced from pluripotent stem cells. Specifically, there may be mentioned ectodermal cells or produced from ectoderm cells, mesoderm cells or derived from mesoderm cells, endodermal cells or derived from ectoderm cells, etc.
Made from ectoderm clackamette cells, components of such tissues and organs, as nervous tissue, the pineal gland, the medulla of the adrenal glands, plastids and epidermal tissue, but they are not limited to, tissues and organs. Derived from mesoderm cells are cells comprising such tissues and organs, muscle tissue, connective tissue, bone, cartilage, cardiac tissue, vascular tissue, hematopoietic tissue, dermal tissue, urinary and reproductive systems, but they are not limited to, tissues and organs. Made from endoderm cells are cells comprising such tissues and organs, as the tissue of the digestive tract, respiratory, or thymus, thyroid, parathyroid, bladder, middle ear, the tissue of the liver and pancreas, but they are not limited to, tissues and organs.
Pluripotent stem cells obtained in accordance with this invention, and/or differentiated cells derived from such cells applicable for pharmacological evaluation or assessment activity of various physiologically active substances (such as drugs) or new gene products of unknown function. For example, they can be used for screening substances and drugs involved in the functional regulation of pluripotent STV the new cells or various differentiated cells, and/or substances or drugs with toxicity or inhibitory effect on pluripotent stem cells, or various differentiated cells. Currently, very few ways of screening was established using human cells, and differentiated cells derived from pluripotent stem cells obtained according to this invention, are useful sources of cells for conducting such screening methods.
This invention relates also to a method for generating chimeric embryos or chimeric animals with the use of pluripotent stem cells obtained according to the method described by this invention, and generated chimeric embryos and chimeric animals. Standard protocols have been established to generate chimeric embryos and of chimeric zhivotnyh, and they can be easily obtained with reference, for example, Manipulation of the Mouse Embryo: A Laboratory Manual (Hogan et al. eds., Cold Spring Harbor Laboratory Press, 1994), although there are no particular restrictions on this link.
The invention will now be explained in more detail by the following examples, with the understanding that they are only examples of this invention and are not intended to limit in any way the scope of the invention.
Example 1: Getting recombi is based protein E-cadherin
Methods construction of vectors for expression of fused protein containing the extracellular region of murine E-cadherin and Fc-region of IgG (IgG/Fc) (see SEQ ID NO:25), (hereinafter referred to as "E-cad-Fc"), and for obtaining and purification of this protein based on the methods reported by the authors of the present invention (Nagaoka et al., Biotechnol. Lett. 24:1857, 2002 (non-Patent document 6); Protein Eng. 16:243, 2003 (non-Patent document 7)). First, a DNA fragment encoding the extracellular domain (E-cad-ECD), (corresponding to amino acid residues 1-699)from cDNA of E-cadherin, amplified using cDNA array containing the full sequence for the murine E-cadherin (provided by RIKEN; RDB No. 1184), as a matrix. A DNA fragment encoding the complex mouse IgG/Fc, was isolated from cDNA obtained by the selection of mRNA expressing murine IgG1 of hybridoma and performing reverse transcription using reverse transcriptase. After confirming the nucleotide sequences of both DNA fragments were embedded in expressing vector pRC-CMV (Invitrogen) to construct expressing vector pRC-E-cad-Fc containing the sequence of E-cad-ECD and IgG/Fc.
To obtain protein E-cad-Fc used cells Cho-K1 (obtained from RIKEN, Tsukuba). The linearized plasmid pRC-E-cad-Fc (1,0 µg) was mixed with 5.0 ál of Lipofectamine™ (Invitrogen) and used for gene transfer in the notches of Cho-K1 in accordance with the Protocol, featured in the instructions to the product. Then, to obtain a clone of cells that produce constitutive and large quantities of protein E-cad-Fc, these cells were removed after the second day from gene transfer and were sown at 0.2 cells per well in 96-well tablet (IWAKI). After culturing for 7 days in RPMI medium 1640 containing 400 μg/ml G418 (Invitrogen), cultural supernatant collected from the wells with the surviving cells and measured the protein content of the E-cad-Fc in these cultural supernatant. Clones with the highest production of the protein E-cad-Fc (line 4G7) was isolated and used for the next experiment. These cells were kondicionirovanie in serum-free medium (Cho-S-SFM II; Invitrogen) and then cultured in the form of mass culture using a rotating flask and collected culture supernatant.
This culture supernatant was filtered using a membrane filter of 0.45 μm and then concentrated using ultrafiltration membranes with a pore size of 100 kDa (YM100; Amicon) and stirred cell (Amicon 8200; Amicon). A concentrated solution were dialyzed with 20 mm phosphate buffer (pH 7,2) and then purified in the usual way using Protein A-column (Amersham Biosciences) and used in the following experiment.
Example 2: Adhesion of ES cells with E-cad-Fc-Cup
Investigated the adhesion of ES cells with a Cup to cool the cultivated cells, covered with protein E-cad-Fc (below referred to here as the "E-cad-Fc-Cup"). PBS-diluted protein solution E-cad-Fc was poured into the raw polystyrene cups for cultivation of different sizes and process for coating at 37°C. After washing and before seeding of these cells was performed processing block within 1-2 hours a 0.1% solution of BSA to prevent nonspecific adhesion of these cells. As control was used cups coated with BSA (0.1%)and gelatin (0.1%)and collagen type I (0,01%; KOKEN) or fibronectin (5,0 µg/ml; KOKEN).
Used lines ES-cells were cells EV (provided by Professor Mr. field from RIKEN), cells R1 (Nagy et al., Proc. Natl. Acad. Sci. USA 90:8424, 1993) and cells from 129SV (obtained from Dainippon Pharmaceutical Co. Ltd.), these experimental results did not show differences between the various lines of ES cells. These ES cells were passively in accordance with the methods described in the Manipulation of the Mouse Embryo: A Laboratory Manual (Hogan et al. eds., Cold Spring Harbor Laboratory Press, 1994) and in Embryonic Stem Cells: Methods and Protocols (Turksen, ed., Humana Press, 2002), by using KnockOut-DMEM (Invitrogen)containing 10% FBS, 0.1 mm solution of non-essential amino acids MEM, 2 mm L-glutamine and 0.1 mm 2-mercaptoethanol (hereinafter referred to here ESM), with the addition of 1000 u/ml LIF (ESGRO; Chemicon), while maintaining their undifferentiated state, and they were submitted for experimentation. ES cells passaged when e is their conditions, will be called hereinafter "ES-cells, passaged under normal conditions".
ES cells passaged under normal conditions, washed twice in serum-free medium and treated with 0.25% trypsin solution containing 1 mm EDTA, obtaining the individual cells, which are suspended in the ESM. If not indicated otherwise, the same conditions were used then to separate these ES cells from a Cup, passage and other experiments. Purified protein E-cad-Fc was applied to the untreated 96-well plate (IWAKI) described above and a cell suspension prepared in this way is 3.0×105cells/ml, were sown in it at 100 μl and were cultured for 4 hours. After washing serum-free medium the medium was replaced with medium containing 10% of the dye Alamar Blue (Biosource International), within 4 hours of the reaction, after which the measured optical density as a measure of the number of viable cells.
These results are shown in figure 1. ES cells typically have a low tendency to adhesion in comparison with fibroblasts and epithelial cells, and they essentially are not able to adhere to polystyrene tablet, which is either raw or processed S, and they form an aggregated cell mass in the environment; however, after preprocessing of the tablet for the cultivation of gelatin, collagen or fibronectin who m they can be attached in the same way, as if seeding in a regular Cup for cell culture. When tested for adhesion ES cells using a tablet coated with different concentrations of E-cad-Fc, as the cell line R1, and the cell line I found satisfactory adhesion at concentrations of 5.0 mg/ml and above. Incidentally, ES cells are able to adhere to E-cad-Fc-Cup under serum-free conditions, and adhesion with this Cup is clearly independent of adhesion molecules in the serum, such as fibronectin.
It is known that coupling via E-cadherin is Sa2+dependent (Mareel et al., Int. J. Dev. Biol. 37:227, 1993; Takeichi, Curr. Opin. Cell Biol. 7:619, 1995; Marrs & Nelson, Int. Rev. Cytol. 165:159, 1996). Test steps add chelat forming agent on the adhesion of ES cells with E-cad-Fc-Cup, ES cells, cultured for 4 hours on E-cad-Fc-Cup, was treated in the same way for 30 minutes ethylenediaminetetraacetate (EDTA) or ethylene glycol-bis(2-aminoethylamide ether)leads to compounds, which (EGTA) at 5 mm final concentration after washing these cells PBS cell number was measured using the dye Alamar Blue as described above. When processing with the use of EDTA, which has a low selectivity for metal ions, adhesion of ES cells with E-cad-Fc and fibronectin was broken, but when processed with the use of EGTA, which has the K and CA 2+-selectivity, binding to fibronectin is not inhibited and only linking to the E-cadherine violated. This effect was not prevented even by adding 5 mm of Mg2+for processing of EGTA. These results suggest that the adhesion of ES cells with E-cad-Fc-Cup involves interaction between molecules E-cadherin present on the surfaces of ES cells, and molecules E-cadherin, immobilized on a solid phase surface E-cad-Fc-Cup.
Then the ES cells passaged under normal culture conditions, were sown in E-cad-Fc-Cup or 24-hole tablet (IWAKI), covered with another substrate, and carried out the cultivation. It is known that ES cells usually form closely adjacent one to another, rounded colonies on cells feeders or on a normal Cup for cell culture. In this case, the ES cells formed tight colonies of the same type even if the raw polystyrene cups for cultivation, covered with gelatin, collagen type I or fibronectin (see figure 2). However, it should be noted that ES cells seeded on E-cad-Fc-Cup, regardless of whether they are cells EV or cells R1, essentially failed to form a separate colony even after 2 or 3 days after seeding, and they found instead, the increase in the number of individually dispersion is the R cells.
Example 3: the Cultivation of ES cells with the use of E-cad-Fc-cups
To study the effectiveness of the proliferation of ES cells on E-cad-Fc-Cup ES cells passaged under normal conditions, were removed and 500 of these ES cells were sown on E-cad-Fc-Cup or on the tablet coated with gelatin (96-well-plate), and were cultured for 3-4 days. After washing these cells serum-free medium, the cell number was measured using the dye Alamar Blue as described above. As a result, the number of ES cells cultured on E-cad-Fc-Cup, relative to the number of ES cells cultured on gelatin tablet, after 3 days of cultivation was significantly higher for both cell lines EF and cell line R1 (see figa). The number of cell groups with E-cad-Fc-cups with both lines ES-cells were approximately 2 times higher at day 4 of cultivation. When removing the ES cells after four identical passages and registration numbers of cells, the number of cultured on E-cad-Fc-Cup ES cells were 3-5 times higher than in the case of cells cultured on plain gelatin Cup. There was no difference between the two cups on the degree of adhesion immediately after planting these ES cells, suggesting that ES cells cultured on E-cad-Fc-Cup, have a higher efficiency of prolifera the AI and the ability to survive. When conducting the same experiment with cells F9 was not found differences in the efficiencies of the proliferation of cells cultured on E-cad-Fc-cups groups and cultured in conventional cups group.
Then investigated the synthesis of DNA from these ES cells using the absorption of 5-bromo-2'-dose irradiation on neurogenesis (BrdU) as an indicator. ES cells cultured for 3 days in the above-described conditions, was recovered in the form of individual cells after labeling of BrdU (10 μm) for 30 minutes and was perseval in 96-well plate. After 4 hours, adherent ES cells were fixed with FixDenat solution (Roche Applied Science) and were washed in PBS, and then gave them to react with anti-BrdU-antibody (BMG 6H8; Roche Applied Science) (100-fold dilution) and stained using CYANOGEN 3(Cy3)-labeled antibody (Jackson Immunoresearch Laboratory) (dilution 1:1000). Cell nuclei were stained with a solution of 4',6-diamidino-2-phenylindole (DAPI) (0.1 ág/ml). Image of antibodies and staining dye was observed using the ArrayScan™ (Cellomics). As a result, cultivated on E-cad-Fc-Cup group showed significantly higher uptake of BrdU in comparison with cultivated on a regular cups of the group (see figv).
It is known that ES cells usually undergo spontaneous differentiation after the formation of the large, close-fitting colonies. Thus, when the cultivation of three different mouse lines is x ES cells, used in this experiment, a normal Cup, these colonies grow to extremely large sizes and produce differentiated cells of various morphologies, if not passedout every 2 or 3 days. However, cultivation on E-cad-Fc-Cup enough to passivate only once every 5-7 days, if you reduce the initial sowing quantity, and in these culture conditions did not observe excessive formation of colonies or differentiated cells. Thus, to verify maintained whether the ES cells cultured on E-cad-Fc-Cup, their undifferentiated state or if there is a dispersed state and the undifferentiated state of these ES cells even after multiple passage, they were passively several times on this Cup and investigated the properties of the obtained ES cells.
First explored the ALP activity and protein expression of Oct-3/4 as indicators of undifferentiated ES cells to confirm the differentiation state of ES cells. The ALP activity was detected using a set of Sigma Diagnostics Alkaline Phosphatase (Sigma). After washing the cultured ES cells (cell lines EF and R1) PBS, they were fixed with a solution of citrate containing 66% acetone/3% formalin, and washed with PBS, after which they were processed within 15 minutes of alkaline dye solution in the naphthol AS-BI-phosphate, on the Chennai this set for color reaction (see figa).
The protein expression of Oct-3/4 was investigated by immunostaining. Specifically, cultured ES cells were fixed with 8% formaldehyde (Waco Pure Chemical Industries, Co., Ltd.) and were washed in PBS and then gave them to react with anti-Oct-3/4-antibody (product of Santa Cruz) (dilution 1:200) and were stained with Alexa Fluor-labeled antibody (Alexa-488; Molecular Probes) (dilution 1:1000). Cell nuclei were stained with DAPI solution (0.1 ág/ml). Image of antibodies and staining dye was observed using a fluorescent microscope (see figv).
As a result, the high activity of ALP (figa) and protein expression of Oct-3/4 (pigv) were confirmed with ES cells, cultured for 14 days on E-cad-Fc-Cup, like ES cells, cultured on plate coated with gelatin, used as a control group (hereinafter referred to as "gelatin Cup").
Then examined the gene expression of Oct-3/4, Rex-1/Zfp42 as markers of undifferentiated ES cells. ES cells cultured on E-cad-Fc-Cup and gelatin Cup within 14 days, were removed and total RNA was obtained using 1 ml TRIZOL (Invitrogen). Then cDNA was synthesized in the usual way using reverse transcriptase M-MLV (Invitrogen) and used as template for polymerase chain reaction (PCR) using the following primers for amplification of each gene fragm the NTA.
Oct-3/4 [size amplification: 528 BP]
5'-primer: 5'-GAAGTTGGAGAAGGTGGAACC-3' (SEQ ID NO:3)
3'-primer: 5'-GCCTCATACTCTTCTCGTTGG-3' (SEQ ID NO:4)
Rex-1 [size amplification: 930 BP]
5'-primer: 5'-AAAGTGAGATTAGCCCCGAG-3' (SEQ ID NO:5)
3'-primer: 5'-TCCCATCCCCTTCAATAGCA-3' (SEQ ID NO:6)
Nanog [size amplification: 710 BP]
5'-primer: 5'-GAGGAAGCATCGAATTCTGG-3' (SEQ ID NO:7)
3'-primer: 5'-AAGTTATGGAGCGGAGCAGC-3' (SEQ ID NO:8)
GAPDH (glyceraldehyde-3-phosphatedehydrogenase) [size amplification: 858 BP]
5'-primer: 5'-GGAAGCTTGTCATCAACGG-3' (SEQ ID NO:9)
3'-primer: 5'-CTCTTGCTCAGTGTCCTTGC-3' (SEQ ID NO:10)
PCR was performed using a thermal cycler TaKaRa PCR Thermal Cycler MP (TaKaRa) using DNA polymerase TaKaRa Taq (TAKARA) as a thermostable DNA polymerase. First, cDNA-containing solution for the PCR reactions were heated at 94°C and then the cycle of heating to 94°C:30 seconds → 59°C:30 seconds → 72°C. for 60 seconds was repeated 22 times with subsequent final heating at 72°C for 5 minutes and then cooled to 4°C. the PCR Product was subjected to electrophoresis on a 1.5% agarose gel, stained with SYBR Green I (TAKARA) and were detected using a Typhoon 8600 (Amersham Biosciences).
These results are shown in figure 5. ES cells cultured on plain gelatin Cup, found high expression of Oct-3/4, Rex-1 and Nanog in the presence of LIF with a significant decrease in expression in the absence of LIF. Similar results were found with the ES cells cultured on E-cad-Fc-Cup, and the high expression of Oct-3/4, Rex-1 and Nanog were detected in the presence of LIF. The same samples used for testing gene expression differentiation markers for neurons, mesoderm cells and endodermal cells, such as NeuroD3 or Sox-1, T-Brachyury, Flk-1, hemoglobin, α-fetoprotein and transthyretin, but gene expression of differentiation markers was detected in the presence of LIF for ES cells, cultured or gelatin Cup, or E-cad-Fc-Cup. As can be seen from the results in figa, 4B and 5, it was confirmed that cultured on E-cad-Fc-Cup ES cells proliferated without the formation of colonies and found a different morphology than with conventional cultivation, while maintaining their undifferentiated state.
Then researched, changed reactivity cultured on E-cad-Fc-Cup of ES cells in relation to LIF. ES cells passaged at regular gelatin Cup and on E-cad-Fc-Cup, were cultured for 5 days with a concentration of LIF 0-1000 u/ml without passage and then both groups of cells were perseval on gelatin Cup and were cultured for another 3 days (with a constant concentration of LIF during the cultivation period). The activity of ALP educated on these cups colonies of ES-cells were detected by the method described above, and measured the proportion of colonies that retain the undifferentiated state. Colonies with ALP activity detected in m is Nisha least 80% of the cells, was evaluated as "undifferentiated".
ES cells cultured on gelatin Cup, formed excessively large colonies at the end of the first 5 days of cultivation, but did not observe cells, which could be explicitly evaluated as being differentiated cells. ES cells cultured on E-cad-Fc-Cup, proliferated without the formation of colonies and kept their dispersed state, as in the previous experiment. After reseeding ES cells cultured on normal Cup with LIF concentration OF 1000 u/ml, kept the ALP activity in almost all of these colonies, but the proportion of undifferentiated colonies decreased with decreasing concentrations of LIF (see Fig.6). ES cells cultivated before the E-cad-Fc-Cup, were able to preserve the properties of the lack of differentiation in virtually all of these colonies, even at a concentration of LIF 100 u/ml did Not show inhibition efficiency of cell proliferation at low concentrations of LIF. This suggests that the cultivation of using E-cad-Fc-Cup allows you to reduce the number of additional factors necessary for the growth of ES cells, such as LIF, in comparison with the methods of the previous level of technology.
Then investigated whether E-cad-Fc-Cup be used for feeder-independent air-conditioning ES cells. ES/EG cells in the usual way was passively and maintain rivali by co-cultivation of cells feeders, but their feeder-dependent satayana cultivation can be air-conditioned in feeder-independent state. However, this requires a high density ES-cells and repeated passage in medium containing a high concentration of LIF (usually 5-fold or 10-fold). For example, for ES cells (line R1)grown in feeder-dependent condition, were conditioned in a feeder-independent state, it is necessary to sow these cells at a density of cells approximately 5,0×104cells/cm2and to add LIF at a concentration of 1×104E/ml. However, it is possible to condense ES cells in feeder-independent state, as in the previous prior art, seeding ES cells on E-cad-Fc-Cup at a density of 500 cells/cm2and passirovannym with ESM containing 1×103U/ml LIF. It was also confirmed that the ES cells obtained in this way, sufficiently maintain their undifferentiated state and pluripotency.
Example 4: study of the ability for differentiation of ES cells cultured on E-cad-Fc-Cup
It was confirmed that the ES cells passaged multiple number of times on E-cad-Fc-Cup, still have pluripotency. First, these ES cells were cultured in suspension in the absence of LIF for education embryonic bodies (EB), and progression of spontaneous differentiation was studied on the basis of Express and several genes differentiation markers. Specifically, for the formation of S from ES cells, ES cells, passaged at least 3 times on E-cad-Fc-Cup, was recovered in the form of individual cells and then cooked a drop containing 500 cells in 15 μl containing LIF ESM for culture in the hanging drop. EB formed in this culture the hanging drops were periodically collected and obtaining RNA and synthesis of cDNA was performed in the ways described. RT-PCR reaction was performed with this DNA as template using the following primers for the amplification of each fragment of the gene marker.
NeuroD3 [size amplification: 405 BP]
5'-primer: 5'-CATCTCTGATCTCGACTGC-3' (SEQ ID NO:11)
3'-primer: 5'-CCAGATGTAGTTGTAGGCG-3' (SEQ ID NO:12)
Sox-1 [size amplification: 407 BP]
5'-primer: 5'-GCACACAGCGTTTTCTCGG-3' (SEQ ID NO:13)
3'-primer: 5'-ACATCCGACTCCTCTTCCC-3' (SEQ ID NO:14)
T/Brachyury-1 [size amplification: 528 BP]
5'-primer: 5'-TCCAGGTGCTATATATTGCC-3' (SEQ ID NO:15)
3'-primer: 5'-TGCTGCCTGTGAGTCACAAC-3' (SEQ ID NO:16)
Flk-1 [size amplification: 398 BP]
5'-primer: 5'-TAGGTGCCTCCCCATACCCTGG-3' (SEQ ID NO:17)
3'-primer: 5'-TGGCCGGCTCTTTCGCTTACTG-3' (SEQ ID NO:18)
hemoglobin [size amplification: 415 BP]
5'-primer: 5'-AACCCTCAATGGCCTGTGG-3' (SEQ ID NO:19)
3'-primer: 5'-TCAGTGGTACTTGTGGGACAGC-3' (SEQ ID NO:20)
α-fetoprotein [size amplification: 997 BP]
5'-primer: 5'-TGCTCAGTACGACAAGGTCG-3' (SEQ ID NO:21)
3'-primer: 5'-ACTGGTGATGCATAGCCTCC-3' (SEQ ID NO:22)
transthyretin [size amplification: 440 BP]
5'-primer: 5'-AGTCCTGGATGCTGTCCGAG-3' (SEQ ID NO:23)
3'-Prime is R: 5'-TCAGAGGTCGGGCAGCCCAGC-3' (SEQ ID NO:24)
These results are shown in Fig.7. When removing LIF from the environment for the induction of spontaneous EB differentiation of the ES cells cultured on plain gelatin Cup, watched the expression of specific gene markers of the three germ sheets, including the ectoderm (NeuroD3, Sox-1), the mesoderm (T-Brachyury, Flk-1, hemoglobin) and endoderm (α-fetoprotein, transthyretin). Even with the use of ES cells cultured on E-cad-Fc-Cup, expression of marker genes of all three embryonic leaves was confirmed at approximately the same level as in the case of a group of gelatinous Cup.
Then investigated the ability of ES cells for differentiation into neurons and cardiomyocytes. It was reported that co-cultivation of ES cells with cells feeders, which are stromal cells seeded in advance to the Cup for cultivation, could induce the differentiation of ES cells into neurons and/or cardiomyocytes (Yamane et al., Methods Mol. Biol. 184:261, 2002; Schroeder et al., Proc. Natl. Acad. Sci. USA 100:4018, 2003). Thus, the ability of ES cells to differentiate into neurons tested in the system of differentiation using cells RA, and the ability to differentiate into cardiomyocytes experienced in the use of ST2 cells. Cells RA or ST2 cells (both obtained from RIKEN Cell Bank) were sown on 6-hole Cup for cell culture (CORNING) and the cult who were verovali to confluently by using DMEM (Invitrogen), containing 10% FBS, for use as cell-feeders. Then prepared culture medium ES cells as single cells and cell-feeders were washed twice in PBS and seeded at 2000 cells per well. The next day, this culture medium was replaced by medium ESM containing 20% KnockOut Serum Replacement (Invitrogen), for differentiation into neurons (RE-feeder) and ESM containing 10% FBS, for differentiation into cardiomyocytes (ST2-feeder). These cells at day 12 of cultivation recorded a solution of 70% ethanol and gave them to interact with the antibody against associated with microtubule protein-2 (MAP-2) (AV; Chemicon) or with an antibody against myosin of sarcomere (MF20; American type culture Collection) as primary antibodies and then labeled with horseradish peroxidase secondary antibody (Histofine Simple Stain PO (R) or PO (M); Nichirei Biosciences) and, finally, the color reaction was performed using the substrate solution ACE (3-amino-9-ethylcarbazole) (Nichirei Biosciences), after which he was assigned observation using an optical microscope. The results are shown in Fig.
When the ES cells cultured on plain gelatin Cup, were sown on cells RA under these culture conditions, were formed colonies to the size observed with the naked eye within a few days of cultivation, and at approximately day 7 stump is investing observed morphological changes in the differentiated cells, detecting the structure of the neurite (axon nerve cells). These cells were strongly positive for the marker of neurons MAR 2, which suggests that these ES cells were differentiated into neurons. ES-stands planted on ST2 cells that formed colonies exhibiting Autonomous pulsation with day 12 of culture, and these colonies of cells were strongly positive for the marker of cardiomyocytes myosin of sarcomere, clearly showing that these ES cells were differentiated into cardiomyocytes. Differentiation into neurons and cardiomyocytes was confirmed also with the use of ES cells cultured on E-cad-Fc-Cup, similar to the control group. These experimental results demonstrate that ES cells cultured on E-cad-Fc-Cup, retain their pluripotency in vitro.
Then tested the ability of education teratoid tumors of these ES cells. Teratoma is a tumor containing fetal tissue and developed the cloth of the three embryonic leaves endoderm, mesoderm and ectoderm, which is formed by transplantation of ES cells in an animal, such as a mouse, and the power of education teratoid tumors used as an indicator of pluripotency of ES cells.
ES cells (line IV) were sown on gelatin Cup and on E-cad-Fc-Cup and passively 5 times every three days. These ES cells injec is listed in the ovaries glabrous mouse Balb/c mice (approximately 200 cells in each) using conventional methods, and at day 60 education teratoid tumors were detected in all of the transplanted ES cells ovaries without noticeable differences in tumor size between cultured on gelatin Cup group and cultured on E-cad-Fc-Cup group. In addition, after preparation of tissue sections in the usual way and observation of histology teratoid tumors of each group had education ectodermal tissues/cells, including skin-like tissues/cells and neurons that were positive for the different markers of neurons (βIII-tubulin, GFA, neurofilament M, GAP-43), mesoderm tissues/cells, including tissue, like bone, cartilage and tissue skeletal muscle, and endodermal tissues/cells, including tissue that is similar to the intestinal tissue and bronchomediastinal tissue, and therefore it was confirmed that the ES cells cultured on E-cad-Fc-Cup, retained the ability of education teratoid tumors.
Example 5: to Study the ability of the formation of chimeras ES cells cultured on E-cad-Fc-Cup
Determined whether ES cells passaged many times on E-cad-Fc-Cup, the ability of the formation of chimeras. ES cells (cell line I), taken from the same batch of frozen cells of the original stock, for which it was confirmed that he has the ability, the formation of chimeras, were sown on gelatin Cup of the E-cad-Fc-Cup and passively 5 times every three days. These ES cells were injected with the mouse blastocyst 57BL/6 (approximately 100 cells each) using conventional method and transplanted into the uterus of loebermann ICR mice (aged 8-10 weeks) and brought to birth. Mouse 57BL/6 usually have black hair, but some newborn specimens were obtained through the transplantation of ES cells wool color agouti (with light ring on the dark hair on body parts (5-80%); a total of four such chimeric mice were obtained from ES cells cultured on gelatin Cup, and a total of seven were derived from ES cells cultured on E-cad-Fc-Cup.
Then these chimeric mice were crossed with normal ICR mice to obtain offspring, to confirm that you purchased from ES cells wool dyeing was transferred to the next generation. Two chimeric male mice that received transplantation of ES cells cultured on E-cad-Fc-Cup, were obtained 14 and 17 calves, respectively, of which 5 and 6, respectively, showed originating from ES cells coat color among individuals, detecting the coat color of mice 57BL/6 blastocysts which were used as hosts. Analysis of microsatellite markers specific for mouse strain, confirmed that these individuals had genotypes originating from these ES cells (Fig.9). Specifically, obtained genomic DNA from whom every young individuals accepted way and four microsatellite marker D4Mit72, D4Mit116, D7Mit276 and D10Mit186, were detected to detect genetic differences between 129SV mice derived from the ES cells and C57BL/6 mice used to generate chimeric mice and breeding. As a result, individuals who believed, do not have the genetic contribution of the ES cells, on the basis of coat colour (No. 1-No. 4 on the pictures), it was found the same distribution for all microsatellite markers, as in the case of mice 57BL/6. On the other hand, individuals who, as believed to have a genetic contribution of the ES cells, on the basis of coat colour (No. 5 to No. 8 on the pictures), found as the distribution of mouse 57BL/6, and distribution specific to ES cells, which confirmed that the genes of ES-cells were transferred into these individuals.
Example 6: a Study of the extent of gene transfer into ES cells cultured on E-cad-Fc-Cup
The conventional way with Lipofectamine 2000 (Invitrogen) was used to transfer expressing GFP vector pEGFP-N2 (Clontech) in ES cells, which were cultured for 3 days on gelatin Cup and on E-cad-Fc-Cup. Individual cells were removed after one day and was perseval them in 96-well pad. After 4 hours, adherent E3-cells were fixed for 10 minutes in 8% formalin solution and then treated with 0.2% solution of Triton X-100/PBS and signal amplifier FX Image-iT (Invitrogen). After washing PBS and the reaction with monoclonal the th anti-cGy-antibody (Nacalai Tesque) was performed staining using Alexa Fluor 546-labeled antibodies against rat IgG. Cell nuclei were stained with DAPI solution (0.1 ág/ml). Images antibody and the dye was observed using the ArrayScan™ (Cellomics). As a result, a significantly higher expression of GFP was detected in cultured on E-cad-Fc-Cup group than in cultivated on a normal Cup group, which suggests that cultured on E-cad-Fc-Cup ES cells had a higher efficiency of gene transfer than usual cultured ES cells (see figure 10).
Example 7: Getting protein E-cad-Fc man and a usability study
To construct a vector expressing a protein of the extracellular region of E-cadherin of human rights and IgG/Fc (see SEQ ID NO:26), (hereinafter referred to here as the hE-cad-Fc), cDNA from line A431 squamous cell carcinoma of man was used as template for amplification of the DNA fragment (corresponding to amino acid residues 1-697)encoding the extracellular domain of E-cadherin man (hE-cad-ECD). After confirming the nucleotide sequence, it was built in expressing the vector containing the sequence IgG/Fc, referred to in Example 1, to construct pRC-hE-cad-Fc. Construction and purification of the vector hE-cad-Fc was performed in accordance with the method described in Example 1.
Investigated the adhesion and proliferation of mouse ES cells on plates for culturing cells, covered with protein hE-cad-Fc (gave the e called "hE-cad-Fc-cups"). The method of preparation of the hE-cad-Fc-cups was the same as the method described in Example 2 above. Specifically, the PBS-diluted protein solution hE-cad-Fc was poured into the raw polystyrene cups for cultivation and processed to cover over night at 37°C, for use as a hE-cad-Fc-cups. When seeding ES cells (lines I and R1) in these cups were discovered such as strong adhesion, which was found using the cups coated with mouse protein E-cad-Fc. ES cells, seeded in hE-cad-Fc-cups, also could not form a separate colony even after 2 or 3 days after seeding, and observed individual cells that were in dispergirovannom condition and actively proliferating state (11). Undifferentiated state and pluripotency of these ES cells were also preserved, as in the cultivation with the use of E-cad-Fc-cups.
Using the production method of the present invention can be obtained pluripotent stem cells such as ES cells, efficiently and on a large scale without the use of cell-feeders. In addition, because these pluripotent stem cells can be cultured in dispergirovannom condition, greatly facilitated the passage and extracting cells. May be a reduced number of factors such as LIF, which until ablaut to liquid medium for cultivation. Furthermore, the method of this invention makes possible the efficient transfer of desired genes in pluripotent stem cells such as ES cells, and allows to obtain high levels of expression. Thus obtained pluripotent stem cells can be used to obtain different types of functionally differentiated cells using suitable known for inducing differentiation of systems and are useful for pharmacological evaluation or assessment activity of various physiologically active substances or new gene products of unknown function.
1. Pic is b cultivation of pluripotent stem cells characterized by the cultivation of these pluripotent stem cells in dispergirovannom condition with maintaining their undifferentiated state and pluripotency, with the use of liquid media and the culture vessel having immobilized or supported on solid-phase surface of the substrate molecule that is adhesive with respect to these pluripotent stem cells without the use of cell-feeders, where this molecule is a protein containing the extracellular domain of E-cadherin and Fc-region of an immunoglobulin.
2. The method of growing pluripotent stem cells according to claim 1, in which the said stage of cultivation should gene transfer in these pluripotent stem cells.
3. The method according to claim 1 or 2, where the molecule that is adhesive with respect to these pluripotent stem cells, is any molecule that is expressed these pluripotent stem cell, or a molecule that is structurally homologous to the specified molecule and has the ability homophiles binding with the specified pluripotent stem cells.
4. The method according to claim 3, where the molecule that is adhesive with respect to these pluripotent stem cells is a molecule belonging to the family cadg is Renov.
5. The method according to claim 4, where the specified molecule belonging to the family of catherinew is E-cadherin or molecule that has structural homology with the specified molecule, which contains the EC1 domain and one or more domains of the EC2 domain, domain EES, domain ES and domain AS E-cadherin and which has the ability homophiles binding with the specified pluripotent stem cells.
6. The method according to claim 5, where the specified E-cadherin obtained from a mammal.
7. The method according to claim 6, where the specified E-cadherin derived from human or mouse.
8. The method according to claim 1 or 2, where the molecule that is adhesive with respect to these pluripotent stem cells, fused with the Fc region of immunoglobulin and immobilized on a solid phase surface a specified substrate through a specified Fc-area
9. The method according to claim 1 or 2, where these pluripotent stem cells are embryonic stem cells (ES-cells) of the mammal or human embryonic germ cells (EG cells) of the mammal.
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology, specifically to production of new cell lines, and can be used for making anti-tumour vaccines. A novel human melanoma cell line mel Gus is disclosed, which has stable cultural and morphological characteristics and is stored in the Special collection of cell cultures of the Institute of Cytology RAS under number RKKK (P) 707D. The obtained cell line is characterised by expression of melanoma (differentiation) markers - CD63, HMW, MelanA, HMB45, as well as cancerous-testicular marker MAGE-3, presence of first and second class histocompatibility antigens.
EFFECT: increased effectiveness of anti-tumour (whole cell, genetically engineered) vaccines, used for treating melanoma and other malignant growths.
1 tbl, 2 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology, specifically to production of new cell lines, and can be used for making anti-tumour vaccines. A novel human melanoma cell line mel Ch is disclosed, which has stable cultural and morphological characteristics and is stored in the Special collection of cell cultures of the Institute of Cytology RAS under number RKKK (P) 706D. The obtained cell line is characterised by expression of melanoma (differentiation) markers - CD63, HMW, as well as cancerous-testicular marker MAGE-3, presence of first and second class histocompatibility antigens.
EFFECT: increased effectiveness of anti-tumour (whole cell, genetically engineered) vaccines, used for treating melanoma and other malignant growths.
1 tbl, 2 ex
SUBSTANCE: invention concerns method for preparing autologous tuberculosis vaccines. Substance of the invention consists in activation in vitro of mononuclear fractions of leukocyte recovered from the patient's blood of the patient suffering from tuberculosis by bedding on density gradient of Ficoll urographine. After centrifugation, mononuclear leukocytes collected from interphase are washed, resuspended in a culture medium DMEM environment and cultivated therein with tuberculine and Roncoleukine added. Then cells are counted, diluted to concentration 2 million cells per ml and cultivated at 5% carbon dioxide concentration. Then cells are resuspended in physiologic saline with Roncoleukine.
EFFECT: application of the method allows preparing high-effective autologous tuberculosis vaccine.
3 tbl, 2 ex
SUBSTANCE: invention concerns obtainment of cells of monocyte origin, inducing transplant acceptance, expressing antigens CD3 and CD14, and can be applied in transplantology. Monocytes are extracted from blood, reproduced in cultural medium with 1-20 mcg/l of cell growth factor M-CSF and cultivated for 24-72 hours in cultural medium with 0.1-20 ng/ml of interferon γ-IFN. Further the cells are extracted from cultural medium. For cell CD3 and CD14 expression antibodies produced by hybridoma DSM ACC2542 are used. If necessary, obtained cells are suspended and freezed.
EFFECT: obtainment of cells of monocyte origin, inducing transplant acceptance and suppressing transplant rejection reaction.
26 cl, 29 dwg, 1 tbl, 13 ex
SUBSTANCE: there is offered new source of cardiac stem self-cell (CSC) that represents a tissue of surgically removed cardiac aneurysms, and there is developed method of CSC separation and cultivation from specified source, as well as method for induction of differentiation of these cells within formation of cardiac hystiocytes, endothelial, smooth muscle and neural cells.
EFFECT: CSC cultures produced according to the invention can be used in clinic environment to repair myocardial conditions in a certain category of patients.
4 cl, 6 dwg, 1 tbl, 5 ex
SUBSTANCE: cultured embryo cell viability is evaluated by the content of regulatory peptide HGF in a culture medium.
EFFECT: invention can be used in cell viability evaluation for transplantology.
SUBSTANCE: invention concerns medicine, namely ophthalmology, and involves use of suspended cultivated neuronal self-cells precipitated from olfactory region of nasal mucosa, or cultivated mesenchymal self-cells precipitated from bone marrow. Stem cells are introduced suprachoroidally in amount 500 thousand per 0.02 ml of physiologic saline or retrobulbary 1.5 million per 0.3 ml of physiologic saline. The introduction is repeated 20-30 days later.
EFFECT: method allows for prolonged and adequate improvement of capacity and reparative activity of retina with various pathologies.
2 cl, 6 dwg, 1 ex
SUBSTANCE: invention relates to field of biotechnology, namely, to obtaining genetically modified cell lines and can be applied in medicine for immunotherapy and immuno-prophylaxis in patients with malignant neoplasms. By means of recombinant method line of cells of human melanoma KG is obtained, which secretes recombinant granulocytic-macrofagal colony-stimulating human factor. Obtained line is deposited with Specialised cell culture collection of vertebrates of Russian cell culture collection under number RCCC ("П") 699"Д".
EFFECT: line of human melanoma cells KG possesses stable cultural, morphological and immunological characteristics and possesses ability to secrete recombinant human GM-CSF, remaining after cell inactivation with ionising irradiation.
SUBSTANCE: human haemopoetic stem cells are separated either from bone marrow or mobilised peripheral blood, or blood separated from umbilical vein. The fractionation procedure is used to prepare in Ficoll gradient from total cell mass the suspension of mononuclear cells wherefrom enriched suspension of cells CD133+ and CD34+ is separated. The prepared suspension is cultivated in the environment Dulbecco M (IMDM) with insulin and transferrin, as well as the stem cell growth factor (SCF), tyrosine kinase ligand 3 of fetal liver (Flt3L), thrombopoietin (TPO) and ionomycin (Ca2+ ionophore). After 7-10 days of cultivation, the grown cell suspension contains 80-92% of CD34+ cells.
EFFECT: invention allows for short-term cultivation of higher number of low-differentiated haemopoetic stem cells to be used in therapeutic purposes.
8 dwg, 4 tbl, 11 ex
SUBSTANCE: there proposed is the method for obtaining endotheliocytes from human embryonic stem cells, which involves differentiation of human embryonic stem cells on the substrate and in synthetic medium. Method allows obtaining endotheliocytes with high efficiency, choosing pure population of functional endotheliocytes that gained the same epigenetic status of key factors of transcription and homeostasis of genes sustaining as their analogues did, which are differentiated in embryo in vivo.
EFFECT: obtained endotheliocytes can be widely used in medical practice.
2 cl, 11 dwg, 4 tbl, 5 ex
SUBSTANCE: invention is related to biotechnology, namely to method for production of chondro-osteogenous cells in vitro and their application. Chondro-osteogenous cells are produced from mesenchyme stem cells of a human being. Mesenchyme stem cells of a human being are cultivated in medium with additives of human blood serum and beta -1 transforming factor of growth, having molecular domain, which provides for interaction with collagen I (TGF-β1-CBD). Then stem cells are exposed to further proliferation by means of addition of human blood serum and TGF-β1-CBD. In the end chondro-osteogenous induction is carried out by dexamethasone and -β-glycerophosphate. Chondro-osteogenous cells may be used in composition that is able to induce osteogenesis.
EFFECT: invention makes it possible to make medical preparations for recovery of bone and/or chondral tissue.
20 cl, 3 dwg, 3 tbl, 7 ex
SUBSTANCE: invention concerns medicine, particularly method of cardiomyocyte selection from cardiomyocyte-containing cell mix without genetic alteration incardiomyocytes. Method of cardiomyocyte content boost in cardiomyocyte-containing cell mix without genetic alteration. Method of cardiomyocyte obtainment without genetic alteration to cardiomyocytes. Method of cardiomyocyte content assessment in cardiomyocyte-containing cell mix.
EFFECT: possible efficient selection of cardiomyocytes from cardiomyocyte-containing cell mix without genetic alterations.
20 cl, 16 dwg, 14 ex
SUBSTANCE: connective or supporting tissue cells fit for transplantation are cultivated in bioreactor (1) which includes main case closed by sterile sealing cap (21) and forms at least one reactor space where transplant surface (11) and mini actuator (14) are placed. Bioreactor (1) is equipped with at least two coupling connections for hoses (19) of nutrient medium and gas feed and discharge.
EFFECT: obtaining cells with mechanical endurance to self-tissue transplantation for application as substitute tissue in treatment of connective and supporting tissues, direct injury of joints, rheumatism and degenerative diseases of joints.
22 cl, 27 dwg, 11 ex
SUBSTANCE: there is proposed cultural medium for culture of anchorage-dependent cells of animals that does not contain exogenic components of primary and secondary animal origin. There is also proposed method for production of anchorage-dependent cells of animals in this medium and the method for production of virus in specified cells.
EFFECT: invention can be applied for production of viruses, viral vaccine etc.
25 cl, 13 dwg, 3 tbl, 10 ex
SUBSTANCE: cultured embryo cell viability is evaluated by the content of regulatory peptide HGF in a culture medium.
EFFECT: invention can be used in cell viability evaluation for transplantology.
SUBSTANCE: invention relates to field of biotechnology and virology. Cell line is obtained from primary cells which are transformed with at least two virus or cellular genes, one of which causes cell type progression, while the other blocks natural protective cell mechanisms induced by replication dysregulation.
EFFECT: described is obtaining said cell lines and their application for obtaining biological preparations or viruses for vaccination.
13 cl, 6 dwg, 1 tbl, 5 ex
FIELD: medicine; biotechnology.
SUBSTANCE: method of fresh-mater shellfish cell and tissue cultivation allows for long-term cultivation of fresh-water shellfish pallium tissue in sterile conditions and complete prevention of tissue culture contamination with yeast and mold fungi, along with preservation of functional tissue activity.
EFFECT: application in cultivation of tissues of invertebrates in biotechnology, medicine and research for cell and tissue culture obtainment.
SUBSTANCE: present invention relates to biotechnology and molecular biology. Proposed is a medium for redifferentiation of dedifferentiated chondrocyte, as well as a method of redifferentiating dedifferentiated chondrocyte into the initial chondrocyte.
EFFECT: obtaining transplantation materials used in transplantology, plastic surgery, as well as cartilage regenerative therapy.
7 cl, 15 dwg
SUBSTANCE: invention can be used in experimental and clinical surgery for obtaining biomatrices used for closing wounds with mechanical injuries of connective tissue. Method includes growing fibroblasts on the base from synthetic material covered with wheat lectin. As base, used is polypropylene net which is kept in 0.05% lectin solution for 3 hours at room temperature before cell application. After that propylene net is placed in suspension of freshly isolated primary or subcultivated embryonic cells of rat in culture medium and incubated at temperature 37°C during 7-9 days, changing culture medium first time after a day and later - every three days.
EFFECT: obtaining biomatrix on the base of propylene net and embryonic fibroblast culture, which can be used for closing and stimulation of efficient healing of connective tissue mechanical injuries in experimental and clinical surgery.
3 cl, 1 ex, 4 dwg
SUBSTANCE: claimed is line of multipotent mesenchymal stem cells of human subcutaneous fatty tissue (panniculus adiposus homo sapiense) for cell and tissue engineering. Invention can be used in laboratories engaged in tissue engineering and novel cell technologies aimed at regeneration and reparation of tissues and organs of mammals including human being.
EFFECT: obtained diploid strain of reserve stem cells represents perspective material for obtaining bone and cartilage tree-dimensional tissue transplants and their testing in animal organism.
SUBSTANCE: method involves deactivation of definite VGC2 DNA sequence of Salmonella typhimurium positioned between ydhE and pykF genes or its part containing at least 50 nucleotides, or the DNA version of at least 85% identity, representing VGC2 DNA of any microbe out of Salmonella aberdeen, Salmonella gallinarum, Salmonella cubana and Salmonella typhi.
EFFECT: obtainment of microbe with reduced adaptability to specific environmental conditions.
6 cl, 12 dwg, 8 ex