WO2011055886A1 - Method for cultivating human embryonic stem cells or dedifferentiated pluripotent stem cells - Google Patents

Abstract

The present invention relates to a method for cultivating human embryonic stem cells or dedifferentiated pluripotent stem cells from among culture mediums for stem cell cultivation, wherein the culture medium, comprised of supporting cells, includes a porous film that is a floor to which a cell growth inhibitor attaches untreated human adipose stem cells. The present invention also relates to a method for recovering human embryonic stem cells or dedifferentiated pluripotent stem cells using the cultivation method.

Description

Cultivation method of human embryonic stem cells or pluripotent stem cells

The present invention relates to a method for culturing human embryonic stem cells or dedifferentiated pluripotent stem cells using non-treated human adipose derived stem cells (hASCs) as support cells. The present invention also relates to a method for recovering human embryonic stem cells or pluripotent stem cells using the culture method.

Human embryonic stem cells have a pluripotency that can differentiate into trioderm (endoderm, mesoderm, and ectoderm) constituting the human body and have a self-renewal that is constantly dividing, such as human incurable diseases. Attention is being paid to the possibility of cell therapy for the treatment of diseases. Since the successful cultivation of human embryonic stem cells by Thomson in 1998, further expectations for cell therapy studies using human embryonic stem cells are expected (Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM , Embryonic stem cell lines derived from human blastocysts Science (1998) 282:. 1145-7)

Despite the success of the method of culturing human embryonic stem cells, there are many problems that must be solved in order to be applied in practical clinical practice. In particular, animal-derived support cells used for culturing human embryonic stem cells, by-products of various animal-derived proteins and growth factors, and the use of anti-cancer agents for cell growth inhibition are major obstacles to the clinical application of human embryonic stem cells. It is becoming.

Various methods have been reported to develop a method for culturing human embryonic stem cells that can solve these problems. For example, in order to exclude the use of xeno-component in a culture method using mouse embryonic fibroblast (MEF), which is an animal-derived support cell, as a support cell, various kinds of human-derived cells (eg, Culture methods using human epidermal fibroblasts, human bone marrow cells, etc. as supporting cells have been reported (Hovatta O, Mikkola M, Gertow K, et al . A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells.Hum Reprod 2003; 18 : 1404-1409; Cheng L, Hammond H, Ye Z, et al . Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture.Stem Cells 2003; 21 : 131-142; And Inzunza J, Gertow K, Stromberg MA, et al . Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cells.Stem Cells 2005; 23 : 544-549)

However, even when using a variety of human-derived support cells as described in the above report, the use of cell growth inhibitors to prevent the support cells from dividing can cause a huge problem, which is a barrier to clinical application. Mitomycin-C, which is mainly used as a cell growth inhibitor of support cells, is an anticancer agent and prevents cell division so that support cells are maintained at an appropriate concentration. The cell growth inhibitor serves to provide an environment in which human embryonic stem cells are maintained in an undifferentiated state and grow appropriately by eliminating a situation in which embryonic stem cell growth, which may be caused by overgrowth of support cells, is prevented. . However, these cell growth inhibitors must be removed in order to use human embryonic stem cells as cell therapy. Recently, it has been reported that treatment with cell growth inhibitors to support cells is not suitable for stably culturing human embryonic stem cells that can be clinically applied by inducing necrosis or apoptosis of support cells (A. Nieto, CM Cabrera, A. Concha et al. Effect of mitomycin-C on human foreskin fibroblasts used as feeders in human embtyonic stem cells: Immunocytochemistry MIB1 score and DNA ploidy and apoptosis evaluated by flow cytometry.Cell Biology International 2007; 31: 269 -278).

Meanwhile, induced pluripotent stem cells (iPS cells) refer to cells having pluripotency by differentiating from differentiated cells and capable of differentiating into various organ cells. iPS cells can be obtained by reprogramming cells differentiated by dedifferentiation inducers, thus allowing the generation of patient immunocompatible pluripotent cell lines without somatic cell transfer. Thus, iPS cells can be derived from the cells of the patient to avoid immune rejection in clinical applications. In addition, iPS cells do not use eggs or embryos, so there is no bioethical controversy or religious criticism. Takahashi, K., and Yamanaka, S., et al., Present for the first time the possibility of iPS cells formation by reverse differentiation through external gene transfer using mouse cells (Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell 126 , 663-676), Takahashi, K. et al. And Yu, J. et al. The formation of iPS cells by differentiation methods has been reported (Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. ( Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell 131 , 861-872; and Yu, J., Vodyanik, MA, Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, JL , Tian, S., Nie, J., Jonsdottir, GA, Ruotti, V., Stewart, R. , et al. (2007) .Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells.Science New York, NY). Takahashi, K. et al. Found that dedifferentiation inducers of Sox2, Oct3 / 4, and Klf4 are necessary to obtain iPS cells, and additionally c-Myc plays a role in promoting the formation of iPS cells. In addition, Yu, J. et al. Found that reverse differentiation inducers of Sox2, Oct3 / 4, and Nanog are necessary for obtaining iPS cells, and the formation of iPS cells increases when Lin28 is present.

Therefore, there is a need in the art to develop a method for stably culturing human embryonic stem cells or pluripotent stem cells to enable clinical application. The present inventors report that when human embryonic stem cells are adhered to the bottom of the porous membrane to culture human embryonic stem cells on the membrane, the embryonic stem cells can be easily and effectively separated and high purity human embryonic stem cells can be obtained. (International Patent Publication No. WO2007 / 139357, Sinae Kim, Seong Eun Ahn, Jae Ho Lee et al., A Novel Culture Technique for Human Embryonic Stem Cells Using Porous Membranes. Stem Cells 2007; 25: 2601-2609) . Embryonic stem cell culture method using the porous membrane provides a suitable space between the support cells and embryonic stem cells, by reducing the contamination from the support cells during subculture and can be isolated without treatment of enzymes, a more pure state It is a way to culture embryonic stem cells.

The inventors of the present invention provide a method for culturing human embryonic stem cells that can solve the problems caused by the use of cell growth inhibitors to induce necrosis or apoptosis in support cells and the problems in clinical application due to the use of animal-derived cells or animal-derived proteins. Various studies were conducted to develop. As a result, when culturing human embryonic stem cells by using human adipose stem cells as supporting cells and attaching to the bottom surface of the porous membrane, the treatment of cell growth inhibitors such as mitomycin C is unnecessary. In addition to avoiding the problem, it has been found that embryonic stem cells can be cultured with high purity without contamination of animal-derived cells or animal-derived proteins. In particular, since it is possible to use adipose stem cells, which can be easily and largely obtained from the patient's own fat, as supporting cells, it has been found that they can be suitably used for the culture of dedifferentiated pluripotent stem cells.

Accordingly, the present invention provides a method for culturing human embryonic stem cells or dedifferentiated pluripotent stem cells using a porous membrane in which non-treated human adipose stem cells are adhered to the bottom surface as support cells.

The present invention also provides a method for recovering human embryonic stem cells or pluripotent stem cells from the culture cultured by the culture method.

According to an aspect of the present invention, in the method for culturing human embryonic stem cells or dedifferentiated pluripotent stem cells in a culture medium for stem cell culture, the medium is human adipocyte stem cells treated with cell growth inhibitors non-treated as support cells There is provided a culture method comprising a porous membrane attached to the bottom surface.

The medium comprising a porous membrane to which the cell growth inhibitor is non-treated human adipose stem cells attached to the bottom surface, by adding a medium for culture of human adipose stem cells and support cell non-treated cell growth inhibitor to one side of the porous membrane After culturing, it can be obtained by dipping in the culture medium for stem cells so that the porous membrane to which the human adipose stem cells are attached is directed downward. Preferably, the support cell culture medium does not include an animal-derived protein. For example, the support cell culture medium may be α-MEM (Minimum Essential Medium) to which antibiotics, glutamine, and serum substitutes are added.

The porous membrane is a cell consisting of polyethylene terephthalate, polyethersulfone, polyvinylidene fluoride, cellulose, nylon, polyethylene, polypropylene, polycarbonate, polyurethane, polyacrylate, polycaprolactone and copolymers thereof It may be selected from the tacky polymer, it is preferred that the pores of the porous membrane has a diameter of 0.1 to 3 ㎛. In addition, the porous membrane is preferably coated with gelatin, collagen, fibronectin, lamidine, or metrigel.

According to another aspect of the invention, the step of culturing human embryonic stem cells or dedifferentiated pluripotent stem cells by the culture method; And isolating human embryonic stem cells or pluripotent stem cells from the porous membrane.

Separating human embryonic stem cells or pluripotent stem cells from the porous membrane may be performed by scraping human embryonic stem cells or pluripotent stem cells from the porous membrane.

In the culture method of the present invention, by using human adipose stem cells not treated with cell growth inhibitors such as mitomycin-C as support cells, problems caused by the use of cell growth inhibitors, that is, inducing necrosis or apoptosis of the support cells, etc. The problem of the present invention can be blocked, and also the problem in clinical application according to the use of animal-derived cells or animal-derived proteins can be solved. Furthermore, the culturing method of the present invention is particularly useful for stable culture of dedifferentiated pluripotent stem cells by using, as support cells, fat stem cells which can be easily obtained from the fat of the patient and can be obtained in a large amount compared to other stem cells. May be suitably used.

Figure 1 is a graph showing the adhesion rate of human embryonic stem cells according to the concentration of human adipose stem cells attached to the bottom of the porous membrane, Figure 1 A shows the adhesion rate when incubated for 3 days (n = 3) 1B shows the adhesion rate when incubated for 6 days (n = 3).

Figure 2 is an optical micrograph of human embryonic stem cells cultured through a porous membrane attached to the bottom surface of human adipose stem cells not treated with mitomycn-C (fluorescence staining) showing the expression of undifferentiated cells to be.

Figure 3 is a result of analyzing the undifferentiated transcription factor expressed in human embryonic stem cells cultured through a porous membrane attached to the bottom surface of human adipose stem cells not treated with mitomycn-C (mitomycn-C).

4 is a chromosome photograph of human embryonic stem cells cultured through a porous membrane attached to the bottom surface of human adipose stem cells not treated with mitomycn-C (mitomycn-C).

FIG. 5 is a fluorescence staining photograph showing the expression of undifferentiated cells of dedifferentiated pluripotent stem cells cultured through a porous membrane attached to the bottom surface of human adipose stem cells not treated with mitomycn-C (mitomycn-C).

FIG. 6 shows the results of analyzing undifferentiated transcription factors expressed by dedifferentiated pluripotent stem cells cultured through a porous membrane attached to the bottom surface of human adipose stem cells not treated with mitomycin-C (mitomycn-C).

As used herein, "human embryonic stem cells" refer to omnipotent cells derived from the internal cell mass of human morula. The human embryonic stem cells, for example, CHA-hES3 (Ahn SE, Kim S, Park KH, Moon SH, Lee HJ, Kim GJ, Lee YJ, Park KH, Cha KY, Chung HM.Primary bone-derived cells induce osteogenic differentiation without exogenous factors in human embryonic stem cells.Biochem Biophys Res Commun. (2006) 10; 340 (2): 403-408) may be used, but is not limited to these examples. Human embryonic stem cells can be readily constructed by those skilled in the art.

In addition, "induced pluripotent stem cells" (iPS cells) are also referred to as "reprogrammed pluripotent stem cells" and reprogrammed (i.e., dedifferentiate) differentiated cells to pluripotency. It refers to a cell induced to have. Retrodifferentiated pluripotent stem cells can be readily constructed by those skilled in the art according to known methods.

The present invention provides a method of culturing human embryonic stem cells or dedifferentiated pluripotent stem cells in a stem cell culture medium, wherein the medium is a support cell to which non-treated human adipose stem cells are attached to the bottom surface. It provides a culture method comprising a porous membrane.

In the culture method of the present invention, by using human adipose stem cells not treated with cell growth inhibitors such as mitomycin-C as support cells, problems caused by the use of cell growth inhibitors, that is, inducing necrosis or apoptosis of the support cells, etc. Of the animal-derived cells or animal-derived proteins (xeno-component) such as mouse Embryonic Fibroblast (MEF) and mouse fibroblasts (STO cell). Problems in clinical applications of use (eg, infections, immune rejection, etc.) can be addressed.

In particular, the culturing method of the present invention can replace human embryonic stem cells, since not only can be easily obtained from the fat of the patient itself, but can also use adipose stem cells, which can be obtained in a large amount compared to other stem cells, as support cells. It can be suitably used for stable culture of known differentiated pluripotent stem cells. In other words, the use of stem cells obtained from the patient's own fat can fundamentally solve the problem of infection or immune rejection.

Human adipose stem cells used as support cells in the culture method of the present invention can be obtained by using adipose tissue discarded during the liposuction process that is commonly performed. Adipose stem cells are derived from liposuction and sedimentation, collagenana, as disclosed in known methods (e.g., WO2000 / 53795 and WO2005 / 042730) from human adipose tissue or adipocytes. It can be obtained through a process such as enzymatic treatment of collagenase and the like and removal of floating cells such as red blood cells by centrifugation. The adipose tissue includes brown or white tissue derived from subcutaneous, retinal, visceral, breast gonad or other adipose tissue sites and can be readily obtained from conventional liposuction.

In the culture method of the present invention, the medium comprising a porous membrane in which the cell growth inhibitor is attached to the bottom surface of the non-treated human adipose stem cells, the human adipose stem cells in which the cell growth inhibitor is non-treated on one side of the porous membrane And after culturing by adding a support cell culture medium, it may be obtained by dipping in the stem cell culture medium so that the porous membrane to which the human adipose stem cells are attached is directed downward.

The support cell culture medium preferably does not contain an animal-derived protein (for example, fetal bovine serum). For example, the support cell culture medium may be α-MEM (Minimum Essential Medium), more preferably 1% penicillin stereptomycin (P / S), 1% glutamax, to which antibiotics, glutamine and serum substitutes are added. (GlutaMAX), Minimum Essential Medium (α-MEM) with 10% Serum Replacement (SR) added, but is not limited thereto. Cultivation of non-treated human adipose stem cells in the support cell culture medium may be performed for 12 to 48 hours, preferably about 24 hours. The density of the supporting cells attached to the porous membrane may be selected in an appropriate range by calculating the cell concentration after 6-7 days of culturing embryonic stem cells, preferably 1.2 X 10 ³ to 4.8 X 10 ³ cells per porous membrane area. / cm ² , and more preferably about 2.4 X 10 ³ cells / cm ² It can be. The porous membrane obtained as described above is used to put the stem cell culture medium inverted so that the support cell attachment surface facing down.

The culturing method of the present invention uses a porous membrane to which support cells are attached, and maintains nutrient supply and maintenance of undifferentiated state of human embryonic stem cells through pores in the porous membrane. That is, through the porous membrane, the cell-cell interaction between the support cells and the embryonic stem cells is constantly spatially provided, that is, without treatment of the growth inhibitor (mitomycin-C) that prevents the support cell division, that is, continuous Human embryonic stem cells can be cultured by using support cells capable of dividing, and thus the embryos can be cultured in a more pure state by reducing contamination from the support cells and separating without treatment of enzymes.

As the material of the porous membrane, as a membrane to which cells such as support cells can be adhered, any polymer having a porous property can be used without limitation. Examples of porous membranes usable in the culture method of the present invention include polyethylene terephthalate, polyethersulfone, polyvinylidene fluoride, cellulose, nylon, polyethylene, polypropylene, polycarbonate, polyurethane, polyacrylate, polycapro Cell-adhesive polymers such as lactones or copolymers thereof. Among the cell-adhesive polymers, polyethylene terephthalate may be more preferably used, and commercially available BD Falcon ™ (BD Bioscience, USA) made to fit the size of the culture well may be used. The pores of the porous membrane preferably have a diameter of 0.1 to 3 μm, preferably 1 to 2 μm.

As the culture medium for stem cell culture, any known human embryonic stem cell culture medium or a medium for reverse differentiated pluripotent stem cell culture may be used. For example, serum substitute (SR), penicillin stereptomycin (P / S) ), Dulbecco's modified Eagle's medium / F-12 (DMEM / F-12) supplemented with mercaptoethanol, non-essential amino acids, and basic Fibroblast Growth Factor (bFGF).

The culture method of the present invention may be used by coating a variety of natural or synthetic materials, such as collagen, fibronectin, lamidine, or metrigel in addition to gelatin on the porous membrane as necessary. For example, by coating collagen, fibronectin, and the like on a porous membrane to which support cells are attached, human embryonic stem cells or pluripotent stem cell cultures can be induced more effectively in the presence of support cells (Heidi Hakala, M). Sc., Kristiina Rajala, M.Sc., Marisa Ojala, et al, .Comparison of Biomaterials and Extracellular Matrices as a Culture Platform for Multiple, Independently Derived Human Embtyonic Stem Cell Lines, Tissue Engineering: Part A 2009; 15: 1 -11).

The present invention comprises the steps of culturing human embryonic stem cells or pluripotent stem cells in the culture method; And a method for recovering human embryonic stem cells or pluripotent stem cells, the method comprising separating human embryonic stem cells or pluripotent stem cells from the porous membrane.

The step of separating human embryonic stem cells or pluripotent stem cells from the porous membrane is preferably scraping human embryonic stem cells or pluripotent stem cells from the porous membrane using a mechanical separation method, for example, a glass rod or the like. Can be carried out. In other words, by using a mechanical separation method, it is possible to eliminate the separate enzyme treatment, it is possible to avoid the problem of contamination by the enzyme. In particular, since the porous membrane has sufficient strength to scrape the cultured cells mechanically, it is possible to simply recover human embryonic stem cells or pluripotent stem cells.

Human embryonic stem cells and pluripotent stem cells recovered as described above have the characteristics of normal karyotype and undifferentiated cells. In other words, the human embryonic stem cells and the pluripotent stem cells recovered as described above were normally expressed in Nanog, Oct-4, Sox-2, and confirmed by immunochemical staining, RT-PCR, human embryonic stem cells The characteristics of human embryonic stem cells and markers of pluripotent stem cells, such as AP (Alkaline phosphatase), Oct4 (POU5F1 (POU class 5 homoeobox 1)), SSEA3 / SSEA4 (stage specific embryo antigen3 / 4), TRA-1-60 / TRA-1-81 (Tumor rejection antigen 1-60 / 1-81) and the like were expressed.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1 Culture of Human Embryonic Stem Cells

Adipose stem cells untreated with mitomycin-C after gelatin-coated BD Falcon ^™ (BD Bioscience, USA) porous membrane with pores 1 μm in diameter 2.4 X 10 ³ cells / cm ² and Support cell culture media (1% P / S, 1% GlutaMAX, α-MEM supplemented with 10% SR) was added and incubated for 24 hours. The porous membrane to which the adipose stem cells adhered was separated and washed twice with phosphate buffered saline. The resulting membrane is directed to a human embryonic stem cell culture medium (20% serum substitute (SR), 1% penicillin stereptomycin (P / S), 0.1 mM mercaptoethanol, 1% non-essential amino acids so that the supporting cells are directed downward). Gibco), and medium consisting of 80% DMEM / F-12 supplemented with 4 ng / ml bFGF). Clumped human embryonic stem cells (CHA-hES15) were finely divided and sprinkled about 60 on the medium. After 48 hours, it was confirmed that human embryonic stem cells adhered well to the membrane, and cultured for 7 days while changing the human embryonic stem cell culture medium every day. Human embryonic stem cell clumps grown by the above method were finely divided and sprinkled on a porous membrane to which newly prepared supporting cells were attached, followed by subculture.

Example 2. Recovery of Human Embryonic Stem Cells

Human embryonic stem cells were recovered without enzymatic treatment by scraping human embryonic stem cells cultured on a porous membrane from the culture medium of Example 1 (medium obtained by 10th passage) using a glass rod.

Example 3. Cultivation of Retrodifferentiated Pluripotent Stem Cells

The medium after finely dividing the pluripotent stem cell colony (provided by the Chai University of Medicine, Cardiovascular Stem Cell Laboratory and Tumor Stem Cell Laboratory) on the porous membrane containing the adipose stem cells prepared in the same manner as in Example 1 About 60 plants were sprayed on the stomach, and 48 hours later, the pluripotent stem cells were confirmed to adhere well to the membrane. As a medium for dedifferentiated pluripotent stem cells, a medium for culturing human embryonic stem cells was used. The medium was changed every 24 hours and incubated for 7 days. The dedifferentiated pluripotent stem cell clumps grown by the above method were finely divided and sprinkled on a porous membrane to which newly prepared supporting cells were attached, and passaged in this manner.

Example 4. Recovery of Retrodifferentiated Pluripotent Stem Cells

The reverse differentiated pluripotent stem cells were recovered without enzymatic treatment by scraping off the differentiated pluripotent stem cells cultured on the porous membrane using a glass rod from the culture medium of Example 3 (the medium obtained by the 15th passage).

Test Example 1.

Human embryonic stem cells were cultured in the same manner as in Example 1, except that 1.2 × 10 ³ and 4.8 × 10 ³ cells / cm ² were used as initial concentrations of human adipose derived stem cells used as support cells. . As can be seen in Figure 1 it can be seen that the adhesion rate of human embryonic cells on the porous membrane is the highest at 2.4 X 10 ³ .

Test Example 2 Characterization of Cultured Stem Cells-Confirmation of Undifferentiated State

To confirm the undifferentiated state of human embryonic stem cells, human embryonic stem cells on the porous membrane in the culture medium of Example 1 were fixed for 4 minutes with 4% paraformaldehyde and 0.1% Triton X-100 (Triton X-100). Was permeated for 5 minutes. After treatment with 1% BSA at room temperature, human embryonic stem cells were diluted 1: 100 with human specific antibodies Oct4, SSEA3, SSEA4, Tra-1-60, and Tra-1-81, and then incubated at 4 ° C for 12 hours. It was. After washing the sample, FITC-conjugated goat anti-mouse IgG (1: 1000) secondary antibody was added to incubate for 1 hour at room temperature to detect the primary antibody. The stained samples were washed again, and DAPI (1: 500) was placed at the bottom of the porous membrane and incubated for 5 minutes at room temperature to stain the nuclei of the support cells. Images were analyzed by fluorescence microscopy (ApoTome, Carl Zeiss, Jena, Germany).

In addition, undifferentiated transcription factors of human embryonic stem cells cultured by the culture method were confirmed at the gene level. First, the culture medium of the well plate was removed, the porous membrane was washed with phosphate buffered saline, and human embryonic stem cell colonies were separated by a glass pipette. Human embryonic stem cell colonies were collected in 1.7 ml Eppendorf test tubes and RNA was extracted by treating 1 ml of TRIzol (invitrogen). RNA was quantified using Nanodrop (Thermo scientific, USA) and 1 μg of RNA was reverse transcribed into cDNA using RT premix (Bioneer, Korea). PCR was performed using cDNA, primers and PCR premix (Bioneer). The primers used are shown in Table 1 below.

Table 1

gene	direction	order
Nanog	Forward direction	5'-CAG CCC CGA TTC TTC CAC CAG TCC C-3 '
	Reverse	5'-CGG AAG ATT CCC AGT CGG GTT CAC C-3 '
Oct4	Forward direction	5'-GAC AGG GGG AGG GGA GGA GCT AGG-3 '
	Reverse	5'-CTT CCC TCC AAC CAG TTG CCC CAA AC-3 '
Sox2	Forward direction	5'-GAC CAG CTC GCA GAC CTA CA-3 '
	Reverse	5'-GAA GAG GTA ACC ACA GGG GG-3 '
β-actin	Forward direction	5'-ACT CTT CCA GCC TTC CTT CC-3 '
	Reverse	5'-ACT CGT CAT ACT CCT GCT TG-3 '

PCR products were loaded on 1.5% agarose gel, stained with ethidium bromide, and irradiated with UV to observe gene expression.

Figure 2 is a photograph of the culture of human embryonic stem cells on the porous membrane attached to the support cells for 4 days. (A)-(e) in FIG. 2 are photographs of human embryonic stem cells under an optical microscope, and (f)-(j) show nuclei staining of human embryonic stem cells through DAPI (blue) staining. In addition to the photographs, the nuclei of the supporting cells at the bottom of the porous membrane were stained by DAPI staining. (K) to (o) are SSEA3, SSEA4, Tra-1-60, Tra-1-81, and Oct4, respectively. Photographed using a fluorescence microscope, (p) is a photograph confirming the undifferentiation of embryonic stem cells by AP (Alkaline Phosphatase) staining. Figure 3 shows the results of confirming the differentiation-related transcription factors Nanog, Oct4, Sox2 by RT-PCR after passage 10 times the human embryonic stem cells on the porous membrane attached to the support cells. In the same way as in the undifferentiated state of the initial culture, it can be confirmed that after the subculture, the undifferentiated transcription factors are strongly expressed. Figure 4 is a chromosome picture of the human embryonic stem cells recovered in Example 2, showing that the obtained embryonic stem cells have a normal karyotype.

In order to confirm the undifferentiated state of pluripotent stem cells, immunostaining and RT-PCR were performed in the same manner as that of embryonic stem cells. Figure 5 is a photograph of the culture for 4 days the reverse differentiated pluripotent stem cells on the support membrane attached porous membrane. (A)-(c) is a photograph which stained the nucleus by DAPI for the pluripotent stem cell in DAPI, (d)-(f) shows SSEA4, Tra-1-60, and Oct4 using the fluorescence microscope, respectively. (G) ~ (i) is a picture showing the staining of the nucleus of the support cells through DAPI (blue) staining with fluorescence pictures of each undifferentiated marker, (j) is the AP (Alkaline Phosphatase) Undifferentiated pluripotent stem cells through staining confirmed the differentiation. Figure 6 shows the results of confirming the differentiation-related transcription factors Nanog, Oct4, Sox2 by RT-PCR after passage 15 times the pluripotent stem cells on the porous membrane attached to the support cells. In the same way as in the undifferentiated state of the initial culture, it can be confirmed that after the subculture, the undifferentiated transcription factors are strongly expressed.

Claims (9)

1. In the method of culturing human embryonic stem cells or pluripotent stem cells in a stem cell culture medium,

   And wherein the medium comprises a porous membrane having adhered to the bottom surface non-treated human adipose stem cells as a support cell.
2. The medium of claim 1, wherein the medium comprising a porous membrane to which the cell growth inhibitor is attached to the bottom surface of the non-treated human adipose stem cells is a human adipose stem cell to which the cell growth inhibitor is non-treated and to the support. After culturing by adding a cell culture medium, the culture method, characterized in that obtained by immersing in the culture medium for stem cells so that the porous membrane to which the human adipose stem cells adhere.
3. The culture method according to claim 2, wherein the support cell culture medium does not contain an animal-derived protein.
4. The culture method according to claim 2, wherein the support cell culture medium is α-MEM (Minimum Essential Medium) to which antibiotics, glutamine and serum substitutes are added.
5. The method of claim 1, wherein the porous membrane is polyethylene terephthalate, polyethersulfone, polyvinylidene fluoride, cellulose, nylon, polyethylene, polypropylene, polycarbonate, polyurethane, polyacrylate, polycaprolactone and their Cultivation method characterized in that the cell adhesive polymer consisting of a copolymer of.
6. The method of claim 1, wherein the pores of the porous membrane has a diameter of 0.1 to 3 ㎛.
7. The method of claim 1, wherein the porous membrane is coated with gelatin, collagen, fibronectin, lamidine, or metrigel.
8. Culturing human embryonic stem cells or dedifferentiated pluripotent stem cells by the culture method according to any one of claims 1 to 7; And separating human embryonic stem cells or pluripotent stem cells from the porous membrane.
9. The human embryonic stem of claim 8, wherein the step of separating human embryonic stem cells or pluripotent stem cells from the porous membrane is performed by scraping human embryonic stem cells or pluripotent stem cells from the porous membrane. Recovery of Cells.

Images