KR101395214B1 - Placenta-derived cells conditioned media and animal-free, feeder-free culture method for maintaining undifferentiated stem cells using the same - Google Patents

Abstract

The present invention relates to a culture medium for conditioned stem cells to placenta-derived cells, and a method for culturing animal cells and mesenchymal stem cells using the same. According to the present invention, it is possible to prevent contamination by heterologous proteins or cells, and there is an advantage that human embryonic stem cells can be economically cultured while maintaining undifferentiated state in vitro for a long period of time.

Description

[0001] The present invention relates to a method for culturing a placenta-derived cell in a conditioned culture medium, and a method for culturing an animal and an animal cell,

The present invention relates to a conditioned culture medium for placenta-derived cells and a method for culturing animal and non-myeloid stem cells using the same. More specifically, the present invention relates to human embryonic stem cells and induced pluripotent stem cells (hereinafter referred to as " iPS " or " iPS cells ") capable of avoiding animal serum addition and support cell utilization by using a conditioned culture medium of human placenta- iPSC ") and a culture medium thereof.

Human embryonic stem cells are capable of differentiating into all embryonic stem cells (endoderm, ectoderm, mesodermal lobe) that constitute the human body and have the ability to differentiate. These stem cells are used for the treatment of intractable diseases such as diabetes, cirrhosis, heart failure, It is a source cell that can be developed as a therapeutic agent. In order to develop a cell therapy agent using human embryonic stem cells, human embryonic stem cells must be able to be cultured for a long period of time while maintaining their undifferentiated state and ensuring safety. As a result of successful development of a technology for culturing human embryonic stem cells in vitro into undifferentiated state by Thomsom in 1998 (Non-Patent Document 0001), development of cell therapy using human embryonic stem cells seemed to be visualized, Many problems have been pointed out in in vitro culture techniques. Some of these have been overcome, but they have not yet been resolved.

In vitro culture of human embryonic stem cells developed by Thomson was performed by adding FBS (fetal bovine serum) to the culture medium and culturing human embryonic stem cells using MEF (mouse embryonic fibroblast) as a supporting cell. Both FBS and MEF are body fluids and cells originating from non-human heterozygous animals. Human embryonic stem cells are inevitably contaminated with animal proteins and cells in an environment where human embryonic stem cells are cultured in contact with them. Development of a cell therapy agent using human embryonic stem cells cultured in this environment has the potential to inject heterologous proteins and cells into the body of the patient, which can lead to unpredictable and serious side effects. Therefore, in view of stability, many studies have been conducted to develop a culture system of human embryonic stem cells excluding animal proteins and cells.

First, chemically modified serum replacement (SR) was developed (20% Knockout Serum Replacement, KSR ^® , Invitrogen) to replace FBS (Non-Patent Document 0002). However, since KSR can not maintain the undifferentiated state of human embryonic stem cells without supporting cells, culture techniques using human origin supporting cells have been developed. Human endothelial cells (HEC), human bone marrow mesenchymal stem cells (HMSC), human placental cells (HPC), human placental cells ) And so on. However, although allogeneic protein and cell contamination can be avoided by using homologous human-derived supporting cells, allogenic protein and cell contamination can not be avoided because it is derived from a different individual from human embryonic stem cells . If a cell therapy agent originating from human embryonic stem cells is contaminated with a heterologous protein and a cell, a rejection reaction may occur due to the immune system of the patient, so that a feeder-free culture system is ultimately required. Development of culture media used in feeder-free culture systems has been started from MEF-conditioned media (Non-Patent Document 0003). A method for culturing a cell culture containing only a compound essential for cell culture for a predetermined period of time in a MEF, collecting it again, and using it as a culture medium for human embryonic stem cell culture to carry out a feeder-free culture, Not an animal-free culture system. Because of the use of culture medium exposed to MEF, there is always the possibility of contamination by heterologous proteins and cells. In recent years, cultures have been developed that completely block the heterologous protein and cell byproducts and are commercially available (TeSR ^TM 2, STEMCELL TECHNOLOGIES).

However, current feeder-free culture systems have other limitations besides culture. In the case of feeder-free culture, a contact material such as gelatin is required at the bottom of the cell culture dish in order to allow the cells to stably adhere to the culture dish instead of the supporting cell. In the case of human embryonic stem cell culture, Gelatin that can not be used can not be used and a special gelatin-like substance that is conditioned on mouse sarcoma cells should be used. It is understood that various cytokines and proteins which are useful for maintenance and survival of the undifferentiated state of human embryonic stem cells in the conditioning process are contained in the gel. This conditioned gel is currently commercially available (Matrigel®, BD Biosciences). However, since Matrigel is also produced by contact with xenogeneic cells, it can not be called an animal-free culture system even when using a culture solution (TeSR ^TM 2) in which a heterologous protein and cell byproducts are completely blocked, There is always a concern of contamination by proteins and cells.

The existing feeder-free culture system has many limitations in terms of economy. Production of MEF-conditioned media or Matrigel requires continuous sacrifice of mouse embryos and embryos, which is costly. In addition, when human embryonic stem cells are cultured in vitro using MEF-conditioned media or Matrigel, basic fibroblast growth factor (bFGF) must be continuously added to the culture medium or supernatant to maintain undifferentiated state, which is very costly Do. Human embryonic stem cells for the development of clinically applicable cell therapy drugs should be mass-producible. The current feeder-free culture system is considered to be economically inexpensive in mass production .

In the meantime, development of a cell therapy agent using embryonic stem cells is an ethical problem because the embryo must be destroyed during the process of obtaining embryonic stem cells. Differentiation of embryonic stem cells into cells required for treatment, And there is a risk of tumor formation when transplanted cells are not completely differentiated. In order to solve this problem, it has been suggested that induced pluripotent stem cells (ESCs), which induce pluripotent stem cells similar to embryonic stem cells through reprogramming in differentiated cells, ; iPS cell) (Cell 132, 567-582, 2008) technology has been developed. The iPS culture also has a problem in mass production of the embryonic stem cells described above.

As a background of the present invention, Korean Patent Laid-Open Publication No. 10-2007-0079586 (2007.08.07) discloses a method for growing human embryonic stem cells using human placental stromal cells, a method for differentiating embryoid bodies, and a method for differentiating into hematopoietic stem cells . Korean Patent Laid-Open No. 10-2010-0006452 (Jan. 19, 2010) describes a method for culturing human embryonic stem cells using human umbilical cord fibroblasts as support cells. However, in this case, it is labor-intensive to cover the supporting cells, so the economy is low and the possibility of contamination due to the heterogeneous protein is still a problem.

Therefore, there is a continuing need for a new stem cell in vitro culture technique capable of economically producing a large amount of embryonic stem cells or embryonic stem cell-like cells while avoiding contamination with different proteins or cells such as immunological rejection.

Throughout this specification, numerous articles or patent documents are referenced and citations are indicated. The disclosure of the cited article or patent document is incorporated herein by reference in its entirety to more clearly describe the state of the art to which the present invention pertains and the content of the present invention.

Korean Patent Publication No. 10-2007-0079586 (2007.08.07) Korean Patent Publication No. 10-2010-0006452 (Jan. 19, 2010)

1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science < / RTI > 1998 Nov 6; 282 (5391): 1145-7. 2. Wiles MV, Johansson BM. Embryonic stem cell development in a chemically defined medium. Exp Cell Res 1997, 25: 247 (1): 241-8. 3. Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001 Oct; 19 (10): 971-4. 4. Rosler ES, Fisk GJ, Ares X, Irving J, Miura T, Rao MS, et al. Long-term culture of human embryonic stem cells in feeder-free conditions. Dev Dyn2004 Feb; 229 (2): 259-74. 5. Park Y, Choi IY, Lee SJ, Lee SR, Sung HJ Kim JH, et al. Undifferentiated propagation of the human embryonic stem cell lines, H1 and HSF6, on human placenta-derived feeder cells without basic fibroblast growth factor supplementation. Stem Cells Dev Nov; 19 (11): 1713-22. 6. Park Y, Kim JH, Lee SJ, Choi IY, Park SJ, Lee SR, et al. Human Feeder Cells Can Support the Undifferentiated Growth of Human and Mouse Embryonic Stem Cells Using Their Own Basic Fibroblast Growth Factors. Stem Cells Dev Feb 25. 7. Genbacev O, Krtolica A, Zdravkovic T, Brunette E, Powell S, Natha, et al. Serum-free derivation of human embryonic stem cell lines on human placental fibroblast feeders. Fertil Steril 2005 May; 83 (5): 1517-29. 8. McDonagh S, Maidji E, Ma W, Chang HT, Fisher S, Pereira L. Viral and bacterial pathogens at the maternal-fetal interface. J Infect Dis 2004 Aug 15; 190 (4): 826-34.

To solve these problems, the present inventors have developed a technique for culturing human embryonic stem cells using only conditioned medium for human placenta-derived supporting cells without Matrigel. The human placenta is an organ that provides an essential environment for the fetus to survive in the adult body during pregnancy and is now being classified as a medical waste after childbirth. However, as the fact that cells constituting the chorion of the human placenta are extracted and used as supporting cells in the culture of human embryonic stem cells (non-patent document 0005), its utility is emerging. In addition, bFGF, which is essential for maintaining the undifferentiated state of human embryonic stem cells, is produced in the human placenta-derived supporting cells, and when the human placenta-derived supporting cells are used, the undifferentiated state of human embryonic stem cells can be maintained without additional supply of bFGF (Non-Patent Document 0006). The present inventors focused on this characteristic of human placenta-derived cells, and cultured medium in which no cytokine was added was exposed to human placenta-derived supporting cells for 24 hours (HPC conditioning) conditioned media. We succeeded in maintaining a large amount of human embryonic stem cells or induced pluripotent stem cells (iPS) in an undifferentiated state for a long period of time in a cell culture dish coated with gelatin alone.

Accordingly, an object of the present invention is to provide a conditioned medium and a preparation method thereof for placenta-derived cells prepared by collecting the cells after exposure to placenta-derived cells (PC conditioning).

It is another object of the present invention to provide a method for culturing an animal and non-myeloid stem cell culture that does not use support cells in a state in which the direct and indirect contact of animal xenogeneic proteins and cells is completely eliminated, Method.

It is yet another object of the present invention to provide a method for producing embryonic stem cells which does not require Matrigel or bFGF and which is more economical than a conventional serum-free, feeder-free culture system Culturing technique.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

In order to achieve the above object, the present invention provides a method for producing placenta-derived cells, comprising the steps of: (a) inoculating placenta-derived cells onto a gelatin-coated well plate; (b) culturing the placenta-derived cells by adding a cell culture solution; And (c) collecting only the culture medium. The present invention also provides a method for preparing conditioned medium for placenta-derived cells.

The present invention also provides a conditioned cell culture medium for the placenta-derived cells produced by the above-described method.

The present invention relates to a method for preparing a placenta-derived cell, comprising the steps of: adding the conditioned cell culture medium to a cell culture dish coated with gelatin; And inoculating stem cells into the cell culture dish.

Hereinafter, the present invention will be described in detail.

(A) inoculating placenta-derived cells onto a gelatin-coated well plate; (b) culturing the placenta-derived cells by adding a cell culture solution; And (c) collecting only the culture medium. The present invention also provides a method for preparing conditioned medium for placenta-derived cells.

In the present invention, the placenta means a placenta separated from a mother after birth, and the placenta can be stored in a sterilized container and ice after being separated.

Although not limited thereto, in the step (a), the placenta-derived cells may be separated from the chorionic plate and subcultured in a DMEM culture medium, and the placenta-derived cells may be treated with trypsin and irradiated.

Although not limited thereto, it is preferable that the placenta-derived cells in the step (a) are placenta-derived fibroblast-like cells cultured separately from the human chorionic plate.

In the step (b), the cell culture solution may be a conventional culture solution in which fecal serum and the like are excluded. But is not particularly limited to, DMEM / F-12 containing a serum substitute, a sulfating agent and an antibiotic. In a more specific embodiment, 10 ml of DMEM / F-12 supplemented with 20% Knockout Serum Replacer (GIBCO), 0.1 mM β-mercaptoethanol, and 1% penicillin-streptomycin (Sigma) can be used.

Although it is not limited thereto, it is preferable to cultivate in step (b) for 20-30 hours, more preferably for 24 hours.

In the present invention, stem cells are all cells capable of dividing indefinitely and capable of differentiating into specific cells under suitable environmental conditions, and include adult stem cells, embryonic stem cells, and embryonic stem cell-like cells . That is, it includes both stem cells having pluripotency, multipotency, or unipotency. Although not limited thereto, the stem cells are preferably embryonic stem cells or inducible pluripotent stem cells (iPS).

In addition, the present invention provides the placenta-derived cells produced by the above method with a conditioned stem cell culture medium.

In addition to bFGF, which is essential for maintaining the undifferentiated state of human embryonic stem cells secreted by the placenta-derived cells, conditioned medium for the placenta-derived cells according to the present invention is also characterized by at least one of the following: atypical methylation of the interleukin- .... the metastatic potential of breast carcinoma cells Proc Natl Acad Sci USA 100:... 13988-13993), austenite-off Rotterdam gerin (Osteoprotegerin, Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation Cell 93, 165-176 (1998)), uPAR (uPAR: a versatile signaling orchestrator. Nat Rev Mol Cell Biol 3: 932-943 (2002)), Identification of Tapr (an airway hyperreactivity regulatory locus) Nat Immunol 2001; 2:. 1109- .16), TIM-2 (The TIM gene family: emerging roles in immunity and disease Nat. Rev Immunol 3:.. 454-462), IGFBP-6 (Insulin-like growth factor binding protein-6 activates programmed cell death in non-small cell lung cancer cells Oncogene 2000; 19: 4432-4436.), ICAM-1 (Structural plasticity in Ig superfamily domain 4 ICAM-1 mediates cell surface dimerization. Proc Natl Acad Sci USA 104: 15358-15363.), Angiogenin, ANG mutations segregate with familial and sporadic amyotrophic lateral sclerosis. Nat . Genet 2006, 38: 411- .413), BDNF (Cell Type-Specific Loss of BDNF Signaling Mimics Optogenetic Control of Cocaine Reward Science 2010; 330-385), IGFBP-4 (IGFBP-4), insulin-like growth factor-4 and -5 in vascular smooth muscle cells, J. Biol . Chem . 273, 16836- 2 (insulin-like growth factor binding protein 2 is a growth inhibitory protein conserved in zebrafish. Proc Natl Acad Sci USA 96: 15274-15279), IL-6 (Impaired immune and acute-phase responses in interleukin-6-deficient mice, Nature 368, 339-342), GLP-2 (the proglucagon- like peptide-2, is a neurotransmitter involved in the regulation of food intake. Nat Med , 6 (2000), pp. 802-807; Glucagon-like Peptide (GLP) -2 Reduces Chemotherapy-associated Mortality and Enhancement Cell Survival in Cells Expression of MCP-1 as a key regulator of proliferation and MCP-1 secretion in synoviocytes from patients with rheumatoid arthritis. Ann Rheum Dis. 2011 ; 61: 687-693. Mar; 70 Suppl 1: i88-91), GRO (Growth-regulated oncogene is pivotal in thrombin-induced angiogenesis. Cancer Res. 2006 Apr 15; 66 (8): 4125-32) regulates human embryonic stem cell self-renewal or adoption of a neuronal fate. Differentiation 81 (2011) 222-232) is a group contains one or more cytokines selected from the consisting of (14 ~ Reference 17) (Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol . 2006 Feb; 24 (2): 160-1.)

Preferably, the culture medium for conditioned stem cells of the placenta according to the present invention comprises at least one cytokine selected from the group consisting of IL-8, MCP-1, TIMP-2, and GRO-a .

Further, the present invention relates to a method for preparing a cell culture, comprising the steps of: adding placenta-derived cells prepared by the above method to a cell culture dish coated with gelatin; And inoculating stem cells into the cell culture dish.

The stem cell culture method can be used for culturing stem cells of various animals such as human, dog, sheep, and the like. Preferably, the placenta is a human placenta, and the stem cell is an embryonic stem cell or inducible pluripotent stem cell (iPS), and the embryonic stem cell is more preferably a human embryonic stem cell.

Conventional conditioned media generally have been contaminated with xenogeneic proteins or cells using animal cells (MEF) derived from mice. The present inventors first established a culture technique using placenta-derived cells in human cells using conditioned media (HPC-conditioned media). Therefore, it is possible to prevent contamination with different proteins or cells, and thus to improve the medical clinical application efficiency of human embryonic stem cells or induced pluripotent stem cells (iPS).

In addition, the conditioned medium for the placenta-derived cells according to the present invention contains various cytokines and proteins that help maintain and survive the undifferentiated state of the stem cells. Thus, the conventional feeder-free culture system It is not necessary to apply Matrigel to the cell culture dish. The present invention is capable of culturing stem cells such as human embryonic stem cells in a state in which only gelatin is applied without expensive Matrigel, which is very economical.

Furthermore, the conventional feeder-free culture system is complicated and costly because continuous supply of bFGF to the culture medium is essential for maintaining the undifferentiated state of human embryonic stem cells. However, The medium contains bFGF, which is essential for maintaining the undifferentiated state of human embryonic stem cells secreted by placenta-derived cells, and is economical because it does not require an additional supply of bFGF.

As shown in FIGS. 3 to 10, the undifferentiation of stem cells cultured using the conditioned culture medium for the animal-derived and non-animal-derived placenta-derived cells according to the present invention was confirmed by measuring the activity of alkaline phosphatase (ALP) . It was also confirmed that the stem cells cultured according to the present invention express typical SSEA4, TRA-60 and TRA-81 cell surface markers on undifferentiated cells. Furthermore, it was confirmed that the undifferentiated markers Rex1, Nanog, and Oct4 were well expressed in the embryonic stem cell line according to the present invention regardless of whether bFGF was added or not.

Embryonic stem cells have the ability to differentiate into trichomes (ectoderm, mesoderm, endoderm) in vivo or in vitro. According to the present invention, in a gelatin-coated plate, placenta-derived cells were cultured in a conditioned medium of stem cell culture to induce differentiation in vitro, (Fig. 11 and Fig. 12). In addition, the ability to differentiate was confirmed once again by quantitative real-time PCR analysis for quantitative gene expression analysis (FIG. 13).

Therefore, according to the present invention, stable and undifferentiated stem cells can be cultured in vitro for a long time in a large amount.

The present invention also relates to the use of a compound of formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of IL-8, osteoprotegerin, uPAR, TIMP-1, TIMP-2, IGFBP-6, ICAM-1, Angiogenin, BDNF, IGFBP- Derived cells comprising placenta-derived cells comprising at least one cytokine selected from the group consisting of IL-2, IL-6, GCP-2, MCP-1, GRO and GRO-a. Preferably, but not exclusively, the conditioned medium of the placenta-derived cells according to the present invention comprises at least one cytokine selected from the group consisting of IL-8, MCP-1, TIMP-2, and GRO- .

In order to determine the composition of conditioned medium for the placenta-derived cells according to the present invention, a total of 120 cytokines were detected through a human cytokine array developed by RayBiotech Inc., USA (FIG. 14 ) Was compared with the mTeSR of STEM CELL TECHNOLOGIES Co., which is sold as a control group. As a result, it was possible to select 17 cytokines with 5 times or more difference in expression. Among them, bFGF, which is essential for maintaining the undifferentiated state of human embryonic stem cells, was added in a considerable amount in mTeSR sold, but embryonic stem cells It was confirmed that the embryonic stem cells retained undifferentiated state and succeeded in long-term culture even though the expression was not relatively high in the culture medium (Fig. 15). MMP-1, TIMP-2, and GRO-a were the four cytokines that showed the greatest difference in the culture conditions that maintained the undifferentiated state of human embryonic stem cells. The exact amount of the components was measured by an enzyme linked immunosorbent assay (ELISA) (FIGS. 16 and 17).

As described above, according to the present invention, there is provided a method for culturing an animal and an avian stem cell, which does not use a supporting cell, in a state in which direct contact or indirect contact of an animal xenogeneic protein or cell is completely eliminated, Can be prevented, and human embryonic stem cells can be cultured for a long period of time while maintaining undifferentiated state in vitro.

In addition, according to the present invention, human embryonic stem cells can be cultured using only cultured conditioned media on human placenta-derived supporting cells without Matrigel, and there is no need to continuously add expensive bFGF, thereby remarkably reducing the cost of cultivation, Human stem cells can be mass produced in vitro and stably for a long period of time.

Therefore, the present invention is a very useful invention in the field of cell therapy using stem cells such as embryonic stem cells.

Fig. 1 is a photograph showing a super-culturing process of human placenta-derived cells. (A) Four days after inoculation into cell culture dishes: Several types of cell populations that are not identical yet are observed. (B) Cell culture plate Two weeks after inoculation: Fibroblast-like cells can be used to identify homogeneously organized cell populations. Magnification (AB): X40
FIG. 2 is a photograph showing the effect of addition of bFGF on placenta-derived cells according to the present invention to PC conditioned media (hereinafter, used in combination with PC-CM). The human placenta-derived cells were cultured in HPC conditioned media without the addition of bFGF to H1 embryonic stem cell lines. It can be seen that the human embryonic stem cell line community remains undifferentiated even if bFGF is not added to the conditioned medium of the placenta-derived cells (picture taken at passage 10).
FIG. 3 is a photograph showing a result of confirming that the stem cell marker ALP is expressed well in the H1 human embryonic stem cell line community cultured with PC-conditioned media only in placenta-derived cells according to the present invention (10 Photographs taken in the subway).
4 is well expressed in H1 human embryonic stem cell line populations cultured in PC-conditioned media in which the undifferentiated markers of human embryonic stem cells, SSEA4, TRA60, TRA81 and OCT4, were placenta-derived cells according to the present invention This is a photograph showing the result of confirming the existence of the image (this is a photograph taken on the 10th transmission line).
FIG. 5 shows that H1 human embryonic stem cell lines cultured using PC-conditioned media for placenta-derived cells according to the present invention exhibit undifferentiated markers Rex1, Nanog and Oct4 regardless of bFGF addition (RT-PCR result in 10 passages).
FIG. 6 shows the results of comparative incubation of H1 cultured with placenta-derived cells according to the present invention using conditioned medium (PC-conditioned media) under different conditions. It can be seen that the gelatin-coated culture dish, which is the condition of the present invention, maintains the most undifferentiated state in the culture condition (PCCM-bFGF (G)) of the culture solution in which bFGF is not separately added. (PCCM + bFGF (G)) on the MATRIGEL, the group cultured in the same culture medium (PCCM-bFGF (M)) sold on the same culture medium, the group to which bFGF was added on gelatin (PCCM + bFGF ). The cells were cultured in the same cell line H1 under the above four conditions. The number of cells in each culture dish was different from that of n = 4, and the number of undifferentiated cells in the total number of cells was calculated as a percentage (%) Indicate.
FIG. 7 is a photograph taken at a magnification x40 of a human embryonic stem cell line cultured under the respective experimental conditions of FIG. 6 (photograph taken at 10 passages). the degree of differentiation between the group to which bFGF was added and the group to which no bFGF was added is clearly distinguished, and it can be seen that the gelatin-coated culture dish according to the present invention maintains the most undifferentiated state in the culture condition of the culture solution without addition of bFGF. Were examined by alkaline phosphatene active staining.
FIG. 8 is a photograph showing a culture of a human induced pluripotent stem cell iPSC cultured with a culture medium (PC-CM-bFGF) conditioned only in a placenta-derived cell to which a bFGF was not added in a gelatin-coated culture dish according to the present invention (Taken at 8 magnifications with a magnification of X40).
FIG. 9 shows that human embryonic stem cell lines (H1) and human induced pluripotent stem cells (H1) were cultured in a gelatin-coated culture dish according to the present invention after culturing the placenta-derived cells in which the bFGF was not separately added, (IPSC) expresses the well-expressed Rex1, Nanog, and Oct4 genes that are stem cell undifferentiated markers.
FIG. 10 shows that human embryonic stem cell line (H1) and human induced pluripotent stem cell (HGF) cells were cultured on a gelatin-coated culture dish with culture medium (PC-CM-bFGF) conditioned only in placenta- (SSEA-1) is a well-expressed expression of stromal cells (iPSC), staining with stem cells undifferentiated markers Nanog, TRA-60, TRA-81, SSEA-4 and OCT- Negative control, DAPI is nuclear stained for presence of cells, taken at magnification x40).
FIG. 11 is a graph showing the results obtained by culturing a placenta-derived cell in which bFGF was not separately added in a gelatin-coated culture dish with a conditioned culture medium (PC-CM-bFGF) alone, (Embryoid bodies) for 2 weeks in vitro, and then incubated for 10 days on gelatin-coated culture plates. Then, AFP (endoderm), a marker for trichotomies, DESMIN (mesodermal), TUJ1 (ectodermal) were stained with immunostaining method and then photographed at a magnification of 40 at a fluorescence microscope. The pluripotent stem cells were differentiated into pluripotent stem cells.
Fig. 12 shows that human induced pluripotent stem cells (iPSC) were cultured under the conditions of Fig. 11 only in a culture medium (PC-CM-bFGF) conditioned with placenta-derived cells in which bFGF was not separately added to a gelatin-coated culture dish according to the present invention This is a photograph showing the result. Human induced pluripotent stem cells were also cultured by the present invention as well as embryonic stem cell lines without losing the inherent characteristics of the stem cells.
FIG. 13 shows the results of quantitative real-time PCR analysis for quantitative gene expression analysis of cells cultured according to the present invention. RNA was extracted from the embryoid - like cell masses induced for 2 weeks to synthesize complementary DNA, and the expression of each gene was examined. In the control group, human embryonic stem cell lines which did not induce differentiation were used, and GAPDH, a house keeping gene, was used to confirm and standardize the complementary DNA synthesis. As a result, it was confirmed that the expression of genes encoding endoderm, mesoderm, and ectoderm are normally performed in the group that induces undifferentiated markers such as OCT-4 and NANOG.
FIG. 14 is a graph showing the relationship between the total amount of 120 proteins and the total amount of the proteins, using a cytokine antibody array kit sold by Raybiotec (Raybiotech Inc., www.raybiotech.com) in the United States to confirm the composition of the conditioned culture medium of the placenta- And the degree of expression was confirmed. As a control group, a basic culture solution not exposed to human placenta derived cells and mTeSR of a stem cell technologies company currently sold were used. The results of the experimental group were normalized to the results of the basal culture used as the control group. Among them, cytokines were selected which differ significantly from the mTeSR group.
FIG. 15 shows a result of measuring the membrane array results of FIG. 14 using density. On the left side, the values of each spot were quantified by using a program for measuring optical density (sicon), and the average value was used to group the cytokines with the expression difference of at least 5 times. And the yellow squares indicate that the expression of the test group is extremely high. On the right, five graphs with the highest expression are shown in red, which are graphically displayed for easy comparison.
FIG. 16 shows the ENTREZE ID and protein name of the top four cytokines showing significant expression differences in FIG.
FIG. 17 is a graph showing the results of an enzyme-linked immunosorbent assay (ELISA) performed to measure the precise amount of the upper four cytokines showing a remarkable expression difference in FIG. As a control group, a basic culture solution (Con1) and a commercially available mTeSR (Con2) which were not exposed to the placenta-derived cells were used. In the experimental group, five randomly selected samples of the present invention were repeated three times (Unit = pg / ml).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  One: Of human placental derived cells  production

Chorionic plates were surgically removed from the placenta of healthy women and incubated at 37 ° C for 30 min in 0.25% trypsin-EDTA (GIBCO-Invitrogen, Carlsbad, Calif.). After that, the cells were cultured in DMEM (Dulbecco's modified Eagle medium) supplemented with 20% fetal bovine serum (FBS; HyClone Laboratories Inc, Logan, UT, 100 U / ml penicillin, and 100 μg / ml streptomycin) ₂ , and 95% humidity.

The medium was changed every 3-4 days to the passage. During incubation, we removed cellular debris floating in the culture medium and found placenta-derived fibroblast-like cells attached to the plate. At about 2 weeks of inoculation, colonies of fibroblast-like cells attached to the plate were formed (see Fig. 1).

HPCs are cultured to 12 ^th passage, which was stored (stock) to all the cells. The frozen stock was subjected to trypsin treatment and irradiation (1500 cGy) followed by an amount of 1 x ¹⁰⁶ cells per cryovial. Placenta-infected pathogens were tested by RT-PCR prior to freezing into stock. These include cytomegalovirus, herpes simplex virus types 1 and 2, Chlamydia trachomatis , Chlamydia spp , Mycoplasma genitalium, Mycoplasma hominis , and Ureaplasma ureaticum . The disclosed primer sequences were used (Non-Patent Document 0007). HPCs positive for pathogenicity were discarded.

Example  2: Placenta-derived cells  Conditioned medium ( PC - conditioned media ) Production

The stored HPCs were thawed and inoculated onto 0.1% gelatin-coated 35-mm well plates at 1 x ¹⁰⁶ cells / well. 10 ml of DMEM / F-12 supplemented with 20% Knockout Serum Replacer (GIBCO), 0.1 mM β-mercaptoethanol, and 1% penicillin-streptomycin (Sigma) was added to the culture dish inoculated with HPC, After culturing at 5% CO ₂ and 95% humidity, only the culture broth was separately extracted and refrigerated at 4 ° C.

Example  3: Placenta-derived cells  Conditioned Culture media (PC-conditioned media)  Long-term in vitro maintenance and culture of undifferentiated human embryonic stem cells using

Human placenta-derived cells were seeded on gelatin-coated cell culture dishes with conditioned medium (HPC-conditioned media), and H1 human embryonic stem cell lines were inoculated into cell culture dishes at 37 ° C, 5% CO ₂ , and 95% Lt; / RTI > Human placenta-derived cells were transfected with conditioned medium (HPC-conditioned media) every 48 hours. H1 human embryonic stem cells were subcultured on a new gelatin-coated cell culture dish every week, and it was confirmed that they were cultured in vitro while maintaining undifferentiated state to at least 20 passages.

In order to confirm the undifferentiated state, the cells were observed with an inverted microscope every day (Fig. 2) and the expression of stem cell markers was examined every 5 passages. (3), (4), (7), and (10) of the stem cell markers, respectively, were alkaline phosphatase (ALP), Oct-4, stage specific embryonic antigen (SSEA) -4, tumor rejection antigen (TRA) , Oct-4, Nanog, and Rex-1 were confirmed by RT-PCR (Fig. 5).

The primers used for RT-PCR were as follows;

Oct-4, 577bp:

CGACCATCTGCCGCTTTGAG (forward, SEQ ID NO: 1)

CCCCCTGTCCCCCATTCCTA (reverse, SEQ ID NO: 2),

Nanog, 149bp:

AAAGAATCTTCACCTATGCC (forward, SEQ ID NO: 3),

GAAGGAAGAGGAGAGACAGT (reverse, SEQ ID NO: 4),

Rex-1, 306 bp:

CAGATCCTAAACAGCTCGCAGAAT (forward, SEQ ID NO: 5),

GCGTACGCAAATTAAAGTCCAGA (reverse, SEQ ID NO: 6).

Co-cultures used for immunocytochemistry were established in 6-well plates. Prior to analysis, adherent cell layers were fixed by addition of 10% formalin at room temperature (15 min), permeabilized with 0.1% Triton X-100 / PBS for 10 min and incubated with primary antibody at 4 ° C Lt; / RTI > The primary antibody to SSEA-4 was purchased from Hybridoma Bank (Hybricoma Bank, IA), and other antibodies were purchased from Chemicon (Chemicon, CA). ALP activity was detected using a commercial kit (Sigma). RT-PCR for stem cell markers was also performed. Total RNA was prepared using a QIAGEN RNeasy kit (Qiagen-Hilden, Germany) and reverse transcription was performed by priming 500 ng of total RNA with a random hexamer using AMVreverse transcriptase (Roche Molecular Biochemicals, Germany). After PCR, the products were analyzed on 1.5% agarose gel and visualized with ethium bromide.

Further, the placenta-derived cells according to the present invention were subjected to a comparative experiment using a conditioned medium of the stem cell culture and matrigel which was commercialized as a control. Human embryonic stem cell line H1, which has been sold in wicell research institute in USA, was used. Measurement of differentiation of stem cells was confirmed through measurement of alkaline phosphatase (ALP) activity (Fig. 7). As a result, in the gelatin-coated plate according to the present invention, the placenta-derived cells of the group (PCCM-bFGF (G)) cultured with the conditioned embryonic stem cell culture medium were undifferentiated to the 17th passage (period of about 4 to 5 months) (Figure 6 and Figure 7).

Example  4: Placenta-derived cells  Conditioned Culture media (PC-conditioned media)  Of undifferentiated state Induced pluripotent stem cells (iPS)  culture

The human embryonic stem cell line H1 and the human induced pluripotent stem cell line iPS (Foreskin-1) were tested in the wicell research institute of the United States. The human induced pluripotent stem cell line iPS was also subcultured in the undifferentiated state as in the human embryonic stem cell line H1 (FIG. 8). As shown in Example 3, the differentiation of stem cells was evaluated by gene amplification technology (FIG. 9) and SSEA4, TRA-60 and TRA-81, Rex-1, Nanog and oct-4, undifferentiated markers of stem cells, (Fig. 10).

Example  5: Placenta-derived cells  Conditioned Culture media (PC-conditioned media)  Human embryonic stem cells and undifferentiated human embryonic stem cells Induced pluripotent stem cells (iPS)  In vitro culture Ability to distinguish  Confirm ( Differentiation in vitro )

Human embryonic stem cells are capable of differentiating into trichomes (ectoderm, mesoderm, endoderm) in vivo or in vitro. In the gelatin-coated plate according to the present invention, human placenta-derived cells The cells cultured with the conditioned embryonic stem cell culture medium were all differentiated into the trichomes when inducing differentiation in vitro.

1) Immunohistochemical staining method

According to the present invention, in a culture condition in which human stem cells or iPSCs growing in an undifferentiated state are cultured in an amount of 14 or more passages and then subjected to enzymatic treatment (dispase) to separate colonies and form an embryoid body, (suspension culture), followed by transfer to a gelatin-coated dish and adherent culture for 10 days. The differentiation-induced culture medium conditions included 80% DMEM-F12, 20% KONCK-OUT SERUM, 0.1 mM β-mercaptoethanol, and 1% nonessential amino acids. The attached cells were stained with immunocytochemistry to determine whether they could differentiate into endoderm, mesoderm, and ectoderm. Adherent cell layers were fixed at room temperature by addition of 4% formalin (15 min), permeabilized with 0.1% Triton X-100 / PBS for 10 min and over-knit with primary antibody at 4 ° C. The primary antibody for AFP and Desmin was purchased from SANTA CRUZ BIOTECHNOLOGY, Inc. and TUJ1 was purchased from Chemicon (Chemicon, CA). The results are shown in Fig. 11 and Fig.

2) Quantitative PCR analysis:

Total RNA isolation of human embryonic stem cells, human embryoid bodies, and human induced pluripotent stem cells was performed using High Pure RNA Isolation kit (Qiagen), 200 units of reverse transcriptase and 500 ng of oligo (dT) Were synthesized. The cells were treated with 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl ₂ , 10 mM dithiothreitol, and 1 mM dNTPs and reacted at 42 ° C for 1 hour. Real-time polymerisation was performed using 1 x SYBR Green mix (Invitrogen) with iCYCLER (BioRad). Denaturation was carried out at 94 ℃ for 30 sec, annealing at 55 ℃ for 30 sec and extension at 72 ℃ for 1 min. The results are shown in Fig.

Example  6: Placenta-derived cells  Conditioned The culture medium (PC-conditioned media)  Analysis of composition and quantitative analysis of content, identification of key candidate substances for maintenance of undifferentiated human stem cell culture

In order to confirm the composition of the conditioned medium of the stem cell culture medium according to the present invention as compared with the conventional commercialized human embryonic stem cell culture materials, the human cytokine array developed by RayBiotech Inc. of USA was used as follows A total of 120 cytokine expression levels were confirmed and compared with mTeSR from STEM CELL TECHNOLOGIES Co., Ltd., which was sold as a control.

1) Human cytokine antibody array:

The conditioned medium of the present invention and the medium used as a control are each subjected to two duplicate spots on a nitrocellulose membrane printed with 60 cytokines, followed by blocking, incubation, washing, and biotin-conjugated antibodies After addition, streptavidin-conjugated peroxidase was reacted with ECL chemiluminescence reagent and developed using autoradiographic film (BioMax Lite, Kodak). Randomly selected 5 samples were repeatedly tested and a total of 120 cytokines were measured. The results of the experimental group were normalized to the results of the basal culture used as the control group. Among them, cytokines were selected which differ significantly from the mTeSR group. The results are shown in Fig. 14 to Fig.

2) ELISA (Enzyme-linked immunosorbent assay):

Experiments were conducted using a Humna ELISA kit developed by Raybiotech to determine the precise contents of the candidates obtained through the cytokine measurement experiment in the present invention. As in the previous experiment, control and experimental groups were used identically and standardization of four cytokines was established. For the procedure, 100 μl of the test group and the control sample were added, followed by overnight reaction at 4 ° C., followed by addition of biotin antibody and incubation for 1 hour. After reacting with Streptavidin solution and TMB One-Step Substrate Reagent, the reaction was stopped with 50 μl Stop Solution and absorbance was measured at 450 nm using a microplate reader. The controls and samples of each cytokine were triplicated. The results are shown in Fig.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. It will be clear to those who have.

Attach an electronic file to a sequence list

Claims (10)

(a) inoculating human placenta-derived cells onto a gelatin-coated well plate;
(b) culturing the placenta-derived cells by adding a cell culture solution; And
(c) collecting only the culture medium. 2. The method of claim 1, wherein the cultured human embryonic stem cell or human induced pluripotent stem cell culture medium is cultured. The method according to claim 1, wherein the placenta-derived cells in step (a) are placenta-derived fibroblast-like cells cultured separately from a human chorionic plate, A method for producing a stem cell culture medium. The method according to claim 1, wherein in step (b), the cell culture medium is DMEM / F-12 containing a serum substitute, wherein the cultured human embryonic stem cell or human induced pluripotent stem cell culture medium Way. The method according to claim 1, wherein the step (b) is performed for 20 to 30 hours. The method for producing conditioned human embryonic stem cells or human induced pluripotent stem cell culture medium. 4. A human embryonic stem cell or human induced pluripotent stem cell culture medium conditioned on a placenta-derived cell prepared by the method according to any one of claims 1 to 4. 6. The method of claim 5, wherein the stem cell culture medium is selected from the group consisting of IL-8, osteoprotegerin, uPAR, TIMP-1, TIMP-2, IGFBP-6, ICAM-1, Angiogenin, BDNF Derived cells comprising placenta-derived cells comprising at least one cytokine selected from the group consisting of IGFBP-4, IGFBP-2, IL-6, GCP-2, MCP-I, GRO, and GRO- Human induced pluripotent stem cell culture medium. Adding placenta-derived cells prepared by the method according to any one of claims 1 to 4 to conditioned cells of human embryonic stem cell or human induced pluripotent stem cell culture medium to a cell culture dish coated with gelatin; And
And inoculating said cell culture dish with human embryonic stem cells or human induced pluripotent stem cells. delete delete IL-8, osteoprotegerin, uPAR, TIMP-1, TIMP-2, IGFBP-6, ICAM-1, Angiogenin, BDNF, IGFBP-4, IGFBP- , GCP-2, MCP-1, GRO, and GRO-a. The human embryonic stem cell or human induced pluripotent stem cell culture medium is conditioned on human placenta derived cells.

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