WO2021233116A1 - Chemically-defined and xeno-free culture system for pluripotent stem cells - Google Patents

Abstract

Provided is a chemically-defined and xeno-free cell culture medium composition system. Extended pluripotent stem cells under this new system can be stably amplified in vitro for a long time; and the pluripotency, the cell characteristics, and the genomic stability are maintained.

Description

Pluripotent stem cells have a clear, heterogeneous culture system Technical field

The invention relates to a culture system and a culture method of an expanded pluripotent stem cell, in particular to a culture system and a culture method of the expanded pluripotent stem cell with clear components and no heterogeneity.

Background technique

Pluripotent stem cells have strong application value due to their self-renewal and ability to differentiate into various types of cells. How to cultivate and differentiate into functionally mature cells in vitro is a challenge for pluripotent stem cells. In order to promote its further clinical application, it is necessary to establish a culture system with a defined composition and no animal origin. At present, there are many media developed for culturing pluripotent stem cells, but there are problems such as unstable genome in long-term culture.

Expanded pluripotent stem cells (EPS cells) are a stem cell line with intra-embryonic and extra-embryonic developmental potential established by the present inventors in 2017, which provide a new tool for studying the regulation of cell developmental potential. In addition, due to the expanded developmental potential of EPS cells, it has stronger chimerism and germline transmission capabilities. A single mouse EPS cell can obtain a mouse developed from a single EPS cell through the tetraploid compensation technology. Human EPS cells also have strong cross-germline (human-mouse chimerism) chimerism ability, which helps to explore the human lineage differentiation process in vitro. It is worth noting that the latest research on EPS cells shows that it can form a blastocyst-like structure in vitro, providing an in vitro platform for the study of early embryonic development. In summary, the current research results related to EPS cells show the great advantages of EPS cells in basic and transformational research.

Recently, Wang et al. found that hEPS cells can generate functionally mature liver cells through directed differentiation. The study found that compared with liver cells derived from traditional pluripotent stem cells, liver cells derived from EPS cells are highly similar to human primary liver cells in terms of gene regulation network. Through a simple two-step method, high-efficiency targeted differentiation can be achieved. First, hEPS cells are pretreated to turn them into primed pluripotent stem cells, and then the traditional PSCs differentiation protocol can be used for liver-induced differentiation. From a methodological point of view, only hEPS cells need to be pretreated, which can be applied to the differentiation of hEPS cells to produce cells of other lineages. Therefore, hEPS cells are expected to produce a variety of functional cell types for clinical applications. However, the clinical transformation of cell types derived from PSCs requires a heterogeneous culture system. Therefore, in order to promote the wide application of hEPS cells, a heterologous-free EPS cell culture system must be developed, and this problem still needs to be solved urgently.

Summary of the invention

The purpose of the present invention is to provide an expanded pluripotent stem cell with a clear composition and no heterologous culture system, under the culture system, the expanded pluripotent stem cell can be expanded in vitro for a long time and stably without losing its Pluripotency and cell characteristics and genome stability. Preferably, the composition, kit, culture system and method provided by the present invention can be used for culturing pluripotent stem cells under feeder-free conditions, for example, culturing expanded pluripotent stem cells under feeder-free conditions.

Specifically, the present invention relates to the following aspects:

1. A composition or kit, preferably, it can be a cell culture medium composition or kit, preferably, it can be used in an expanded pluripotent stem cell culture system, characterized in that it comprises the following components : Catalase, Vitamin C (VC), Activin A and Laminin 521 (Laminin 521).

2. The composition or kit according to item 1, characterized in that it comprises the following components:

(1)ITSX;

(2) Catalase,

(3) Vitamin C,

(4) Activin A,

(5) Cytokines,

(6) GSK3b inhibitor,

(7) GPCR inhibitors,

(8) PARP inhibitor,

(9) ROCK inhibitor,

(10) Laminin 521, and

Optionally, (11) WNT inhibitor.

3. A composition or kit, preferably, it can be a cell culture medium composition or kit, preferably, it can be used in an expanded pluripotent stem cell culture system, characterized in that it comprises the following components :

(1)ITSX;

(4) Activin A,

(5) Cytokines,

(6) GSK3b inhibitor,

(7) GPCR inhibitors,

(8) PARP inhibitor,

(9) ROCK inhibitor,

(10) Laminin 521, and

Optionally, (11) WNT inhibitor;

Preferably, the composition or kit further includes (2) catalase and/or (3) vitamin C.

4. The composition or kit according to item 2 or 3, wherein the ITSX is a combination of insulin, transferrrin, sodium selenite and ethanolamine;

Preferably, the transferrin includes apo-transferrin (apo-transferrin) or holo-transferrin (holo-transferrin);

Optionally, the concentration of the insulin is 10-50 μg/ml; optionally, the concentration of the transferrin is 5.5-27.5 μg/ml; optionally, the concentration of the sodium selenite is 1- 10ng/ml; optionally, the concentration of the ethanolamine is 20000x-50000x.

5. The composition or kit according to any one of items 1-3, wherein the basal medium of the composition or kit is a mixed medium of DMEM/F12 and Neurobasal;

Preferably, the volume ratio of the DMEM/F12 and the Neurobasal is (0.8-1.2):(0.8-1.2); more preferably, the volume ratio is 1:1.

6. The composition or kit according to item 2 or 3, wherein the (5) cytokine is selected from the group consisting of: human leukemia inhibitory factor (LIF, "L"), interleukin (IL) -6, IL-11, IL-27, IL-31, leukemia inhibitory factor, oncostatin M, cardiotrophin-1, neuropoietin and cardiotrophin-like cytokine 1;

Preferably, the (5) cytokine is human LIF.

7. The composition or kit according to item 2 or 3, wherein the (6) GSK3b inhibitor is selected from the group consisting of: CHIR99021 ("C") [6-[[2-[[4- (2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile]; Bio[ (2'Z,3'E)-6-bromoisatin-3'-oxime]; TWS119; BIO-acetoxime; GSK 3I inhibitor XV; SB-216763; CHIR 99021 trihydrochloride; GSK-3 inhibition Agent IX[((2Z,3E)-6'-bromo-3-(oximino)-[2,3'-dihydroindolyl]-2'-one]; GSK 3IX[6-bromoindigo Red-3'-oxime]; GSK-3β inhibitor XII [3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]phenol ]; GSK-3 inhibitor XVI[6-(2-(4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-pyrimidin-2-yl Amino)ethyl-amino)-nicotinonitrile]; or SB-415286[3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole- 2,5-Diketone];

Preferably, the (6) GSK3b inhibitor is at least one of CHIR99021, Bio, and TWS119.

8. The composition or kit according to item 2 or 3, wherein the (7) GPCR inhibitor is (S)-(+)-dimethindidine maleate, tripinamin hydrochloride, or dimethicone At least one of loratadine.

9. The composition or kit according to item 2 or 3, wherein the (8) PARP inhibitor is at least one of minocycline hydrochloride, BSI-201 or nicotinamide.

10. The composition or kit according to item 2 or 3, wherein the (9) ROCK inhibitor is at least one of Y-27632 or fasudil.

11. The composition or kit according to item 2 or 3, wherein the (11) WNT inhibitor is at least one of IWR-1-endo or XAV939.

12. The composition or kit according to any one of items 1-3, wherein the composition or kit is further added with 1%-5% of xeno-free culture base.

13. The composition or kit according to item 2 or 3, wherein the concentration of (2) catalase is 1000x-5000x; optionally, the concentration of (3) vitamin C is 50- 200μg/ml; optionally, the concentration of (4) Activin A is 5-100 ng/ml; optionally, the concentration of (5) cytokine (preferably human LIF) is 10-15 ng/ml; Optionally, the concentration of the (6) GSK3b inhibitor is 0.8-1.2 μM; optionally, the concentration of the (7) GPCR inhibitor is 1-5 μM; optionally, the (8) PARP inhibitor Optionally, the concentration of (9) ROCK inhibitor is 2-10 μM; optionally, the concentration of (10) Laminin 521 is 2.5-10 μg/ml (for example, 2.5 μg/ ml); Optionally, the concentration of the (11) WNT inhibitor is 0.5 μM.

14. A medium (preferably used for culturing expanded pluripotent stem cells), which comprises the composition according to any one of items 1-13;

Preferably, the basic medium of the medium is a mixed medium of DMEM/F12 and Neurobasal;

Preferably, the volume ratio of the DMEM/F12 and the Neurobasal is (0.8-1.2):(0.8-1.2); more preferably, the volume ratio is 1:1.

15. A method for culturing expanded pluripotent stem cells, characterized in that the expanded pluripotent stem cells are cultured in the medium described in item 14.

16. A method for establishing a line of expanded pluripotent stem cells, characterized in that the somatic cells are reprogrammed and then seeded into the medium described in item 14; preferably, the somatic cells are fibroblasts.

Description of the drawings

Figure 1 shows the development of a heterogeneous culture system for human expanded pluripotent stem cells.

(a) Flow cytometric analysis of the proportion of OCT4-Tdtomato+H1-EPS cells treated with multiple factors for 2 generations. n=2. None means LCDM medium.

(b) is an analysis of the number of human EPS cells cultured in feeder-free cells under the action of vitamin C and catalase and their combinations. Among them, VC, vitamin C. n=3. Use ES1-EPS cells.

(c) Analysis of the number of human EPS cells cultured with feeder-free cells treated with different basal media. n=4. Use ES1-EPS cells.

(d-f) shows the analysis of the effect of different matrix proteins on human EPS cells cultured without feeder cells. Among them, (d) is the adhesion ratio of human EPS cells inoculated for 1.5 hours. (e) is the survival efficiency of human EPS cells 24 hours after inoculation. (f) Proliferation efficiency of human EPS cells inoculated for 72 hours. Among them, the index in (e-f) represents the number of cells at a specific time point divided by the number of inoculated cells. (d-f), n=3. Use ES1-EPS cells.

(g) shows the representative clonal morphology of non-heterologous human EPS cells of different genetic backgrounds.

Error bars represent standard errors. *P<0.05. Ruler, 100μm.

The experiments in (a-g) were all repeated independently at least twice, with similar results.

Figure 2 shows the characteristics of human EPS cells without allogenes.

(a) The doubling time analysis of feeder cell co-culture and heterologous human EPS cells. n=3.

(b) shows the single cell clone formation efficiency of feeder cell co-culture and heterologous human EPS cells. n=32.

(c) shows the karyotype analysis of heterologous human EPS cells with different genetic backgrounds.

(d) shows the ratio of OCT4+ or NANOG+ in human EPS cells without heterologous analysis by flow cytometry.

(e) shows that feeder cell co-culture and heterologous human EPS cells mainly utilize OCT4 distal enhancer elements. The primed human PSC (H1 and iPS) was used as a control. In the designated cell line, the luciferase reporter system is used to detect the reporter activity of the distal enhancer to evaluate OCT4 transcriptional regulation. Empty vector transfection as the basic activity. n=3.

(f) is a representative picture of in vivo teratomas produced by human EPS cells without heterologous origin. Use XF H1-EPS cells.

(g) is a 4.5-day chimeric embryo image of human EPS cells without allogeneic. mclover is an anti-mclover antibody. Arrows indicate mClover+ cells with OCT4 and CDX2 expression, respectively.

(h) The picture shows the chimerism of human EPS cells without heterologous in E6.5 chimera. Use mclover-XF ES1-EPS cells.

Among them, in (a, b and e), F-LCDM means human EPS cells co-cultured with feeder cells; XF-LCDM means human EPS cells without heterologous origin. Error bars represent standard errors. *P<0.05. Ruler, 100μm. The experiments in (a-h) were all repeated independently at least twice, with similar results.

Figure 3 shows the important factors that determine the culture of feeder-free human EPS cells.

(a) is the expression of OCT4 and Tdtomato in H1-EPS cells knocked into OCT4-Tdtomato reporter gene.

(b) The picture shows the morphology and expression of OCT4-Tdtomato H1-EPS cells treated with different factors. The cells were treated for 2 passages under feeder-free conditions. Among them, BF is bright field. None means LCDM medium.

(c) The immunostaining image shows that OCT4 and NANOG are expressed in OT H1-EPS cells without feeder cells treated with Activin A.

(d) The picture shows the morphology of feeder-free H1-EPS cells cultured with vitamin C, catalase, and a combination thereof. Among them, VC is vitamin C. The simplified LCDM means that N2 and B27 are replaced with ITSX, and Activin A is added at the same time.

Ruler, 100μm. (a-d) The experiments in (a-d) are all repeated independently at least twice, and the results are similar.

Figure 4 shows that Laminin 521 supports the cultivation of heterologous human EPS cells.

(a) is crystal violet staining showing the viability of H1-EPS cells cultured on different matrix proteins for 72 hours.

(b) The picture shows the morphology of OT H1-EPS cells seeded on Laminin 521 for 24, 48 and 72 hours. The picture shows the expression of OCT4-tdtomato 72 hours after inoculation.

(c) Flow cytometry analysis of Neu5Gc expression in human EPS cell culture medium under different conditions. Use ES1-EPS cells.

Among them, F-LCDM means that human EPS cells are cultured on feeder cells, using LCDM medium. F-LCDM+10% FBS means that human EPS cells are cultured on feeder cells, using LCDM plus 10% FBS medium. FF-LCDM said that human EPS cells are cultured on feeder-free cells, using LCDM medium without allogeneic sources. Isotype control means using isotype antibodies instead of anti-Neu5GC antibodies and human EPS cells without feeder cells cultured with heterologous LCDM. NC said that no antibodies are used, and feeder-free human EPS cells cultured with heterologous LCDM are used.

Scale bar: 100μm. The experiments in (a-c) were all repeated independently at least twice, with similar results.

Figure 5 shows the characteristics of human EPS without heterologous sources

(a-b) Flow cytometric analysis of the ratio of EDU+ in feeder cell cultured and heterologous human EPS cells.

Among them, (a) is the flow analysis graph, and (b) is the flow analysis statistics graph. n=2.

(c-d) Survival and proliferation of heterologous human EPS cells treated with Rock inhibitor. Use XF H1-EPS cells. The index represents the number of cells at a specific time point divided by the number of inoculated cells. (c-d), n=3.

(e) Use different cell digestion reagents to treat the cells. Use XF ES1-EPS cells. Cells are cultured in 24-well plates. Count the cells after digestion. n=4.

(f) Insert the mclover reporter gene into the AAVS1 site in human EPS cells without heterologous origin. Use XF ES1-EPS cells. Above, the picture represents the expression of mclover in ES1-EPS cells without allogenes. BF, bright field. In the figure below, genomic PCR verified that the mclover reporter gene was inserted into the AAVS1 site in ES1-EPS cells without heterologous origin. CK1 and CK2 are primers that specifically detect the homologous recombination of the 3'arm and 5'arm of the targeting vector.

(g) Karyotype analysis of EPS of early generation non-heterologous humans with different genetic backgrounds.

(h) Flow cytometric analysis of the signal of γ-H2AX in feeder cell culture and heterologous human EPS cells. n=2.

Among them, F-LCDM means that human EPS cells are cultured on feeder cells; XF-LCDM means that there is no heterologous human EPS cells.

Error bars represent standard errors. *P<0.05. Scale bar, 100μm. (a-h) The experiments in (a-h) are all repeated independently at least twice, and the results are similar.

Figure 6 shows the molecular characteristics of non-heterologous human EPS cells

(a) The immunostaining image shows the expression of pluripotency marker genes in non-heterologous human EPS cells.

(b) Q-PCR analysis of the expression of pluripotency marker genes in non-heterologous human EPS cells. n=3.

(c) The immunostaining image shows the expression of H3K27me3 in female human EPS cells that are not heterologous.

(d) Correlation analysis of full gene expression between EPS cells cultured in feeder cells and non-heterologous EPS cells.

F-LCDM, human EPS cells are cultured on feeder cells; XF-LCDM, no heterologous human EPS cells.

Error bars represent standard errors. The experiments in SEM. scale, 100μm. (a-c) were all repeated independently at least twice, and the results were similar.

Figure 7 shows the differentiation potential of human EPS cells without allogenes

(a) The immunostaining image shows the expression of differentiation marker genes in the derivative of non-heterologous human EPS cells after EB differentiation. Use XF ES1-EPS cells.

(b) The picture shows the histological analysis of teratomas produced by human EPS cells without heterologous origin.

(c) The picture shows the FACS analysis of the expression of KRT7 and GATA3 in human EPS derivatives without heterologous trophoblast differentiation. NC, no secondary antibody is added. Use XF H1-EPS cells.

(d) Q-PCR analysis of the expression of trophoblast marker genes in the trophoblast differentiation of non-heterologous human EPS cells. Use XF H1-EPS cells. n=3. The gene expression at different time points was standardized by D0.

(e) The immunostaining image shows the expression of HLA-G and hCGβ in trophoblast-like cells derived from human EPS without heterologous origin. Use XF H1-EPS cells.

(f) The picture shows the chimerism of human EPS cells without heterologous in 4.5-day blastocysts. The table below the figure is the statistical table of the chimeric experiment.

Error bars represent standard errors. *P<0.05. Scale bar, 100μm. (a-f) The experiments in (a-f) are all repeated independently at least twice, and the results are similar.

Figure 8 shows the production of heterologous human EPS cells from human fibroblasts by reprogramming

(a) The picture shows the process of producing heterologous human EPS cells from human fibroblasts through reprogramming

(b) The picture shows the changes in cell morphology during the reprogramming process. The image on the right shows the morphology of human EPS cells without allogeneity at the first generation.

(c) The immunostaining image shows the expression of OCT4 and SOX2 in selected human EPS-like clones produced from human fibroblasts.

(d-e) The picture shows the morphological ruler of human EPS clone formed after inoculation of clone (d) and single cell (e), 100μm. The experiments in (b-d) were all repeated independently at least twice, with similar results.

Figure 9 shows the characteristics of establishing heterologous human EPS cells from human fibroblasts

(a) The picture shows the morphology of non-heterogeneous human EPS cells derived from human fibroblasts.

(b) The immunostaining image shows the expression of pluripotency marker genes in non-heterologous human EPS cells produced in human fibroblasts.

(c) The immunostaining image shows the expression of differentiation marker genes in non-heterologous human EPS in EB differentiated cells. Human EPS cells without heterologous origin were established from human fibroblasts.

(d) The picture shows the histological analysis of teratomas produced by human fibroblasts without heterologous human EPS cells.

(e) Karyotype analysis of heterologous human EPS cells produced by human fibroblasts.

(f) The correlation analysis of full gene expression between human EPS cells cultured in feeder cells and non-heterologous human EPS cells produced by human fibroblasts. F-LCDM, human EPS cells are cultured on feeder cells; XF-LCDM, no heterologous human EPS cells.

Ruler, 100μm. The experiments in (a-d) were all repeated independently at least twice, with similar results.

Figure 10 shows that different concentrations of components in the culture system of the present invention can support the culture of feeder-free EPS.

(a) is the crystal violet staining of ITSX with different concentrations.

(b) EPS morphology images and OCT4-Tdtomato fluorescence images of different concentrations of Actinvin A.

(c-e) is the morphological map of EPS cultured with different concentrations of catalase, vitamin C and Y 27632.

Ruler, 100μm.

Figure 11 shows the in vitro and in vivo extra-embryonic development ability of human EPS cells without heterologous

(a) is the morphology of non-heterogeneous human EPS cells in the trophoblast stem cell culture medium. Ruler, 100μm.

(b) The immunofluorescence image shows that trophoblast stem cell-specific transcription factors (KRT7/GATA3/NR2F2) and pluripotent transcription factors (OCT4/SOX2) are used in non-exogenous human EPS cells and similar trophoblast stem cells transformed from them. Express the situation. Ruler, 100μm.

(c) Q-PCR analysis of the expression of trophoblast stem cell marker genes and pluripotency marker genes in non-heterologous human EPS cells and similar trophoblast stem cells transformed from them. n=3.

(d) is a 4.5-day chimeric embryo image of human EPS cells without heterologous. mclover is an anti-mclover antibody. Arrows indicate mClover+ cells with OCT4 and CDX2 expression, respectively. Ruler, 100μm.

(e) The chart shows the chimerism and chimerism experiment statistics of human EPS cells without heterologous in E6.5 chimera. Use mclover-XF ES1-EPS cells. Ruler, 500μm.

(f) is a 6.5-day chimeric embryo image of human EPS cells without allogeneic. GFP is an anti-mclover antibody. Arrows indicate mClover+ cells with OCT4 and CK18 expression, respectively. Ruler, 50μm.

(g) Mitochondrial Q-PCR analysis of the chimerism ratio of human EPS cells without heterologous in the 10.5-day human-mouse chimera. Different color blocks represent the mosaic ratio in different tissues (embryo/placenta/yolk sac). The red dotted line indicates that there is one human-derived cell among 10,000 mouse cells. n=2.

The experiments in (a-g) were all repeated independently at least twice, with similar results.

Detailed ways

In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

In the following examples, unless otherwise specified, the methods used are all conventional methods, and all reagents used can be obtained from commercial sources.

I. Definition

In the present invention, the pluripotent stem cells include expanded pluripotent stem cells and induced pluripotent stem cells. The term "pluripotent" (or multipotent) refers to having differentiation into three germ layers: endoderm (e.g., internal stomach wall, gastrointestinal tract, lung), mesoderm (e.g., muscle, bone, blood, genitourinary system) ) Or ectoderm (e.g., epidermal tissue and nervous system). The term "non-pluripotent" means that the cell does not have the potential to differentiate into all three germ layers.

In the present invention, the EPS cell is an expanded pluripotent stem cell, which means that when compared with the type of pluripotent stem cell from which it is derived, by contacting the donor cell with a chemical compound, it has an improved ability to produce extra-embryonic lineage in vivo The pluripotent stem cells have the following characteristics: ①have the dual ability to develop into endo-embryonic tissues and extra-embryonic tissues, ②have some properties similar to naive pluripotent stem cells, ④can produce teratomas, ⑤not obvious The differentiation bias of ⑥ has the ability of single cells to form clones. Among them, for the preparation method of EPS cells, see, for example, CN108884436 (or PCT/CN2015/086854), which is incorporated herein by reference in its entirety. Specifically, it can be obtained by using commercially available reprogramming kits or other methods to transfer donor cells (such as blood-derived cells, embryo-derived cells, skin-derived cells, fibroblasts, adipocytes, epithelial cells, endothelial cells). , Mesenchymal cells, parenchymal cells, nerve cells and connective tissue cells) are cultured in a reprogramming medium comprising the cell culture medium composition of the present invention or the cell culture medium composition described in CN108884436 for a sufficient period of time to make Obtained by cell reprogramming. Stem cells may be stem cells isolated or obtained from human embryos that have not undergone in vivo development within 14 days of fertilization.

The method for establishing an expanded pluripotent stem cell line is the method for preparing an expanded pluripotent stem cell line.

As used herein, the term "induced pluripotent stem cell" (iPSC) is a type of pluripotent stem cell artificially derived from non-pluripotent cells. Induced pluripotent stem cells can be prepared by reprogramming the cells to be induced. Reprogrammed cells can be obtained by inducing partially or fully differentiated cells obtained from mammals, such as any mammals (such as cows, sheep, pigs, dogs, cats, horses, primates), preferably humans. Sources include: bone marrow, fibroblasts, embryonic tissues (such as tissues isolated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development), peripheral blood, cord blood, pancreas, skin or any organ or tissue. Useful cell types that can be induced include but are not limited to: pluripotent stem cells, blood-derived cells, embryo-derived cells, skin-derived cells, fibroblasts, adipocytes, epithelial cells, endothelial cells, mesenchymal cells , Parenchymal cells, nerve cells and connective tissue cells.

As used herein, the concentration unit of the liquid components such as ethanolamine is calculated as "x", which is related to the volume of the added component, that is, the volume of the added component x the concentration of the added component = the culture medium total capacity. For example, when preparing a 20 ml medium, if the concentration of the component is 20000x, the addition volume of the component is 1 μl.

II. Composition

About expanded pluripotent stem cell culture composition

In the expanded pluripotent stem cell culture system of the present invention, the insulin is preferably human insulin with a concentration of 10-50 μg/ml. Specifically, the concentration of the insulin may be 10-15 μg/ml, 10-20 μg/ml, 10-25 μg/ml, 10-30 μg/ml, 10-35 μg/ml, 10-40 μg/ml, 10-45 μg/ml. ml, 10-50μg/ml, 15-20μg/ml, 15-25μg/ml, 15-30μg/ml, 15-40μg/ml, 15-50μg/ml, 20-25μg/ml, 20-35μg/ml, 20-45μg/ml, 30-40μg/ml, 30-50μg/ml or 40-50μg/ml.

In the culture system of the expanded pluripotent stem cells of the present invention, the transferrin is apotransferrin or hototransferrin, preferably human-derived transferrin, with a concentration of 5.5-27.5 μg/ml. Specifically, the concentration of the transferrin may be 5.5-6.0 μg/ml, 5.5-6.5 μg/ml, 5.5-7.0 μg/ml, 5.5-7.5 μg/ml, 5.5-8.0 μg/ml, 5.5-8.5 μg/ml, 5.5-9.0μg/ml, 5.5-10.0μg/ml, 5.5-11.0μg/ml, 5.5-12.0μg/ml, 5.5-13.0μg/ml, 5.5-15.0μg/ml, 5.5-20.0μg /ml, 5.5-27.5μg/ml, 6.0-7.0μg/ml, 6.0-8.0μg/ml, 6.0-9.0μg/ml, 6.0-10.0μg/ml, 6.0-15.0μg/ml, 7.0-8.0μg/ ml, 7.0-10.0μg/ml, 7.0-15.0μg/ml, 7.0-20.0μg/ml, 10.0-15.0μg/ml, 10.0-20.0μg/ml, 10.0-25.0μg/ml, 15.0-20.0μg/ml , 15.0-25.0μg/ml.

In the expanded pluripotent stem cell culture system of the present invention, the concentration of the sodium selenite is 1-10 ng/ml. Specifically, its concentration can be 1-2ng/ml, 1-3ng/ml, 1-4ng/ml, 1-5ng/ml, 1-6ng/ml, 1-7ng/ml, 1-8ng/ml, 1 -9ng/ml, 2-3ng/ml, 2-4ng/ml, 2-5ng/ml, 2-6ng/ml, 2-10ng/ml, 3-4ng/ml, 3-5ng/ml, 3-6ng /ml, 3-8 ng/ml, 3-10ng/ml, 4-6ng/ml, 4-8ng/ml, 4-10ng/ml, 5-8ng/ml, 5-10ng/ml, 6-8ng/ ml, 6-10ng/ml.

In the expanded pluripotent stem cell culture system of the present invention, the concentration of the ethanolamine is 20000x-50000x. Specifically, the concentration may be 20000x-30000x, 20000x-40000x, 20000x-50000x, 30000x-40000x, 40000x-50000x.

In the expanded pluripotent stem cell culture system of the present invention, the catalase is human or bovine catalase, and its concentration is 1000x-5000x. Specifically, the concentration may be 1000x-2000x, 1000x-3000x, 1000x-4000x, 1000x-5000x, 2000x-3000x, 2000x-4000x, 2000x-5000x, 3000x-4000x, 3000x-5000x, 4000x-5000x.

In the expanded pluripotent stem cell culture system of the present invention, the concentration of the vitamin C is 50-200 μg/ml. Specifically, the concentration may be 50-100 μg/ml, 80-120 μg/ml, 100-120 μg/ml, 100-150 μg/ml.

In the expanded pluripotent stem cell culture system of the present invention, the concentration of Activin A is 5-100 ng/ml. Specifically, its concentration can be 5-20ng/ml, 5-30ng/ml, 5-40ng/ml, 5-50ng/ml, 5-60ng/ml, 5-80ng/ml, 5-100ng/ml, 10 -20ng/ml, 10-40ng/ml, 20-40ng/ml, 20-50ng/ml, 30-40ng/ml, 40-50ng/ml.

In the expanded pluripotent stem cell culture system of the present invention, the preferred cytokine is human leukemia inhibitory factor (LIF) ("L) used in a concentration range of 10-15ng/ml, preferably 10ng/ml, that is, leukemia Type 6 cytokines, which can act as JAK-STAT signaling pathway activating molecules to function in cell culture. IL-6 is a prototype four-helix bundle cytokine, which is neuropoietin (a group of structurally related Founding members of cytokines, including IL-6, IL-11, IL-27, IL-31, leukemia inhibitory factor, oncostatin M, cardiotrophin-1 (cardiotrophin-1), neuropoietin and cardiotrophin Protein-like cytokine 1 (also known as neoneutrophin 1 and B cell stimulating factor-3) and two viral analogs of IL-6. These members of the interleukin 6 family of cytokines can be disclosed for LIF Equivalent concentrations are used in the compositions disclosed herein.

In the expanded pluripotent stem cell culture system of the present invention, the GSK3b inhibitor is selective for GSK3. Suitable GSK3b inhibitors are CHIR99021, Bio or TWS119, which are glycogen synthase kinase 3 inhibitors. The concentration range is 0.8-1.2 μM, preferably 1.0-1.2 μM, more preferably 1.0 μM.

Other available GSK3b inhibitors are commercially available and can be used in the compositions disclosed herein. Examples include, but are not limited to, BIO-acetoxime; GSK 3I inhibitor XV; SB-216763; CHIR 99021 trihydrochloride; GSK-3 inhibitor IX[((2Z,3E)-6'-bromo-3-( Oxime)-[2,3'-dihydroindolyl]-2'-one]; GSK 3 IX[6-bromoisatin-3'-oxime]; GSK-3β inhibitor XII[3- [[6-(3-Aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]phenol]; GSK-3 inhibitor XVI[6-(2-(4- (2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-pyrimidin-2-ylamino)ethyl-amino)-nicotinonitrile]; or SB-415286 [3-[(3-Chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione].

In the expanded pluripotent stem cell culture system of the present invention, the (7) GPCR inhibitor refers to a G protein coupled receptor (GPCR) inhibitor. A preferred GPCR inhibitor is (S)-(+)-Dimethindene maleate ((S)-(+)-Dimethindene maleate) used in a concentration range of 1-5 μM, more preferably 2 μM. Dimethinidine maleate is an enantiomer, which is a subtype selective mAChR (muscarinic acetylcholine receptor) M2, mAChR M1, mAChR M3 and mAChR M4 antagonist and histamine H1 receptor antagonist . However, other GPCR inhibitors may be included in the CEP composition disclosed herein, and they include, but are not limited to, ethylenediamines, for example, tripelennamine HCL (competitively blocks central and peripheral histamine H1 receptor histamine H1 antagonist), mepyramine and antazoline; tricyclic or tetracyclic, such as loratidine or its metabolite desloret Desloratadine (selective histamine H1 antagonist).

In the expanded pluripotent stem cell culture system of the present invention, the (8) PARP inhibitor is preferably minocycline hydrochloride used at 2-5 μM, more preferably 2 μM. Other PARP inhibitors include but are not limited to BSI-201201 (4-iodo-3-nitrobenzamide) or nicotinamide.

In the preferred expanded pluripotent stem cell culture system of the present invention, the cytokine is leukemia inhibitory factor LIF ("L"); the GSK3b inhibitor is aminopyrimidine with the chemical name [6-[[2-[ [4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile] CHIR99021 ("C"); G protein-coupled receptor (GPCR) inhibitors are acetylcholine receptor antagonists, preferably (S)-(+)-dimethindine maleate ("D"); and The PARP inhibitor is minocycline hydrochloride ("M"). This preferred mixture is referred to herein as LCDM and can be used to condition pluripotent cells in vitro, thereby expanding their ability to generate embryonic and extra-embryonic lineages.

In the expanded pluripotent stem cell culture system of the present invention, the (9) ROCK inhibitor refers to an inhibitor of coiled-coil protein kinase (ROCK), and its concentration is 2-10 μM, preferably 5 μM. Specifically, the concentration can be 2-5μM, 2-8μM, 3-5μM, 3-8μM, 3-10μM, 4-6μM, 4-8μM, 4-10μM, 5-6μM, 5-8μM, 6-8μM . The ROCK inhibitor used in the present invention is preferably Y-27632[(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide+ ++ dihydrochloride)] or fasudil, more preferably Y-27632.

In the culture system for expanded pluripotent stem cells of the present invention, the concentration of the laminin 521 (Laminin 521) is 2.5-10 μg/ml. Specifically, the concentration may be 2.5-4.0 μg/ml, 2.5-5.0 μg/ml, 2.5-6.0 μg/ml, 2.5-7.0 μg/ml, 2.5-8.0 μg/ml, 2.5-9.0 μg/ml, 2.5 -10.0μg/ml, 4.0-5.0μg/ml, 4.0-6.0μg/ml, 4.0-7.0μg/ml, 4.0-8.0μg/ml, 4.0-9.0μg/ml, 4.0-10.0μg/ml, 5.0- 7.0μg/ml, 5.0-8.0μg/ml, 5.0-10.0μg/ml, 7.0-8.0μg/ml, 7.0-10.0μg/ml, 8.0-10.0μg/ml.

Exogenous-free culture system for determining the composition of extended pluripotent stem cells

Experimental materials and operating methods

1. Preparation of non-heterogeneous human EPS cell culture medium

Take 50ml as an example: 25ml DMEM/F12, 25ml Neurobasal, 10μg/ml human insulin, 5.5μg/ml transferrin, 1ng/ml sodium selenite, 20000x ethanolamine, 5000x human catalase, 100μg/ml vitamin C , 5-40ng/ml Activin A, 10ng/ml human LIF, 1μM CHIR99021, 2μM(S)-(+)-Dimethindene maleate, 2μM Minocycline hydrochloride, 5μM Y-27632 and 2.5μg/ml Laminin521. For most cell lines, 0.5μM IWR-endo-1 is recommended. If it is not necessary to be free of heterologous sources, cattle-derived catalase (Sigma, C1345) can be used to replace human-derived catalase in the culture medium. The prepared medium can be placed at 4°C for 1 week.

2. Prepare the cell plate coated with extracellular matrix Laminin 521.

XF-EPS cells were cultured on Laminin 521 matrix, Laminin 521 was diluted 1:40 with DPBS (Thermo Fisher Scientific, 2069089) with calcium and magnesium ions, and 350μl of coated culture plate was added according to one well of each 24-well plate, and then Place the culture plate at 37°C and incubate for at least 2 hours or 4°C overnight for coating. Note that the coated culture plate cannot be dried, because drying will inactivate the protein coated on the bottom of the plate. The coated board can be stored at 4°C for a week.

3. Culture and passage of heterologous human EPS cells.

The heterogeneous EPS is cultured in a constant temperature incubator with 20% O2, 5% CO2, and 37°C. It needs to be passaged every 3-4 days (or when the confluence reaches 85%-95%), and pass it at a ratio of 1:6-1:10. Generally, there is no need to change the medium, unless the density of inoculated cells is greater than 2 million per plate. When passaging, first wash the EPS cells with DPBS (Thermo Fisher Scientific, 14190136) without calcium and magnesium ions, and then add an appropriate amount of 0.5X TrypLE Select (ThermoFisher Scientific, 12563029) to cover the cells, and place them in a 37℃ incubator for digestion 3 Take it out after a minute, remove the digestion solution, blow it into single cells with culture medium, and then inoculate directly. Other digestive enzymes can also be used, such as Trypsin-EDTA (Thermo Fisher Scientific, 25300-062) or Accutase (Millipore, SCR005).

4. EPS cells cultured in the feeder layer are transformed into a heterogeneous EPS culture system.

After digestion, EPS cells were resuspended in an appropriate amount of EPS medium with feeder layer (N2B27-LCDM), and inoculated on Laminin 521 culture plate, and switched to XF-EPS medium after 24 hours. In the first 3-5 passages, it is best to use a high-density passage ratio (1:3) to improve cell viability and reduce screening pressure. With passage, the cells will proliferate better and better. In addition, for cell lines with poor adaptability, it is recommended to add 1%-5% non-heterogeneous KSR (xeno-free KSR, ThermoFisher Scientific, 12618013) to help improve cell survival and proliferation. Ordinary KSR also has a similar effect.

5. Switch the traditional hPSCs to the XF hEPS culture system.

First, remove the hPSC medium and wash away cell debris and non-adherent cells with DMEM/F12 medium. Traditional hPSCs were digested with ReleSR enzyme into small clones and inoculated on Laminin 521-coated culture plates, cultured in mTeSR1 medium for the first 24 hours, and then switched to XF-EPS medium. Similarly, the addition of 1-5% non-heterogeneous KSR can improve the survival and proliferation rate of cells, and the first 3 passages are best to be passaged at high density. After 10 passages, the cell culture system can be switched.

6. Cryopreservation of XF-EPS cells without animal origin

After XF-EPS cell digestion, centrifuge at 300g for 5 minutes, remove the supernatant, and add 1 mL of pre-chilled cryopreservation solution (

D10Cryopreservation Medium, Biological Industries, 05-713) resuspend the cell pellet, then transfer the cell suspension to a cryotube, place the cryotube in a gradient cryostat box, and immediately store it at -80°C for 24 hours, then Transfer to a liquid nitrogen tank for long-term storage.

7. Proliferation experiment

In order to analyze the proliferation rate of EPS cells with or without feeder culture, both EPS cells were digested and re-plated at 80,000/well. After 72 hours, 5μM EDU was added to the detection well and incubated at 37°C for 2.5 hours. There are 3 multiple holes in the experiment, and the control group refers to the condition without adding EDU. After incubation, the cells were digested, and the cells were fixed and stained according to the standard of the kit (Thermo Fisher Scientific, C10424), and finally tested on the machine (BD FACS Verse), using FlowJo software (Ashland) for data analysis.

8. Double time detection

The experiment was carried out in a 24-well plate. EPS cells and XF-EPS cells under Feeder conditions were digested and seeded on feeder cells or Laminin 521-coated culture plates at 40,000 cells/well, and counted every 24 hours. The doubling time calculation formula: DT=48*[log2/(lgNt (total number of cells on the fourth day)-lgNo (total number of cells on the second day))].

9. Analyze the cloning efficiency of single cells

In order to analyze the single cell cloning efficiency of XF-EPS cells, the cells were digested into single cells and seeded on a feeder layer or a 96-well culture plate coated with Laminin 521 at a density of 1, 10, 100 per well. After 5 days, count the number of clones in each well, and finally count the average value of the cloning efficiency of EPS cells in the three densities as the final result.

10. Immunofluorescence

EPS cells were fixed with 4% paraformaldehyde (DingGuo, AR-0211) at room temperature for minutes, and then used with PBS (Corning, 21-040-CVR), 0.25% Triton X-100 (Sigma-Aldrich, T8787), 3% Donkey serum (Jackson Immuno Research, 017-000-121) was blocked at room temperature for 45 minutes. The primary antibody diluted according to a certain ratio was incubated overnight at 4°C, washed with PBS three times the next day, and the secondary antibody diluted solution was added to incubate at room temperature for 1 hour. The nucleus was stained with DAPI (Roche Life Science, 0236276001). The antibody information is shown in Table 1 below.

Table 1 Antibodies used for immunofluorescence and flow cytometry analysis

11. Streaming analysis

After the cells are digested, fix the cells with BD Cytofix/Cytoperm solution (BD, 554714) at 4°C for 20 minutes, then wash three times with PBS, add the primary antibody and incubate for at least 4 hours, and incubate the secondary antibody for 1 hour. Then wash the cells, test on the machine (BD FACS Verse), and use FlowJo software to analyze the experimental data. In the experiment, each processing condition is set up with multiple wells. Among them, the used antibody information is shown in Table 1.

12. EB formation experiment

XF-EPS digested cells into a single cell, a density of 5 x ¹⁰⁵ seeded on low attachment 6-well plate using IMDM medium + 15% serum (VISTECH, VIS8618005) in the week of culture, medium was changed every 3 days. Then inoculate it into Matrigel-coated 24-well plates and continue culturing for one week, changing the medium every 3 days. Then fix the EB on the culture plate, perform immunofluorescence staining and detect.

13. Teratoma and immunohistochemical experiments

Approximately 5*10 ⁶ EPS cells were resuspended in 50μl culture medium, and then mixed with an equal volume of Matrigel previously melted on ice. The cell mixture was injected subcutaneously into NPG mice, and after 4-8 weeks, the formation of subcutaneous teratomas in the mice could be seen. When the diameter of the tumor body exceeded 1.5 cm, the mice were sacrificed and the teratoma removed was fixed. Then dehydration, embedding, subsequent sectioning and staining are performed.

14. Karyotype analysis

The sample was collected when the cell growth confluence reached 50±70%, and after incubating in fresh medium for 2 hours, 0.02 mg/ml colchicine was added and incubated for 1 hour at room temperature. Use PBS to wash cells, digest cells, and settle. In order to obtain a single cell suspension, resuspend it with a hypotonic solution (0.56% KCl) and place it at room temperature for 6 minutes. After centrifugation, the hypotonic solution was removed, and 5 mL (3:1 methanol:acetic acid) was added in the next step, and allowed to settle for 5 minutes at room temperature. The fixation step was repeated 3 times. Finally, add 1mL fixative to resuspend. Then the cell suspension was added dropwise to the pre-chilled 5% acetic acid±ethanol solution and stained with Giemsa. Each cell line analysis will detect 30 ± 40 metaphases. Simultaneously detect the number of chromosomes and abnormal chromosome structure.

15. Trophoblast differentiation

XF-EPS cells were digested and seeded into Matrigel-coated 24-well plates at a cell density of 25,000 per well, and XF-EPS cell culture medium was used for the first 24 hours. Start differentiation the next day and switch to differentiation medium: 40ml DMEM/F12+40ml IMDM (Thermo Fisher Scientific, 12440-053) medium with 20% KSR (Thermo Fisher Scientific, A3181502), 10ng/ml BMP4, 1μM A83 -01 (Tocris, 2939), 0.1μM PD173074 (Selleck, S1264). Samples were collected and analyzed on the 1-8th day of differentiation. The FACS sample was fixed as described above, and the mRNA was extracted with Trizol reagent.

16.OCT4 remote enhancer detection

First, the OCT4-DE luciferase plasmid and its internal reference plasmid pGL4.74 were co-transformed into different types of cells (hPSC, hEPS with and without feeder layer). After transfection, the cells were respectively seeded on the corresponding medium and matrix-coated 96-well plates, and seeded at a density of 5000 cells per well, requiring three replicate wells. After 3 days, the cells were lysed and tested with a dual luciferase detection kit.

17. Chimera Experiment

Cells required for chimerization experiments need to ensure the best culture condition. On the day of digestion, the cell confluence needs to reach 70%. After the cells are digested, filter them with a 40um screen to ensure a single cell suspension. Centrifuge at 450-600g for 3 minutes, remove the supernatant, and resuspend in the culture medium to a cell density ^{of 2-6*10 5 /mL.} Place the cells on ice for 20-30 minutes. After that, the injection is started, usually 10-12 cells/8-cell embryos are injected. If blastocysts are cultured in vitro, the embryos injected with cells need to be cultured in KSOM for 48 hours. If it is a chimera that develops in the body, it is necessary to transfer the embryo after the injection of the cells to a pseudopregnant female mouse, and then perform analysis and testing.

18. Establish EPS cell line from fibroblasts

The separated fibroblasts were seeded into a 6-well plate at ^{1*10 5} /cm ^{2, and then cultured for 1-2 days.} CytoTuneTM-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific, A16517) was used to transfect fibroblasts. 5-7 days after transfection, the transfected fibroblasts were re-seeded on a Laminin 521-coated 6-well plate at a rate of ^{1*10 5} /cm ^2. After 24 hours, the medium was changed to a heterologous-free EPS medium supplemented with 1-5% non-heterologous KSR. After about 14 days, hEPS-like clones can be seen. At this time, clones can be picked or digested into single cells for inoculation. After 4-7 days, clones can be observed to grow up. In order to improve the survival rate and the efficiency of line establishment, it is recommended that the first three generations adopt mechanical method for passage. After the third generation, the EPS cell line established from fibroblasts can proliferate well.

19. Quantitative PCR

Use Direct-zol RNAKits (ZYMO Research, R2051) to isolate total RNA. TransScript first-strand cDNA synthesis SuperMix (TransGen Biotech, AT301) was used to convert RNA into cDNA. Quantitative PCR analysis was performed using the KAPA SYBR FAST qPCR kit (KAPA Biosystems, KK4601) with the BioRAD CFX Connect real-time system. The primers used for Q-PCR analysis (SEQ ID NO: 1-24) are listed in Table 2.

Table 2

20. Mitochondrial PCR detection

Use DNeasy Blood&Tissue Kit (QIAGEN, 69506) to extract total DNA. For human-specific mitochondrial DNA fragments, a total amount of 18 ng is used for the DNA sample, and mitochondrial DNA fragments common to humans and mice are used as internal controls. Finally, the relative expression value was calculated using the δδCT method, and finally the relative expression value was calculated with the mouse pregnancy tissues that were not injected with XF-EPS cells. Table 2 lists the primers used for mitochondrial Q-PCR analysis (SEQ ID NO: 25-28).

21. Similar to the acquisition of trophoblast stem cells

XF-EPS was digested and seeded on a 6-well plate coated with feeder cells at a density of 500,000 per well. Cultivate in XF-EPS medium for 24 hours, then change to trophoblast stem cell medium, add 0.1mM 2-mercaptoethanol, 0.2% fetal bovine serum, 0.5% double antibody, 0.3% bovine serum albumin to DMEM/F12 , 1% insulin-transferrin-sodium selenite-ethanolamine supplement, 1.5μg/ml L-ascorbic acid, 50ng/ml hEGF, 2μM CHIR99021, 0.5μM A83-01, 1μM SB431542, 0.8mM valproic acid and 5μM Y-27632. The trophoblast stem cells are similar to cells with the cell digestion solution ACCUTASE or TrypLE according to the ratio of 1:3 to 1:4, and passage once every four days. The fluid needs to be changed every day. The antibodies used for immunofluorescence analysis are shown in Table 1.

Example 1. Screening system for stable culture of EPS cells

In order to develop a heterogeneous EPS cell culture system, we first identified the key cytokines secreted by feeder cells for maintaining the stemness of hEPS cells. By using the OCT4-Tdtomato hEPS cell line (Figure 3a), we found that under feeder-free culture conditions, Activin A can effectively maintain the proportion of OCT4+hEPS cells above 80% (Figure 1a and Figure 3b) and express The key stem transcription factor (Figure 3c). Subsequently, we adopted a simplest culture combination to optimize the feeder-free culture system, namely LCDM+ITSX+Activin A, with the purpose of replacing N2 and B27 additives. Under this simplified culture condition, we found that the combination of catalase and vitamin C can further promote the proliferation of hEPS cells (Figure 1b and Figure 3d). In addition, we also found that DMEM/F12 and Neurobasal medium prepared in a 1:1 volume is the best combination of basic medium for hEPS cell proliferation (Figure 1c).

Next, through comparison, we determined a matrix protein that could replace Matrigel (large batch differences, containing unknown animal components). Studies have found that Laminin521 can continuously promote the attachment, survival and proliferation of hEPS cells (Figure 1d-f and Figure 4a-b). After a series of screenings (see Figure 10), the inventors obtained a hEPS cell culture system with clear ingredients and no heterologous sources.

Under such optimal culture conditions, hEPS cells of different genetic backgrounds can be well expanded in vitro (Figure 1g). Moreover, detection of the negative expression of Neu5Gc showed that hEPS cells under this condition lacked the expression of non-human mammalian components (Figure 4c). The above results show that we have established a well-defined, heterologous hEPS cell culture system (see Table 3).

Table 3 Composition of EPS medium without feeder layer

Component	concentration	Item No.
DMEM/F12(liquid)	2x	11330-032(ThermoFisher Scientific)
Neurobasal(liquid)	2x	21103-049(ThermoFisher Scientific)
insulin	10-50μg/ml	91077C(Sigma)
Transferrin	5.5-27.5μg/ml	T1147(Sigma)
Sodium selenite	1-10ng/ml	S5261(Sigma)
Ethanolamine (liquid)	20000x-50000x	398136(Sigma)
Catalase (liquid)	1000x-5000x	C3556(Sigma)
Vitamin C	50-200μg/ml	A8960(Sigma)
Activin A	5-100ng/ml	HST-A-1000(Stemimmune LLC)
Human LIF	10ng/ml	300-05(Peprotech)
CHIR99021	1μM	S1263(Selleck)
(S)-(+)-Dimethindene maleate	2μM	1425(Tocris)
Minocycline,Hydrochloride	2μM	sc-203339(Santa Curz)
Y-27632	2-10μM	S1049(Selleck)
Laminin 521	2.5-10μg/ml	77004(Stem cell technologies)
Optional components	To	To
IWR-1-endo	0.5μM	S7086(Selleck)

Example 2. Features of XF-EPS

In order to determine the characteristics of hEPS cells in a heterogeneous culture system, we first analyzed and compared their proliferation ability. The results of the study showed that the doubling time of XF-EPS cells was about 15 hours (Figure 2a), and the proportion of EDU+ cells was about 50%. (Figure 5a-b). Subsequently, we also found that the efficiency of single-cell clone formation of XF-EPS cells was 50% (Figure 2b), and the above results were not significantly different from hEPS cells in the original culture system. It is worth noting that the addition of low-dose (2-10μM) ROCK inhibitor (Y27632 or Fasudil) is essential for single cell passage and proliferation of hEPS cells (Figure 5c-d). In addition, hEPS cells can be passaged using different digestion solutions (Figure 5e). Importantly, due to the rapid proliferation ability of XF-EPS cells and the efficient single-cell cloning ability, this cell line can successfully perform gene targeting (Figure 5f).

Example 3. Genomic stability of XF-EPS

Subsequently, we studied the stability of the genome of XF-EPS cells after long-term culture. The results of karyotype analysis showed that XF-EPS cells of different genetic backgrounds still maintained a normal karyotype after more than 20 passages (Figure 2c and Figure 5g). The flow staining results of rH2AX are consistent with this. As a marker of gene damage, rH2AX has no significant difference in expression levels in XF-EPS nuclei and F-EPS cells (Figure 5h). The key molecular characteristics of XF-EPS cells were further analyzed, and the expression of key stemness marker genes was determined (Figure 2d and Figure 6a-b). We have also tested the XF-EPS cells

The characteristics were tested: the activity of OCT4 DE and the activation of X chromosome in female cells. The analysis showed that the level of F-EPS can still be maintained in XF-EPS cells (Figure 2e and Figure 6c). The analysis showed that the full gene expression profile of XF-EPS cells was similar to that of F-EPS under the culture conditions of feeder cells (Figure 6d). The above results show that XF-EPS cells can maintain the stability of the genome in long-term culture in vitro, and express the molecular characteristics of hEPS cells.

Example 4. In vitro and in vivo development potential of XF-EPS

The study first tested the developmental potential of XF-EPS cells by in vitro EB differentiation. XF-EPS cells have a strong ability to differentiate into three germ layers (Figure 7a), which is consistent with the differentiation results of teratomas in vivo (Figure 2f and Figure 7b). Immediately after the identification of the extra-embryonic differentiation potential of XF-EPS cells in vitro, we found that XF-EPS cells can differentiate into trophoblast-like cells through induction, and the results of FACS, Q-PCR, and immunofluorescence detection are shown in the figure (Figure 7c- e) as shown. More importantly, by culturing XF-EPS under the culture conditions of trophoblast stem cells, XF-EPS can be transformed into trophoblast stem cells similar to cells (Figure 11a). The results of immunofluorescence and Q-PCR detection are shown in the figure (Figure 11a). 11b-c). In summary, XF-EPS cells can generate intra-embryonic and extra-embryonic lineages in vitro.

Subsequently, we focused our research on evaluating the developmental potential of XF-EPS cells in vivo. Firstly, XF-EPS cells labeled with mClover reporter gene were injected into mouse 8-cell stage embryos and placed in embryo culture medium for 48 hours in vitro culture. By observing the injected embryos at day E4.5, it was found that about 50% of the mouse embryos had hEPS cell-derived cells in the inner cell mass and trophoblastic ectoderm (Figure 2g and Figure 7f, Figure 11d). Furthermore, E4.5-day embryos were transferred back to the uterus of pseudo-pregnant mice. It was detected that 60.4% (32/53) of E6.5-day mouse embryos could detect hEPS cell-derived cells (Figure 2h, Figure 11e). ). Immunofluorescence results showed that XF-EPS-derived cells can be co-stained with the intra-embryonic transcription factor OCT4 and the extra-embryonic transcription factor CK18 (Figure 11f). Further detection by mitochondrial PCR revealed that XF-EPS cells could be detected in the chimeric pregnancy tissues at 10.5 days, embryos, placenta and yolk sacs (Figure 11g). The above results show that XF-EPS cells have the potential for intra-embryonic and extra-embryonic development in vivo.

The EPS conditions established by the present invention with definite ingredients and non-heterogeneous can be applied to cells from multiple sources (such as H1 cultured under other conditions, EPS cells derived from blastocysts, EPS cells established by reprogramming of fibroblasts). These cell lines can be expanded stably for a long period of time under the condition of the EPS with the determined composition of the present invention and without heterologous, and maintain the basic properties of EPS, such as stemness and differentiation ability; it can also maintain the characteristics of EPS and bidirectional chimerism. Ability, see Table 4 for details.

Table 4 Characteristics of expanded pluripotent stem cells in XF-LCDM

Example 5. Establishment of XF-EPS

Finally, we tried to use cell reprogramming on the basis of the XF-EPS culture system to generate hEPS cell lines (Figure 8a). XF-EPS cell culture medium is used in the reprogramming process, and 5% XF-KSR (KSR without heterologous) is added to promote cell proliferation during the line establishment process. 10-14 days after transfection, the hEPS cell clones produced can be expanded in the XF-EPS cell culture system (Figure 8b). We also confirmed the co-expression of OCT4 and SOX2 genes in the XF-EPS cell line by immunofluorescence (Figure 8c). Lines can be established by picking single clones and single cells (Figure 8d-e). The morphology of the hEPS cell line derived from fibroblasts is similar to that of embryonic cell lines (Figure 9a), and maintains the expression and differentiation potential of stemness marker genes (Figure 2d and Figure 9b-d). In addition, the genome-wide transcription profile analysis structure of fibroblast-derived hEPS cells is very similar to F-EPS cells (Figure 9f). Based on the above results, it can be seen that the XF-EPS cell culture system can support the establishment of hEPS cell lines derived from somatic cells.

Summarize

In the present invention, a hEPS cell culture system with clear components and no heterologous is established, which supports the long-term culture and establishment of hEPS cells, and can maintain the bidirectional chimerism ability of hEPS cells in mice. Moreover, the system supports gene targeting of hEPS cells and long-term maintenance of the expression of modified genes. More importantly, XF-EPS cells cultured in vitro for a long time can maintain the normal karyotype, and the results are similar to traditional PSCs cultured under feeder-free cell conditions. The ability of the XF-EPS cell line to maintain genome integrity in vitro may be related to the combination of catalase and vitamin C, an antioxidant, which is reported to reduce ROS-induced gene mutation. This culture system has the potential to widely apply hEPS cells to directed differentiation, disease modeling and developmental biology, and pave the way for the application of EPS technology in clinical practice.

The specific embodiments described above further describe the purpose, technical solutions and beneficial effects of the present invention in further detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Within the spirit and principle of the present invention, any modification, equivalent replacement, improvement, etc., shall be included in the protection scope of the present invention.

Claims (16)

1. A composition or kit, preferably, it can be a cell culture medium composition or kit, preferably, it can be used in an expanded pluripotent stem cell culture system, characterized in that it includes the following components: Catalase, Vitamin C, Activin A and Laminin 521.
2. The composition or kit according to claim 1, characterized in that it comprises the following components:

   (1)ITSX;

   (2) Catalase,

   (3) Vitamin C,

   (4) Activin A,

   (5) Cytokines,

   (6) GSK3b inhibitor,

   (7) GPCR inhibitors,

   (8) PARP inhibitor,

   (9) ROCK inhibitor,

   (10) Laminin 521, and

   Optionally, (11) WNT inhibitor.
3. A composition or kit, preferably, it can be a cell culture medium composition or kit, preferably, it can be used in an expanded pluripotent stem cell culture system, characterized in that it comprises the following components:

   (1)ITSX;

   (4) Activin A,

   (5) Cytokines,

   (6) GSK3b inhibitor,

   (7) GPCR inhibitors,

   (8) PARP inhibitor,

   (9) ROCK inhibitor,

   (10) Laminin 521, and

   Optionally, (11) WNT inhibitor;

   Preferably, the composition or kit further includes (2) catalase and/or (3) vitamin C.
4. The composition or kit according to claim 2 or 3, wherein the ITSX is a combination of insulin, transferrin, sodium selenite and ethanolamine;

   Preferably, the transferrin includes apotransferrin or hototransferrin;

   Optionally, the concentration of the insulin is 10-50 μg/ml; optionally, the concentration of the transferrin is 5.5-27.5 μg/ml; optionally, the concentration of the sodium selenite is 1- 10ng/ml; optionally, the concentration of the ethanolamine is 20000x-50000x.
5. The composition or kit according to any one of claims 1 to 3, wherein the basic medium of the composition or kit is a mixed medium of DMEM/F12 and Neurobasal;

   Preferably, the volume ratio of the DMEM/F12 and the Neurobasal is (0.8-1.2):(0.8-1.2); more preferably, the volume ratio is 1:1.
6. The composition or kit according to claim 2 or 3, wherein the (5) cytokine is selected from the group consisting of: human leukemia inhibitory factor (LIF, "L"), interleukin (IL)- 6, IL-11, IL-27, IL-31, leukemia inhibitory factor, oncostatin M, cardiotrophin-1, neuropoietin and cardiotrophin-like cytokine 1;

   Preferably, the (5) cytokine is human LIF.
7. The composition or kit according to claim 2 or 3, wherein the (6) GSK3b inhibitor is selected from the group consisting of: CHIR99021 ("C") [6-[[2-[[4-( 2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile]; Bio[( 2'Z,3'E)-6-bromoisatin-3'-oxime]; TWS119; BIO-acetoxime; GSK 3I inhibitor XV; SB-216763; CHIR 99021 trihydrochloride; GSK-3 inhibitor IX[((2Z,3E)-6'-bromo-3-(oximino)-[2,3'-dihydroindolyl]-2'-one]; GSK 3 IX[6-bromoindigo Red-3'-oxime]; GSK-3β inhibitor XII[3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]phenol ]; GSK-3 inhibitor XVI[6-(2-(4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-pyrimidin-2-yl Amino)ethyl-amino)-nicotinonitrile]; or SB-415286[3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole- 2,5-Diketone];

   Preferably, the (6) GSK3b inhibitor is at least one of CHIR99021, Bio, and TWS119.
8. The composition or kit according to claim 2 or 3, wherein the (7) GPCR inhibitor is (S)-(+)-dimethylindene maleate, tripinamin hydrochloride, or dichloride At least one of Leitadine.
9. The composition or kit according to claim 2 or 3, wherein the (8) PARP inhibitor is at least one of minocycline hydrochloride, BSI-201 or nicotinamide.
10. The composition or kit according to claim 2 or 3, wherein the (9) ROCK inhibitor is at least one of Y-27632 or fasudil.
11. The composition or kit according to claim 2 or 3, wherein the (11) WNT inhibitor is at least one of IWR-1-endo or XAV939.
12. The composition or kit according to any one of claims 1 to 3, wherein 1%-5% of KSR medium is added to the composition or kit.
13. The composition or kit according to claim 2 or 3, wherein the concentration of (2) catalase is 1000x-5000x; optionally, the concentration of (3) vitamin C is 50-200 μg /ml; optionally, the concentration of (4) Activin A is 5-100 ng/ml; optionally, the concentration of (5) cytokine (preferably human LIF) is 10-15 ng/ml; optionally Preferably, the concentration of the (6) GSK3b inhibitor is 0.8-1.2 μM; optionally, the concentration of the (7) GPCR inhibitor is 1-5 μM; optionally, the concentration of the (8) PARP inhibitor The concentration is 2-5 μM; optionally, the concentration of (9) ROCK inhibitor is 2-10 μM; optionally, the concentration of (10) Laminin 521 is 2.5-10 μg/ml (for example, 2.5 μg/ml ); Optionally, the concentration of the (11) WNT inhibitor is 0.5 μM.
14. A medium (preferably used for culturing expanded pluripotent stem cells), which comprises the composition according to any one of claims 1-13;

    Preferably, the basic medium of the medium is a mixed medium of DMEM/F12 and Neurobasal;

    Preferably, the volume ratio of the DMEM/F12 and the Neurobasal is (0.8-1.2):(0.8-1.2); more preferably, the volume ratio is 1:1.
15. A method for culturing an expanded pluripotent stem cell, characterized in that the expanded pluripotent stem cell is cultured in the medium according to claim 14.
16. The method for establishing a line of expanded pluripotent stem cells is characterized in that the somatic cells are reprogrammed and then seeded into the medium according to claim 14; preferably, the somatic cells are fibroblasts.

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