CBA culture medium and application thereof in in-vitro induced differentiation of embryonic ectomesoderm cells

Abstract

The invention relates to the technical field of stem cells, in particular to a CBA culture medium and application of the CBA culture medium in in-vitro induced differentiation of embryonic ectomesoderm cells. The invention provides a culture medium for rapidly (5 days) and efficiently (more than 90%) inducing the differentiation of the original state (Naive) and primed state (Primed) human pluripotent stem cells (hPSCs) into the extraembryonic mesoderm cells (ExMs) with self-renewal ability, the culture medium has universality, and simultaneously supports the induction of the rapid and efficient differentiation of the Naive and Primed hPSCs into the ExMs; the ExMs obtained by differentiating the hPSCs are highly similar to the ExMs in human embryos in the periimplantation stage and the periintestinal stage, and can be stably cultured and amplified for a long time in vitro. The culture medium and the induction method have the advantages of being short in period, high in yield and low in cost, can promote the industrialization of ExMs, and have wide application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cells, in particular to a CBA culture medium and an application thereof in inducing differentiation of extraembryonic mesodermal cells in vitro. Background Art

[0002] Human extraembryonic mesoderm (ExM) is an important component of the placenta, amnion, yolk sac, blood islands and umbilical cord. It can produce rich extracellular matrix and plays a key role in the development of human embryos. In addition, human ExM is also the common developmental origin of mesenchymal stem cells in tissues such as the placenta, amnion and umbilical cord, as well as the first wave of the human hematopoietic system. Therefore, human extraembryonic mesoderm cells (ExM cells, ExMs) have broad application prospects in developmental biology, regenerative medicine and medical beauty industries.

[0003] At present, some progress has been made in the research on the in vitro culture and acquisition of human ExMs, but there are still some obvious defects, as follows:

[0004] 1. In 2022, Pham et al. unexpectedly discovered that the trophoblast stem cell culture on the 30th day after differentiation contained some mesenchymal cells (16.3%) in the process of differentiating naive hPSCs to produce trophoblast stem cells. After flow sorting and identification, it was determined that these mesenchymal cells were ExMs. The ExMs obtained in this study can be expanded for at least 14 generations (70 days) under trophoblast stem cell culture conditions. The shortcomings of this study are: (1) ExMs can only be obtained from Naive hPSCs, not from Primed hPSCs; (2) It is time-consuming and requires 30 days of in vitro differentiation; (3) The efficiency is low, with the proportion of ExMs being only 16.3%.

[0005] 2. In 2023, Ai et al. developed a new culture system that can efficiently induce Naive hPSCs to differentiate into ExMs quickly and efficiently. The shortcomings of this study are: only Naive hPSCs are induced to obtain ExMs, and it is uncertain whether it can support the induction of Primed hPSCs to produce ExMs; and it is uncertain whether the obtained ExMs can be stably cultured and expanded in vitro.

[0006] 3. In 2023, Farkas and Ferretti first differentiated Naive hPSCs into primitive endoderm cells in vitro, and then further differentiated the primitive endoderm cells into ExMs through a newly developed induction system. The shortcomings of this study are as follows: (1) The operation is cumbersome, and it is necessary to first differentiate Naive hPSCs into primitive endoderm cells, and then induce the obtained primitive endoderm cells to differentiate into ExMs through a two-step method; (2) It is time-consuming. It takes 7-8 days for Naive hPSCs to differentiate into primitive endoderm, and 15 days for primitive endoderm cells to differentiate into ExMs; (3) The process of differentiation of primitive endoderm cells into ExMs is accompanied by a large number of cell deaths; (4) This method does not support the acquisition of ExMs through Primed hPSCs; (5) It is uncertain whether the ExMs obtained by this method have the ability to self-renew.

[0007] 4. In 2024, Wang et al. established a new method to obtain ExMs by differentiating Primed hPSCs under suspension conditions. The shortcomings of this study are: (1) low differentiation efficiency, a large number of miscellaneous cells remain in the cell culture, and only about 24% of the cells are ExMs; (2) this method only obtains ExMs by differentiating Primed hPSCs, and it is uncertain whether ExMs can be obtained through Naive hPSCs; (3) it takes a long time and requires 7 days of in vitro differentiation.

[0008] 5. The inventors previously disclosed a method for inducing differentiation of extraembryonic endoderm and / or extraembryonic mesoderm in CN116875538A. However, this method efficiently induces Naive hPSCs into extraembryonic mesoderm cells, while the efficiency of producing ExMs by differentiating Primed hPSCs is low.

[0009] In summary, for the application of human ExMs in basic research and clinical transformation, it is crucial to develop a new culture medium system that can simultaneously induce Naive and Primed hPSCs to quickly and efficiently produce ExMs that can be expanded in vitro. Summary of the invention

[0010] In order to solve the above technical problems, the present invention provides a fast and efficient induction The culture medium for in vitro expansion of hPSCs to produce ExMs is CBA culture medium, including: DMEM / F12 culture medium, Neurobasal culture medium, 0.25-1% N2, 0.5-2% B27, 0.1-1% GlutaMAX, 0.1-1% non-essential amino acids, 0.01-0.1mM β-mercaptoethanol, 0.001%~0.01% human serum albumin, 1-100μg / ml L-ascorbic acid-2-phosphate magnesium salt, 0.05-0.2% chemical lipid concentrate, 1-20μg / ml insulin, 0.2%~0.5% sodium pyruvate, 0.01-0.05μg / ml progesterone, 5-50ng / ml recombinant human fibroblast growth factor 4 (FGF4), 0.5-2μg / ml heparin sodium, 2-10μΜ Y27632, 1-3μΜ CHIR99021, 1-10ng / ml recombinant human bone morphogenetic protein 4 (BMP4) and 2-20ng / ml recombinant human activin A (Activin-A).

[0011] The above % represents volume percentage, that is, (v / v).

[0012] In some embodiments, the volume ratio of DMEM / F12 medium to Neurobasal medium is 1:0.8-1.2, preferably 1:1.

[0013] In some embodiments, the volume ratio of DMEM medium to F12 medium is 1:0.8-1.2, preferably 1:1.

[0014] Furthermore, the present invention provides the use of CBA culture medium in inducing differentiation of extraembryonic mesoderm cells in vitro.

[0015] Specifically, the present invention provides a method for inducing differentiation of extraembryonic mesoderm cells in vitro, comprising: digesting Primed hPSCs or Naive hPSCs into single cells, resuspending the Primed hPSCs with the CBA culture medium and inoculating them in a culture dish or culture plate coated with an extracellular matrix, and then placing them in a CO2 incubator for culture.

[0016] Preferably, the extracellular matrix comprises matrigel, type IV collagen, vitronectin or laminin.

[0017] Preferably, during the culture process in a CO2 incubator, the medium is changed every two days.

[0018] Preferably, the culture is placed in a CO2 incubator for less than 5 days, preferably 5 days.

[0019] Preferably, on the 5th day of culture, more than 91.4% of Naive hPSCs are specialized into extraembryonic mesoderm cells.

[0020] Preferably, on the 5th day of culture, more than 98.8% of the Primed hPSCs are specialized into extraembryonic mesoderm cells.

[0021] Preferably, the inoculation density is 1 to 1.5×10 4 Cells / cm 2 .

[0022] Preferably, before being digested into single cells, the Primed hPSCs or Naive hPSCs are cultured in AIC, E8, mTeSR, AIC-N or PXGL system.

[0023] In the specific implementation process, including but not limited to the above-mentioned AIC, E8 or PXGL system, other culture systems for Primed hPSCs or Naive hPSCs are applicable to the above-mentioned method of the present invention.

[0024] In a specific implementation process, on the fifth day of culture, the induced extraembryonic mesoderm cells are digested and inoculated into trophoblast stem cell culture medium or CB culture medium, and the extraembryonic mesoderm cells can be stably cultured and expanded (for more than 10 generations).

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides a culture medium for rapidly (5 days) and efficiently (>90%) inducing naive and primed hPSCs to differentiate into ExMs with self-renewal ability. The culture medium of the present invention has universal applicability and supports the rapid and efficient differentiation of naive and primed hPSCs into ExMs. "Rapid" means more simulation and better function, because in human embryos, ExMs specialization only takes about 5 days. These ExMs obtained by differentiating hPSCs are highly similar to ExMs in peri-implantation and peri-gastrulation human embryos, and can be cultured and expanded stably for a long time in vitro. Therefore, the culture medium and induction method of the present invention have the advantages of short cycle, high yield and low cost, and can promote ExMs to industrialization. The present invention provides a new cell model for the study of the developmental dynamics and developmental principles of the extraembryonic mesoderm, and has broad application prospects in the field of regenerative medicine and the medical beauty industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The results of rapid and efficient differentiation of Naive hPSCs into ExMs in CB culture medium are shown in the figure:

[0028] (a) Schematic diagram and bright field image of ExMs differentiated in CB medium and extended culture in trophoblast stem cell medium, the scale bar represents 100 μm; (b) Immunofluorescence staining detection shows the expression of markers in Naive hPSCs after 4 days of differentiation in CB medium, the scale bar represents 100 μm; (c) Principal component analysis of bulk RNA sequencing data of Naive hPSCs differentiated for different days in CB medium; (d) Heat map showing the expression dynamics of pluripotency and ExM marker genes in Naive hPSCs differentiated for different days in CB medium and ExMs extended culture; (e) GO (Geneontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis based on differentially expressed genes between the starting Naive hPSCs and Naive hPSCs after 4 days of differentiation in CB medium; (f) UMAP (Uniform Manifold Approximation and Genomes) of single-cell transcriptome data from Naive hPSCs differentiated for 4 days in CB medium (g) Bubble plot of marker gene expression in specific cell populations as shown; (h) Spearman correlation analysis showing cell types; (i) RNA rate analysis of single-cell transcriptome data; (j,k) integrated analysis of single-cell transcriptome data from naive hPSCs after 4 days of differentiation in CB medium with publicly published embryonic data and in vitro differentiated cell data.

[0029] Figure 2The results of identifying and expanding the culture of ExMs produced by Naive hPSCs induced by CB medium: (a) Immunofluorescence staining shows the expression of markers in Naive hPSCs after 4 days of differentiation in CB medium, and the scale represents 100 microns; (b) Flow cytometry analysis of the expression of GATA6 and SNAIL in Naive hPSCs after 4 days of differentiation in CB medium; (c) Spearman correlation analysis between the initial Naive hPSCs, Naive hPSCs differentiated for different days in CB medium, and ExMs of expanded culture; (d) Volcano plot analysis of differentially expressed genes between the initial Naive hPSCs and Naive hPSCs differentiated for 4 days in CB medium; (e) UMAP map showing the expression of hrGFP, mCherry-WPRE and Y chromosome-specific gene RPS4Y1; (f) UMAP map showing the expression of specific cell type markers; (g) UMAP analysis of Naive hPSCs differentiated for 4 days in CB medium using E-Cadherin. hPSCs were analyzed by flow cytometry; (h,i) ExMs expanded in CB medium for 5 generations were identified by immunofluorescence staining of display markers, the scale bar represents 100 μm; (j,k) ExMs expanded in trophoblast stem cell medium for 30 generations were identified by immunofluorescence staining of display markers, the scale bar represents 100 μm; (l) GO (Gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis based on the differentially expressed genes between Naive hPSCs after 4 days of differentiation in CB medium and ExMs expanded in trophoblast stem cell medium for 30 generations; (mo) Naive hPSCs cultured under the PXGL system can also efficiently differentiate into ExMs in CB medium.

[0030] Figure 3The results are as follows: (a) Schematic diagram and bright field image of Primed hPSCs differentiation in CB medium, the scale bar represents 100 μm; (b) Principal component analysis of bulk RNA sequencing data of Naive hPSCs, Primed hPSCs, and Naive hPSCs and Primed hPSCs differentiated in CB medium for 4 days; (c) Spearman correlation analysis of bulk RNA sequencing data of Naive hPSCs, Primed hPSCs, and Naive hPSCs and Primed hPSCs differentiated in CB medium for 4 days; (d) Heat map showing the expression of pluripotency, ExM, amnion and trophoblast cell marker genes in Naive hPSCs, Primed hPSCs, and Naive hPSCs and Primed hPSCs differentiated in CB medium for 4 days; (e) Principal component analysis of bulk RNA sequencing data of Naive hPSCs, Primed hPSCs, and Naive hPSCs and Primed hPSCs differentiated in CB medium for different days. (f) shows the expression dynamics of markers in Naive hPSCs, Primed hPSCs, and Naive hPSCs and Primed hPSCs differentiated for different days in CB medium; (g) UMAP visualization and cell type clustering based on single-cell transcriptome data from Primed hPSCs cultured for different days in CB medium; (h) shows the proportion of cell types; (i) bubble plot showing the expression of marker genes in specific cell types; (j) UMAP plot showing the expression of specific genes; (k) developmental trajectory plot predicted by Slingshot; (l) shows the proportion of cell types.

[0031] Figure 4The fate of Primed hPSCs in CB medium is different from that of Naive hPSCs. Result figures: (a) Immunofluorescence staining shows that Primed hPSCs are mainly specialized into amniotic cells in CB medium, and the scale represents 100 μm; (b) Volcano plot showing differentially expressed genes between Naive hPSCs and Primed hPSCs after 4 days of differentiation in CB medium; (c) UMAP clustering plot based on single-cell transcriptome data of Primed hPSCs after 4 days of differentiation in CB medium; (d) Bubble plot showing the expression of marker genes in specific cell types; (e) UMAP plot showing the expression of marker genes in Primed hPSCs after 4 days of differentiation in CB medium; (f) Spearman correlation analysis showing cell types; (g) RNA velocity plot showing cell developmental trajectory; (h) Principal component analysis of bulk RNA sequencing data of Primed hPSCs differentiated for different days in CB medium; (i) Representative pluripotency and specific lineage marker genes in Primed hPSCs differentiated for different days in CB medium Heat map of expression in hPSCs; (j) Expression dynamics of primitive streak marker genes in Primed hPSCs differentiated for different days in CB medium; (k) Differentiation of Primed hESCs cultured in E8 system in CB medium, the scale bar represents 100 μm.

[0032] Figure 5Activin-A was added to the CB culture medium to enable Primed hPSCs to efficiently specialize into ExMs. Result figures: (a) Schematic diagram of the composition of CBA culture medium and its treatment of Primed (AIC) hPSCs; (b) Bright field images of Primed hPSCs after 5 days of differentiation in CB culture medium and CBA culture medium, respectively, with the scale bar representing 100 μm; (c) Immunofluorescence staining of TFAP2A and GATA6 in Primed hPSCs after 5 days of differentiation in CB and CBA culture medium, respectively, with the scale bar representing 100 μm; (d) The proportion of TFAP2A-positive and GATA6-positive cells; (e) Flow cytometry analysis of TFAP2A-positive and GATA6-positive cells; (f) Principal component analysis of gene expression profiles of Primed hPSCs differentiated for different times in CB and CBA culture medium; (g) Heat map of the expression of different cell lineage marker genes in Primed hPSCs differentiated for different times in CB and CBA culture medium; (h) UMAP visualization and cell type clustering of single-cell transcriptome data of hPSCs; (i) UMAP graph showing gene expression in Primed hPSCs after 5 days of differentiation in CBA medium; (j) Schematic diagram, bright field image and immunofluorescence staining image of switching Primed hPSCs after 5 days of differentiation in CBA medium to human trophoblast stem cell medium for extended culture, with the scale bar representing 100 μm; (k) Staining image of Primed hPSCs cultured in the E8 system that are efficiently differentiated into ExMs in CBA medium, with the scale bar representing 100 μm.

[0033] Figure 6Figure 1 shows the results of efficient differentiation of Primed hPSCs into ExMs in CBA medium: (a) Bright field image and staining image showing markers after Primed hPSCs differentiated in CBA medium for 4 days, the scale bar represents 100 microns; (b) Staining image showing markers after Primed hPSCs differentiated in CBA medium for 5 days, the scale bar represents 100 microns; (c) Spearman correlation analysis of gene expression profiles of Primed hPSCs differentiated for different days in CB and CBA medium; (d) Expression dynamics of marker genes in Primed hPSCs cultured in CBA medium for different days; (e) GO and KEGG enrichment analysis of differentially expressed genes between Primed hPSCs and Primed hPSCs treated with CBA medium for 5 days; (f) UMAP image showing the expression of hrGFP, mCherry-WPRE and Y chromosome-specific gene RPS4Y1; (g) Bubble plots showing the expression of marker genes in specific cell types in hPSCs; (h) Heat map of the expression of early and late ExM marker genes in Primed hPSCs treated with CBA for different periods of time and in ExMs cultured for extended periods of time; (i,j) show the expression dynamics of markers in Primed hPSCs cultured in CBA medium for different days, with the scale bar representing 100 μm.

[0034] Figure 7 The following are the results of efficient differentiation of Naive hPSCs into ExMs in CBA medium: (a) Schematic diagram of the composition of CBA medium and its treatment of Naive hPSCs; (b) Bright field image of Naive hPSCs after 5 days of differentiation in CBA medium, with the scale bar representing 100 μm; (c) Immunofluorescence staining of markers in Naive hPSCs after 5 days of differentiation in CBA medium, with the scale bar representing 100 μm; (d) Heat map showing the expression dynamics of representative marker genes in Naive hPSCs treated with CBA for different days; (e) UMAP visualization and cell type clustering of single-cell transcriptome data of Naive hPSCs after 5 days of differentiation in CBA medium; (f) UMAP map showing gene expression in Naive hPSCs after 5 days of differentiation in CBA medium; (g) Immunofluorescence staining of early hematopoietic markers in Naive and Primed hPSCs after 5 days of differentiation in CBA medium, with the scale bar representing 100 μm.

[0035] Figure 8Figure 1 is the result of CBA medium inducing efficient differentiation of Naive hPSCs into ExMs: (a) showing the expression dynamics of marker genes in Naive hPSCs differentiated in CBA for different times; (b) heat map showing the expression of representative genes in Naive and Primed hPSCs differentiated in CBA for different times; (c) bubble map showing the expression of marker genes in specific cell types in Naive hPSCs treated with CBA for 5 days; (d) UMAP map showing the expression of early hematopoietic marker genes in Primed hPSCs treated with CBA for 5 days; (e) schematic diagram of transferring Naive hPSCs differentiated in CBA medium for 5 days to trophoblast stem cell medium for extended culture; (f) Naive hPSCs differentiated in CBA medium for 5 days were transferred to trophoblast stem cell medium for extended culture, and immunofluorescence staining was performed with antibodies showing markers after 10 generations of culture, and the scale bar represents 100 microns; (g) shows the expression of early and late ExM marker genes in Naive hPSCs at different differentiation stages Heat map of expression in hPSCs; (h) Staining image showing the efficient differentiation of PXGL-cultured Naive hPSCs into ExMs under the CBA system, the scale bar represents 100 μm.

[0036] Fig. 9 The ExMs produced by differentiation of Naive and Primed hPSCs are highly similar to ExMs in embryos. Result Figure: (a) Based on Bulk RNA-seq data, correlation analysis was performed on Naive and Primed hPSCs differentiated and cultured under different conditions; (b) Based on single-cell transcriptome data, correlation analysis was performed on cells from different sources, different culture conditions and their different cell subpopulations; (c) Based on single-cell transcriptome data, integrated analysis was performed on single-cell datasets from different sources, different culture conditions and their different cell types. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. % represents volume percentage, i.e. (v / v). If the specific technology or conditions are not specified in the embodiments, they are all conventional methods or carried out according to the technology or conditions described in the literature of this field, or according to the product manual. The reagents and instruments used, etc., which do not indicate the manufacturer, are all conventional products that can be purchased through regular channels.

[0038] The CB culture medium formula in the following examples is: mN2B27 culture medium supplemented with 50 ng / ml recombinant human fibroblast growth factor 4 (FGF4) (Peprotech, 100-31-25), 1 μg / ml heparin sodium (Sigma, H3149), 10 μΜ Y27632 (a selective ROCK1 and ROCK2 inhibitor) (Selleck, S1049), 2 μΜ CHIR99021 (a WNT signaling pathway agonist) (Selleck, S2924) and 10 ng / ml recombinant human bone morphogenetic protein 4 (BMP4) (R&D Systems, 314-BP-050).

[0039] The formula of mN2B27 culture medium is as follows: basal culture medium (DMEM / F12 (volume ratio 1:1) and Neurobasal mixed in equal volume ratio) supplemented with 1% N2 (Thermo Fisher Scientific, 17502-048), 2% B27 (Thermo Fisher Scientific, 17504-044), 0.5% GlutaMAX (Thermo Fisher Scientific, 35050-061), 1% non-essential amino acids (NEAA, Thermo Fisher Scientific, 11140-050), 0.1 mM β-mercaptoethanol (β-ME) (Sigma, M7522), 0.006% human serum albumin (Sigma, A1933), 50 μg / ml L-ascorbic acid-2-phosphate magnesium salt (Vc) (Sigma, A8960), 0.1% chemical lipid concentrate ...0.1% Scientific, 11905-031), 12.5 μg / ml insulin (Roche, 11376497001), 0.25% sodium pyruvate (Thermo Fisher Scientific, 11360-070), 0.02 μg / ml progesterone (Sigma, P8783).

[0040] In the following examples, three naive hPSCs (AIC-N1, AIC-N2 and AIC-N4) were used. These three cell lines were from our previous study (Ai et al., 2023). Naive hPSCs were cultured in AIC-N or PXGL medium, the medium was changed every two days, and 50% TrypLE was used to digest the single cells for subculture every 4-5 days, with a subculture ratio of 1:5 to 1:10. The formula of AIC-N medium is as follows: 10 ng / ml activin A, 2 μM IWP2 (a WNT inhibitor), 0.3 μM CHIR99021 (a WNT signaling pathway agonist), 1 μM PD0325901 (an inhibitor of the MEK signaling pathway), 2 μM (a PKC inhibitor), 10 ng / ml hLIF (recombinant human leukemia inhibitory factor) and 10 μM Y27632 (a selective ROCK1 and ROCK2 inhibitor). PXGL is a public culture system and will not be described in detail here.

[0041] In the following examples, three strains of Primed hPSCs (hES1, h1 and H9) were used. These three cell lines were from our previous study (Ai et al., 2020). Primed hPSCs were cultured in AIC medium, the medium was changed every two days, and 50% TrypLE was used to digest single cells for passage every 3-4 days, with a passage ratio of 1:10 to 1:20. The formula of AIC medium is as follows: 10 ng / ml activin A, 2 μM IWP2, and 0.6 μM CHIR99021 were added to the basal medium mN2B27.

[0042] Example 1 confirms that CB medium can induce Naive hPSCs to differentiate into ExMs efficiently without feeder conditions

[0043] The CB medium previously developed by the inventors can rapidly induce Naive hPSCs to differentiate into ExMs efficiently on a mouse embryonic fibroblast feeder layer (feeder). Since feeders contain animal-derived components, they are not conducive to the conversion and use of ExMs. In order to test whether CB medium can also induce Naive hPSCs to differentiate into ExMs rapidly without feeders, we digested Naive hPSCs into single cells with 50% TrypLE, removed the supernatant after centrifugation, resuspended Naive hPSCs with CB medium, and cultured at 1-1.5×10 4 Cells / cm 2The cells were inoculated at a density of 100 μg / mL into TC-treated culture dishes / plates coated with extracellular matrix (matrigel, type IV collagen, vitronectin and laminin, etc.), and then cultured in a CO2 incubator. The medium was changed every two days, and the cells were tested and analyzed after the fourth day of culture.

[0044] After identification by immunofluorescence staining, flow cytometry, bulk RNA sequencing, and single-cell transcriptome sequencing, it was found that on the fourth day, 93.5% of the cells were induced into ExMs, and the induced ExMs were highly similar to the ExMs in the peri-implantation and peri-gastrulation embryos ( Figure 1 and Figure 2 ).

[0045] Naive hPSCs differentiated in CB medium for 4 days were digested into single cells with 50% TrypLE, and then the cells were stained for live cells with E-Cadherin flow cytometry antibodies. Finally, E-Cadherin-negative cells (90.2%) were sorted out by flow cytometry and inoculated into CB medium or publicly reported trophoblast stem cell medium (Okae et al., 2018; Pham et al., 2022) for extended culture. The medium was changed every two days, and single cells were passaged with 50% TrypLE every 3-5 days, with a passage ratio of 1:3 to 1:5.

[0046] The results showed that ExMs can be stably expanded for at least 5 generations in CB culture medium (due to time constraints, the present invention only cultured for 5 generations, and it is expected that more generations can be passed), and can be stably expanded for at least more than 30 generations in trophoblast stem cell culture medium ( Figure 1 and Figure 2 We further confirmed that Naive hPSCs cultured in the PXGL system can also efficiently differentiate into ExMs in CB medium ( Figure 1 and Figure 2 ).

[0047] Example 2 The efficiency of CB medium in inducing the differentiation of primed hPSCs to produce ExMs is much lower than that of naive hPSCs under feeder-free conditions

[0048] In order to test whether Primed hPSCs can also differentiate into ExMs quickly and efficiently in CB medium like Naive hPSCs under feeder-free conditions, we digested Primed hPSCs cultured in AIC or E8 system into single cells with 50% TrypLE, removed the supernatant after centrifugation, resuspended Primed hPSCs in CB medium, and cultured at 1-1.5×10 4 Cells / cm 2The cells were seeded at a density of 100 μg / mL into TC-treated culture dishes / plates coated with extracellular matrix, and then placed in a CO2 incubator for culture, with the medium changed every two days.

[0049] Through immunofluorescence staining, flow cytometry, bulk RNA sequencing and single-cell transcriptome sequencing, it was found that on the fourth day, 18.8% (AIC) and about 50% (E8) of the differentiated cells were ExMs, and the remaining cells were almost all amniotic cells ( Figure 3 and Figure 4 That is, compared with Naive hPSCs, the efficiency of Primed hPSCs in differentiating into ExMs in CB medium was significantly lower than that of Naive hPSCs ( Figure 3 and Figure 4 ).

[0050] Example 3 CBA medium induces primed hPSCs to differentiate into ExMs efficiently under feeder-free conditions

[0051] In order to improve the efficiency of Primed hPSCs differentiation into ExMs, the present invention screened a large number of growth factors, cytokines and chemical small molecules and found that adding Activin-A (recombinant human activin A), an activator of Activin / Nodal signaling, to CB culture medium can effectively inhibit the specialization of Primed hPSCs into amniotic cells and promote the specialization of Primed hPSCs into ExMs. Therefore, Activin-A (5ng / ml) was added to the CB culture medium, and this CB culture medium with added Activin-A was called CBA culture medium ( Figure 5 ).

[0052] Under feeder-free conditions, we digested the primed hPSCs cultured in AIC or E8 medium into single cells using 50% TrypLE, removed the supernatant after centrifugation, resuspended the primed hPSCs in CBA medium, and cultured at 1-1.5×10 4 Cells / cm 2 The cells were inoculated into TC-treated culture dishes / plates coated with Matrigel at a density of 100 μg / mL and then placed in a CO2 incubator for culture, with the medium changed every two days.

[0053] Through immunofluorescence staining, flow cytometry, bulk RNA sequencing and single-cell transcriptome sequencing, it was found that on the fourth day, although most of the primed hPSCs were specialized into ExMs, a small number of pluripotent cells remained in the culture ( Figure 6), so we extended the differentiation time to the fifth day. The test results showed that after five days of differentiation of Primed hPSCs in CBA medium, 98.8% of the cells were specialized into ExMs ( Figure 5 and Figure 6 Primed hPSCs cultured in other systems, such as E8, can also efficiently differentiate into ExMs in CBA medium ( Figure 5 and Figure 6 ).

[0054] On the fifth day, the Primed hPSCs that had been differentiated in CBA medium for 5 days were digested into single cells with 50% TrypLE and then directly inoculated into the publicly reported trophoblast stem cell medium for adherent culture. Since then, the medium was changed every two days, and single cells were passaged with 50% TrypLE every 3-5 days, with a passage ratio of 1:3 to 1:5. At least more than 10 generations can be passaged. After 10 generations, the present invention did not continue to subculture and analyze the ExMs. It was found that these cells can be stably cultured and expanded in the trophoblast stem cell medium ( Figure 5 and Figure 6 ).

[0055] Example 4 CBA medium induces efficient differentiation of Naive hPSCs into ExMs under feeder-free conditions

[0056] In order to further determine whether Naive hPSCs can also differentiate into ExMs quickly and efficiently in CBA medium like Primed hPSCs under feeder-free conditions, we digested Naive hPSCs cultured in AIC-N or PXGL medium into single cells with 50% TrypLE, removed the supernatant after centrifugation, resuspended Naive hPSCs in CBA medium, and cultured at 1-1.5×10 4 Cells / cm 2 The cells were inoculated at a density of 100 μg / mL into TC-treated culture dishes / plates coated with extracellular matrix Matrigel, and then placed in a CO2 incubator for culture, with the medium changed every two days.

[0057] Through immunofluorescence staining, flow cytometry, bulk RNA sequencing and single-cell transcriptome sequencing, it was found that on the fifth day, 91.4% of the differentiated cells were specialized into ExMs ( Figure 7 and Figure 8 In addition, naive hPSCs cultured in other systems, such as PXGL, can also efficiently differentiate into ExMs in CBA medium ( Figure 7 and Figure 8 ).

[0058] Naive hPSCs differentiated in CBA medium for 5 days were digested into single cells with 50% TrypLE and then directly inoculated into publicly reported trophoblast stem cell medium for adherent culture. Since then, the medium was changed every two days, and single cells were passaged with 50% TrypLE every 3-5 days, with a passage ratio of 1:3 to 1:5. At least more than 10 generations can be passaged. After 10 generations, the present invention did not continue to subculture and analyze the ExMs. It was found that these cells can be stably cultured and expanded ( Figure 7 and Figure 8 ).

[0059] Example 5 ExMs differentiated from naive and primed hPSCs are highly similar to ExMs in embryos

[0060] In order to identify the cellular characteristics of ExMs produced by differentiation of Naive or Primed hPSCs induced by CB or CBA system, we collected samples of Naive or Primed hPSCs differentiated by CB or CBA system for different days and obtained their transcriptome data. We compared and analyzed the transcriptome data with that of human early postimplantation embryos and found that ExMs produced by differentiation of Naive hPSCs and Primed hPSCs under CB and CBA conditions were similar to ExMs in human early postimplantation embryos ( Fig. 9 ).

[0061] In summary, under feeder-free conditions, CB and CBA medium have different induction effects on Naive hPSCs and Pimed hPSCs, specifically: (1) CB medium induces Naive hPSCs to differentiate into ExMs rapidly (4 days) and efficiently (93.5%), but the effect of inducing Pimed hPSCs to differentiate into ExMs is poor (4 days is accompanied by the generation of a large number of amniotic cells, about 81.2% of amniotic cells, and only a small number of about 18.8% of cells differentiate into ExMs); (2) CBA medium induces both Naive hPSCs and Pimed hPSCs to differentiate into ExMs rapidly (5 days) and efficiently (greater than 90%, 91.4% for Naive hPSCs and 98.8% for Pimed hPSCs). That is to say, CBA medium is more compatible and universal in inducing the differentiation of Naive hPSCs and Pimed hPSCs into ExMs; (3) ExMs induced by CB and CBA can be stably cultured and expanded in vitro (for more than 10 generations). (4) ExMs differentiated from Naive hPSCs and Pimed hPSCs induced by CB and CBA medium are highly similar to ExMs in post-implantation embryos and can be stably cultured and expanded in CB medium or trophoblast stem cell medium for at least 10 generations.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. CBA culture medium, characterized in that include: DMEM / F12 medium, Neurobasal medium, 0.25-1% N2, 0.5-2% B27, 0.1-1% GlutaMAX, 0.1-1% non-essential amino acids, 0.01-0.1 mM β-mercaptoethanol, 0.001%~0.01% human serum albumin, 1-100 μg / ml L-ascorbic acid-2-phosphate magnesium salt, 0.05-0.2% chemical lipid concentrate, 1-20 μg / ml insulin, 0.2%~0.5% sodium pyruvate, 0.01-0.05 μg / ml progesterone, 5-50 ng / ml recombinant human fibroblast growth factor 4 (FGF4), 0.5-2 μg / ml heparin sodium, 2-10 μΜ Y27632, 1-3 μΜ CHIR99021, 1-10 ng / ml recombinant human bone morphogenetic protein 4 (BMP4) and 2-20 ng / ml recombinant human Activin-A.

2. Application of CBA culture medium in inducing differentiation of extraembryonic mesoderm cells in vitro.

3. A method for inducing differentiation of extraembryonic mesoderm cells in vitro, characterized in that: include: After digesting the Primed hPSCs or Naive hPSCs into single cells, the Primed hPSCs are resuspended with the CBA culture medium of claim 1 and inoculated into a culture dish or culture plate coated with an extracellular matrix, and then placed in a CO2 incubator for culture.

4. The method according to claim 3, characterized in that During the culture process in a CO2 incubator, the medium was changed every two days.

5. The method according to claim 3, characterized in that: Place in a CO2 incubator and culture for no more than 5 days, preferably 5 days.

6. The method according to claim 5, characterized in that On the 5th day of culture, more than 91.4% of Naive hPSCs specialized into extraembryonic mesoderm cells.

7. The method according to claim 5, characterized in that On the fifth day of culture, more than 98.8% of the Primed hPSCs specialized into extraembryonic mesoderm cells.

8. The method according to claim 3, characterized in that The seeding density is 1~1.5×10 4 Cells / cm 2 .

9. The method according to claim 3, characterized in that: Before being digested into single cells, the Primed hPSCs or Naive hPSCs were cultured in AIC, E8, mTeSR, AIC-N or PXGL system.

10. The method according to claim 5, characterized in that On the fifth day of culture, the induced extraembryonic mesoderm cells are digested and inoculated into trophoblast stem cell culture medium or CB culture medium, and the extraembryonic mesoderm cells can be stably cultured and expanded.

Images