CBA medium and its use in the in vitro induction of differentiation of extraembryonic mesoderm cells

Abstract

This invention relates to the field of stem cell technology, and particularly to CBA culture medium and its application in in vitro induction of extraembryonic mesodermal cell differentiation. This invention provides a culture medium for rapidly (5 days) and efficiently (>90%) inducing the differentiation of naive and primed human pluripotent stem cells (hPSCs) into self-renewing extraembryonic mesodermal cells (ExMs). The culture medium of this invention is universally applicable, simultaneously supporting the rapid and efficient differentiation of naive and primed hPSCs into ExMs. These ExMs obtained through hPSC differentiation are highly similar to ExMs in peri-implantation and peri-gastrulanial human embryos and can be stably cultured and expanded in vitro for a long period. The culture medium and induction method of this invention have the advantages of short cycle time, high yield, and low cost, which can promote the industrialization of ExMs and have broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of stem cell technology, and more particularly to CBA culture medium and its application in in vitro induced differentiation of extraembryonic mesodermal cells. Background Technology

[0002] Human extraembryonic mesoderm (ExM) is an important component of the placenta, amnion, yolk sac, blood islands, and umbilical cord, producing abundant extracellular matrix and playing a crucial role in human embryonic development. Furthermore, human ExM is 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 hematopoiesis in the human body. Therefore, human extraembryonic mesoderm cells (ExM cells, ExMs) have broad application prospects in developmental biology, regenerative medicine, and the medical aesthetics industry.

[0003] Currently, some progress has been made in the in vitro culture and acquisition of human ExMs, but there are still some obvious shortcomings, as follows:

[0004] 1. In 2022, Pham et al., during the differentiation of trophoblast stem cells from naive hPSCs, unexpectedly discovered that the trophoblast stem cell culture on day 30 after differentiation contained some mesenchymal cells (16.3%). After flow cytometry sorting and identification, these mesenchymal cells were identified as ExMs. The ExMs obtained in this study can be expanded for at least 14 generations (70 days) under trophoblast stem cell culture conditions. The limitations of this study are: (1) ExMs can only be obtained from naive hPSCs, not from primed hPSCs; (2) It is time-consuming, requiring 30 days of in vitro differentiation; (3) It is inefficient, 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 rapidly and efficiently into ExMs. However, this study has limitations: it only induces naive hPSCs to acquire ExMs, and it is uncertain whether this supports the induction of ExMs from Primed hPSCs; furthermore, it is uncertain whether the obtained ExMs can be stably cultured and amplified 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 using a newly developed induction system. The shortcomings of this study are: (1) The operation is cumbersome, requiring Naive hPSCs to be differentiated into primitive endoderm cells first, and then the obtained primitive endoderm cells to be induced to differentiate into ExMs in two steps; (2) It is time-consuming, Naive hPSCs to differentiate into primitive endoderm cells takes 7-8 days, and primitive endoderm cells to differentiate into ExMs takes 15 days; (3) A large number of cells die during the differentiation of primitive endoderm cells into ExMs; (4) This method does not support obtaining ExMs from Primed hPSCs; (5) It is uncertain whether the ExMs obtained by this method have self-renewal capabilities.

[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, with a large number of contaminating cells remaining in the cell culture, and only about 24% of the cells being ExMs; (2) this method only obtains ExMs by differentiating Primed hPSCs, and it is uncertain whether ExMs can be obtained from Naive hPSCs; (3) it is time-consuming, requiring 7 days of in vitro differentiation.

[0008] 5. The inventor 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 generating ExMs through differentiation of Primed hPSCs is relatively low.

[0009] In summary, for the application of human ExMs in basic research and clinical translation, it is crucial to develop a new culture medium system that can simultaneously induce naive and primed hPSCs to rapidly and efficiently generate ExMs that can be expanded in vitro. Summary of the Invention

[0010] To address the aforementioned technical challenges, this invention provides a rapid and efficient induction method. The culture medium for in vitro amplification of ExMs from (primitive) and Primed (initial) hPSCs, i.e., CBA medium, includes: DMEM / F12 medium, Neurobasal medium, 0.25-1% N2, 0.5-2% B27, and 0.1-1% N2. 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, 2-10μM Y27632, 1-3μM CHIR99021, 1-10ng / ml recombinant human bone morphogenetic protein 4 (BMP4), and 2-20ng / ml recombinant human activin A (Activin-A).

[0011] The percentage mentioned above represents volume percentage, i.e. (v / v).

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

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

[0014] Furthermore, this invention provides the application of CBA culture medium in the in vitro induction of differentiation into extraembryonic mesodermal cells.

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

[0016] Preferably, the extracellular matrix includes matrix gel, type IV collagen, vitrin, or laminin.

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

[0018] Preferably, the culture is placed in a CO2 incubator for no more than 5 days, and more preferably 5 days.

[0019] Preferably, by day 5 of culture, more than 91.4% of the naive hPSCs have specialized into extraembryonic mesodermal cells.

[0020] Preferably, by day 5 of culture, more than 98.8% of the Primed hPSCs have specialized into extraembryonic mesodermal cells.

[0021] Preferably, the inoculation density is 1–1.5 × 10⁻⁶. 4 cells / cm 2 .

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

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

[0024] In the specific implementation process, on day 5 of culture, the induced extraembryonic mesodermal cells are digested and inoculated into trophoblast stem cell culture medium or CB culture medium. The extraembryonic mesodermal cells can be stably cultured and expanded (more than 10 generations).

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] This invention provides a culture medium for rapidly (within 5 days) and efficiently (>90%) inducing the differentiation of naive and primed hPSCs into ExMs with self-renewal capabilities. The culture medium of this invention is universally applicable, simultaneously supporting the rapid and efficient differentiation of naive and primed hPSCs into ExMs. "Rapid" implies greater realism and better functionality, as ExMs specialization in human embryos takes only about 5 days. These ExMs obtained through hPSC differentiation are highly similar to ExMs in peri-implantation and peri-gastrulanation human embryos and can be stably cultured and expanded in vitro for a long period. Therefore, the culture medium and induction method of this invention have the advantages of short cycle, high yield, and low cost, which can promote the industrialization of ExMs. This invention provides a new cell model for the study of the developmental dynamics and principles of extraembryonic mesoderm, and has broad application prospects in the fields of regenerative medicine and the medical aesthetics industry. Attached Figure Description

[0027] Figure 1 The following is a graph showing the rapid and efficient differentiation of Naive hPSCs into ExMs in CB medium:

[0028] (a) Schematic and bright-field plot of ExMs differentiating in CB medium and expanding in trophoblast stem cell medium, scale bar represents 100 μm; (b) Immunofluorescence staining showing the expression of biomarkers in Naive hPSCs differentiated for 4 days in CB medium, 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) Heatmap showing pluripotency and ExM biomarker gene expression dynamics in Naive hPSCs differentiated for different days in CB medium and in expanded ExMs; (e) GO (Geneontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses based on differentially expressed genes between initial Naive hPSCs and Naive hPSCs differentiated for 4 days in CB medium; (f) UMAP (Uniform Manifold Approximation and Microbiome Mapping) data from single-cell transcriptome data of Naive hPSCs differentiated for 4 days in CB medium. (g) Projection visualization and clustering; (h) Bubble plot showing the expression of marker genes in specific cell populations; (i) Spearman correlation analysis showing cell types; (j) RNA rate analysis of single-cell transcriptome data; (j,k) Integrated analysis of single-cell transcriptome data from Naive hPSCs differentiated in CB medium for 4 days with published embryonic and in vitro differentiated cell data.

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

[0030] Figure 3The results show that Primed hPSCs primarily differentiated into amniotic cells in CB medium: (a) Schematic and bright-field plot of Primed hPSC differentiation in CB medium, scale bar represents 100 micrometers; (b) Principal component analysis of bulk RNA sequencing data of Naive hPSCs, Primed hPSCs, and Naive hPSCs and Primed hPSCs after 4 days of differentiation in CB medium; (c) Spearman correlation analysis of bulk RNA sequencing data of Naive hPSCs, Primed hPSCs, and Naive hPSCs and Primed hPSCs after 4 days of differentiation in CB medium; (d) Heatmap showing the expression of pluripotency, ExM, amniotic, and trophoblast cell marker genes in Naive hPSCs, Primed hPSCs, and Naive hPSCs and Primed hPSCs after 4 days of differentiation in CB medium; (e) Naive hPSCs, Primed hPSCs, Naive hPSCs and Primed hPSCs after different differentiation days in CB medium. Principal component analysis of bulk RNA sequencing data from hPSCs and extended-cultured ExMs; (f) showing the expression dynamics of biomarkers 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) showing the proportion of cell types; (i) showing bubble plots of biomarker gene expression in specific cell types; (j) showing UMAP plots of specific gene expression; (k) developmental trajectory plots predicted by Slingshot; (l) showing the proportion of cell types.

[0031] Figure 4The following figures illustrate the fate specialization of Primed hPSCs in CB medium compared to Naive hPSCs: (a) Immunofluorescence staining shows that Primed hPSCs in CB medium primarily specialize as amniotic cells, with the scale bar representing 100 micrometers; (b) Volcano plot showing differentially expressed genes between Naive and Primed hPSCs after 4 days of differentiation in CB medium; (c) UMAP clustering based on single-cell transcriptome data of Primed hPSCs after 4 days of differentiation in CB medium; (d) Bubble plot showing marker gene expression in specific cell types; (e) UMAP plot showing marker gene expression 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 trajectories; (h) Principal component analysis of bulk RNA sequencing data of Primed hPSCs after different days of differentiation in CB medium; (i) Representative pluripotency and lineage-specific marker genes in Primed hPSCs after different days of differentiation in CB medium. (j) Expression dynamics of original marker genes in Primed hPSCs differentiated for different days in CB medium; (k) Differentiation of Primed hESCs cultured in E8 system in CB medium, with the scale bar representing 100 micrometers.

[0032] Figure 5The results of adding Activin-A to CB medium to efficiently differentiate Primed hPSCs into ExMs are shown in the following figures: (a) Schematic diagram of the composition of CBA medium and its treatment of Primed (AIC) hPSCs; (b) Bright field images of Primed hPSCs after 5 days of differentiation in CB and CBA media, with the scale bar representing 100 micrometers; (c) Immunofluorescence staining images of TFAP2A and GATA6 in Primed hPSCs after 5 days of differentiation in CB and CBA media, with the scale bar representing 100 micrometers; (d) 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 at different times in CB and CBA media; (g) Heatmap of marker gene expression in different cell lineages of Primed hPSCs differentiated at different times in CB and CBA media; (h) Primed hPSCs after 5 days of differentiation in CBA medium. UMAP visualization and cell type clustering of single-cell transcriptome data of hPSCs; (i) UMAP showing gene expression in Primed hPSCs after 5 days of differentiation in CBA medium; (j) Schematic diagram, bright field diagram and immunofluorescence staining of Primed hPSCs after 5 days of differentiation in CBA medium switched to human trophoblast stem cell medium for extended culture, with the scale bar representing 100 micrometers; (k) Staining diagram of Primed hPSCs cultured in the E8 system efficiently differentiating into ExMs in CBA medium, with the scale bar representing 100 micrometers.

[0033] Figure 6The results show the efficient differentiation of Primed hPSCs into ExMs in CBA medium: (a) Bright field plot and staining plot showing markers of Primed hPSCs after 4 days of differentiation in CBA medium, with the scale bar representing 100 micrometers; (b) Staining plot showing markers of Primed hPSCs after 5 days of differentiation in CBA medium, with the scale bar representing 100 micrometers; (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 for different days in CBA medium; (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 plot showing the expression of hrGFP, mCherry-WPRE, and the Y chromosome-specific gene RPS4Y1; (g) Primed hPSCs cultured for 5 days in CBA medium. (h) Bubble plot showing the expression of marker genes in specific cell types in hPSCs; (h) Heatmaps of early and late ExM marker gene expression in Primed hPSCs treated with CBA for different time periods and in ExMs cultured in extended culture; (i,j) Dynamics of marker expression in Primed hPSCs cultured in CBA medium for different number of days, with the scale bar representing 100 micrometers.

[0034] Figure 7 The results show the 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 micrometers; (c) Immunofluorescence staining image of biomarkers in Naive hPSCs after 5 days of differentiation in CBA medium, with the scale bar representing 100 micrometers; (d) Heatmap showing the expression dynamics of representative biomarker 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 image showing gene expression in Naive hPSCs after 5 days of differentiation in CBA medium; (g) Immunofluorescence staining image of early hematopoietic biomarkers in Naive and Primed hPSCs after 5 days of differentiation in CBA medium, with the scale bar representing 100 micrometers.

[0035] Figure 8The results of CBA-induced high-efficiency differentiation of Naive hPSCs into ExMs are shown in the following diagrams: (a) showing the expression dynamics of marker genes in Naive hPSCs differentiated at different time points in CBA; (b) showing the heatmap of representative gene expression in Naive and Primed hPSCs differentiated at different time points in CBA; (c) showing the bubble map of marker gene expression in specific cell types in Naive hPSCs treated with CBA for 5 days; (d) showing the UMAP map of early hematopoietic marker gene expression in Primed hPSCs treated with CBA for 5 days; (e) a schematic diagram of transferring Naive hPSCs differentiated in CBA for 5 days to trophoblast stem cell culture medium for extended culture; (f) transferring Naive hPSCs differentiated in CBA for 5 days to trophoblast stem cell culture medium for extended culture, and performing immunofluorescence staining with antibodies displaying markers after 10 passages, with the scale bar representing 100 micrometers; (g) showing the expression of early and late ExM marker genes in Naive hPSCs at different differentiation stages. Heatmap of expression in hPSCs; (h) shows the staining map of efficient differentiation of Naive hPSCs cultured in PXGL into ExMs in the CBA system, with the scale bar representing 100 micrometers.

[0036] Figure 9 The results show the high similarity between ExMs differentiated from Naive and Primed hPSCs and ExMs in embryos: (a) Correlation analysis of Naive and Primed hPSCs differentiated and cultured under different conditions based on Bulk RNA-seq data; (b) Correlation analysis of different sources, different culture conditions and different cell subpopulations based on single-cell transcriptome data; (c) Integrated analysis of single-cell datasets from different sources, different culture conditions and different cell types based on single-cell transcriptome data. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. % represents volume percentage, i.e., (v / v). Unless otherwise specified, specific techniques or conditions in the embodiments are conventional methods or performed according to techniques or conditions described in the literature in this field, or according to product instructions. Reagents and instruments used without specified manufacturers are all conventional products that can be purchased through legitimate channels.

[0038] The CB medium formulation in the following examples is as follows: mN2B27 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 μM Y27632 (a selective ROCK1 and ROCK2 inhibitor) (Selleck, S1049), 2 μM 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 formulation of mN2B27 medium is as follows: basal medium (DMEM / F12 (volume ratio 1:1) mixed with Neurobasal 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), and 0.1% chemical lipid concentrate (Thermo Fisher). 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 derived from our previous study (Ai et al., 2023). Naive hPSCs were cultured in AIC-N or PXGL medium, with medium changes every two days. Every 4-5 days, cells were digested into single cells using 50% TrypLE for passage at a ratio of 1:5 to 1:10. The AIC-N medium formulation was 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 (a MEK signaling pathway inhibitor), and 2 μM [unclear - possibly a specific compound or ingredient] were added to the basal medium mN2B27. (A PKC inhibitor), 10 ng / ml hLIF (recombinant human leukemia inhibitory factor) and 10 μMY27632 (a selective ROCK1 and ROCK2 inhibitor). PXGL is a publicly available culture system and will not be described in detail here.

[0041] In the following examples, three Primed hPSCs (hES1, h1, and H9) were used, derived from our previous study (Ai et al., 2020). Primed hPSCs were cultured in AIC medium, with medium changes every two days. Every 3-4 days, cells were digested into single cells using 50% TrypLE and passaged at a ratio of 1:10 to 1:20. The AIC medium formulation was as follows: 10 ng / ml activator A, 2 μM IWP2, and 0.6 μM CHIR99021 were added to the basal medium mN2B27.

[0042] Example 1 demonstrates that CB medium can induce Naive hPSCs to differentiate into ExMs efficiently under feeder-free conditions.

[0043] The inventors previously developed CB medium, which can rapidly induce naive hPSCs to differentiate efficiently into ExMs on a mouse embryonic fibroblast feeder. However, because the feeder contains animal-derived components, it is not conducive to the conversion and use of ExMs. To test whether CB medium can also induce naive hPSCs to differentiate rapidly into ExMs under feeder-free conditions, we digested naive hPSCs into single cells using 50% TrypLE, centrifuged them, discarded the supernatant, resuspended the naive hPSCs in CB medium, and then... 4 cells / cm 2Cells were seeded at the specified density into TC-treated culture dishes / plates containing extracellular matrix (Matrix gel, type IV collagen, hydrin, and laminin, etc.), and then incubated in a CO2 incubator. The medium was changed every two days, and the cells were analyzed after the fourth day of incubation.

[0044] Identification using immunofluorescence staining, flow cytometry, bulk RNA sequencing, and single-cell transcriptome sequencing revealed that on day four, 93.5% of the cells were induced to become ExMs, and the induced ExMs were highly similar to those found in peri-implantation and peri-gastrulan embryos. Figure 1 and Figure 2 ).

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

[0046] The results showed that ExMs could be stably amplified for at least 5 generations in CB medium (due to time constraints, this invention only cultured for 5 generations, but it is expected that more generations could be passaged), and could be stably amplified for more than 30 generations in trophoblast stem cell 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 Primed hPSCs to differentiate into ExMs under feeder-free conditions was much lower than that of Naive hPSCs.

[0048] To test whether Primed hPSCs could differentiate into ExMs as rapidly and efficiently as Naive hPSCs in CB medium under feeder-free conditions, we digested Primed hPSCs cultured in AIC or E8 systems into single cells using 50% TrypLE, centrifuged, removed the supernatant, resuspended the Primed hPSCs in CB medium, and then... 4 cells / cm 2Cells were seeded at the specified density 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] Identification using immunofluorescence staining, flow cytometry, bulk RNA sequencing, and single-cell transcriptome sequencing revealed that on day four, approximately 18.8% (AIC) and 50% (E8) of the differentiated cells were ExMs, while the remaining cells were almost entirely amniotic cells. Figure 3 and Figure 4 That is, compared with naive hPSCs, primed hPSCs had a significantly lower efficiency in differentiating to produce ExMs in CB medium. Figure 3 and Figure 4 ).

[0050] Example 3: CBA medium induced efficient differentiation of Primed hPSCs into ExMs under feeder-free conditions.

[0051] To improve the efficiency of Primed hPSC differentiation into ExMs, this invention screened a large number of growth factors, cytokines, and small chemical molecules. It was found that adding Activin-A (recombinant human activin A), an activator of the Activin / Nodal signaling pathway, to CB medium effectively inhibited the differentiation of Primed hPSCs into amniotic cells and promoted their differentiation into ExMs. Therefore, Activin-A (5 ng / ml) was added to CB medium, and this CB medium with added Activin-A was named CBA medium. Figure 5 ).

[0052] Under feeder-free conditions, we digested Primed hPSCs cultured in AIC or E8 medium into single cells using 50% TrypLE, centrifuged, discarded the supernatant, resuspended the Primed hPSCs in CBA medium, and sputtered at 1-1.5 × 10⁻⁶ cells / mL. 4 cells / cm 2 Cells were seeded at the appropriate density into Matrigel-coated TC-treated culture dishes / plates and then incubated in a CO2 incubator, with the medium changed every two days.

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

[0054] On day 5, Primed hPSCs differentiated for 5 days in CBA medium were digested into single cells using 50% TrypLE and directly seeded into a publicly reported trophoblast stem cell culture medium for adherent culture. Thereafter, the medium was changed every two days, and single-cell passages were performed every 3-5 days using 50% TrypLE at a passage ratio of 1:3 to 1:5. At least 10 passages were performed; after 10 passages, the ExMs were not further passaged or analyzed. The results showed that these cells could be stably cultured and expanded in trophoblast stem cell culture medium. Figure 5 and Figure 6 ).

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

[0056] To further determine whether naive hPSCs could differentiate into ExMs rapidly and efficiently in CBA medium under feeder-free conditions, similar to primed hPSCs, we digested naive hPSCs cultured in AIC-N or PXGL medium into single cells using 50% TrypLE. After centrifugation, the supernatant was discarded, and the naive hPSCs were resuspended in CBA medium at a concentration of 1–1.5 × 10⁻⁶ cells / mL. 4 cells / cm 2 Cells were seeded at the specified density into TC-treated culture dishes / plates coated with Matrigel extracellular matrix, and then incubated in a CO2 incubator with the medium changed every two days.

[0057] Identification using immunofluorescence staining, flow cytometry, bulk RNA sequencing, and single-cell transcriptome sequencing revealed that on day five, 91.4% of the differentiated cells had specialized into ExMs (ExMs). Figure 7 and Figure 8 Furthermore, 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 for 5 days in CBA medium were digested into single cells with 50% TrypLE and then directly seeded into a publicly reported feeder stem cell culture medium for adherent culture. The medium was changed every two days, and single cells were passaged every 3-5 days with 50% TrypLE at a passage ratio of 1:3 to 1:5. At least 10 passages were performed; after 10 passages, the ExMs were not further passaged or analyzed. The results showed that these cells could 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 the embryo.

[0060] To identify the cellular characteristics of ExMs generated by the differentiation of Naive or Primed hPSCs induced by CB or CBA systems, we collected samples of Naive or Primed hPSCs after different days of differentiation induced by CB or CBA systems and obtained their transcriptome data. These data were then compared with transcriptome data from early post-implantation human embryos. The results showed that, under CB and CBA conditions, the ExMs generated by the differentiation of Naive and Primed hPSCs were generally similar to those in early post-implantation human embryos. Figure 9 ).

[0061] In summary, under feeder-free conditions, CB and CBA media had different induction effects on Naive hPSCs and Pimed hPSCs, specifically: (1) CB media induced Naive hPSCs to differentiate into ExMs rapidly (4 days) and efficiently (93.5%), but the effect of inducing Pimed hPSCs to differentiate into ExMs was poor (at 4 days, a large number of amniotic cells were generated, about 81.2% of which were amniotic cells, and only a small number of cells, about 18.8%, differentiated into ExMs); (2) CBA media induced 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). In other words, CBA medium is more compatible and universal in inducing Naive hPSCs and Pimed hPSCs to differentiate into ExMs; (3) ExMs induced by CB and CBA can be stably cultured and expanded in vitro (more than 10 generations). (4) ExMs induced by CB and CBA medium to differentiate from Naive hPSCs and Pimed hPSCs 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, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for inducing differentiation of extraembryonic mesodermal cells in vitro, characterized in that, include: After Primed hPSCs were digested into single cells, they were resuspended in CBA medium and seeded in culture dishes or plates with extracellular matrix, and then placed in a CO2 incubator for culture. The CBA culture medium includes: 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, 0.5-2 μg / ml heparin sodium, 2-10 μM Y27632, 1-3 μM CHIR99021, 1-10 ng / ml recombinant human bone morphogenetic protein 4 and 2-20 ng / ml recombinant human activin A.

2. The method according to claim 1, characterized in that, During the incubation process in a CO2 incubator, the medium should be changed every two days.

3. The method according to claim 1, characterized in that, Incubate in a CO2 incubator for no more than 5 days.

4. The method according to claim 3, characterized in that, Incubate in a CO2 incubator for 5 days.

5. The method according to claim 1, characterized in that, The inoculation density is 1~1.5×10⁻⁶. 4 cells / cm 2 .

6. The method according to claim 1, characterized in that, Prior to digestion into single cells, the Primed hPSCs were cultured in AIC, E8, mTeSR, AIC-N, or PXGL systems.

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