Methods for the derivation culture of embryonic and extra-embryonic stem cells

Abstract

The present disclosure relates to culture conditions and methods of deriving embryonic stem cells (ESCs), extraembryonic endoderm stem cells (XENs) and trophoblast stem cells (TSCs) from reproductive cells or naïve pluripotent stem cells (PSCs), simultaneously. Aspects of the method comprise use of a medium that comprises a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF-β) activator, and a WNT activator for culturing the cells. Methods of obtaining growth restricted ESCs and multilineage stem cells are also described. Also provided are in vitro culture systems for producing the stem cells.

Description

METHODS FOR THE DERIVATION CULTURE OF EMBRYONIC AND EXTRA- EMBRYONIC STEM CELLSCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 488,401 , filed March 3, 2023, and titled “METHODS FOR THE DERIVATION CULTURE OF EMBRYONIC AND EXTRA-EMBRYONIC STEM CELLS,” which is incorporated by reference herein in its entirety.ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with support under Grant No. GM 138565 awarded by the National Institutes of Health. The government has certain rights in the invention.INCORPORATION OF SEQUENCE LISTING

[0003] This present application contains a Sequence Listing that which has been submitted in .XML format via Patent Center and is hereby incorporated by reference in its entirety. Said WIPO Sequence Listing was created on February 28, 2024, XML copy is named 106546-788644 Sequence listing. xml, and is 98000 bytes in size.BACKGROUND

[0004] 1 . Field

[0005] The present invention relates to derivation of stem cells from embryonic and extraembryonic tissues using the culture conditions from reproductive cells and methods for establishing co-cultures of embryonic and extraembryonic stem cells.

[0006] 2. Discussion of Related Art

[0007] Faithful embryogenesis requires precise coordination between embryonic and extraembryonic tissues. Although stem cells from embryonic and extraembryonic origins have been generated for several mammalian species, they are grown in different culture conditions with diverse media composition, which makes it difficult to study cross-lineage communication. Formulating culture conditions that establish embryonic and extraembryonic stem cell cocultures are therefore necessary, to dissect lineage crosstalk during early mammalian development. Such co-cultures of embryonic and extraembryonic stem cells can be applied to uncover several conserved and previously unrecognized growth and signaling pathways of pluripotent cells.SUMMARY OF INVENTION

[0008] In some aspects, the disclosure provides a method for derivation of embryonic stem cells (ESCs), extraembryonic endoderm stem cells (XENs), and / or trophoblast stem cells (TSCs) in vitro, the method comprising obtaining reproductive cells or naive pluripotent stem cells (PSCs); culturing the reproductive cells or naive human PSCs in a culture medium that comprises a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF- |3) activator, and a WNT activator; and simultaneously deriving stable ESCs, XENs, and TSCs.

[0009] In some aspects, the method further comprises, isolating the epiblast-like, endoderm-like, and / or trophoblast-like cells from the cell outgrowth; and culturing and passaging the isolated cells to obtain stable ESCs, XENs, or TSCs.

[0010] In some aspects of the method, the ESCs, XENs, and TSCs are produced from reproductive cells. In some aspects of the method, the reproductive cells comprise blastocyst. In further aspects of the method, the derivation is from a single or multiple blastocysts. In some aspects, the reproductive cells are obtained from a mammal. In some aspects, the reproductive cells are obtained from a human, a rodent, or a non-human primate.

[0011] The disclosure further provides a method of restricting growth of ESCs in a cell culture, the method comprising, obtaining a population of ESCs derived from reproductive cells or PSCs; culturing ESCs in a culture medium that comprises a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF- ) activator, and a WNT activator; adding a growth restricting agent to the culture medium, wherein the agent is selected from laminin, collagen, XENs, or any combination thereof; and maintaining the cells in the culture for at least 5 days to obtain a population of growth restricted ESCs. In some aspects ESCs and XENs are obtained using a method of derivation provided in the disclosure.

[0012] In some aspects, the growth restricting agent is laminin and the laminin is at a concentration of 1 - 200 pg / ml in the culture medium. In some aspects, the growth restricting agent is collagen and the collagen is at a concentration of 1 - 200 pg / ml in the culture medium. In some aspects of the method, the growth restricting agent is XENs and the XENs is added at an initial ESC to XEN ratio of about 1 :2 to about 8:1 to the culture medium.

[0013] In some aspects of the method, the growth restricted ESCs comprise ESCs with reduced cell density, and / or reduced expression of one or more Lyz2, Gpnmb, Saa3, B2m, H2-D1 , Thbsl , Fbln2, Ctsl, Bgn, Plau, Marco, Col1a2, Ctss, Timp2, C1qa, Lilrb4a, Csf1 , Col1a1 , Tnfaip2, Den, Lox, Thbs2, Il1 rn, Plek, Adgrel , Mpegl , Msr1 , Serpinel Adam8, H2- M2, Cybb, C1qc, Mmp3, Ccl3, Anxal , Postn, Cd274, Vim, Xist, Mmp9, Lgals3bp, Cxcll , Wfdc17, Sdc3, lfi202b, Clec4e, Acssl , Vcaml , C1qb, Ccl2, Col5a3, Egr1 , Fcgr3, C3, C3ar1 , 111 a, Fcgr2b, Lilr4b, Ccl6, Plk2, Clec4n, Ccl9, Pf4, Csf2rb, Axl, Atp6v0d2, Ms4a6d, Cd44, App,Zeb2, Ms4a7, Greml, Ptgs2, Tnc, Slfn2, Itgb2, Col6a1, Aebpl, Mmp12, Col5a1, Slc7a2, Mmp2, Tyrobp, Mmp13, Cxcl2, Trem2, S100a6, Id2, Ecm1, Ltbp2, Cd53, Bmp1, Serpinfl, Ptges, Plxndl, Feerig, Fxyd5, Cd36, Arhgdib, Ccl5, Cyr61, Sirpa, Rgs1, Slc15a3, Rac2, AI607873, Apbblip, Xdh, Clec4d, Ccl7, Slc11a1, Bst1 , Alcam, Tgfbi, Col5a2, Glrx, Tnfrsf23, Slfn5, Gas7, Gas6, Tnfaip3, Ctla2a, 111 rl1, Atf3, Itpripl2, Ddr2, 6720427l07Rik, Cd84, Ptprc, Itgb3, Prrxl, Fabp4, Pmp22, Cav1, Mical2, Ctgf, Col6a2, Inhba, Igfbp7, Tm4sf1, Lum, Hk3, Fam46c, Cd33, Adamtsl, Clec7a, Lcn2, Serpinb2, Mir17hg, Col8a1, Il4ra, Csf2rb2, Adssll, Irg1, Gsap, Ccrl2, Cd300lf, Tmem176b, Egr2, Rgs16, Rab7b, Rhob, Grem2, Col12a1, Col6a3, Lgals3, lfi204, Rassf4, Rnd3, Fam198b, Cd14, Pcdh7, Marchl, Cp, Ncf4, lfi203, Ptger4, Csflr, Smpdl3a, Havcr2, H2-T23, Hclsl, Sigled, Naip2, Emp1, WIs, Ier5, Rps2-ps13, Cd93, Serpina3g, Penk, Serpina3h, Layn, Cryab, Thbd, Olr1, Nlrc5, Acta2, Col16a1, Pik3ap1, Thy1, Ncf1, AF251705, Mmp10, Synpo, Igf1, Ly9, Ptx3, Npy, II33, Tns1, Gda, Mafb, Casp4, Tfpi2, Cd180, Mr1, Lmxlb, Ets1, Ccdc80, Ugt1a7c, Gm15487, Gm8995, Rnasel, Cav2, Il7r, Mrc2, Itpr2, C5ar1, Tgfbr2, Cyp1b1, P2ry6, Gbp9, Dusp8, Acpp, Ifi44, Antxr2, Cxcl3, Ly6a, Nrp1, Rgl1, Oaf, Ccl8, Clec2d, Ptpre, Pdgfra, Arhgap25, Arsb, S1pr1, Adgrg6, Fam20c, Sh3kbp1, Runxl, Kitl, Gab2, Col3a1, Rspo4, H10ra, 111 b, Arhgap24, Ikbke, Emilin2, Cd34, Itga4, Hacd4, Nptxl, Steap4, Ccbel, Mmp8, Caspl, Entpdl, Sqrdl, Efnb3, Cd302, Gem, Dkk3, Rnase4, DuspIO, Mnda, Psmb8, Cd82, Sod3, Osbpl5, Lats2, Fgd3, Arhgap31, Cd86, Ms4a14, Mcoln2, Pik3cg, Cyp4v3, Slc25a24, Tagln, Cebpa, Rsph4a, Fas, Ereg, Klhl6, Col4a5, 181001101 ORik, Itgam, Arg2, Tmem176a, Tlr4, Flrt2, Efempl, Pla2g4a, 01 s1, B430306N03Rik, Mn1, Procr, Stabl, Plat, Cd200, Nos2, Rasgeflb, Wnt5a, Ccr1, Sept5, Gap43, Mef2c, Creb5, Ifitl, Negri, Parva, Gpr176, Orai2, S100a8, Pappa, 111 r1 , Prl2c3, Raetld, Hspalb, Adamts5, Evi2a, C1ra, Zfp703, Filipll, Fgf7, Dclkl, Nov, Heph, Sema3a, Pcdh19, Pgm5, Slc2a9, Irf2, Vgll3, Fcgr4, Cyth4, Mgstl, Gbp8, Emp3, H20rb, Fhl2, Casp12, Dkk2, Fill, 4930523C07Rik, Ngf, Maspl, Lrrc15, Cxcl5, Scarf2, Lrrc18, Nbl1, Gm10143, Pdzrn3, Pdlim4, Osmr, Slc22a4, Mmp19, Myo18b, Ctso, Angpt4, Mxd4, Fads3, Adgrg2, Fam78b, Hgf, Cpq, Serpina3n, Prl2c2, Rbms3, Pcdh9, Tfec, Ldlrad4, Rgs2, Cd40, Fbxo32, Mir22hg, Sparcl 1, Tgfb2, Pde4b, Cspg4, Cd200r1, Abca8b, Ctsk, Tap2, Ehd2, Svepl, Rcan2, Vax2os, Dchsl, Gm14453, Zfpm2, Slc22a13b-ps, Gm42531, Gng11, Pla2g4c, Isg15, Serpinb6b, Cdh13, Gm33280, Milrl, Slc8a1, Maf, Cnripl, Slpi, Fam13a, Runx2, Bdkrb2, Till, Itga11, Gbp4, Gm15559, IHOrb, Il15ra, Rab32, Mctpl, Sspn, Dachl, Pamrl, Lat2, Mcpt8, Msrb3, Serpinb9, Cd52, Rgma, Tbxasl, Itga2, Osr2, Nedd9, Gdnf, Nol4, Ust, Eya4, Slc16a2, C920025E04Rik, Lcelg, Nr2f2, Foxd, II7, Pcp4l1, Ednra, 9330175E14Rik, Lama2, Plxna4, Serpinbla, Nipall, Mfap4, Irx1, Rspo3, Prss23, Nsg1, Dnm3os, Osginl, Pxdd, Fat4, Fat3, Snai2, Thsd7a, Gm26809, Tgfb3, Cxxc4, Meis3, Rbm24, Cplx2, Vax2, Areg, Gabrbl, Lancl3, PI15, Lrrc32, Fgf8, RP24-439I22.8, Scara3, Fgf2, Kcnn3, 4933427D06Rik, Rnf150, or any combination thereof, when compared to derived ESCs.

[0014] According to some aspects of the disclosure, provided herein is a method for obtaining multi-lineage stem cells in a shared cell culture condition, the method comprising, obtaining a population of ESCs derived from reproductive cells or PSCs; obtaining a population of XENs and / or TSCs derived from reproductive cells or PSCs; co-culturing ESCs, XENs, and / or TSCs in a culture medium that comprises a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF-|3) activator, and a WNT activator; maintaining the mixed cells in the culture medium for at least 5 days to obtain a population of the multi-lineage stem cells.

[0015] In some aspects of the method, the multi-lineage stem cells comprise ESCs and XENs cells. In some aspects, the multi-lineage stem cells comprise ESCs and TSCs cells. In some aspects, the multi-lineage stem cells comprise ESCs, XENs, and TSCs cells.

[0016] Various aspects of the method further comprises adding laminin at a concentration of 1 - 200 pg / ml in the culture medium. In some aspects, the method further comprises adding collagen at a concentration of 1 - 200 pg / ml in the culture medium.

[0017] In some aspects of the method the ESCs, XENs, and / or TSCs are obtained using a method of derivation provided in the disclosure.

[0018] In some aspects the method comprises combining ESCs and XENs at an initial ESC to XEN ratio of about 1 :2 to about 8:1. In certain aspects, the method comprises combining ESCs and TSCs at an initial ESC to TSC ratio of about 1 :2 to about 8:1. In some aspects, the method comprises combining ESCs, XENs, and TSCs at an initial ESC to XEN to TSC ratio of about 1 :2:2 to about 8:1 :1.

[0019] In some aspects, the culture medium used in the methods provided further comprises neurobasal medium. In some aspects, the FGF activator used in any of the methods provided herein is FGF2. In some aspects, the TGF- activator used in any of the methods provided herein is Activin-A. In some aspects, the WNT activator used in any of the methods provided herein is a GSK3 inhibitor. In some aspects, the GSK3 inhibitor is CHIR99021.

[0020] In some aspects of the methods the XENs and / or TSCs express extraembryonic endoderm or trophoblast markers, respectively. In further aspects of the methods the XENs and TSCs can form XEN-tumors and TSC-tumors, respectively.

[0021] Some aspects of the disclosure provides an in vitro co-culture system comprising, ESCs derived from reproductive cells or PSCs; XENs, TSCs derived from reproductive cells or PSCs; a growth restricting agent for the culture medium, wherein the agent is selected fromlaminin, collagen, or any combination thereof; and a culture medium comprising a FGF activator, a TGF- activator, and a WNT activator.

[0022] In some aspects of the system the mixed population of ESCs, XENs, and / or TSCs form multi-lineage stem cells. In some aspects, the system comprises ESCs and XENs cells. In some aspects, the ESCs and XENs are present in the system at an initial ESC to XEN ratio of about 1 :2 to about 8:1. In some aspects, the system comprises ESCs and TSCs cells. In some aspects, ESCs and TSCs are present in the system at an initial ESC to TSC ratio of about 1 :2 to about 8:1. In some aspects, the system comprises ESCs, XENs, and TSCs cells. In some aspects, the ESCs, XENs, and TSCs are present in the system at an initial ESC to XEN to TSC ratio of about 1 :2:2 to about 8:1 :1.

[0023] In some aspects, the system comprises laminin. In some aspects, the laminin is at a concentration of 1 - 200 pg / ml in the culture medium. In some aspects, the system comprises collagen. In some aspects, the collagen is at a concentration of 1 - 200 pg / ml in the culture medium.

[0024] In some aspects of the system the ESCs, XENs, and / or TSCs are obtained using a method of derivation provided in the disclosure.

[0025] In some aspects of the system the culture medium further comprises neurobasal medium. In some aspects of the system the FGF activator is FGF2. In some aspects of the system the TGF-fJ activator is Activin-A. In some aspects of the system the WNT activator is a GSK3 inhibitor. In some aspects, the GSK3 inhibitor is CHIR99021.

[0026] In some aspects of the system the XENs and / or TSCs express extraembryonic endoderm or trophoblast markers, respectively. In further aspects, the XENs and TSCs can form XEN-tumors and TSC-tumors, respectively.

[0027] Another aspect of the disclosure provided an in vitro co-culture system comprising, ESCs derived from blastocysts, wherein the ESCs have a mutation or deletion in one or more integrin gene and / or, XENs derived from blastocysts, wherein the XENs have a mutation or deletion in one or more laminin gene; and a culture medium comprising a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF-|3) activator, and a WNT activator.

[0028] Further aspect of the disclosure provides an embryo model produced by differentiation of cells produced by the any of the disclosed methods or any of the disclosed systems.

[0029] In some aspects, a method for cellular analysis is provided, the method comprising using cells produced by the any of the disclosed methods or the any of the disclosed systems.In some aspects, the cellular analysis comprise drug assessments, toxicity assessments, cell growth assessments, cell proliferation assessments, cell differentiation assessments, tissue development assessments, cell culture assessments or any combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1A-1G illustrate derivation and characterization of embryonic and extraembryonic stem cells from mouse blastocyst. FIG. 1A illustrates schematic of derivation of FTW embryonic and extraembryonic stem cell lines from mouse blastocyst. FIG. 1 B shows representative bright field (BF) images of colonies of FTW-mXENs, FTW-mTSCs and FTW- mESCs. Scale bar, 100 pm. FIG. 1C displays representative immunofluorescence (IF) images of extraembryonic endoderm (GATA6 and SOX17), trophoblast (EOMES and CDX2), and epiblast (SOX2 and OCT4) lineage markers in FTW-mXENs (top), FTW-mTSCs (middle), and FTW-mESCs (bottom), respectively. Scale bar, 100 pm. FIG. 1 D and FIG. 1 F show representative BF and fluorescence images showing chimera contribution of GFP-labeled FTW-mXENs (FIG. 1 D) and FTW-mTSCs (FIG. 1 F) to E11.5 mouse conceptuses. Scale bar, 1 mm. FIG. 1E depicts IF staining of a chimeric yolk sac membrane for GFP, GATA6 and GATA4. FIG. 1G depicts IF staining of a chimeric sagittal placental section for CK8 and GFP. Different layers of the placenta are delineated by dotted lines. Scale bar, 100 pm.

[0031] FIG. 2A-2T show derivation, characterization of FTW-mXENs, FTW-mTSCs and FTW- mESCs and developmental potential of FTW-mXENs and FTW-mTSCs. FIG. 2A is representative BF images of an E4.5 mouse blastocyst (upper left), day 8 outgrowth (bottom left) and passage 1 (right) during FTW stem cells derivation. Higher magnification images of the boxed areas are shown in the middle panel. Scale bars, 100 pm. FIG. 2B depicts flow cytometry results showing the expression of GATA6 and EOMES in FTW-mXENs (left) and FTW-mTSCs (right). FIG. 2C illustrates qRT-PCR results showing the relative expression levels of lineage markers in FTW-mESCs, FTW-mXENs and FTW-mTSCs. (mean ± SD, n = 3, biological replicates). FIG. 2D shows representative IF images of PDGFRa and SOX17 of FTW-mXENs. Scale bar, 100 pm. FIG. 2E is a representative IF image of TFAP2c of FTW- mTSCs. Scale bar, 100 pm. FIGs 2F-2I display representative BF (FIG. 2F and FIG. 2H) and IF images (FIG. 2G and FIG. 2I) of conventional mXENs (FIG. 2F and FIG. 2G) and mTSCs (FIG. 2H and FIG. 2I). Scale bars, 100 pm. FIG. 2J shows RT-PCR results showing the expression of lineage markers in conventional mXENs and mTSCs and converted FTW- mXENs and FTW-mTSCs. GAPDH, loading control. FIG. 2K displays doubling time of conventional mXENs (n = 5, biological replicates), mTSCs (n = 5, biological replicates), converted FTW-mXENs (n = 8, biological replicates), and converted FTW-mTSCs (n = 5, biological replicates). Box-and-whisker plots showing the median value (bar inside box), 25thand 75thpercentiles (bottom and top of box, respectively), and minimum and maximum values(bottom and top whisker, respectively). FIG. 2L illustrates quantification of cell numbers for mESCs and FTW-mESCs, as well as mXENs and FTW-mXENs, after 5 days of treatment with various inhibitors. PD17, PD173074, an FGF inhibitor; SB43, SB431542, a TGF-beta inhibitor; LDN19, LDN193189, a BMP inhibitor; EC359, a LIF inhibitor; IWR1 , a WNT inhibitor (mean ± SD, n = 3 biological replicates; *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 , N.S., not significant; p values were calculated using two-tailed Student’s t test).. FIG. 2M shows qRT- PCR results showing the relative expression levels of several PE and VE markers in randomly differentiated FTW-mXENs at indicated time points (mean ± SD, n = 3 biological replicates; *p < 0.05, ***p < 0.001. N.S., not significant; p values were calculated using two-tailed Student’s t test). FIG. 2N shows representative images showing the appearance and H&E staining of a XEN-teratoma. A higher magnification image of the boxed area is shown on the right. FIG. 20 shows representative IF images of AFP, GATA6, PAX6 and T in XEN-teratoma sections. Blue, DAPI. Scale bars, 100 pm. FIG. 2P is a representative IF image of FOXA1 and COL6A1 in a XEN-teratoma section. FIG. 2Q depicts Representative BF and DAPI staining image of randomly differentiated FTW-mTSCs at day 12. Yellow dashed lines indicate multinucleated cells. Scale bar, 100 pm. FIG. 2R shows representative images showing the appearance and H&E staining of a TSC-teratoma. A higher magnification image of the boxed area is shown in the right panel. FIG. 2S shows qRT-PCR results showing the relative expression levels of trophoblast differentiation-associated genes in random differentiated FTW-mTSCs at indicated timepoints (mean ± SD, n = 3, biological replicates). FIG. 2T is representative BF and fluorescence images showing chimeric contribution of mKO-labeled FTW-mXENs (left) and FTW-mTSCs (right) to E11.5 mouse conceptuses. Scale bars, 1 mm.

[0032] FIG. 3A-3L illustrate transcriptomic profiling of mouse FTW stem cells. FIG. 3A displays LIMAP visualization of integrative analysis of FTW-mXENs, FTW-mTSCs, FTW- mESCs and published datasets of mPSCs, mTSCs and mXENs. FIG. 3B depicts correlation analysis of FTW-mXENs, FTW-mTSCs and FTW-mXENs with published scRNA-seq dataset of in vivo mouse embryos from different stages. FIG. 3C shows LIMAP analysis of mouse blastocysts, blastocyst outgrowths, and stable (passage 10) FTW-mESCs, FTW-mXENs and FTW-mTSCs. FIG. 3D-3F display heatmaps and GO terms analysis of stage-specific (blastocyst, blastocyst outgrowth and stable FTW stem cells) genes from three lineages during FTW stem cells derivation. FIG. 3G shows uniform manifold approximation (LIMAP) visual representation captures the single cell populations within FTW-mXENs, FTW-mTSCs, and FTW-mESCs. FIG. 3H shows violin plots illustrating the expression levels of FTW-mESC, FTW-mXEN, and FTW-mTSC marker genes. FIG. 3I shows a heatmap visualizes the Pearson correlation matrix, comparing FTW-mXENs, FTW-mTSCs, FTW-mESCs, and different mouse datasets. FIG. 3J shows a diagram detailing the derivation timeline for FTW-mXENs, FTW-mTSCs, and FTW-mESCs in the top panel and LIMAP visual representation integrating all scRNA-seq data, which include ICM / EPI, TE, and PrE of blastocysts; ELCs, TLCs, and XLCs from day 8 outgrowth; and established FTW-mESCs, FTW-mTSCs, and FTW-mXENs in the bottom panel. FIG. 3K shows pseudotime trajectory depicts the scRNA-seq progression of distinct cell lines in mice in the top panel, a heatmap show casing the pseudotime-dependent gene expression changes, notably, C1 , C4, and C7 represent early-stage patterns; C2, C5, and C8 represent mid-stage patterns; and C3, C6, and C9 capture late-stage patterns in the bottom left and a kinetics plot visualizes the relative expression trends of marker genes throughout developmental pseudotime in the bottom right panel. FIG. 3L shows a geneexpression heatmap details the differentially expressed genes for each identified cluster in the left panel and. Associated Gene ontology (GO) terms for each cluster in the right panel.

[0033] FIG. 4A-4H depict transcriptomic analyses of mouse FTW stem cells. FIG 4A shows correlation analysis of FTW-mXENs, FTW-mTSCs, FTW-mESCs and published datasets of mPSCs, mTSCs and mXENs. FIG. 4B shows the expression levels (Iog2 [FPKM\10+1]) of epiblast, trophoblast and extraembryonic endoderm markers in FTW-mESCs, FTW-mTSCs, and FTW-mXENs obtained from RNA-seq datasets. FIG. 4C displays correlation analysis of FTW-mXENs, FTW-mTSCs, FTW-mESCs and published datasets from mouse embryos and stem cell embryo models. FIG. 4D depicts PCA plots of RNA-seq datasets from FTW-mESCs, FTW-mTSCs, and FTW-mXENs and in vivo E3.5-E6.5 mouse conceptuses. Inner cell mass (ICM), Epiblast (Epi), Primitive endoderm (PrE), Visceral endoderm (VE), Extraembryonic endoderm (ExE). FIG. 4E illustrates LIMAP analysis of scRNA-seq datasets of FTW-mESCs, FTW-mTSCs, FTW-mXENs and in vivo E5.5 mouse conceptuses (EPI, PrE and TE cells). FIG. 4F shows LIMAP representation of single cell populations from FTW-mXENs, FTW- mTSCs, and FTW-mESCs. FIG. 4G shows LIMAP visualization illustrating the expression of representative markers in the mouse FTW stem cells: FTW-mESCs (Nanog, Pou5f1 , Sox2), FTW-XENs (Gata6, Sox17, Pdgfra), and FTW-TSCs (Cdx2, Gata3, Krt7). FIG. 4H shows a pie chart illustrating the proportion of different marker gene expression in different cell clusters.

[0034] FIG. 5A-5K illustrate proliferation restriction of FTW-mESCs by FTW-mXENs. FIG. 5A is a schematic of the establishment of FTW stem cell co-cultures to study crosslineage communications. FIG. 5B shows representative fluorescence and BF merged images of day 1 to day 5 separately cultured FTW-mESCs (green) and FTW-mESCs co-cultured with FTW-mXENs (red) and / or FTW-mTSCs (blue arrowheads), or proliferative mouse embryonic fibroblast. Scale bar, 100 pm. FIG. 5C depicts violin plot showing area multiplied by GFP intensity for each FTW-mESC colonies in day 5 separate and different co-cultures. FIG. 5D illustrates growth curves of separate (FTW-mESCs) and co-cultures (mESCs / mXENs) from days 1 to 5 (mean ± SD, day 1 , n = 2, day 2-5, n = 5, biological replicates). FIG. 5E is aschematic of teratoma formation with FTW-mESCs only and FTW-mESCs co-injected with FTW-mXENs (mESCs:mXENs = 4:1). The same number (1 x 106) of FTW-mESCs were injected in each condition. FIG. 5F show images of teratomas generated from FTW-mESCs injected with (bottom) and without (top) FTW-mXENs. FIG. 5G illustrates lengths and widths of teratomas generated from FTW-mESCs injected with (orange) and without (green) FTW- mXENs. FIG. 5H depicts weight of teratomas generated from FTW-mESCs injected with (orange) and without (green) FTW-mXENs. (mean ± SD, n = 5, biological replicates). FIG. 5I is a schematic of tissue dissection of E6.5-6.75 mouse conceptus. FIG. 5J displays representative BF images showing ex vivo culture of EPI+VE (VE+) and EPI (VE-) tissues isolated from E6.5-6.75 mouse conceptuses at indicated time points. Scale bar, 100 pm. FIG. 5K shows total cell number of VE+ and VE- tissues after 48 h ex vivo culture (mean ± SD, n = 10, biological replicates). N.S. not significant, ****P-value < 0.0001 , P-values were calculated using two-tailed Student’s t test.

[0035] FIG. 6A-6R illustrate proliferation restriction of FTW-mESCs by FTW-mXENs. FIG. 6A depicts bar plot showing the cell densities of FTW-mESCs in day 5 separate and different co-cultures. FIG. 6B depicts bar plot showing the cell densities of FTW-mESCs in separate and co-cultures (different mESCs: mTSCs and / or mESCs:mXENs ratios). FIG. 6C is representative BF and fluorescence merged images of day 5 3D separate and co-cultures (different mESCs:mXENs ratios). Green, FTW-mESCs. Scale bar, 100 pm. FIG. 6D depicts bar plot showing the total cell number of day 5 3D separate and co-cultures (different mESCs:mXENs ratios) (mean ± SD, n = 2, biological replicates). FIG. 6E shows representative IF images showing the expression of Activated Caspase 3 in separately cultured FTW-mESCs (bottom) and FTW-mESCs co-cultured with FTW-mXENs (top). Scale bars, 100 pm. FIG. 6F depicts bar plot showing the percentages of Caspase3+ cells in separately cultured FTW- mESCs (left) and FTW-ESCs co-cultured with FTW-mXENs (right), (mean ± SD, n = 3, biological replicates, N.S., not significant). FIG. 6G shows representative IF images showing the expression of GATA6 and SOX2 in co-cultured FTW-mXENs (mKO) and FTW-mESCs (GFP), respectively. Scale bars, 100 pm. FIG. 6H depicts bar plot showing the cell densities of FTW-mESCs in day 5 separate, contact and non-contact (transwell) co-cultures (mean ± SD, n = 3, biological replicates). FIG. 6I shows representative BF and fluorescence merged images of FTW-mESCs and FTW-mXENs in micropatterned co-cultures from day 1 to day 4. Scale bars, 100 pm. Yellow arrowhead, a standalone FTW-mESCs colony. Red arrowheads, FTW-mESCs colonies adjacent to FTW-mXENs. FIG. 6J shows representative H&E staining images of a teratoma generated from FTW-mESCs alone (top) and FTW-mESCs co-injected with FTW-mXENs (bottom). FIG. 6K illustrates weight of teratoma formed by FTW-mESCs and FTW-mESCs co-injected with FTW-mXENs at different mix ratios (mean ± SD, n = 3,biological replicates). FIG. 6L depicts bar plot showing the area of each in vitro cultured E6.5- 6.75 mouse epiblast with (red) or without (green) VE (mean ± SD, n = 10, biological replicates). *P-value < 0.01 , **P-value < 0.001. P-values were calculated using two-tailed Student’s t test. FIG. 6M shows flow cytometry analysis of apoptotic cells (Annexin V+ / PI+) for separately cultured (top) and co-cultured (bottom) FTW-mESCs from day 3 to day 5. FIG. 6N shows quantification of the results from FIG. 6M (mean ± SD, n = 3 biological replicates, N.S., not significant). FIG. 60 shows bar chart illustrating the distribution of cell cycle phases in separately cultured and co-cultured FTW-mESCs from day 3 to day 5 (mean ± SD, n = 3 biological replicates; **p < 0.01 , ***p < 0.001 , ****p < 0.0001 ; p values were calculated using two-tailed Student’s t test). FIG. 6P is a schematic of PrE complementation experiments. FIG. 6Q shows representative IF images displaying GATA6 and OCT4 in WT ex vivo E5.5 mouse embryos (top), ex vivo E5.5 mouse embryos treated with 0.5 pM PD (middle), and ex vivo E5.5 mouse embryos injected with FTW-mXEN (GFP+) cells after 0.5 pM PD treatment (bottom). Scale bars, 25 pm. FIG. 6R shows bar chart displaying the number of OCT4+ and GATA6+ cells in ex vivo E5.5 mouse embryos under various conditions (mean ± SD, n = 9 biological replicates; **p < 0.01 , ***p < 0.001 , ****p < 0.0001 , N.S., not significant; p values were calculated using two-tailed Student’s t test).

[0036] FIG. 7A-7H illustrate mechanistic insights of growth inhibition. FIG. 7A is a schematic of RNA-seq experiments. FIG. 7B displays bar graphs showing the number (left) and strength (right) of cell-cell interactions in co-cultures and separate cultures. FIG. 7C depicts circle plots showing the ratios of number (left) and strength (right) of cell-cell interactions between co-cultured and separately cultured samples. Red lines, increased interactions; blue lines, decreased interactions. FIG. 7D shows heatmaps of outgoing signaling patterns (left) and incoming signaling patterns (right) in co-cultured mouse FTWstem cells. FIG. 7E shows violin plot showing area multiplied by GFP intensity for each FTW-mESC colonies in day 5 separate and co-cultures (mESCs: mXENs = 2:1 or 1 :1) as well as separate cultures supplemented with different ECM proteins. Matrigel_L: 0.5% (v / v), Matrigel_H: 2% (v / v), Laminin_L: 30 pg / ml, Laminin_H: 120 pg / ml, Collagen_L: 15 pg / ml, Collagen_H: 60 pg / ml, Vitronectin_L: 5 pg / ml, Vitronectin_H: 30 pg / ml. N.S. not significant. FIG. 7F depicts violin plot showing area multiplied by GFP intensity for each FTW-mESC colonies in different conditions. FIG. 7G is a schematic summary of mechanistic insights of the observed proliferation inhibition of FTW-mESCs by FTW-mXENs. N.S. not significant, ****P- value < 0.0001. P-values were calculated using two-tailed Student’s t test. FIG. 7H shows a heatmap illustrating the Pearson correlation among FTW-mESCs, FTW-mXENs, and FTW- mTSCs in separate cultures and co-cultures. The displayed numbers correspond to Pearson correlation coefficients.

[0037] FIG. 8A-8P illustrate mechanistic insights of growth inhibition and transcriptomic analyses of co-cultured FTW stem cells. FIG. 8A illustrates joint LIMAP embedding of singlecell transcriptomes from separately- and co-cultured FTW-mESCs, FTW-mXENs or FTW- mTSCs. FIG. 8B shows percentage of cells in each cluster derived from separately and co- cultured FTW-mESCs, FTW-mXENs or FTW-mTSCs. FIG. 8C shows representative BF and fluorescence merged images of separately- and co-cultured FTW-mESCs, as well as separately cultured FTW-mESCs supplemented with different ECM proteins. Scale bar, 100 I.Lm. FIG. 8D depicts bar plot showing the cell densities of FTW-mESCs in day 5 separate and co-cultures, as well as separate cultures supplemented with different ECM proteins (mean ± SD, n = 3, biological replicates, N.S., not significant). FIG. 8E shows representative BF and fluorescence merged images of separately- and co-cultured FTW-mESCs (with WT or Lamc~ FTW-mXENs [clones #4 and #6]). FIG. 8F depicts bar plot showing the cell densities of FTW- mESCs in day 5 separate and co-cultures (with WT of Lam / n / n' / 'FTW-mXENs) (mean ± SD, n = 3, biological replicates). FIG. 8G shows representative BF and fluorescence merged images of separately and co-cultured FTW-mESCs (WT or / fgb7' / '[clones #2 and #6]) Scale bar, 100 I.Lm. FIG. 8H depicts bar plot showing the relative cell densities of FTW-mESCs (WT or lntegrin-fi ~) in day 5 separate and co-cultures (mean ± SD, n = 3, biological replicates). FIG. 8I illustrates a Venn diagram showing shared 502 DEGs (492 down-regulated; 10 up- regulated) between separately cultured FTW-mESCs and FTW-mESCs co-cultured with FTW- mXENs or with both FTW-mXENs and FTW-mTSCs. FIG. 8J is a heatmap showing the expression levels of AP-1 family members in separately (FTW-mESCs) and co-cultured (FTW- mESCs-co) FTW-mESCs. FIG. 8K and FIG. 8L show enriched GO terms (FIG. 8K) and Bioplanet terms (FIG. 8L) for 492 down-regulated genes from (FIG. 8I). FIG. 8M depicts MMP expression levels (FPKM value) in separately cultured FTW-mESCs, FTW-mESCs co- cultured with FTW- mXEN (top), and FTW-mESCs co-cultured with FTW-mXENs and FTW- mTSCs (bottom). FIG. 8N shows LIMAP visualization of monkey FTW stem cells from both separately cultured and co-cultured conditions. Colors indicate the cell source, distinguishing between separately cultured and co-cultured cells. FIG. 80 shows bar chart illustrating the cell densities of FTW-mESCs on day 5 when supplemented with COLLEGEN IV, LAMININ, or a combination of COLLEGEN IV and LAMININ (mean ± SD, n = 3 biological replicates, ****p < 0.0001 ; p values were calculated using two-tailed Student’s t test). FIG. 8P shows bar plot illustrating the cell densities of mESCs (under 2iLIF and serum / LIF conditions), FTW- mESCs, and EpiSCs on day 5, when supplemented with COLLEGEN IV, LAMININ, or a combination of COLLEGEN IV and LAMININ (mean ± SD, n = 3 biological replicates, **p < 0.01 , ****p < 0.0001 , N.S., not significant; p values were calculated using two-tailed Student’s t test).

[0038] FIG. 9A-9N illustrate cynomolgus monkey embryonic and extraembryonic stem cells derivation. FIG. 9A is a schematic of derivation of FTW embryonic and extra-embryonic stem cell lines from cynomolgus monkey blastocyst. FIG. 9B shows Representative BF images of a 10 d.p.f monkey blastocyst, day 12 outgrowth and stable FTW-cyXENs (passage 14). Scale bars, 50 pm. FIG. 9C shows representative BF images of a 10 d.p.f monkey blastocyst, day 12 outgrowth and stable FTW-cyTSCs (passage 10). Scale bars, 50 pm. FIG. 9D shows representative BF images of a 7 d.p.f monkey blastocyst, day 7 outgrowth, FTW- cyESCs, passage 7. Scale bars, 50 pm. FIG. 9E shows Representative IF images of monkey extra-embryonic endoderm (GATA6 and GATA4), trophoblast (GATA3 and CK7), and epiblast (OCT4 and SOX2) lineage markers in FTW-cyXENs (top), FTW-cyTSCs (middle), and FTW- cyESCs (bottom), respectively. Scale bar, 100 pm. FIG. 9F shows representative IF costaining images of COL6A1 , FOXA1 , and GATA4 in differentiated FTW-cyXENs at day 9. Blue, DAPI. Scale bars, 100 pm. FIG. 9G shows representative IF co-staining images of GATA3 with the EVT maker HLAG in SCT-like cells differentiated from FTW-cyTSCs. FIG. 9H shows representative IF co-staining images of GATA3 with the EVT makers HCG and HCGB in EVT- like cells differentiated from FTW-cyTSCs. FIG. 9I shows LIMAP visualization of all scRNA- seq cells from monkey FTW stem cells in the left panel and LIMAP plot, showing the expression of representative markers in the different clusters / cell lines: FTW-cyESC (SOX2, POU5F1 , NANOG), FTW-cyPS (MIXL1 , LHX1 , GSC), FTW-cyXEN (SOX17, GATA6), FTW- cyVE / YE (IHH, MIXL1), FTW-cyEXMC (COL6A1 , HAND2), FTW-cyTSC (GNR2F2, LRP2), FTW-cyCTB (C1QBP, COMMD6), and FTW-cySTB (CGA, CRH) in the right panel. FIG. 9J shows heatmap illustrating the Pearson correlation across various cell types with FTW cells and correlated with in vivo monkey datasets. The numbers represent Pearson correlation coefficients. FIG. 9K shows heatmaps of TF enrichment scores comparing FTW-XENs between monkeys and mice. FIG. 9L shows gene knockout of HMGA 1, NFE2L1, and SALL1 in monkey FTW-cyXENs and the representative BF images. FIG. 9M shows qRT-PCR analysis of GATA4, PDGFRA, and NFE2L1 expression in NFE2L1 knockout FTW-cyXENs (mean ± SD, n = 2 biological replicates; ****p < 0.0001 ; p values were calculated using a two- tailed Student’s t test). FIG. 9N shows representative IF co-staining images of GATA4 and GATA6 in wild-type and NFE2L1 knockout FTW-cyXENs. Blue, DAPI. Scale bar, 100 pm.

[0039] FIG. 10A-10Q illustrate derivation and characterization of FTW-cyXENs, FTW- cyTSCs and FTW-cyESCs. FIG. 10A depicts growth curve of FTW-cyXENs (passage 26) (n =9, biological replicates). FIG. 10B illustrates karyotype analysis of FTW-cyXENs. FIG. 10C displays RT-PCR results showing the expression of several hypoblast markers in FTW- cyXENs. M, DNA ladder. N.C., non-template control. FIG. 10D is representative IF images showing GATA4, SOX17, FOXA2, GATA3 and OCT4 expression patterns in FTW-cyXENs.Blue, DAPI. Scale bars, 50 m. FIG. 10E depicts growth curve analysis of FTW-cyTSCs (passage 16) (n = 2, biological replicates). FIG. 10F shows karyotype analysis of FTW- cyTSCs. FIG. 10G shows RT-PCR results showing the expression of several trophoblast markers in FTW-cyTSCs. M, DNA ladder. N.C., non-template control. FIG. 10H shows representative IF images showing TFAP2C expression in FTW-cyTSCs. Blue, DAPI. Scale bar, 50 pm. FIG. 101 shows RT-PCR results showing the expression of VE / YE and EXMC related genes in differentiated FTW-cyXENs at day 9. FIG. 10J shows representative BF and H&E staining images of a TSC-teratoma generated from FTW-cyTSCs at day 7 post-injection. FIG. 10K shows representative IF images showing the expression patterns of EVT (HLAG), SCT (HCG), and TSC (GATA3 and TFAP2C) marker genes in FTW-cyTSCs derived TSC- teratoma sections. Blue, DAPI. Scale bars, 50 pm. FIG. 10L depicts growth curve of FTW- cyESCs (passage 9) (n = 3, biological replicates). FIG. 10M shows representative IF images showing the expression of OCT4 and SOX2 in FTW-cyESCs. Blue, DAPI. Scale bars, 50 pm. FIG. 10N shows representative H&E staining images of an XEN-tumor generated from FTW- cyXENs (3 months post-injection). Scale bars, 100 pm (left) and 20 pm (right). FIG. 10O shows representative images showing the appearance and H&E staining of a teratoma generated from FTW-cyESCs (2 months post-injection). Scale bars, 200 pm (left) and 50 pm (right). FIG. 10P shows violin plots illustrating gene set signature scores for in vivo monkey Post-paTE, PreE-TE, PreL-TE, alongside in vitro human Early-EVTs, Early-STBs, Post-CTBs, STBs, EVTs, and Pre-CTBs in FTW-cyTSCs. FIG. 10Q shows heatmaps showing the eight regulon groups (G1-8) across all FTW stem cell clusters, with representative TF regulons indicated (numbers of target genes predicted by SCENIC are provided in brackets).

[0040] FIG. 11A-11D show transcriptomic analyses of cynomolgus monkey FTW stem cells. FIG. 11A illustrates LIMAP analysis of FTW-cyXENs, FTW-cyTSCs and FTW-cyESCs. FIG. 11B depicts bubble plot showing the expression patterns of lineage makers in FTW- cyXENs, FTW-cyTSCs and FTW-cyESCs. FIG. 11C is a heatmap showing the DEGs of FTW- cyESCs, FTW-cyXENs and FTW-cyTSCs, Selected genes from the DEGs and top two enriched GO terms were shown on the right. FIG. 11D illustrates correlation analysis of FTW- cyXENs, FTW-cyTSCs, FTW-cyESCs and published datasets from cynomolgus monkey conceptuses.

[0041] FIG. 12A-12E show monkey embryonic and extraembryonic stem cells co-cultures. FIG. 12A shows representative fluorescence and BF merged images of day 1 to day 5 separately cultured FTW-cyESCs (green) and FTW-mESCs co-cultured with FTW-cyXENs (red arrowheads) and / or FTW-cyTSCs (blue arrowheads), or proliferative fibroblast. Scale bar, 100 pm. FIG. 12B depicts violin plot showing area multiplied by GFP intensity for each FTW- mESC colonies in different conditions. FIG. 12C shows heatmaps of outgoing signalingpatterns (left) and incoming signaling patterns (right) in co-cultured cynomolgus FTW stem cells. FIG. 12D illustrates violin plot showing area multiplied by GFP intensity for each FTW- cyESC colonies in day 5 separate and co-cultures (cyESCs: cyXENs = 1 :3.5 or 1 :7) as well as separate cultures supplemented with different ECM proteins. Matrigel_L: 0.5% (v / v), Matrigel_H: 2% (v / v), Laminin_L: 2.5 pg / ml, Laminin_H: 10 pg / ml, Collagen_L: 15 pg / ml, Collagen_H: 60 g / ml. *P-value < 0.05, ****P-value < 0.0001. P-values were calculated using two-tailed Student’s t test. FIG. 12E shows heatmap showing the Pearson correlation between separately cultured and co-cultured FTW-cyESCs, FTW-cyXENs, and FTW-cyTSCs. The numbers represent Pearson correlation coefficients.

[0042] FIG. 13A-13K illustrate lineage crosstalk among co-cultured cynomolgus monkey FTW stem cells. FIG. 13A shows growth curves of separately and co-cultured (with FTW- cyXENs) tFTW-cyESCs (n=3, biological replicates). FIG. 13B-13D depict DEGs of separately cultured and co-cultured FTW-cyESCs (FIG. 13B), FTW-cyXENs (FIG. 13C), and FTW- cyTSCs (FIG. 13D). Top enriched GO terms were shown on the right. FIG. 13E depicts heatmaps showing top 10 outgoing (left) and incoming (right) signaling in co-culture of FTW- cyESCs and FTW-cyXENs. FIG. 13F shows representative BF and fluorescence merged images of separately- and co-cultured FTW-cyESCs, as well as separately cultured FTW- cyESCs supplemented with different ECM proteins. Scale bar, 100 pm. FIG. 13G is a bar plot showing the cell densities of FTW-cyESCs in day 5 separate and co-cultures, as well as separate cultures supplemented with different ECM proteins (n=3, biological replicates). FIG. 13H shows flow cytometry analysis of apoptotic cells (Annexin V+ / PI+) for separately cultured in the top panel and co-cultured in the bottom panel FTW-cyESCs from day 3 to day 5. FIG. 131 shows quantification of the results from FIG. 13H (mean ± SD, n = 3 biological replicates, N.S., not significant). FIG. 13J shows LIMAP visualization of monkey FTW stem cells from both separately cultured and co-cultured conditions. Colors indicate the cell source, distinguishing between separately cultured and co-cultured cells. FIG. 13K shows bar chart displaying the distribution of cell cycle phases in separately cultured versus co-cultured monkey FTW stem cells.

[0043] FIG. 14A-14D show cross-species comparison. FIG. 14A-14C show SCENIC analysis showing divergent (left) and conserved (middle) TF-driven regulons in each mouse and monkey stem cell. Right, Venn plots showing common and distinct targets (between mice and cynomolgus monkeys) of well-known lineage TFs including NANOG, SOX17 and GATA3. Red dots represent the gene regulatory networks regulated by corresponding transcription factor related to the RBPS. FIG. 14D illustrates cross-species comparison of signaling crosstalk among embryonic and extraembryonic cells.

[0044] FIG. 15A-15F show derivation of human FTW-XENs and cross-species comparisons. FIG. 15A shows a schematic illustrating the derivation of FTW-hXENs from human naive ESCs or EPSCs. FIG. 15B shows representative BF images of human naive ESCs and FTW-hXENs (P11). Scale bars, 100 pm. FIG. 15C shows representative IF images showing the expression of XEN cell markers (GATA6, GATA4, SOX17, and FOXA2) in FTW- hXENs. Scale bars, 100 pm. FIG. 15D shows Left: a Venn diagram showing the conserved and species-specific expressed genes in FTW-mXENs, FTW-cyXENs, and FTW-hXENs on the left and GO term enrichment analysis of conserved genes (n = 2,608) in FTW-mXENs, FTW-cyXENs, and FTW-hXENs on the right. FIG. 15E shows Hub-gene-network analysis predicts several core FTW-XEN regulators. FIG. 15F shows qRT-PCR analysis of Gata4, Gata6, Sox17, Foxa2, Laminin, and Pdgfra expression in FTW-mXENs and FTW-cyXENs after knockdown of Src, Pdgfr, and Jak2 (*p < 0.05, **p < 0.01 , ***p < 0.001 , N.S., not significant; p values were calculated using two-tailed Student’s t test).

[0045] FIG. 16A-16N show human XEN derivation and cross-species comparison. FIG. 16A shows representative time-lapse IF co-staining images of SOX2 and GATA6 showing the conversion of human naive ESCs into FTW-hXENs. Scale bars, 100 pm. FIG. 16B shows representative BF (left) and merged BF and fluorescence (right) images of the EPSC-derived FTW-hXENs (SOX17-tdTomato reporter) at different passages (P3, P16, and P25). Scale bar, 100 pm. FIG. 16C shows IF co-staining images of hypoblast markers (GATA4, GATA6, SOX17, and FOXA2) in EPSC-derived FTW-hXENs. Scale bars, 100 pm. FIG. 16D shows qRT-PCR results showing the relative expression levels of lineage-specific markers in FTW- hXENs compared with FTW-hiPSCs (mean ± SD, n = 3 biological replicates; **p < 0.01 , ***p < 0.001 ; p values were calculated using two-tailed Student’s t test). FIG. 16E shows PCA plot of RNA-seq data from separately cultured and co-cultured FTW-hXENs, differentiated FTW- hXEN at day 9 (FTW-hXENs-dif), and published in vivo datasets of hypoblasts from D6 to D14. FIG 16F shows IF co-staining images of FTW-hXENs VE / YE-like marker (FOXA1+ GATA6-) (left) and EXMC-like marker (COL6A1 + GATA6-) (right) in differentiation cultures. Scale bars, 100 pm. FIG. 16G shows heatmap showing the Pearson correlation among human, monkey, and mouse FTW-XENs. The numbers represent Pearson correlation coefficients. FIG 16H shows heatmap of differentially expressed genes in human (n = 2,132), monkey (n = 2,667), and mouse (n = 1 ,767) FTW-XENs. The color key from blue to red indicates low to high expression levels. Shown on the right panel are the enriched GO terms. FIG. 161 shows scaled expression patterns of representative TFs in mouse, monkey, and human FTW-XENs. FIG. 16J shows a Venn diagram showing the conserved and species-specific DEGs (XENs vs. EPls) among human, monkey, and mouse FTW-XENs. FIG. 16K shows qRT-PCR results showing the expression of SRC, PDGFR, and JAK2 in different knockdown (KD) clones in mouse (top) and monkey (bottom) FTW-XENs (mean ± SD, n = 3 biological replicates; *p <0.05, **p < 0.01 , ***p < 0.001 j **** p < 0.0001 , N.S., not significant; p values were calculated using two-tailed Student’s t test). FIG. 16L illustrates correlation analysis of FTW- hXEN-LCs with epiblast, cytotrophoblast, syncytiotrophoblast and the sub-clusters of hypoblast cells FIG. 16M shows a representative BF image of a mouse twin-embryo after blastocyst injection of human FTW-XEN-LCs. FIG. 16N shows a table summarizing the outcomes of different injection experiments.

[0046] FIG. 17 shows that a common culture condition for mouse and monkey embryonic and extraembryonic stem cells.

[0047] The drawing figures do not limit the present inventive concept to the specific embodiments disclosed and described herein. The drawings are not necessarily to scale, emphasis instead being placed on clearly illustrating principles of certain embodiments of the present inventive concept.DETAILED DESCRIPTION

[0048] The following detailed description references the accompanying drawings that illustrate various aspects of the present inventive concept. The drawings and description are intended to describe aspects of the present inventive concept in sufficient detail to enable those skilled in the art to practice the present inventive concept. Other components can be utilized, and changes can be made without departing from the scope of the present inventive concept. The following description is, therefore, not to be taken in a limiting sense.

[0049] Provided herein are culture conditions and methods of deriving embryonic stem cells (ESCs), extraembryonic endoderm stem cells (XENs) and trophoblast stem cells (TSCs) from reproductive cells or naive pluripotent stem cells (PSCs), simultaneously, and in vitro culture systems comprising such. The present disclosure is based, at least in part, on the surprising discovery that culture condition that activates fibroblast growth factor (FGF), transforming growth factor beta (TGF- ) and Wingless-related integration site (WNT) signaling pathways enabled simultaneous derivation of stable ESCs, XENs, and TSCs from reproductive cells especially blastocysts. Further provided in the disclosure are methods to establish ESCs, XENs, and / or TSCs stem cell co-cultures. A method of establishing growth restricted ESCs is further demonstrated herein.I. Terminology

[0050] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred aspects and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure asillustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

[0051] As used in the specification, articles “a” and “an” are used herein to refer to one or to more than one (i.e. , at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0052] “About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result. The term “about” in association with a numerical value means that the numerical value can vary plus or minus by 5% or less of the numerical value.

[0053] Throughout this specification, unless the context requires otherwise, the word “comprise” and “include” and variations (e.g., “comprises,” “comprising,” “includes,” “including”) will be understood to imply the inclusion of a stated component, feature, element, or step or group of components, features, elements, or steps but not the exclusion of any other integer or step or group of integers or steps.

[0054] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0055] As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”

[0056] Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0057] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise-indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowestvalue and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0058] As used herein, “effective amount,” “effective dose,” or an “amount effective to”, refers to an amount that is effective in providing at least one desirable biological result.

[0059] To perform the culturing methods described herein, a suitable population of cells can be obtained from any suitable source. In some aspects, the population of cells can be obtained from a subject, for example, from tissue (e.g., embryotic tissue), bone (e.g., bone marrow), blood (e.g., peripheral blood or umbilical cord blood), bodily fluid (e.g., tear, urine, or saliva), serum, plasma, or protein, via any means known in the art.

[0060] In some aspects, the population of cells comprises reproductive cells, e.g., female germline stem cells and progeny thereof. Examples of reproductive cells include, but are not limited to, embryos, oocytes, zygotes, blastomeres, morulae, and blastocysts. Reproductive cells, such a blastocyst may be human, non-human primates (e.g., monkey), rodent (e.g., rat, mouse), ungulate (e.g., sheep, goat, pig, cow and horse) blastocyst. A population of cells comprising blastocysts can be produced by natural mating, in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT), and retrieved using any known surgical or non-surgical (e.g., uterine flushing) methods in the art. The blastocysts may be fresh, frozen, or vitrified.

[0061] In some aspects, the population of cells comprise naive pluripotent stem cells. Naive pluripotent stem cells (PSC) resemble the embryonic epiblast at an earlier time-point in development than conventional 'primed' PSC. Naive pluripotent stem cells can be obtained using any known methods in the art and can include human, non-human primates (e.g., monkey), rodent (e.g., rat, mouse), sheep, goat, pig, or an ungulate (e.g., horse) PSCs. In some aspects, the population of cells comprise naive human pluripotent stem cells (hPSCs).

[0062] As used herein, “FGF activator” refers to a molecule that partially or fully enhances, increases, or stimulates a biological activity of a FGF protein. FGFs are a family of cell signaling proteins that play a role in a wide range of cellular processes. FGF family members comprise FGF proteins (e.g., FGF1 , FGF2, FGB, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11 , FGF12, FGF13, FGF14, FGF15, FGF16, FGF 17, FGF 18, FGF 19, FGF20, FGF21 , FGF22, and FGF23) and FGF receptor (FGFR) proteins (e.g., FGFR1 , FGFR2, FGFR3, FGFR4, and FGFRL1). Suitable FGF activators include, but are not limited to, proteins, nucleic acids, small molecules, or any combination thereof. Additionally, FGF activator can be a molecule of any type that enhances the signaling associated with at least one FGF family members (e.g., FGFs or FGFRs) in a cell, for example, either by increasing transcription or translation of a FGF family member, or by increasing FGF activity, or both. Insome embodiments, the FGF activator may act directly by interacting with a FGF receptor or indirectly by interacting with one or more intracellular components of the FGF signaling pathway. In some aspects, FGF activators as described herein may increase FGF signaling in cells (e.g., reproductive cells or PSCs) by at least 20% or more, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or above. The increased FGF signaling in the presence of at least one FGF activator can be determined by conventional methods, e.g., using protein assays such as ELISA or Western blot.

[0063] As used herein, the term “FGF” refers to a FGF polypeptide having the same or similar bioactivity of a wild-type FGF polypeptide. A FGF polypeptide may have an amino acid sequence that is at least 70% or more (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type FGF polypeptide, and is capable of trigger FGF signaling pathway.

[0064] As used herein, “TGF-P” refers to a molecule that partially or fully enhances, increases, or stimulates a biological activity of a TGF-|3 protein. TGF-P is a multifunctional cytokine belonging to the transforming growth factor superfamily that comprises TGF- ps (e.g., TGF-p1 , TGF-p2, and TGF-p3), Activins (e.g., Activin-A, Activin-B, and Activin-C), bone morphogenetic proteins (BMPs), and TGF-P receptors (TGFRs) (e.g., type I, type II, and type III receptors). Sequences of genes and proteins in the TGF-p pathway are known in the art and may be obtained from publicly available databases. Suitable TGF-p activators include, but are not limited to, proteins, nucleic acids, small molecules, or any combination thereof. Methods for identifying activators of TGF-p may comprise contacting TGF-p with a candidate TGF-p activator and measuring a detectable change in one or more biological activities typically associated with TGF-p. A TGF-P activator can be a molecule of any type that enhances the signaling associated with at least one TGF-P family members (e.g., TGF-P, Activin, BMP, or FGFR) in a cell, for example, either by increasing transcription or translation of a TGF-P family member, or by increasing TGF-P activity, or both. In some embodiments, the TGF-P activator may act directly by interacting with a TGF-P receptor or indirectly by interacting with one or more intracellular components of the TGF-P signaling pathway. In some aspects, TGF-P activator as described herein may increase TGF-P signaling in cells (e.g., reproductive cells or PSCs) by at least 20% or more, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or above. The increased TGF-P signaling in the presence of at least one TGF-P activator can be determined by conventional methods, e.g., using protein assays such as ELISA or Western blot.

[0065] As used herein, “TGF-P” refers to a TGF-p polypeptide having the same or similar bioactivity of a wild-type TGF-p polypeptide. A TGF-p polypeptide may have an amino acid sequence that is at least 70% or more (e.g., at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type TGF-P polypeptide and is capable of trigger TGF-p signaling pathway.

[0066] As used herein, the term “activin” refers to an activin polypeptide having the same or similar bioactivity of a wild-type activin polypeptide. An activin polypeptide may have an amino acid sequence that is at least 70% or more (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type activin polypeptide and is capable of trigger TGF-P signaling pathway.

[0067] As used herein, “WNT activator” refers to a molecule that partially or fully enhances, increases, or stimulates a biological activity of a WNT protein. WNTs are a family of secreted proteins important for a wide array of developmental and physiological processes. WNT family members comprise WNT1 , WNT2, WNT2b (also called WNT13), WNT3, WNT3a, WNT4, WNT5a, WNT5b, WNT6, WNT7a, WNT7b, WNT8a, WNT8b, WNT9a, WNT9b, WNT10a, WNT10b, WNT11 , and WNT16. Sequences of genes and proteins in the WNT pathway are known in the art and may be obtained from publicly available databases. Suitable WNT activators include, but are not limited to, proteins, nucleic acids, small molecules, or any combination thereof. Methods for identifying activators of WNT may comprise contacting WNT with a candidate WNT activator and measuring a detectable change in one or more biological activities typically associated with WNT. A WNT activator can be a molecule of any type that enhances the signaling associated with at least one WNT family members (e.g., WNT1 , WNT2, WNT3, or WNT3a) in a cell, for example, either by increasing transcription or translation of a WNT family member, or by increasing WNT activity, or both. In some aspects, the WNT activator may act directly by interacting with a WNT receptor or indirectly by interacting with one or more intracellular components of the WNT signaling pathway such as P-catenin, a kinase or phosphatase that acts on P-catenin, a transcription factor that assembles with P-catenin.

[0068] As used herein, “WNT” refers to a WNT polypeptide having the same or similar bioactivity of a wild-type WNT polypeptide. A WNT polypeptide may have an amino acid sequence that is at least 70% or more (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type WNT polypeptide and is capable of trigger WNT signaling pathway.

[0069] As used herein, “GSK3” refers to either or both isoforms of GSK3 (GSK3a and GSK3P). GSK3 is a serine / threonine kinase that has been identified as a regulator of glucose metabolism. Inhibitors that inhibit either or both of these isoforms are of contemplated for use in the disclosure.

[0070] As used herein “laminin” refers to family of glycoproteins of the extracellular matrix and components of the basal lamina, and can include laminin, laminin protein fragments and / or laminin polypeptides. Laminin may further include, but are not limited to, laminin A (or A1) chain, laminin B1 chain, laminin B2 chain, laminin A2 chain (merosin), laminin G1 chain, the globular domain repeats within the laminin A1 chain, or any functional fragments thereof. A laminin protein molecule comprises one a-chain subunit, one -chain subunit, and one y- chain subunit, all joined together in a trimer through a coiled-coil domain. The twelve known laminin subunit chains can form at least 15 trimeric laminin types in native tissues. Within the trimeric laminin structures are identifiable domains that possess binding activity towards other laminin and basal lamina molecules, and membrane-bound receptors. There exist five different alpha chains, three beta chains and three gamma chains that in human tissues have been found in at least fifteen different combinations. These molecules are termed laminin-1 to laminin- 15 based on their historical discovery, but an alternative nomenclature describes the isoforms based on their chain composition, e.g., laminin-111 (laminin-1) that contains alpha-I , beta-1 and gamma-1 chains. Four structurally defined family groups of laminins have been identified. The first group of five identified laminin molecules all share the p1 and y1 chains and vary by their a-chain composition (a1 to a5 chain). The second group of five identified laminin molecules, including laminin-521 , all share the p2 and y1 chain, and again vary by their a-chain composition. The third group of identified laminin molecules has one identified member, laminin-332, with a chain composition of a3p3y2. The fourth group of identified laminin molecules has one identified member, laminin-213, with the newly identified y3 chain (a2p1y3). At least 15 subtypes (or "isoforms") of laminin (LN) have been identified, based on their chain composition, including LN-111 (a1p1y1), LN-121 (aip2y1), LN-211 (a2piy1), LN- 213 (a2p1y3), LN-221 (a2p2y1), LN-311 (a3p3y1), LN-321 (a3p2y1), LN-332 (a3p3y2), LN- 411 (a4p1y1), LN-421 (a4p2y1), LN-423 (a4p2y3), LN-511 (a5piy1), LN-521 (a5p2y1), LN- 522 (a5p2y2), and LN-523 (a5p2y3). Laminin may be a human laminin, a mouse laminin, or a rat laminin. As disclosed herein, the laminin may be a wild-type or a recombinant laminin.

[0071] As used herein “collagen” refers to the main structural protein in the extracellular matrix found in various connective tissues. Collagen can be Collagen I, Collagen IV, CollagenII, Collagen III, or any combination thereof. The collagen can be from vertebrates, including bovine, ovine, porcine, chicken, and human collagens. The collagen disclosed herein can be a type specifically selected from the group consisting of type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type DC, type X, type XI, type XII, type XIII, type XIV, type XV, type XVI, type XVII, type XVIII, type XDC, and type XX. Collagen is a triple helical protein composed of three separate a-chains. Collagen appears in heterotrimeric and homotrimeric forms, depending on collagen type. Type I collagen is typically a heterotrimeric complex containingtwo a1(l) and one a2(l) chains, although some tissues contain trace amounts of a1(1) homotrimer, while type III collagen is a homotrimeric complex containing three a1 (I II) chains in triple helical formation, etc. Collagen can encompass variants and fragments thereof, and functional equivalents and derivatives thereof, which preferably retain at least one structural or functional characteristic of collagen, for example, a (Gly-X-Y)ndomain. As disclosed herein, collagen can be a wild type or a recombinant collagen. In some aspects, the collagen used is of one type substantially free from collagen of any other type.

[0072] As used herein “integrin” refers to glycoprotein cell adhesion receptors that mediate cell-cell (via counter receptors on other cells) and cell-substrate (i.e., cell-extra cellular matrix) interactions. As disclosed herein, integrin can include either the alpha or beta subunit of an integrin, or both. By “subunit” herein is meant either the alpha or the beta subunits of the protein, one of each subunit is required to achieve functionality for the wild-type integrin protein. There are currently 19 alpha and 8 beta subunits known, generating 24 heterodimers. Genes encoding integrins in humans can include ITGA1 , ITGA2, ITGA2B, ITGA3, ITGA4, ITGA4, ITGA5, ITGA6, ITGA7, ITGA8, ITGA9, and ITGA10. The wild type sequences of integrins are well known in the art and may be obtained from publicly available databases. For e.g., nucleotide sequence for ITGA1 is available at NCBI database under accession number NM_181501.2 and the protein sequence under accession number P56199.2.

[0073] As used herein “NRG1” or “Neuregulinl” is a protein which is frequently used in the maintenance of stem cells. NRG1 is also known as Also known as: ARIA, GGF, GGF2, HGL, HRG, HRG1 , HRGA, MST131 , MSTP131 , NDF, NRG1-IT2, or SMDF. As disclosed herein, NRG1 can be an isolated full-length protein, polypeptide, a bioactive domain of NRG1 , variants and fragments thereof, and functional equivalents and derivatives thereof, which retain at least one structural or functional characteristic of NRG1. NRG1 can be a wild type or a recombinant NRG1. The NRG1 can be from vertebrates, including bovine, ovine, porcine, chicken, and human NRG1. The wild type sequences of NRG1 are well known in the art and may be obtained from publicly available databases. For e.g., nucleotide sequence for human NRG1 is available at NCBI database under accession number NC_000008.11 and the protein sequence under accession number NP_039258.1.

[0074] As used herein “Rock inhibitor” refers to Rho-kinase inhibitors (rho-associated protein kinase inhibitor) and is a compound that target rho kinase (ROCK) and inhibit the ROCK pathway. Rock inhibitor as described herein may decrease Rock signaling in cells (e.g., stem cells) by at least 20% or more, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater. Rock inhibitors used in the methods and culture medium described herein, can be selected from AT-13148, BA-210, -Elemene, Belumosudil, Chroman, DJ4, Fasudil, GSK- 576371 , GSK429286A, H-1152 Inhibitor, Hydroxyfasudil, Ibuprofen, LX-7101, Netarsudil, RKI-1447, Ripasudil, TCS-7001 , Thiazovivin, Verosudil (AR-12286), Y-27632, Y-30141 , Y-33075, and / or Y-39983.

[0075] As used herein “ALK inhibitor” refers to inhibitor of ALK especially, TGF-p type I receptor ALK5 kinase, type I activin / nodal receptor ALK4 and / or type I nodal receptor ALK7. ALK inhibitor as described herein may decrease ALK signaling in cells (e.g., stem cells) by at least 20% or more, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater. Non-limiting example of ALK5 / 4 / 7 inhibitor includes A83-01.

[0076] As used herein “multi-lineage cells” or “cross-lineage cells” refers to multiple cells cultured together which express different lineage markers. By way of a non-limiting example, a culture where some cells express epiblast markers, others express extraembryonic endoderm markers, and / or other cells express trophoblast markers.

[0077] As used herein “DMEM / F-12 medium” refers to Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 is a basal medium and comprises a 1 :1 mixture of DMEM and Ham's F-12.II. Method of derivation and culture of ESCs, XENs, and / or TSCs

[0078] The present disclosure provides a method of derivation of ESCs, XENs, and / or TSCs, simultaneously. Also provided is a shared culture condition that allows for simultaneously derivation of ESCs, XENs, and / or TSCs from reproductive cell or naive pluripotent stem cells (PSCs).

[0079] Combining activators of FGF, TGF-p, and WNT signaling pathways can be used for deriving ESCs, XENs, and / or TSCs from reproductive cells or PSCs, in ex vivo or in vitro culture. In some aspects, the present disclosure provides a method for derivation of ESCs, XENs, and / or TSCs in vitro, the method comprising, obtaining reproductive cells, or PSCs, culturing the cells in a culture medium that comprises a FGF activator, TGF-P activator, and a WNT activator; simultaneously deriving stable ESCs, XENs, and TSCs. In some aspects, the method further comprises isolating the epiblast-like, endoderm-like, and / or trophoblast-like cells from the cell outgrowth (e.g., blastocyst outgrowth), and culturing and passaging the isolated cells to obtain stable ESCs, XENs, and / or TSCs.

[0080] In some aspects, the derived cells comprise ESCs and XENs. In some aspects, the derived cells comprise ESCs and TSCs. In some aspects, the derived cells comprise ESCs, XENs, and TSCs.

[0081] In some aspects, ESCs, XENs, and / or TSCs are derived from reproductive cells. In some aspects, the reproductive cell comprise blastocyst. In some aspects, ESCs, XENs, and / or TSCs are derived from stem cells of a blastocyst. In some aspects, the blastocyst is ahuman blastocyst. In some aspects, the blastocyst is a mouse blastocyst. In some aspects, the blastocyst is a non-human primate blastocyst. In some aspects, the blastocyst is a monkey blastocyst.

[0082] In some aspects, ESCs, XENs, and / or TSCs are derived from a single blastocyst. In some aspects, ESCs, XENs, and / or TSCs are derived from multiple blastocysts.

[0083] In some aspects, derived ESCs, XENs, and TSCs express epiblast, extraembryonic endoderm, and trophoblast markers, respectively. In some aspects, derived ESCs express one or more of Octamer-binding transcription factor 4 (Oct4), SRY-box 2 (Sox2), Nanog, KLF Transcription Factor 2 (Klf2), or any combination thereof, at an elevated level compared to derived XENs and / or TSCs. In some aspects, derived XENs express one or more of GATA Binding Protein 4 (Gata4), GATA Binding Protein 6 (Gata6), Forkhead box protein A2 (Foxa2), SRY-box 17 (Sox17), or any combination thereof, at an elevated level, compared to derived ESCs and TENs. In some aspects, derived TSCs express one or more hepatocyte nuclear factor-4 alpha (Hnf4a), E74 Like ETS Transcription Factor 5 (Elf5), Homeobox protein CDX-2 (Cdx2), Keratin 8 (Krt8), Eomesodermin (Eomes), or any combination thereof, at an elevated level, compared to derived ESCs and XENs.

[0084] In some aspects, the XENs derived using the methods described herein express one or more markers at comparable levels to conventionally isolated or derived XENs (e.g, markers disclosed in FIG. 2J). In some aspects, the XENs derived using the methods described herein express one or more of Gata4, Gata6, Foxa2, Sox17, or any combination thereof, at a similar level as a conventional XENs. In some aspects, the TSCs derived using the methods described herein express one or more markers at comparable levels to conventionally isolated or derived TSCs (e.g, markers disclosed in FIG. 2J). In some aspects, the TSCs derived using the methods described herein express one or more of Hnf4a, Elf5, Cdx2, Krt8, Eomes, or any combination thereof at similar levels as conventionally isolated or derived TSCs.

[0085] In some aspects, the marker having an increased level in the derived cells has an increased expression level of at least about .01%, at least about 0.05%, at least about 0.1 %, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% greater compared to other cells derived simultaneously.

[0086] In some aspects, the marker having elevated level has an elevated expression level having a log 10 value from about 0.1 to about 5 as compared to a reference gene (for e.g., RPLPO). For example, a log 10 fold value can be about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.

[0087] In some aspects, to derive ESCs, XENs, and / or TSCs, blastocyst is cultured in a suitable culture medium (e.g., cell culture medium) which comprises a FGF activator, TGF- activator, and a WNT activator. In some aspects, zona of the blastocyst is removed, and the blastocyst is cultured in the medium for about 6-12 days. Cells in the blastocyst outgrowth can be manually isolated, cultured, and passaged in the disclosed culture medium, to yield derived ESCs, XENs, and / or TSCs. Alternatively, blastocyst outgrowth can be passaged using TrypLE, re-seeded, passaged in the disclosed culture medium, to yield derived ESCs, XENs, and / or TSCs.

[0088] In some aspects, reproductive cells or PSCs can be cultured in a suitable medium (e.g., cell culture medium) in the presence of an effective amount at least one FGF activator, at least one TGF-fJ activator, and at least one WNT activator such as those described herein for a suitable period of time, e.g., at least 18 hours, at least about 24 hour, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, or longer.

[0089] Activators for any of FGF family members can be used in the methods described herein. In some aspects, FGF activators used herein are specific to one FGF family member, e.g., specific to FGF or FGFR. In some aspects, FGF activators are universal to two or more FGF family members, e.g., universal to FGF and FGFR. In some aspects, FGF activators used herein are specific to FGF2. In some aspects, the FGF activator is FGF2.

[0090] Wild-type FGF sequences (e.g., sequences of FGF2) of various species are available on the world wide web from the NCBI, including human, mouse, and rat. For example, the nucleotide sequence encoding an isoform of human FGF2 is available at NCBI under Accession No. NM_002006.4 and its corresponding amino acid sequence is under Accession No. NP_001997.5. FGF proteins may be isolated from naturally occurring sources (e.g., mammalian cells that naturally produce the protein), produced in eukaryotic or prokaryotic cells using recombinant expression technology, or chemically synthesized. Soluble, biologically active FGF proteins may be prepared in purified form using methods known in the art.

[0091] An effective amount of a FGF activator used in methods described herein can be between 0.1 ng / ml and 10,000 ng / ml. In some aspects, the effective amount of the FGF activator for the methods described herein can be between 1 ng / ml and 10,000 ng / ml, between 10 ng / ml and 10,000 ng / ml, between 100 ng / ml and 10,000 ng / ml, between 1 ,000 and 10,000 ng / ml, between 5,000 ng / ml and 10,000 ng / ml, between 0.1 ng / ml and 5,000 ng / ml, between 1 and 5,000 ng / ml, between 10 ng / ml and 5,000 ng / ml, between 1 ,000 ng / ml and 5,000 ng / ml, or between 2,500 and 5,000 ng / ml. In some aspects, FGF activator used in the methods described herein can be between 0.1 ng / ml and 100 ng / ml. In some aspects, the effective amount of the FGF activator used in the methods described herein can be between 1 ng / ml and 100 ng / ml. In some aspects, the effective amount of the FGF activator used in the methods described herein is about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 20 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, or about 100 ng / ml. In some aspect, the FGF activator is added to the culture medium at about 6 ng / ml. In some aspects, the FGF activator is added to the culture medium at about 20 ng / ml. In some aspects, FGF activator added to the culture medium is FGF2 at about 6 ng / ml. In some aspects, FGF activator added to the culture medium is FGF2 at about 20 ng / ml.

[0092] Activators for any of TGF-p family member can be used in the culturing methods described herein. In some aspects, TGF-p activator used herein are specific to one TGF-p family member, e.g., specific to a TGF-p or an Activin (e.g., TGF-P2 or Activin-A). In some aspects, TGF-p activators are universal to two or more TGF- TGF-p family members, e.g., universal to a TGF-p and an Activin (e.g., TGF-p or Activin-A). In some aspects, TGF-p activator used herein is Activin-A.

[0093] Wild-type TGF-P sequences (e.g., sequences of TGF-P1 , TGF-P2, or TGF-P3) of various species are available on the world wide web from the NCBI, including, for example, human, mouse, and rat. For example, the nucleotide sequence encoding an isoform of human TGF-P2 is available at NCBI under Accession No. NM_001024847.2 and its corresponding amino acid sequence is under Accession No. NP_001020018.1.

[0094] Wild-type Activin sequences (e.g., sequences of Activin-A, Activin-B, and Activin- C) of various species are available on the world wide web from the NCBI, including human, mouse, and rat. For example, the nucleotide sequence encoding an isoform of human Activin- A is available at NCBI under Accession No. NM_002192.3 and its corresponding amino acid sequence is under Accession No. NP 002183.1.

[0095] TGF-p proteins may be isolated from naturally occurring sources (e.g., mammalian cells that naturally produce the protein), produced in eukaryotic or prokaryotic cells using recombinant expression technology, or chemically synthesized. Soluble, biologically active TGF-p proteins may be prepared in purified form using methods known in the art.

[0096] An effective amount of a TGF-p activator used in methods described herein can be between 0.1 ng / ml and 10,000 ng / ml. In some aspects, the effective amount of the TGF-P activator for the methods described herein can be between 1 ng / ml and 10,000 ng / ml, between 10 and 10,000 ng / ml, between 100 ng / ml and 10,000 ng / ml, between 1 ,000 ng / ml and 10,000 ng / ml, between 5,000 ng / ml and 10,000 ng / ml, between 0.1 and 5,000 ng / ml, between 1 and 5,000 ng / ml, between 10 and 5,000 ng / ml, between 1 ,000 and 5,000 ng / ml, or between 2,500 and 5,000 ng / ml. In some aspects, TGF-P activator used in the methods described herein can be between 0.1 ng / ml and 100 ng / ml. In some aspects, the effective amount of the TGF-P activator used in the methods described herein can be between 1 ng / ml and 100 ng / ml. In some aspects, the effective amount of the TGF-P activator used in the methods described herein is about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml, about 20 ng / ml, about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, or about 100 ng / ml. In some aspects, TGF-p activator is added to the culture medium at about 20 ng / ml. In some aspect, the TGF- P activator is added to the culture medium at about 25 ng / ml. In some aspects, the TGF-p activator added to the culture medium is Activin-A at about 20 ng / ml. In some aspects, the TGF-P activator added to the culture medium is Activin-A at about 25 ng / ml.

[0097] Activators for any of WNT family members can be used in the methods described herein. In some embodiments, WNT activators used herein are specific to one WNT family member, e.g., specific to WNT1 , WNT2, WNT3, or WNT3a. In some embodiments, WNT activators are universal to two or more WNT family members, e.g., universal to WNT3 and WNT3a. In some embodiments, WNT activators used herein are specific to WNT3a.

[0098] Wild-type WNT sequences (e.g., sequences of WNT1 , WNT2, WNT3, or WNT3a) of various species are available on the world wide web from the NCBI, including human, mouse, and rat. For example, the nucleotide sequence encoding an isoform of human WNT3a is available at NCBI under Accession No. NM_033131.3 and its corresponding amino acid sequence is under Accession No. NP_149122.1.

[0099] In some aspects, the WNT activator is a biologically active WNT protein, e.g., WNT3 a protein. In some aspects, the WNT activator is a glycogen synthase kinase 3 (GSK3) inhibitor. In some aspects, the GSK3 inhibitor specifically inhibits GSK3 and does not substantially inhibit the majority of other mammalian kinases. Any GSK3 inhibitor may be used in methods described herein. Exemplary GSK3 inhibitors include, but are not limited to, BIO, AR-A014418, SB 216763, SB-415286, CHIR98014 (CT98014), CHIR98023 (CT98023), CHIR99021 (CT99021), and CHIR99021 trihydrochloride.

[0100] An effective amount of a WNT activator used in methods described herein can be between 0.1 pM and 10,000 pM. In some aspects, the effective amount of the WNT activator for the methods described herein can be between 1 pM and 10,000 pM, between 10 pM and 10,000 pM, between 100 pM and 10,000 pM, between 1 ,000 and 10,000 pM, between 5,000 and 10,000 pM, between 0.1 and 5,000 pM, between 1 pM and 5,000 pM, between 10 pM and 5,000 pM, between 1 ,000 pM and 5,000 pM, or between 2,500 pM and 5,000 pM. In some aspects, WNT activator used in the methods described herein can be between 0.1 pM and 100 pM. In some aspects, the effective amount of the WNT activator used in the methods described herein can be between 1 pM and 100 pM. In some aspects, the effective amount of the WNT activator used in the methods described herein is about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, or about 100 pM. In some aspect, the WNT activator is added to the culture medium at about 1.5 pM. In some aspects, the WNT activator is added to the culture medium at about 3 pM. In some aspects, the WNT activator added to the culture medium is CHIR99021 at about 1.5 pM. In some aspects, the WNT activator added to the culture medium is CHIR99021 at about 3 pM.

[0101] In some aspects, the culture medium described herein, further comprises at least one additional factor, e.g., knock out serum (KSR) or knockout serum replacement (KOSR), N2 supplement (N2), B27 supplement (B27) or B27 minus insulin supplement, L-glutamine (for example, GlutaMAX), Nonessential amino acids (NEAA), p-Mercaptoethanol, Penicillinstreptomycin, fetal bovine serum (FBS), BSA, ITS-X supplement, Forskolin, EGF, Rock inhibitor, ALK inhibitor, NRG1 , or any combination thereof.

[0102] In some aspects, ESCs, XENs, and / or TSCs are derived in a culture medium in the presence of the at least one additional factor and a FGF activator, a TGF-[3 activator, and a WNT activator. Alternatively, or in addition to, the at least one additional factor may be addedprior to and / or after ESCs, XENs, and / or TSCs derivation in the presence of a FGF activator, a TGF-p activator, and a WNT activator.

[0103] In some aspects, KSR or KOSR used in the described methods can be between 1-30%. In some aspects, the KSR or KOSR is added to the culture medium for the methods described herein can be about 1 %, about 5%, about 10%, about 12%, about 15%, about 17%, about 20%, about 22%, about 25%, about 27%, about 30%. In some aspects, KSR or KOSR is added to the culture medium used in the methods described herein can be about 20%.

[0104] In some aspects, one or more of the additional factors N2 supplement, B27 supplement, L-glutamine, Nonessential amino acids, P-Mercaptoethanol, Penicillinstreptomycin, FBS, BSA, ITS-X supplement, Forskolin, EGF, Rock inhibitor, ALK inhibitor, or NRG1 can be used in the described methods between 0.1% to 10%. In some aspects, the effective amount of one or more of the additional factors N2 supplement, B27 supplement, L- glutamine, Nonessential amino acids, P-Mercaptoethanol, Penicillin-Streptomycin, fetal bovine serum (FBS), BSA, ITS-X supplement, Forskolin, EGF, Rock inhibitor used in the methods described herein is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%,, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In some aspect, the additional factors such as one or more of N2 supplement, B27 supplement, L-glutamine (for example Glutamax), nonessential amino acids are added to the culture medium used in the methods described herein at a range between O.IX to 2X. In some aspects, the additional factors such as one or more of N2 supplement, B27 supplement, GlutaMAX, Nonessential amino acids is added to the culture medium at about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1.2X, about 1.3X, about 1.4X, about 1.5X, about 1.6X, about 1.7X, about 1.8X, about 1.9X, or about 2X. In some aspects, P-Mercaptoethanol can be used in the described methods at between 0.1 mM - 55 mM. In some aspects, P-Mercaptoethanol can be used in culture medium of the described methods at about 0.1 mM, about 0.5 mM, about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, or about 55 mM.

[0105] In some aspects, the culture medium comprises a cell culture medium. In some aspects, the culture medium comprises neurobasal medium. Neurobasal medium is known by those of skill in the art; by way of a non-limiting example, Gibco’s Neurobasal™ medium. In some aspects, the culture medium comprises DMEM / F-12 medium. DMEM / F12 medium can be, by way of non-limiting example, Gibco’s DMEM / F12™ medium. In some aspects, the culture medium comprises a basal medium supplemented with B27. Non-limiting examples of B27 includes Gibco’s B27™. In some aspects, the culture medium comprises a basal mediumsupplemented with N2. Non-limiting examples of N2 includes Gibco’s N2™. In some aspects, the culture medium comprises a basal medium supplemented with B27 and N2. In some aspects, the culture medium comprises DMEM / F12 supplemented with N2. In some aspects, the culture medium comprises neurobasal medium supplemented with B27. In some aspects, a mixture of two or more cell culture medium can be used. By way of non-limiting example, 1 :1 (v / v) mixture of DMEM / F12 and neurobasal medium can be used as a culture medium for derivation of ESCs, XENs, and / or TSCs. In some aspects, the culture medium comprises DMEM / F12 supplemented with N2 medium, neurobasal medium supplemented with B27, or any combination thereof.

[0106] In some aspects, the culture medium disclosed herein can be used for derivation of ESCs, XENs, and / or TSCs. In some aspects, the culture medium disclosed herein can be used for further differentiation of ESCs, XENs, and / or TSCs. In some aspects, the culture medium disclosed herein can be used for co-culture of ESCs, XENs, and / or TSCs. Further, in one aspect, the culture medium disclosed herein can be used for co-culturing of ESCs, XENs, and / or TSCs.

[0107] In some aspects, the culture medium used for derivation in the disclosed methods comprises one or more of DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, p-mercaptoethanol, FGF2, Activin-A, CHIR99021 , or any combination thereof.

[0108] In further aspects, the culture medium used for derivation in the disclosed methods comprises one or more of DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, P-mercaptoethanol, Knockout serum replacement, FGF2, Activin-A, CHIR99021 , or any combination thereof.

[0109] In some aspects, the culture medium used for derivation in the disclosed methods comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, P- mercaptoethanol, FGF2, Activin-A, and CHIR99021.

[0110] In further aspects, the culture medium used for derivation in the disclosed methods comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, P- mercaptoethanol, Knockout serum replacement, FGF2, Activin-A, and CHIR99021.

[0111] In some aspects, the culture medium used for derivation in the disclosed methods comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), P-mercaptoethanol (55mM), FGF2 (20 ng / mL), Activin-A (20 ng / mL), and CHIR99021 (3 pM).

[0112] In some aspects, the medium used for derivation in the disclosed methods comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X),P-mercaptoethanol (55mM), 20% KnockOut Serum Replacement, FGF2 (6 ng / mL), Activin-A (25 ng / mL) and CHIR99021 (1.5 pM).

[0113] In further aspects, after derivation, ESCs, XENs, and / or TSCs can be cultured further in a medium with or without the FGF, TGF-p and WNT activators. For example, derived ESCs, XENs, and / or TSCs can be cultured in a differentiation medium.

[0114] In some aspects, the culture medium used as a differentiation medium comprise one or more of DMEM / F12, neurobasal medium, N2, B27 minus insulin supplement, Glutamax, NEAA, P-mercaptoethanol, Penicillin-Streptomycin, FBS, or any combination thereof.

[0115] In some aspects, the culture medium used as a differentiation medium in the disclosed method comprise DMEM / F12, neurobasal medium, N2, B27 minus insulin supplement, Glutamax, NEAA, P-mercaptoethanol, Penicillin-Streptomycin, and FBS.

[0116] In some aspects, the culture medium used as a differentiation medium in the disclosed methods comprise 1 :1 (v / v) mixture of DMEM / F12 and neurobasal medium, N2 (1X), B27 minus insulin supplement (1X), Glutamax (1X), NEAA (1X), P-mercaptoethanol (0.1 mM), Penicillin-Streptomycin (0.5%), and FBS (10%).

[0117] In some aspects, the derived ESCs, XENs, and / or TSCs are further processed. In some aspects, the method further comprises restricting ESCs growth in cell culture. In some aspects, the method comprises co-culturing ESCs, XENs, and / or TSCs. In some aspects, the method comprises producing multilineage cells using derived ESCs, XENs, and / or TSCs. In some aspects, the ESCs, XENs, and / or TSCs as described herein may be implanted in vivo to form teratomas or tumors.III. Method of restricting ESC growth using co-culture or extracellular matrix protein (Laminin and / or Collagen)

[0118] The disclosure further provides a method of restricting the growth of ESCs in cell culture. The method comprises culturing ESCs derived from reproductive cells or PSCs in a culture medium that includes a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF-P) activator, and a WNT activator, adding a growth restricting agent to the culture medium, and maintaining the cells in the culture for at least 5 days to obtain a population of growth restricted ESCs. In some aspects, the growth restricting agent is selected from laminin, collagen, XENs, or any combination thereof.

[0119] In some aspects, ESCs is derived using a method provided in the disclosure. Briefly, reproductive cells, or PSCs, are cultured in a culture medium that comprises a FGF activator, TGF-p activator, and a WNT activator and deriving stable ESCs. In some aspects,the method further comprises isolating the epiblast-like from the cell outgrowth (e.g., blastocyst outgrowth), and culturing and passaging the isolated cells to obtain stable ESCs.

[0120] In some aspects, growth restricted ESCs comprise ESCs which express one or more of Podxl, Gm26724, Cntnap3, Gm17477, Aqp8, Spockl, Enpep, S1pr5, Nxf7, and Fut1 at an elevated level compared to derived ESCs.

[0121] In some aspects, growth restricted ESCs have an elevated expression of one or more the disclosed genes at a log fold change between 0.1 to 10.0. In some aspects the elevated expression of the disclosed genes in the multilineage cells have a fold change between 1.0 to 5.0. In some aspects, the fold change can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.

[0122] In some aspects, growth restricted ESCs comprise ESCs with reduced expression of one or more Lyz2, Gpnmb, Saa3, B2m, H2-D1, Thbsl, Fbln2, Ctsl, Bgn, Plau, Marco, Col1a2, Ctss, Timp2, C1qa, Lilrb4a, Csf1, Col1a1, Tnfaip2, Den, Lox, Thbs2, Il1rn, Plek, Adgrel, Mpegl, Msr1, Serpinel Adam8, H2-M2, Cybb, C1qc, Mmp3, Ccl3, Anxal, Postn, Cd274, Vim, Xist, Mmp9, Lgals3bp, Cxcll, Wfdc17, Sdc3, lfi202b, Clec4e, Acssl, Vcaml, C1qb, Ccl2, Col5a3, Egr1, Fcgr3, C3, C3ar1, 111a, Fcgr2b, Lilr4b, Ccl6, Plk2, Clec4n, Ccl9, Pf4, Csf2rb, Axl, Atp6v0d2, Ms4a6d, Cd44, App, Zeb2, Ms4a7, Greml, Ptgs2, Tnc, Slfn2, Itgb2, Col6a1, Aebpl, Mmp12, Col5a1, Slc7a2, Mmp2, Tyrobp, Mmp13, Cxcl2, Trem2, S100a6, Id2, Ecm1, Ltbp2, Cd53, Bmp1, Serpinfl, Ptges, Plxndl, Feerig, Fxyd5, Cd36, Arhgdib, Ccl5, Cyr61, Sirpa, Rgs1, Slc15a3, Rac2, AI607873, Apbblip, Xdh, Clec4d, Ccl7, Slc11 a1 , Bst1, Alcam, Tgfbi, Col5a2, Glrx, Tnfrsf23, Slfn5, Gas7, Gas6, Tnfaip3, Ctla2a, 111 rl1 , Atf3, Itpripl2, Ddr2, 6720427l07Rik, Cd84, Ptprc, Itgb3, Prod, Fabp4, Pmp22, Cav1, Mical2, Ctgf, Col6a2, Inhba, Igfbp7, Tm4sf1, Lum, Hk3, Fam46c, Cd33, Adamtsl, Clec7a, Lcn2, Serpinb2, Mir17hg, Col8a1, Il4ra, Csf2rb2, Adssll, Irg1, Gsap, Ccrl2, Cd300lf, Tmem176b, Egr2, Rgs16, Rab7b, Rhob, Grem2, Col12a1, Col6a3, Lgals3, lfi204, Rassf4, Rnd3, Fam 198b, Cd 14, Pcdh7, March 1, Cp, Ncf4, lfi203, Ptger4, Csflr, Smpdl3a, Havcr2, H2-T23, Hclsl, Sigled, Naip2, Emp1, WIs, Ier5, Rps2-ps13, Cd93, Serpina3g, Penk, Serpina3h, Layn, Cryab, Thbd, Olr1, Nlrc5, Acta2, Col16a1, Pik3ap1, Thy1, Ncf1, AF251705, Mmp10, Synpo, Igf1, Ly9, Ptx3, Npy, II33, Tns1, Gda, Mafb, Casp4, Tfpi2, Cd180, Mr1, Lmxlb, Ets1, Ccdc80, Ugt1a7c, Gm15487, Gm8995, Rnasel, Cav2, Il7r, Mrc2, Itpr2, C5ar1, Tgfbr2, Cyp1b1, P2ry6, Gbp9, Dusp8, Acpp, Ifi44, Antxr2, Cxcl3, Ly6a, Nrp1, Rgl1, Oaf, Ccl8, Clec2d, Ptpre, Pdgfra, Arhgap25, Arsb, S1pr1, Adgrg6, Fam20c, Sh3kbp1, Runxl, Kitl, Gab2, Col3a1, Rspo4, H10ra, 111 b, Arhgap24, Ikbke, Emilin2, Cd34, Itga4, Hacd4, Nptxl, Steap4, Ccbel, Mmp8, Caspl, Entpdl, Sqrdl, Efnb3, Cd302, Gem, Dkk3, Rnase4, Dusp10, Mnda, Psmb8, Cd82, Sod3, Osbpl5, Lats2, Fgd3, Arhgap31, Cd86, Ms4a14, Mcoln2, Pik3cg, Cyp4v3, Slc25a24, Tagln,Cebpa, Rsph4a, Fas, Ereg, Klhl6, Col4a5, 181001101 ORik, Itgam, Arg2, Tmem176a, Tlr4, Flrt2, Efempl , Pla2g4a, C1s1 , B430306N03Rik, Mn1 , Procr, Stabl , Plat, Cd200, Nos2, Rasgefl b, Wnt5a, Ccr1 , Sept5, Gap43, Mef2c, Creb5, Ifitl , Negri , Parva, Gpr176, Orai2, S100a8, Pappa, Il1r1 , Prl2c3, Raetld, Hspalb, Adamts5, Evi2a, C1ra, Zfp703, Filipll, Fgf7, Dclkl , Nov, Heph, Sema3a, Pcdh19, Pgm5, Slc2a9, Irf2, Vgll3, Fcgr4, Cyth4, Mgstl , Gbp8, Emp3, H20rb, Fhl2, Casp12, Dkk2, Fill , 4930523C07Rik, Ngf, Maspl , Lrrc15, Cxcl5, Scarf2, Lrrc18, Nbl1 , Gm10143, Pdzrn3, Pdlim4, Osmr, Slc22a4, Mmp19, Myo18b, Ctso, Angpt4, Mxd4, Fads3, Adgrg2, Fam78b, Hgf, Cpq, Serpina3n, Prl2c2, Rbms3, Pcdh9, Tfec, Ldlrad4, Rgs2, Cd40, Fbxo32, Mir22hg, Sparcll , Tgfb2, Pde4b, Cspg4, Cd200r1 , Abca8b, Ctsk, Tap2, Ehd2, Svepl , Rcan2, Vax2os, Dchsl , Gm14453, Zfpm2, Slc22a13b-ps, Gm42531 , Gng11 , Pla2g4c, Isg15, Serpinb6b, Cdh13, Gm33280, Milrl , Slc8a1 , Maf, Cnripl , Slpi, Fam13a, Runx2, Bdkrb2, Till , Itgal 1 , Gbp4, Gm15559, IHOrb, Il15ra, Rab32, Mctpl , Sspn, Dachl , Pamrl , Lat2, Mcpt8, Msrb3, Serpinb9, Cd52, Rgma, Tbxasl , Itga2, Osr2, Nedd9, Gdnf, Nol4, Ust, Eya4, Slc16a2, C920025E04Rik, Lcelg, Nr2f2, Foxd , II7, Pcp4l1 , Ednra, 9330175E14Rik, Lama2, Plxna4, Serpinbla, Nipall , Mfap4, Irx1 , Rspo3, Prss23, Nsg1 , Dnm3os, Osginl , Pxdd , Fat4, Fat3, Snai2, Thsd7a, Gm26809, Tgfb3, Cxxc4, Meis3, Rbm24, Cplx2, Vax2, Areg, Gabrbl , Lancl3, PI15, Lrrc32, Fgf8, RP24-439I22.8, Scara3, Fgf2, Kcnn3, 4933427D06Rik, Rnf150, or any combination thereof, when compared to derived ESCs.

[0123] In some aspects, growth restricted ESCs have a reduced expression of one or more the disclosed genes at a log fold change between -0.1 to -10.0. In some aspects the reduced expression of the disclosed genes in the growth restricted ESCs have a fold change between -1.0 to -5.0. In some aspects, the fold change can be -0.1 , -0.2, -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, -0.9, -1 , -1.1 , -1.2, -1.3, -1.4, -1.5, -1.6, -1.7, -1.8, -1.9, -2, -2.1 , -2.2, -2.3, -2.4, -2.5, -2.6, -2.7, -2.8, -2.9, -3, -3.1 , -3.2, -3.3, -3.4, -3.5, -3.6, -3.7, -3.8, -3.9, -4, -4.1 , -4.2, -4.3, -4.4, -4.5, -4.6, -4.7, -4.8, -4.9, or -5.

[0124] In some aspects, growth restricted ESCs comprise ESCs with reduced cell density in cell culture when compared to cell density of derived ESCs. In some aspects, the growth restricted ESCs comprise a cell density of less than 8X104cells / cm2. In some aspects, the growth restricted ESCs comprise a cell density of less than 7X104cells / cm2. In some aspects, the growth restricted ESCs comprise a cell density of less than 6X104cells / cm2. In some aspects, the growth restricted ESCs comprise a cell density of less than 5X104cells / cm2. In some aspects, the growth restricted ESCs comprise a cell density of less than 4X104cells / cm2. In some aspects, the growth restricted ESCs comprise a cell density of less than 3X104cells / cm2. In some aspects, the growth restricted ESCs comprise a cell density of less than 2X104cells / cm2.

[0125] In some aspects, the growth restricted ESCs has a cell density that is about 20% reduced when compared to cell density of derived ESCs. In some aspects, the growth restricted ESCs has a cell density that is about 30% reduced when compared to cell density of derived ESCs. In some aspects, the growth restricted ESCs has a cell density that is about 40% reduced when compared to cell density of derived ESCs. In some aspects, the growth restricted ESCs has a cell density that is about 50% reduced when compared to cell density of derived ESCs. In some aspects, the growth restricted ESCs has a cell density that is about 60% reduced when compared to cell density of derived ESCs. In some aspects, the growth restricted ESCs has a cell density that is about 70% reduced when compared to cell density of derived ESCs. In some aspects, the growth restricted ESCs has a cell density that is about 80% reduced when compared to cell density of derived ESCs.

[0126] In some aspects, the growth restricting agent used in the method described herein is laminin. In some aspects, the growth restricting agent used in the method described herein is collagen. In some aspects, the growth restricting agent used in the method described herein is XENs. In some aspects, growth restricting agent is a combination of one or more of laminin, collagen, and XENs. In some aspects, the growth restricting agent is laminin and collagen.

[0127] Any laminin family member can be used in the culturing methods described herein, as a growth restriction agent and can include isolated laminin, peptide derived from laminin, polypeptide derived from laminin, or any functional fragments thereof. Laminin sequences (e.g., sequences of laminin A (or A1) chain, laminin B1 chain, laminin B2 chain, laminin A2 chain (merosin), laminin G1 chain) of various species are available on the world wide web from the NCBI, including human, mouse, and rat. For example, the nucleotide sequence encoding an isoform of human laminin B2 chain is available at NCBI under Accession No. NC_000007.14 and amino acid sequence is under Accession No. P07942.2.

[0128] An effective amount of a laminin used as a growth restriction agent in methods described herein can be between 0.1 pg / ml to 1000 pg / ml. In some aspects, the laminin is used can be between 1 pg / ml to 500 pg / ml. In some aspects, the laminin is used can be between 1 pg / ml to 200 pg / ml. In some aspects, the laminin is used can be between 10 pg / ml to 100 pg / ml. In some aspects, the laminin is added to the culture medium as a growth restriction agent at about 1 pg / ml, about 2 pg / ml, about 3 pg / ml, about 4 pg / ml, about 5 pg / ml, about 6 pg / ml, about 7 pg / ml, about 8 pg / ml, about 9 pg / ml, about 10 pg / ml, about 15 pg / ml, about 20 pg / ml, about 25 pg / ml, about 30 pg / ml, about 40 pg / ml, about 50 pg / ml, about 60 pg / ml, about 70 pg / ml, about 80 pg / ml, about 90 pg / ml, about 100 pM, about 110 pg / ml, about 120 pg / ml, about 130 pg / ml, about 140 pg / ml, about 150 pg / ml, about 160 pg / ml, about 170 pg / ml, about 180 pg / ml, about 190 pg / ml, or about 200 pg / ml. In some aspects, the laminin isadded to the culture medium as a growth restriction agent, for the methods described herein, at about 30 pg / ml.

[0129] Any collagen type can be used in the culturing methods described herein, as a growth restriction agent and can include isolated collagen, peptide derived from collagen, polypeptide derived from collagen, a chain, or any functional fragments thereof. Collagen sequences of various species are available on the world wide web from the NCBI, including human, mouse, and rat. For example, the nucleotide sequence encoding an isoform of human collagen type I alpha 1 chain is available at NCBI under Accession No. NC_000017.11 and amino acid sequence is under Accession No. KAI2583922.1.

[0130] An effective amount of a collagen used as a growth restriction agent in methods described herein can be between 0.1 pg / ml to 1000 pg / ml. In some aspects, the collagen is used can be between 1 pg / ml to 500 pg / ml. In some aspects, the collagen is used can be between 1 pg / ml to 200 pg / ml. In some aspects, the collagen is used can be between 10 pg / ml to 100 pg / ml. In some aspects, the collagen is added to the culture medium as a growth restriction agent at about 1 pg / ml, about 2 pg / ml, about 3 pg / ml, about 4 pg / ml, about 5 pg / ml, about 6 pg / ml, about 7 pg / ml, about 8 pg / ml, about 9 pg / ml, about 10 pg / ml, about 15 pg / ml, about 20 pg / ml, about 25 pg / ml, about 30 pg / ml, about 40 pg / ml, about 50 pg / ml, about 60 pg / ml, about 70 pg / ml, about 80 pg / ml, about 90 pg / ml, about 100 pM, about 110 pg / ml, about 120 pg / ml, about 130 pg / ml, about 140 pg / ml, about 150 pg / ml, about 160 pg / ml, about 170 pg / ml, about 180 pg / ml, about 190 pg / ml, or about 200 pg / ml. In some aspects, the collagen is added to the culture medium as a growth restriction agent, for the methods described herein, at about 15 pg / ml.

[0131] In some aspects, the growth restriction agent is derived XENs. XENs are derived from reproductive cells or PSCs using methods described herein. An effective amount of XENs used as a growth restriction agent comprises adding the XENs at an initial ESC to XEN ratio of about 1 :2 to about 8:1 to the culture medium. In some aspects, XENs are added at an initial ESC to XEN ratio of about 1 :2, about 1 :1 , about 2:1 , about 3:1 , about 4:1 , about 5:1 , about 6:1 , about 7:1 , or about 8:1.

[0132] In some aspects, the culture medium used for obtaining growth restricted ESCs comprises one or more of DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, [3-mercaptoethanol, FGF2, Activin-A, CHIR99021 , or any combination thereof.

[0133] In further aspects, the culture medium used for obtaining growth restricted ESCs comprises one or more of DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, p-mercaptoethanol, Knockout serum replacement, FGF2, Activin-A, CHIR99021 , or any combination thereof.

[0134] In some aspects, the culture medium for obtaining growth restricted ESCs comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, - mercaptoethanol, FGF2, Activin-A, and CHIR99021.

[0135] In further aspects, the culture medium for obtaining growth restricted ESCs comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, - mercaptoethanol, Knockout serum replacement, FGF2, Activin-A, and CHIR99021.

[0136] In some aspects, the medium comprise DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), |3-mercaptoethanol (55mM), FGF2 (20 ng / mL), Activin- A (20 ng / mL), CHIR99021 (3 pM).

[0137] In some aspects, the medium comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), |3-mercaptoethanol (55mM), 20% KnockOut Serum Replacement, FGF2 (6 ng / mL), Activin-A (25 ng / mL) and CHIR99021 (1.5 M).IV. Method for obtaining multi-lineage stem cells

[0138] Further provided in the disclosure is a method of obtaining multi-lineage stem cells in a shared cell culture condition. The method comprises obtaining a population of ESCs derived from reproductive cells or PSCs, obtaining a population of XENs and / or TSCs derived from reproductive cells or PSCs and co-culturing ESCs, XENs, and / or TSCs in a culture medium that comprises a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF-p) activator, and a WNT activator. The method further comprises maintaining the mixed cells in the culture medium for at least 5 days to obtain a population of multi-lineage stem cells.

[0139] The derived ESCs, XENs, and / or TSCs can be obtained by culturing reproductive cells, or PSCs in a culture medium that comprises a FGF activator, TGF-p activator, and a WNT activator, and isolating the epiblast-like, endoderm-like, and / or trophoblast-like cells from the cell outgrowth (e.g., blastocyst outgrowth), as described in the disclosure. The isolated cells can be further cultured and passaged to obtain stable ESCs, XENs, and / or TSCs.

[0140] One or more of the derived ESCs, XENs, and / or TSCs can then be cultured together in a shared culture medium that comprises a FGF activator, TGF- activator, and a WNT activator. The cultured mix of cells are then maintained in the medium for at least 5 days to obtain a population of multi-lineage stem cells. The multi-lineage cells thus obtained express epiblast, extraembryonic endoderm, and / or trophoblast markers.

[0141] In some aspects, the method of obtaining multilineage cells further comprises adding a growth restriction agent to the culture medium. In some aspects, the growth restricting agent added to the culture medium is laminin. In some aspects, the growthrestricting agent added to the culture medium is collagen. In some aspects, the growth restricting agent added to the culture medium is a combination of laminin and collagen. Laminin and / or collagen can be added to the culture medium to obtain multilineage cells at a concentration of 1 - 200 pg / ml. In some aspects, laminin is added to the culture medium to obtain multilineage cells at a concentration of 30 pg / ml. In some aspects, collagen is added to the culture medium to obtain multilineage cells at a concentration of 15 pg / ml.

[0142] In some aspects, the culture medium for obtaining multilineage cells comprise a FGF activator, FGF2. In some aspects, the culture medium for obtaining multilineage cells comprise a TGF- activator, Activin-A. In some aspects, the culture medium for obtaining multilineage cells comprise a WNT activator, CHIR99021.

[0143] In some aspects, the multilineage cells comprise ESCs and XENs. In some aspects, the multilineage cells comprise ESCs and TSCs. In some aspects, the multilineage cells comprise ESCs, XENs, and TSCs.

[0144] In some aspects, the method comprises combining ESCs and XENs at an initial ESC to XEN ratio of about 1 :2 to about 8:1. In some aspects, ESCs and XENs are combined at an initial ESC to XEN ratio of about 1 :2, about 1 :1 , about 2: 1 , about 3: 1 , about 4: 1 , about 5: 1 , about 6: 1 , about 7:1 , or about 8: 1.

[0145] In some aspects, the method comprises combining ESCs and TSCs at an initial ESC to TSC ratio of about 1 :2 to about 8:1. In some aspects, ESCs and TSCs are combined at an initial ESC to TSC ratio of about 1 :2, about 1 :1 , about 2:1 , about 3: 1 , about 4: 1 , about 5: 1 , about 6: 1 , about 7:1 , or about 8: 1.

[0146] In some aspects, the method comprises combining ESCs, XENs, and TSCs at an initial ESC to XEN to TSC ratio of about 1 :2:2 to about 8:1 :1. In some aspects, ESCs, XENs, and TSCs are combined at an initial ESC to XEN to TSC ratio of about 1 :2:2, about 1 :1 :1 , about 2:1 :1 , about 3:1 :1 , about 4:1 :1 , about 5:1 :1 , about 6:1 :1 , about 7: 1 : 1 , or about 8:1 :1.

[0147] In some aspects, the multilineages cell express one or more of Podxl, Gm26724, Cntnap3, Gm 17477, Aqp8, Spockl , Enpep, S1pr5, Nxf7, and Fut1 at an elevated level compared to individually cultured ESCs, XENs, and / or TSCs.

[0148] In some aspects, multilineage cells have an elevated expression of one or more the disclosed genes at a log fold change between 0.1 to 10.0. In some aspects the elevated expression of the disclosed genes in the multilineage cells have a fold change between 1.0 to5.0. In some aspects, the fold change can be 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.1 ,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1 , 3.2, 3.3,3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.

[0149] In some aspects, multilineage cells comprise reduced expression of one or more Lyz2, Gpnmb, Saa3, B2m, H2-D1, Thbsl, Fbln2, Ctsl, Bgn, Plau, Marco, Col1a2, Ctss, Timp2, C1qa, Lilrb4a, Csf1, Col1a1, Tnfaip2, Den, Lox, Thbs2, Il1rn, Plek, Adgrel, Mpegl, Msr1, Serpinel Adam8, H2-M2, Cybb, C1qc, Mmp3, Ccl3, Anxal, Postn, Cd274, Vim, Xist, Mmp9, Lgals3bp, Cxcll, Wfdc17, Sdc3, lfi202b, Clec4e, Acssl, Vcaml, C1qb, Ccl2, Col5a3, Egr1 , Fcgr3, C3, C3ar1, 111 a, Fcgr2b, Lilr4b, Ccl6, Plk2, Clec4n, Ccl9, Pf4, Csf2rb, Axl, Atp6v0d2, Ms4a6d, Cd44, App, Zeb2, Ms4a7, Greml, Ptgs2, Tnc, Slfn2, Itgb2, Col6a1, Aebpl, Mmp12, Col5a1, Slc7a2, Mmp2, Tyrobp, Mmp13, Cxcl2, Trem2, S100a6, Id2, Ecm1, Ltbp2, Cd53, Bmp1, Serpinfl, Ptges, Plxndl, Feerig, Fxyd5, Cd36, Arhgdib, Ccl5, Cyr61, Sirpa, Rgs1, Slc15a3, Rac2, AI607873, Apbblip, Xdh, Clec4d, Ccl7, Slc11a1, Bst1, Alcam, Tgfbi, Col5a2, Glrx, Tnfrsf23, Slfn5, Gas7, Gas6, Tnfaip3, Ctla2a, 111 rl1, Atf3, Itpripl2, Ddr2, 6720427l07Rik, Cd84, Ptprc, Itgb3, Prrxl, Fabp4, Pmp22, Cav1, Mical2, Ctgf, Col6a2, Inhba, Igfbp7, Tm4sf1, Lum, Hk3, Fam46c, Cd33, Adamtsl, Clec7a, Lcn2, Serpinb2, Mir17hg, Col8a1, Il4ra, Csf2rb2, Adssll, Irg1, Gsap, Ccrl2, Cd300lf, Tmem176b, Egr2, Rgs16, Rab7b, Rhob, Grem2, Col12a1, Col6a3, Lgals3, lfi204, Rassf4, Rnd3, Fam198b, Cd14, Pcdh7, Marchl, Cp, Ncf4, lfi203, Ptger4, Csflr, Smpdl3a, Havcr2, H2-T23, Hclsl, Sigled, Naip2, Emp1, WIs, Ier5, Rps2-ps13, Cd93, Serpina3g, Penk, Serpina3h, Layn, Cryab, Thbd, Olr1, Nlrc5, Acta2, Col16a1, Pik3ap1, Thy1, Ncf1, AF251705, Mmp10, Synpo, Igf1, Ly9, Ptx3, Npy, II33, Tns1, Gda, Mafb, Casp4, Tfpi2, Cd180, Mr1, Lmxlb, Ets1, Ccdc80, Ugt1a7c, Gm15487, Gm8995, Rnasel, Cav2, Il7r, Mrc2, Itpr2, C5ar1, Tgfbr2, Cyp1b1, P2ry6, Gbp9, Dusp8, Acpp, Ifi44, Antxr2, Cxcl3, Ly6a, Nrp1, Rgl1, Oaf, Ccl8, Clec2d, Ptpre, Pdgfra, Arhgap25, Arsb, S1pr1, Adgrg6, Fam20c, Sh3kbp1, Runxl, Kitl, Gab2, Col3a1, Rspo4, H10ra, 111 b, Arhgap24, Ikbke, Emilin2, Cd34, Itga4, Hacd4, Nptxl, Steap4, Ccbel, Mmp8, Caspl, Entpdl, Sqrdl, Efnb3, Cd302, Gem, Dkk3, Rnase4, DuspIO, Mnda, Psmb8, Cd82, Sod3, Osbpl5, Lats2, Fgd3, Arhgap31, Cd86, Ms4a14, Mcoln2, Pik3cg, Cyp4v3, Slc25a24, Tagln, Cebpa, Rsph4a, Fas, Ereg, Klhl6, Col4a5, 1810011O10Rik, Itgam, Arg2, Tmem176a, TI , Flrt2, Efempl, Pla2g4a, C1s1, B430306N03Rik, Mn1, Procr, Stabl, Plat, Cd200, Nos2, Rasgeflb, Wnt5a, Ccr1, Sept5, Gap43, Mef2c, Creb5, Ifitl, Negri, Parva, Gpr176, Orai2, S100a8, Pappa, 111 r1, Prl2c3, Raetld, Hspalb, Adamts5, Evi2a, C1ra, Zfp703, Filipll, Fgf7, Dclkl, Nov, Heph, Sema3a, Pcdh19, Pgm5, Slc2a9, Irf2, Vgll3, Fcgr4, Cyth4, Mgstl, Gbp8, Emp3, H20rb, Fhl2, Casp12, Dkk2, Fli1, 4930523C07Rik, Ngf, Maspl, Lrrc15, Cxcl5, Scarf2, Lrrc18, Nbl1, Gm10143, Pdzrn3, Pdlim4, Osmr, Slc22a4, Mmp19, Myo18b, Ctso, Angpt4, Mxd4, Fads3, Adgrg2, Fam78b, Hgf, Cpq, Serpina3n, Prl2c2, Rbms3, Pcdh9, Tfec, Ldlrad4, Rgs2, Cd40, Fbxo32, Mir22hg, Sparcll, Tgfb2, Pde4b, Cspg4, Cd200r1, Abca8b, Ctsk, Tap2, Ehd2, Svepl, Rcan2, Vax2os, Dchsl, Gm14453, Zfpm2, Slc22a13b-ps, Gm42531, Gng11, Pla2g4c, Isg15, Serpinb6b, Cdh13, Gm33280, Milrl, Slc8a1, Maf, Cnripl, Slpi, Fam13a, Runx2, Bdkrb2, Till, Itga11, Gbp4, Gm15559, H10rb, Il15ra, Rab32, Mctpl, Sspn, Dachl, Pamrl, Lat2, Mcpt8,Msrb3, Serpinb9, Cd52, Rgma, Tbxasl , Itga2, Osr2, Nedd9, Gdnf, Nol4, list, Eya4, Slc16a2, C920025E04Rik, Lcelg, Nr2f2, Foxd , II7, Pcp4l1 , Ednra, 9330175E14Rik, Lama2, Plxna4, Serpinbla, Nipall , Mfap4, Irx1 , Rspo3, Prss23, Nsg1 , Dnm3os, Osginl , Pxdd , Fat4, Fat3, Snai2, Thsd7a, Gm26809, Tgfb3, Cxxc4, Meis3, Rbm24, Cplx2, Vax2, Areg, Gabrbl , Lancl3, Pi15, Lrrc32, Fgf8, RP24-439I22.8, Scara3, Fgf2, Kcnn3, 4933427D06Rik, Rnf150, or any combination thereof, when compared to individually cultured ESCs, XENS, and / or TSCs.

[0150] In some aspects, multilineage cells have a reduced expression of one or more the disclosed genes at a log fold change between -0.1 to -10.0. In some aspects the reduced expression of the disclosed genes in the growth restricted ESCs have a fold change between -1.0 to -5.0. In some aspects, the fold change can be -0.1 , -0.2, -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, -0.9, -1 , -1.1 , -1.2, -1.3, -1.4, -1.5, -1.6, -1.7, -1.8, -1.9, -2, -2.1 , -2.2, -2.3, -2.4, -2.5, -2.6, -2.7, -2.8, -2.9, -3, -3.1 , -3.2, -3.3, -3.4, -3.5, -3.6, -3.7, -3.8, -3.9, -4, -4.1 , -4.2, -4.3, -4.4, -4.5, -4.6, -4.7, -4.8, -4.9, or -5.

[0151] In some aspects, the culture medium for obtaining multilineage cells comprise one or more of DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, - mercaptoethanol, FGF2, Activin-A, CHIR99021 , or any combination thereof. In further aspects, the culture medium used for obtaining growth restricted ESCs comprise one or more of DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, p-mercaptoethanol, Knockout serum replacement, FGF2, Activin-A, CHIR99021 , or any combination thereof. In some aspects, the culture medium for obtaining multilineage cells comprise DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, p-mercaptoethanol, FGF2, Activin- A, and CHIR99021. In further aspects, the culture medium for obtaining multilineage cells comprise DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, p- mercaptoethanol, Knockout serum replacement, FGF2, Activin-A, and CHIR99021. In some aspects, the culture medium for obtaining multilineage cells comprise DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), P-mercaptoethanol (55mM), FGF2 (20 ng / mL), Activin-A (20 ng / mL), CHIR99021 (3 pM). In some aspects, the medium comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), P- mercaptoethanol (55mM), 20% KnockOut Serum Replacement, FGF2 (6 ng / mL), Activin-A (25 ng / mL), and CHIR99021 (1.5 pM).V. Culture of mutant ESCs and XENs

[0152] According to an aspect of the present disclosure, provided is a method of cell culture comprising engineered derived ESCs and / or XENs. The method comprises obtaining ESCs and / or XENs derived from reproductive cells or PSCs. In some aspects, the ESCs and / or XENs are derived from blastocysts. The derived ESCs are then engineered to have amutation or deletion in one or more integrin, while the derived XENs are engineered to have a mutation or deletion in one or more laminin. The engineered ESCs and / or XENs are cultured in a cell culture medium comprising a FGF activator, TGF-0 activator, and a WNT activator.

[0153] The engineered ESCs can be used to establish cultures with ESCs having uninhibited proliferation, when cultured individually or when co-cultured with XENs, engineered XENs, or TSCs. Similarly, engineered XENs can be used to obtain co-culture with uninhibited proliferation when cultured individually or when co-cultured with ESCs or engineered ESCs. Such cultures can be used to study cell-cell communication in stem cells.

[0154] The derived ESCs and / or XENs can be engineered to have the mutation or deletion, using any known method of art. For e.g., a plasmid carrying the sgRNA specific for integrin or laminin can be transfected into derived ESCs or XENs, respectively, by using an electroporator and positive cells for transfection can be sorted and cultured. Mutations or knockout can be confirmed by sequencing methods.

[0155] In some aspects, the derived ESCs are engineered to exhibit reduced expression levels of one or more integrins. The reduced expression level of one or more integrins in ESCs is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% greater compared to derived non-engineered ESCs.

[0156] In some aspects, the derived XENs are engineered to exhibit reduced expression levels of one or more lamins. The reduced expression level of one or more integrins in XENs is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% greater compared to derived non-engineered XENs.

[0157] In some aspects, the medium for culturing engineered ESCs and / or XENs comprises one or more of DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, [3-mercaptoethanol, FGF2, Activin-A, CHIR99021 , or any combination thereof.

[0158] In further aspects, the medium for culturing engineered ESCs and / or XENs comprises one or more of DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax,NEAA, P-mercaptoethanol, Knockout serum replacement, FGF2, Activin-A, CHIR99021 , or any combination thereof.

[0159] In some aspects, the medium for culturing engineered ESCs and / or XENs comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, - mercaptoethanol, FGF2, Activin-A, and CHIR99021.

[0160] In further aspects, the medium for culturing engineered ESCs and / or XENs comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, P- mercaptoethanol, Knockout serum replacement, FGF2, Activin-A, and CHIR99021.

[0161] In some aspects, the medium for culturing engineered ESCs and / or XENs comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), P-mercaptoethanol (55mM), FGF2 (20 ng / mL), Activin-A (20 ng / mL), and CHIR99021 (3 pM).

[0162] In some aspects, the medium comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), P-mercaptoethanol (55mM), 20% KnockOut Serum Replacement, FGF2 (6 ng / mL), Activin-A (25 ng / mL), and CHIR99021 (1.5 pM).

[0163] In some aspects, the medium for culturing engineered ESCs and XENs further comprises a growth restricting agent, wherein the growth restricting agent is selected from laminin, collagen, XENs, or any combination thereof. The addition of the growth restricting agent, can allow for controlled and / or timed proliferation of the ESCs. In some aspects, the disclosed culture medium comprises a growth restricting agent laminin at a concentration of 1 pg / ml - 200 pg / ml. In some aspects, the disclosed culture medium comprises laminin at a concentration of 30 pg / ml. In some aspects, the disclosed culture medium comprises a growth restriction agent collagen at a concentration of 1 pg / ml - 200 pg / ml. In some aspects, the disclosed culture medium comprises collagen at a concentration of 30 pg / ml. In some aspects, the growth restriction agent in the disclosed system is XENs, wherein the XENs is present in the culture medium at an initial ESC to XEN ratio of about 1 :2 to about 8:1.VI. Culture Medium

[0164] Provided herein are culture medium which can be used in any of the disclosed methods. The disclosed medium, in general, comprises a basal medium, FGF2, Activin-A, CHIR99021 , additional factors, or any combination thereof.

[0165] In some aspects, the culture medium comprises one or more of DMEM / F-12 medium, neurobasal medium, N2, B27, L-glutamine, NEAA, P-mercaptoethanol, FGF2, Activin-A, CHIR99021 , or any combination thereof.

[0166] In further aspects, the culture medium used in the disclosed methods comprises one or more of DMEM / F-12 medium, neurobasal medium, N2, B27, L-glutamine, NEAA, p-mercaptoethanol, Knock out or Knockout serum replacement, FGF2, Activin-A, CHIR99021 , or any combination thereof.

[0167] The basal medium may be any conventional medium used for culturing stem cells. The basal medium may be manually prepared according to conventional methods. Further, the basal medium may be a commercially available medium or a mixture thereof. For example, the basal medium may be selected from the group consisting of DM EM (Dulbecco's Modified Eagle's Medium; GIBCO), MEM (Minimal Essential Medium; GIBCO), BME (Basal Medium Eagle; GIBCO), RPMI 1640 (GIBCO), DMEM / F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12; GIBCO), DMEM / F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10; GIBCO), a-MEM (a-Minimal essential Medium; GIBCO), G-MEM (Glasgow's Minimal Essential Medium; GIBCO), IMDM (Isocove's Modified Dulbecco's Medium; GIBCO), Neurobasal medium (GIBCO), and KnockOut DMEM (GIBCO). The basal medium may contain one or more supplements, which includes, but not limited to, KnockOut Serum Replacement (GIBCO), KnockOut SR XenoFree (GIBCO), KnockOut SR XenoFree Growth Factor Cocktail (GIBCO), N2 supplement (GIBCO), B27 supplement (GIBCO).

[0168] In some aspects, the culture medium may comprise a combination of two or more basal mediums. By way of non-limiting example, 1 :1 (v / v) mixture of DMEM / F12 and neurobasal medium can be used as a culture medium. In some aspects, the culture medium comprises DMEM / F12 supplemented with N2 medium, neurobasal medium supplemented with B27, or any combination thereof.

[0169] In some aspects, the culture medium comprises DMEM / F12 and can be used at a range between 0.1X to 2X. The DMEN / F12 can be, for example, DMEM / F-12 Medium (GIBCO: 11320033). In some aspects, DMEM / F12 medium is used at about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1.2X, about 1.3X, about 1.4X, about 1.5X, about 1.6X, about 1.7X, about 1.8X, about 1.9X, or about 2X.

[0170] In some aspects, the culture medium comprises DMEM / F12 at a concentration from about 0% (v / v) to about 100% (v / v). In some aspects, about 1 % (v / v), about 5% (v / v), about 10% (v / v), about 15% (v / v), about 20% (v / v), about 25% (v / v), about 30% (v / v), about 35% (v / v), about 40% (v / v), 45% (v / v), about 50% (v / v), about 55% (v / v), about 60% (v / v), about 65% (v / v), about 70% (v / v), about 75% (v / v), about 80% (v / v), about 85% (v / v), about 90% (v / v), about 95% (v / v), or about 99% (v / v) of DMEM / F12 can be used in the culture medium. In some aspects, the culture medium comprises DMEM / F12 at a concentration of at least 40% (v / v). In some aspects, the culture medium comprises DMEM / F12 at a concentration of about 100% (v / v).

[0171] In some aspects, the culture medium comprises neurobasal medium. The neurobasal medium can be, for example, Neurobasal Medium (GIBCO: 21103049). The neurobasal medium can be used at a concentration between 0.1X to 2X. In some aspects, neurobasal medium is used in the culture medium at about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1.2X, about 1.3X, about 1.4X, about 1.5X, about 1.6X, about 1.7X, about 1.8X, about 1.9X, or about 2X.

[0172] In some aspects, the culture medium comprises neurobasal medium at a concentration from about 0% (v / v) to about 100% (v / v). In some aspects, about 1% (v / v), about 5% (v / v), about 10% (v / v), about 15% (v / v), about 20% (v / v), about 25% (v / v), about 30% (v / v), about 35% (v / v), about 40% (v / v), 45% (v / v), about 50% (v / v), about 55% (v / v), about 60% (v / v), about 65% (v / v), about 70% (v / v), about 75% (v / v), about 80% (v / v), about 85% (v / v), about 90% (v / v), about 95% (v / v), or about 99% (v / v) of neurobasal medium can be used in the culture medium. In some aspects, the culture medium comprises neurobasal medium at a concentration of at least 40% (v / v). In some aspects, the culture medium comprises neurobasal medium at a concentration of about 100% (v / v).

[0173] In some aspects, the culture medium comprises N2 supplement. The N2 can be, for example, N2 (GIBCO: 17502048). The N2 can be used in the culture medium at a concentration between 0.1X to 2X. In some aspects, N2 is used at about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1.2X, about 1.3X, about 1.4X, about 1.5X, about 1.6X, about 1.7X, about 1.8X, about 1 ,9X, or about 2X.

[0174] In some aspects, the culture medium comprises N2 at a concentration from about 0.1 % (v / v) to about 5% (v / v). In some aspects, about 0.1% (v / v), about 0.5% (v / v), about 1% (v / v), about 1.5% (v / v), about 2% (v / v), about 2.5% (v / v), about 3% (v / v), about 3.5% (v / v), about 4% (v / v), about 4.5% (v / v), or about 5% (v / v) of N2 can be used in the culture medium. In some aspects, the culture medium comprises N2 at a concentration of about 1% (v / v).

[0175] In some aspects, the culture medium comprises B27 supplement. The B27 can be, for example, B27 (GIBCO: 17504044). The B27 can be used in the culture medium at a concentration between 0.1X to 2X. In some aspects, B27 is used at about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1.2X, about 1.3X, about 1.4X, about 1.5X, about 1.6X, about 1.7X, about 1.8X, about 1.9X, or about 2X.

[0176] In some aspects, the culture medium comprises B27 at a concentration from about 0.1% (v / v) to about 5% (v / v). In some aspects, about 0.1% (v / v), about 0.5% (v / v), about 1%(v / v), about 1.5% (v / v), about 2% (v / v), about 2.5% (v / v), about 3% (v / v), about 3.5% (v / v), about 4% (v / v), about 4.5% (v / v), or about 5% (v / v) of B27 can be used in the culture medium. In some aspects, the culture medium comprises B27 at a concentration of about 1% (v / v).

[0177] In some aspects, the culture medium comprises L-glutamine. L-glutamine, for example, can be added to the culture medium as Gibco GlutaMAX™ (GIBCO: 35050061). The Glutamax can be added in the culture medium at a concentration between 0.1X to 2X. In some aspects, Glutamax is used at about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1.2X, about 1.3X, about 1.4X, about 1.5X, about 1.6X, about 1.7X, about 1.8X, about 1.9X, or about 2X.

[0178] In some aspects, the culture medium comprises Glutamax at a concentration from about 0.1% (v / v) to about 5% (v / v). In some aspects, about 0.1% (v / v), about 0.5% (v / v), about 1 % (v / v), about 1.5% (v / v), about 2% (v / v), about 2.5% (v / v), about 3% (v / v), about 3.5% (v / v), about 4% (v / v), about 4.5% (v / v), or about 5% (v / v) of Glutamax can be used in the culture medium. In some aspects, the culture medium comprises Glutamax at a concentration of about 1% (v / v).

[0179] In some aspects, the culture medium comprises NEAA. Non-limiting example of NEAA include MEM Gibco NEAA™ (GIBCO: 11140050). The NEAA can be added in the culture medium at a concentration between 0.1X to 2X. In some aspects, NEAA is used at about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1.2X, about 1.3X, about 1.4X, about 1.5X, about 1.6X, about 1.7X, about 1.8X, about 1.9X, or about 2X.

[0180] In some aspects, the culture medium comprises NEAA at a concentration from about 0.1% (v / v) to about 5% (v / v). In some aspects, about 0.1% (v / v), about 0.5% (v / v), about 1% (v / v), about 1.5% (v / v), about 2% (v / v), about 2.5% (v / v), about 3% (v / v), about 3.5% (v / v), about 4% (v / v), about 4.5% (v / v), or about 5% (v / v) of NEAA can be used in the culture medium. In some aspects, the culture medium comprises NEAA at a concentration of about 1% (v / v).

[0181] In some aspects, the culture medium comprises |3-Mercaptoethanol. In some aspects, P-Mercaptoethanol can be used in the culture medium at a concentration between 0.1 mM - 100 mM. In some aspects, P-Mercaptoethanol can be used in culture medium at about 0.1 mM, about 0.5 mM, about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM. In some aspects, P-Mercaptoethanol can be used in culture medium at about 55 mM.

[0182] In some aspects, FGF activator used in the culture medium herein can be at a concentration between 0.1 ng / ml and 100 ng / ml. In some aspects, the effective amount of the FGF activator in culture medium can be between 1 ng / ml and 100 ng / ml. In some aspects, the effective amount of the FGF activator in the culture medium is about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 20 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, or about 100 ng / ml. In some aspect, the FGF activator is added to the culture medium at about 6 ng / ml. In some aspects, the FGF activator is added to the culture medium at about 20 ng / ml. In some aspects, FGF activator added to the culture medium is FGF2 at about 6 ng / ml. In some aspects, FGF activator added to the culture medium is FGF2 at about 20 ng / ml.

[0183] In some aspects, TGF-|3 activator used in the culture medium described herein can be at a concentration between 0.1 ng / ml and 100 ng / ml. In some aspects, the effective amount of the TGF-P activator used in the culture medium can be between 1 ng / ml and 100 ng / ml. In some aspects, the effective amount of the TGF-p activator in the culture medium is about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 15 ng / ml, about 16 ng / ml, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml, about 20 ng / ml, about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26 ng / ml, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, or about 100 ng / ml. In some aspects, TGF-p activator is added to the culture medium at about 20 ng / ml. In some aspect, the TGF-p activator is added to the culture medium at about 25 ng / ml. In some aspects, the TGF-p activator added to the culture medium is Activin-A at about 20 ng / ml. In some aspects, the TGF-P activator added to the culture medium is Activin-A at about 25 ng / ml.

[0184] An effective amount of a WNT activator used in culture medium described herein can be at a concentration between 0.1 pM and 10,000 pM. In some aspects, WNT activator used in the culture medium can be at a concentration between 0.1 pM and 100 pM. In some aspects, the effective amount of the WNT activator used in the culture medium can be at a concentration between 1 pM and 100 pM. In some aspects, the effective amount of the WNT activator used in the culture medium is about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, or about 100 pM. In some aspect, the WNT activator is added to the culture medium at about 1.5 pM. In someaspects, the WNT activator is added to the culture medium at about 3 pM. In some aspects, the WNT activator added to the culture medium is CHIR99021 at about 1.5 pM. In some aspects, the WNT activator added to the culture medium is CHIR99021 at about 3 pM.

[0185] In some aspects, the culture medium comprises KSR or KOSR. The KOSR, for example, can be Gibco KnockOut™ Serum Replacement (GIBCO: 10828028). In some aspects, KSR or KOSR is used in the culture medium at a concentration between 1%-30%. In some aspects, the KSR or KOSR is added to the culture medium for the methods described herein can be about 1 %, about 5%, about 10%, about 12%, about 15%, about 17%, about 20%, about 22%, about 25%, about 27%, about 30%. In some aspects, KSR or KOSR is added to the culture medium at about 20%.

[0186] In some aspects, the culture medium comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1 X), NEAA (1X), P-mercaptoethanol (55mM), FGF2 (20 ng / mL), Activin-A (20 ng / mL), and CHIR99021 (3 pM).

[0187] In some aspects, the culture medium comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), 0-mercaptoethanol (55mM), 20% KnockOut Serum Replacement, FGF2 (6 ng / mL), Activin-A (25 ng / mL), and CHIR99021 (1.5 pM).

[0188] In further aspects, the culture medium can comprise one or more growth restriction agents. Growth restriction agents used in the culture medium can be selected from laminin, collagen, XENs, or any combination thereof. In some aspects, the culture medium disclosed herein comprises laminin as the growth restriction agent. In some aspects, the culture medium disclosed herein comprises collagen as the growth restriction agent. In some aspects, the culture medium disclosed herein comprises XENs as the growth restriction agent. In some aspects, the culture medium disclosed herein comprises a combination of laminin, collagen and / or XENs as the growth restriction agents.

[0189] An effective amount of a laminin used as a growth restriction agent in the culture medium described herein can be between 0.1 pg / ml to 1000 pg / ml. In some aspects, the laminin used in the culture medium can be between 1 pg / ml to 500 pg / ml. In some aspects, the laminin used in the culture medium can be between 1 pg / ml to 200 pg / ml. In some aspects, the laminin used in the culture medium can be between 10 pg / ml to 100 pg / ml. In some aspects, the laminin is added to the culture medium as a growth restriction agent at about 1 pg / ml, about 2 pg / ml, about 3 pg / ml, about 4 pg / ml, about 5 pg / ml, about 6 pg / ml, about 7 pg / ml, about 8 pg / ml, about 9 pg / ml, about 10 pg / ml, about 15 pg / ml, about 20 pg / ml, about 25 pg / ml, about 30 pg / ml, about 40 pg / ml, about 50 pg / ml, about 60 pg / ml, about 70 pg / ml, about 80 pg / ml, about 90 pg / ml, about 100 pM, about 110 pg / ml, about 120 pg / ml, about 130 pg / ml, about 140 pg / ml, about 150 pg / ml, about 160 pg / ml, about 170 pg / ml, about 180 pg / ml,about 190 pg / ml, or about 200 pg / ml. In some aspects, the laminin is added to the culture medium as a growth restriction agent at about 30 pg / ml.

[0190] An effective amount of a collagen used as a growth restriction agent in the culture medium described herein can be between 0.1 pg / ml to 1000 pg / ml. In some aspects, the collagen used in the culture medium can be between 1 pg / ml to 500 pg / ml. In some aspects, the collagen used in the culture medium can be between 1 pg / ml to 200 pg / ml. In some aspects, the collagen used in the culture medium can be between 10 pg / ml to 100 pg / ml. In some aspects, the collagen is added to the culture medium as a growth restriction agent at about 1 pg / ml, about 2 pg / ml, about 3 pg / ml, about 4 pg / ml, about 5 pg / ml, about 6 pg / ml, about 7 pg / ml, about 8 pg / ml, about 9 pg / ml, about 10 pg / ml, about 15 pg / ml, about 20 pg / ml, about 25 pg / ml, about 30 pg / ml, about 40 pg / ml, about 50 pg / ml, about 60 pg / ml, about 70 pg / ml, about 80 pg / ml, about 90 pg / ml, about 100 pM, about 110 pg / ml, about 120 pg / ml, about 130 pg / ml, about 140 pg / ml, about 150 pg / ml, about 160 pg / ml, about 170 pg / ml, about 180 pg / ml, about 190 pg / ml, or about 200 pg / ml. In some aspects, the collagen is added to the culture medium as a growth restriction agent, at about 15 pg / ml.

[0191] In some aspects, the growth restriction agent used in the culture medium is derived XENs. XENs are derived from reproductive cells or PSCs using methods described herein. An effective amount of XENs used as a growth restriction agent comprises adding the XENs at an initial ESC to XEN ratio of about 1 :2 to about 8:1 to the culture medium. In some aspects, XENs are added at an initial ESC to XEN ratio of about 1 :2, about 1 :1 , about 2:1 , about 3:1 , about 4:1 , about 5:1 , about 6:1 , about 7:1 , or about 8:1.

[0192] In some aspects, the culture medium comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), |3-mercaptoethanol (55mM), FGF2 (20 ng / mL), Activin-A (20 ng / mL), CHIR99021 (3 pM), and laminin (30 pg / ml).

[0193] In some aspects, the culture medium comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), [3-mercaptoethanol (55mM), FGF2 (20 ng / mL), Activin-A (20 ng / mL), CHIR99021 (3 pM), and collagen (15 pg / ml).

[0194] In some aspects, the culture medium comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), |3-mercaptoethanol (55mM), FGF2 (20 ng / mL), Activin-A (20 ng / mL), CHIR99021 (3 pM), and XENs (an initial ESC to XEN ratio of about 1 :1).

[0195] In some aspects, the culture medium comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), P-mercaptoethanol (55mM), 20% KnockOut Serum Replacement, FGF2 (6 ng / mL), Activin-A (25 ng / mL), CHIR99021 (1.5 pM), and laminin (30 pg / ml).

[0196] In some aspects, the culture medium comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), p-mercaptoethanol (55mM), 20% KnockOut Serum Replacement, FGF2 (6 ng / mL), Activin-A (25 ng / mL), CHIR99021 (1.5 pM), and collagen (15 pg / ml).

[0197] In some aspects, the culture medium comprises DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), P-mercaptoethanol (55mM), 20% KnockOut Serum Replacement, FGF2 (6 ng / mL), Activin-A (25 ng / mL), CHIR99021 (1 .5 pM), and XENS (an initial ESC to XEN ratio of about 1 :1).

[0198] Provided further in the disclosure, is a second culture medium which comprises basal medium supplemented with one or more of the additional factors N2 supplement, B27, L-glutamine, NEAA, P-Mercaptoethanol, antibiotics, fetal bovine serum (FBS), Bovine Serum Albumin (BSA), KOSR, Insulin-Transferrin-Selenium-Ethanolamine (ITS-X) supplement, Forskolin, EGF, Rock inhibitor, neuregulinl (NRG1), and / or ALK inhibitor.

[0199] Such medium can comprise a basal medium, for example DMEM / F12, neurobasal medium, or any combination thereof. In some aspects, the culture medium may comprise a combination of two or more basal mediums. By way of non-limiting example, 1 :1 (v / v) mixture of DMEM / F12 and neurobasal medium can be used as a culture medium. In some aspects, the culture medium comprises DMEM / F12 supplemented with N2 medium, neurobasal medium supplemented with B27, or any combination thereof.

[0200] In some aspects, the culture medium comprises DMEM / F12 and can be used at a concentration between O.IX to 2X. The DMEN / F12 can be, for example, DMEM / F-12 Medium (GIBCO: 11320033). In some aspects, DM EM / F12 medium is used at a concentration of about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1.2X, about 1.3X, about 1.4X, about 1.5X, about 1.6X, about 1.7X, about 1.8X, about 1.9X, or about 2X.

[0201] In some aspects, the culture medium comprises DMEM / F12 at a concentration from about 40% (v / v) to about 99% (v / v). In some aspects, about 40% (v / v), about 45% (v / v), about 50% (v / v), about 55% (v / v), about 60% (v / v), about 65% (v / v), about 70% (v / v), about 75% (v / v), about 80% (v / v), about 85% (v / v), about 90% (v / v), about 95% (v / v), or about 99% (v / v) of DMEM / F12 can be used in the culture medium. In some aspects, the culture medium comprises DMEM / F12 at a concentration of at least 40% (v / v).

[0202] In some aspects, the culture medium comprises neurobasal medium. The neurobasal medium can be, for example, Neurobasal Medium (GIBCO: 21103049). The neurobasal medium can be used at a concentration between 0.1X to 2X. In some aspects, neurobasal medium is used in the culture medium at about 0.1X, about 0.2X, about 0.3X,about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1.2X, about 1.3X, about 1.4X, about 1.5X, about 1.6X, about 1.7X, about 1.8X, about 1.9X, or about 2X.

[0203] In some aspects, the culture medium comprises neurobasal medium at a concentration from about 40% (v / v) to about 99% (v / v). In some aspects, about 40% (v / v), about 45% (v / v), about 50% (v / v), about 55% (v / v), about 60% (v / v), about 65% (v / v), about 70% (v / v), about 75% (v / v), about 80% (v / v), about 85% (v / v), about 90% (v / v), about 95% (v / v), or about 99% (v / v) of neurobasal medium can be used in the culture medium. In some aspects, the culture medium comprises neurobasal medium at a concentration of at least 40% (v / v).

[0204] In some aspects, the culture medium comprises N2 supplement. The N2 can be, for example, N2 (GIBCO: 17502048). The N2 can be used in the culture medium at a concentration between 0.1X to 2X. In some aspects, N2 is used at about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1.2X, about 1.3X, about 1.4X, about 1.5X, about 1.6X, about 1.7X, about 1.8X, about 1.9X, or about 2X.

[0205] In some aspects, the culture medium comprises N2 at a concentration from about 0.1 % (v / v) to about 5% (v / v). In some aspects, about 0.1% (v / v), about 0.5% (v / v), about 1% (v / v), about 1.5% (v / v), about 2% (v / v), about 2.5% (v / v), about 3% (v / v), about 3.5% (v / v), about 4% (v / v), about 4.5% (v / v), or about 5% (v / v) of N2 can be used in the culture medium. In some aspects, the culture medium comprises N2 at a concentration of about 1% (v / v).

[0206] In some aspects, the culture medium comprises B27 supplement. The B27 can be, for example, B27 (GIBCO: 17504044). In some aspects, B27 supplement is a B27 minus insulin supplement, for example Gibco™ B-27 Supplement, minus insulin (GIBCO: A1895601). The B27 can be used in the culture medium at a range between 0.1X to 2X. In some aspects, B27 is used at about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1.2X, about 1.3X, about 1.4X, about 1.5X, about 1.6X, about 1.7X, about 1.8X, about 1.9X, or about 2X.

[0207] In some aspects, the culture medium comprises B27 at a concentration from about 0.1 % (v / v) to about 5% (v / v). In some aspects, about 0.1% (v / v), about 0.5% (v / v), about 1% (v / v), about 1.5% (v / v), about 2% (v / v), about 2.5% (v / v), about 3% (v / v), about 3.5% (v / v), about 4% (v / v), about 4.5% (v / v), or about 5% (v / v) of B27 can be used in the culture medium. In some aspects, the culture medium comprises B27 at a concentration of about 1% (v / v). In some aspects, the culture medium comprises B27 minus insulin at a concentration of about 1 % (v / v).

[0208] In some aspects, the culture medium comprises L-glutamine. L-glutamine, for example, can be added to the culture medium as Gibco GlutaMAX™ (GIBCO: 35050061). The Glutamax can be added in the culture medium at a concentration between 0.1X to 2X. In some aspects, Glutamax is used at about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1.2X, about 1.3X, about 1.4X, about 1.5X, about 1.6X, about 1.7X, about 1.8X, about 1.9X, or about 2X.

[0209] In some aspects, the culture medium comprises Glutamax at a concentration from about 0.1% (v / v) to about 5% (v / v). In some aspects, about 0.1% (v / v), about 0.5% (v / v), about 1 % (v / v), about 1.5% (v / v), about 2% (v / v), about 2.5% (v / v), about 3% (v / v), about 3.5% (v / v), about 4% (v / v), about 4.5% (v / v), or about 5% (v / v) of Glutamax can be used in the culture medium. In some aspects, the culture medium comprises Glutamax at a concentration of about 1% (v / v).

[0210] In some aspects, the culture medium comprises NEAA. Non-limiting example of NEAA include MEM Gibco NEAA™ (GIBCO: 11140050). The NEAA can be added in the culture medium at a concentration between 0.1X to 2X. In some aspects, NEAA is used at about 0.1X, about 0.2X, about 0.3X, about 0.4X, about 0.5X, about 0.6X, about 0.7X, about 0.8X, about 0.9X, about 1X, about 1.2X, about 1.3X, about 1.4X, about 1.5X, about 1.6X, about 1.7X, about 1.8X, about 1.9X, or about 2X.

[0211] In some aspects, the culture medium comprises NEAA at a concentration from about 0.1% (v / v) to about 5% (v / v). In some aspects, about 0.1% (v / v), about 0.5% (v / v), about 1 % (v / v), about 1.5% (v / v), about 2% (v / v), about 2.5% (v / v), about 3% (v / v), about 3.5% (v / v), about 4% (v / v), about 4.5% (v / v), or about 5% (v / v) of NEAA can be used in the culture medium. In some aspects, the culture medium comprises NEAA at a concentration of about 1% (v / v).

[0212] In some aspects, the culture medium comprises |3-Mercaptoethanol. In some aspects, P-Mercaptoethanol can be used in the culture medium at a concentration between 0.1 mM - 100 mM. In some aspects, P-Mercaptoethanol can be used in culture medium at about 0.1 mM, about 0.5 mM, about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM. In some aspects, P-Mercaptoethanol can be used in culture medium at about 0.1 mM.

[0213] In some aspects, the culture medium comprises an antibiotic. Antibiotic used in the culture, by way of non-limiting example can be penicillin-streptomycin. In some aspects, the penicillin-streptomycin can be Gibco™ Penicillin-Streptomycin (GIBCO: 15140122). In someaspects, penicillin-streptomycin can be used in the culture medium at a concentration between 0.1-5%. In some aspects, the penicillin-streptomycin is added to the culture medium at about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1 .5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%. In some aspects, penicillin-streptomycin is added to the culture medium at about 0.5%. In some aspects, penicillin-streptomycin is added to the culture medium at about 1%.

[0214] In some aspects, the culture medium comprises KSR or KOSR. The KOSR can be for example, Gibco KnockOut™ Serum Replacement (GIBCO: 10828028). In some aspects, KSR or KOSR is used in the culture medium at a concentration between 0.1-30%. In some aspects, the KSR or KOSR added to the culture medium can be about 0.1 %, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 15%, about 17%, about 20%, about 22%, about 25%, about 27%, or about 30%. In some aspects, KSR or KOSR is added to the culture medium at about 4%.

[0215] In some aspects, the culture medium comprises FBS. In some aspects, FBS is used in the culture medium at a concentration between 1%-30%. In some aspects, the FBS added to the culture medium can be about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 15%, about 17%, about 20%, about 22%, about 25%, about 27%, or about 30%. In some aspects, FBS is added to the culture medium at about 10%.

[0216] In some aspects, the culture medium comprises BSA. In some aspects, BSA can be used in the culture medium at a concentration between 0.1-3%. In some aspects, the BSA is added to the culture medium at about 0.1 %, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1 %, about 1.5%, about 2%, about 2.5%, or about 3%. In some aspects, BSA is added to the culture medium at about 0.3%.

[0217] In some aspects, the culture medium comprises ITS-X supplement. ITS-X, for example can be Gibco™ Insulin-Transferrin-Selenium-Ethanolamine (GIBCO: 51500056). In some aspects, ITS-X supplement can be used in the culture medium at a concentration between 0.1-5%. In some aspects, the ITS-X supplement is added to the culture medium at about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%. In some aspects, ITS-X supplement is added to the culture medium at about 1%.

[0218] In some aspects, the culture medium comprises NRG1. In some aspects, NRG1 can be used in the culture medium at a concentration between 1-500 ng / ml. In some aspects, the NRG1 can be added to the culture medium at about 1 ng / ml, about 5 ng / ml, about 10 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 50 ng / ml, about 55 ng / ml, about 60 ng / ml, about 65 ng / ml, about 70 ng / ml, about 75 ng / ml, about 80 ng / ml, about 85 ng / ml, about 90 ng / ml, about 95 ng / ml, about 100 ng / ml, about 150 ng / ml, about 200 ng / ml, about 250 ng / ml, about 300 ng / ml, about 400 ng / ml, about 450 ng / ml, or about 500 ng / ml. In some aspects, NRG1 is added to the culture medium at about 100 ng / ml.

[0219] In some aspects, the culture medium comprises a Rock inhibitor. Rock inhibitor used in the culture medium, for example, can be Y27632 (CAS No. 146986-50-7). In some aspects, Y27632 can be used in the culture medium at a concentration between 0.1 mM - 5 mM. In some aspects, Y27632 can be used in culture medium at about 0.1 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM. In some aspects, Y27632 can be used in culture medium at about 2.5 mM.

[0220] In some aspects, the culture medium comprises a ALK inhibitor. ALK inhibitor used in the culture medium, for example, can be A83-01 (CAS No. 909910-43-6). In some aspects, A83-01 can be used in the culture medium at a concentration between 0.1 mM - 10 mM. In some aspects, A83-01 can be used in culture medium at about 0.1 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, or about 10 mM. In some aspects, A83-01 can be used in culture medium at about 7.5 mM.

[0221] In some aspects, the culture medium comprises DMEM / F12, neurobasal medium, N2, B27 minus insulin supplement, GlutaMAX, NEAA, |3-mercaptoethanol, penicillinstreptomycin, and FBS.

[0222] In some aspects, the culture medium comprises 1 :1 (v / v) mixture of DMEM / F12 and neurobasal medium, 1X N2 supplement, 1X B27 minus insulin supplement, 1X GlutaMAX, 1X NEAA, 0.1 mM |3-mercaptoethanol, 0.5% penicillin-streptomycin, and 10% FBS.

[0223] In some aspects, the culture medium comprises 1 :1 (v / v) mixture of DMEM / F12 and Neurobasal medium, 0.5X N2 supplement, 0.5X B27 supplement, 1% GlutaMAX, 1% NEAA, 0.1 mM |3-Mercaptoethanol, 1 % penicillin-streptomycin, and 10% FBS.

[0224] In some aspects, the culture medium comprises DMEM / F12, penicillinstreptomycin, BSA, KOSR, |3-Mercaptoethanol, ITS-X supplement, NRG1 , Y27632, and A83- 01.

[0225] In some aspects, the culture medium comprises DMEM / F12, 1% penicillinstreptomycin, 0.3% BSA, 4% KOSR ,0.1 mM p-Mercaptoethanol, 1 % ITS-X supplement, 100 ng / ml NRG1 , 2.5 mM Y27632, and 7.5 mM A83-01.

[0226] In some aspects, the culture medium disclosed herein can be used for derivation of ESCs, XENs, and / or TSCs. In some aspects, the culture medium disclosed herein can be used for further differentiation of ESCs, XENs, and / or TSCs. In some aspects, the culture medium disclosed herein can be used for co-culture of ESCs, XENs, and / or TSCs. Further, in one aspect, the culture medium disclosed herein can be used for co-culturing of ESCs, XENs, and / or TSCs.VII. In vitro systems

[0227] Aspects of the present disclosure provide an in vitro culture system comprising derived, multilineage, or co-cultured or engineered ESCs, XENs, and / or TSCs, which can be prepared by any of the methods described herein.

[0228] In some aspects, the in vitro system comprises a system for deriving ESCs, XENs, and / or TSCs using the methods described herein. Such as system would comprise reproductive cells or PSCs and a culture medium that comprises a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF-p) activator, and a WNT activator.

[0229] In some aspects, the in vitro system for deriving ESCs, XENs, and / or TSCs comprises one or more blastocyst, and a culture medium comprising FGF2, Activin-A, and CHIR99021.

[0230] In some aspects, the system for deriving ESCs, XENs, and / or TSCs comprises one or more blastocyst, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, P-mercaptoethanol, FGF2, Activin-A, CHIR99021 , or any combination thereof.

[0231] In further aspects, the disclosed system for deriving ESCs, XENs, and / or TSCs comprises one or more blastocyst, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, |3-mercaptoethanol, Knockout serum replacement, FGF2, Activin-A, CHIR99021 , or any combination thereof.

[0232] In some aspects, the system for deriving ESCs, XENs, and / or TSCs comprises one or more blastocyst, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, p-mercaptoethanol, FGF2, Activin-A, and CHIR99021.

[0233] In further aspects, the disclosed system for deriving ESCs, XENs, and / or TSCs comprises one or more blastocyst, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, p-mercaptoethanol, Knockout serum replacement, FGF2, Activin-A, and CHIR99021.

[0234] In some aspects, the disclosed system for deriving ESCs, XENs, and / or TSCs comprises one or more blastocyst, and a culture medium with DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), P-mercaptoethanol (55mM), FGF2 (20 ng / mL), Activin-A (20 ng / mL), and CHIR99021 (3 pM).

[0235] In some aspects, the disclosed system for deriving ESCs, XENs, and / or TSCs comprises one or more blastocyst, and a culture medium with DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), P-mercaptoethanol (55mM), 20% KnockOut Serum Replacement, FGF2 (6 ng / mL), Activin-A (25 ng / mL), and CHIR99021 (1.5 pM).

[0236] The present disclosure, in some aspects, further provides an in vitro system for obtaining growth restricted ESCs. The in vitro system for obtaining growth restricted ESCs comprise derived ESCs, a growth restricting agent, wherein the growth restricting agent is selected from laminin, collagen, XENs, or any combination thereof, and a culture medium that comprises a FGF activator, a TGF-p activator, and a WNT activator.

[0237] In some aspects, the in vitro system for obtaining growth restricted ESCs comprises derived ESCs, a growth restricting agent, wherein the growth restricting agent is selected from laminin, collagen, XENs, or any combination thereof, and a culture medium comprising FGF2, Activin-A, and CHIR99021.

[0238] In some aspects, the system for obtaining growth restricted ESCs comprises derived ESCs, a growth restricting agent selected from laminin, collagen, XENs, or any combination thereof, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, P-mercaptoethanol, FGF2, Activin-A, CHIR99021 , or any combination thereof.

[0239] In further aspects, the disclosed system for obtaining growth restricted ESCs comprises derived ESCs, a growth restricting agent selected from laminin, collagen, XENs, or any combination thereof, a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, P-mercaptoethanol, Knockout serum replacement, FGF2, Activin-A, CHIR99021 , or any combination thereof.

[0240] In some aspects, the system for obtaining growth restricted ESCs comprises derived ESCs, a growth restricting agent selected from laminin, collagen or any combinationthereof, XENs, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, p-mercaptoethanol, FGF2, Activin-A, and CHIR99021.

[0241] In further aspects, the system for obtaining growth restricted ESCs comprises derived ESCs, a growth restricting agent selected from laminin, collagen, XENs, or any combination thereof, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, P-mercaptoethanol, Knockout serum replacement, FGF2, Activin-A, and CHIR99021.

[0242] In some aspects, the system for obtaining growth restricted ESCs comprises derived ESCs, a growth restricting agent selected from laminin, collagen, XENs, or any combination thereof, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), P-mercaptoethanol (55mM), FGF2 (20 ng / mL), Activin-A (20 ng / mL), and CHIR99021 (3 pM).

[0243] In some aspects, the system for obtaining growth restricted ESCs comprises derived ESCs, a growth restricting agent selected from laminin, collagen, XENs, or any combination thereof, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), p-mercaptoethanol (55mM), 20% KnockOut Serum Replacement, FGF2 (6 ng / mL), Activin-A (25 ng / mL), and CHIR99021 (1.5 pM).

[0244] In some aspects, the disclosed system comprises a growth restricting agent laminin at a concentration of 1 - 200 pg / ml. In some aspects, the disclosed system comprises laminin at a concentration of 30 pg / ml. In some aspects, the disclosed system comprises a growth restriction agent collagen at a concentration of 1 - 200 pg / ml. In some aspects, the disclosed system comprises collagen at a concentration of 30 pg / ml. In some aspects, the growth restriction agent in the disclosed system is XENs, wherein the XENs is present in the culture medium at an initial ESC to XEN ratio of about 1 :2 to about 8:1.

[0245] The disclosure further provides an in vitro system for obtaining multilineage cells. The in vitro system for obtaining multilineage cells comprise derived ESCs, XENs, and / or TSCs, and a culture medium that comprises a FGF activator, a TGF-P activator, and a WNT activator.

[0246] In some aspects, the in vitro system for obtaining multilineage cells comprises derived ESCs, XENs, and / or TSCs, and a culture medium comprising FGF2, Activin-A and CHIR99021.

[0247] In some aspects, the system for obtaining multilineage cells comprises derived ESCs, XENs, and / or TSCs, and a culture medium comprising DMEM / F-12 medium,neurobasal medium, N2, B27, Glutamax, NEAA, p-mercaptoethanol, FGF2, Activin-A, CHIR99021 , or any combination thereof.

[0248] In further aspects, the disclosed system for obtaining multilineage cells comprises derived ESCs, XENs, and / or TSCs, a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, P-mercaptoethanol, Knockout serum replacement, FGF2, Activin-A, CHIR99021, or any combination thereof.

[0249] In some aspects, the in vitro system for obtaining multilineage cells comprises derived ESCs, XENS and / or TSCs, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, P-mercaptoethanol, FGF2, Activin-A, and CHIR99021.

[0250] In further aspects, the in vitro system for obtaining multilineage cells comprises derived ESCs, XENs, and / or TSCs, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, P-mercaptoethanol, Knockout serum replacement, FGF2, Activin-A, and CHIR99021.

[0251] In some aspects, the in vitro system for obtaining multilineage cells comprises derived ESCs, XENs, and / or TSCs, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), P-mercaptoethanol (55mM), FGF2 (20 ng / mL), Activin-A (20 ng / mL), and CHIR99021 (3 pM).

[0252] In some aspects, the in vitro system for obtaining multilineage cells comprises derived ESCs, XENS, and / or TSCs, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), P-mercaptoethanol (55mM), 20% KnockOut Serum Replacement, FGF2 (6 ng / mL), Activin-A (25 ng / mL) and CHIR99021 (1.5 pM).

[0253] In some aspects, the in vitro system for obtaining multilineage cells further comprises a growth restricting agent, wherein the growth restricting agent is selected from laminin, collagen, XENs, or any combination thereof. In some aspects, the disclosed in vitro system comprises a growth restricting agent laminin at a concentration of 1 - 200 pg / ml. In some aspects, the disclosed in vitro system comprises laminin at a concentration of 30 pg / ml. In some aspects, the disclosed in vitro system comprises a growth restriction agent collagen at a concentration of 1 - 200 pg / ml. In some aspects, the disclosed in vitro system comprises collagen at a concentration of 30 pg / ml. In some aspects, the growth restriction agent in the in vitro disclosed system is XENs, wherein the XENs is present in the culture medium at an initial ESC to XEN ratio of about 1:2 to about 8:1.

[0254] The derived ESCs, XENs, and / or TSCs in the in vitro system for obtaining multilineage cells can obtained using methods described in the disclosure. In some aspects, the system comprises derived ESCs and XENs at an initial ESC to XEN ratio of about 1 :2 to about 8:1. In some aspects, the system comprises derived ESCs and TSCs at an initial ESC to TSC ratio of about 1 :2 to about 8:1. In some aspects, the system comprises derived ESCs, XENs, and TSCs at an initial ESC to XEN to TSC ratio of about 1 :2:2 to about 8:1 :1.

[0255] In some aspects, the disclosure provides an in vitro system for co-culture of derived ESC, XENs, and TSCs. The system for co-culture of derived ESC, XENs, and TSCs can comprise derived ESCs, XENs, and / or TSCs, and a culture medium that comprises a FGF activator, aTGF- activator, and a WNT activator.

[0256] In some aspects, the in vitro system for co-culture of derived ESC, XENs, and TSCs comprises derived ESCs, XENs, and / or TSCs, and a culture medium comprising FGF2, Activin-A and CHIR99021.

[0257] In some aspects, the system for co-culture of derived ESC, XENs, and TSCs comprises derived ESCs, XENs, and / or TSCs, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, p-mercaptoethanol, FGF2, Activin- A, CHIR99021 , or any combination thereof.

[0258] In further aspects, the disclosed system for co-culture of derived ESC, XENs, and TSCs comprises derived ESCs, XENs, and / or TSCs, a culture medium comprising DMEM / F- 12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, p-mercaptoethanol, Knockout serum replacement, FGF2, Activin-A, CHIR99021 , or any combination thereof.

[0259] In some aspects, the system for co-culture of derived ESC, XENs, and TSCs comprises derived ESCs, XENS and / or TSCs, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, P-mercaptoethanol, FGF2, Activin- A, and CHIR99021.

[0260] In further aspects, the system for co-culture of derived ESC, XENs, and TSCs comprises derived ESCs, XENs, and / or TSCs, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, P-mercaptoethanol, Knockout serum replacement, FGF2, Activin-A, and CHIR99021.

[0261] In some aspects, the system for co-culture of derived ESC, XENs, and TSCs comprises derived ESCs, XENs, and / or TSCs, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), P-mercaptoethanol (55mM), FGF2 (20 ng / mL), Activin-A (20 ng / mL), and CHIR99021 (3 pM).

[0262] In some aspects, system for co-culture of derived ESC, XENs, and TSCs comprises derived ESCs, XENS, and / or TSCs, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), p-mercaptoethanol (55mM), 20% KnockOut Serum Replacement, FGF2 (6 ng / mL), Activin-A (25 ng / mL) and CHIR99021 (1.5 pM).

[0263] In some aspects, the system for co-culture of derived ESC, XENs, and TSCs further comprises a growth restricting agent, wherein the growth restricting agent is selected from laminin, collagen, XENs, or any combination thereof. In some aspects, the disclosed system comprises a growth restricting agent laminin at a concentration of 1 - 200 pg / ml. In some aspects, the disclosed system comprises laminin at a concentration of 30 pg / ml. In some aspects, the disclosed system comprises a growth restriction agent collagen at a concentration of 1 - 200 pg / ml. In some aspects, the disclosed system comprises collagen at a concentration of 30 pg / ml. In some aspects, the growth restriction agent in the disclosed system is XENs, wherein the XENs is present in the culture medium at an initial ESC to XEN ratio of about 1 :2 to about 8:1.

[0264] The derived ESCs, XENs, and / or TSCs in the in vitro system can obtained using methods described in the disclosure. In some aspects, the system comprises derived ESCs and XENs at an initial ESC to XEN ratio of about 1 :2 to about 8:1. In some aspects, the system comprises derived ESCs and TSCs at an initial ESC to TSC ratio of about 1 :2 to about 8:1. In some aspects, the system comprises derived ESCs, XENs, and TSCs at an initial ESC to XEN to TSC ratio of about 1 :2:2 to about 8:1 :1.

[0265] In some aspects, the disclosure provides an in vitro system comprising engineered ESC, and / or XENs. The system with engineered ESC, and / or XENs can comprise engineered derived ESCs, and / or XENs with mutation or deletion in one or more integrin or one or more laminin, respectively, and a culture medium that comprises a FGF activator, a TGF- activator, and a WNT activator.

[0266] In some aspects, the in vitro system with engineered ESC, and / or XENs comprises ESCs, and / or XENs with mutation or deletion in one or more integrin or one or more laminin, respectively, and a culture medium comprising FGF2, Activin-A and CHIR99021.

[0267] In some aspects, the in vitro system with engineered ESC, and / or XENs comprises ESCs, and / or XENs with mutation or deletion in one or more integrin or one or more laminin, respectively, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, [3-mercaptoethanol, FGF2, Activin-A, CHIR99021 , or any combination thereof.

[0268] In further aspects, the disclosed in vitro system with engineered ESC, and / or XENs comprises ESCs, and / or XENs with mutation or deletion in one or more integrin or one or more laminin, respectively, a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, p-mercaptoethanol, Knockout serum replacement, FGF2, Activin- A, CHIR99021 , or any combination thereof.

[0269] In some aspects, the in vitro system with engineered ESC, and / or XENs comprises ESCs, and / or XENs with mutation or deletion in one or more integrin or one or more laminin, respectively, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, P-mercaptoethanol, FGF2, Activin-A, and CHIR99021.

[0270] In further aspects, the in vitro system with engineered ESC, and / or XENs comprises ESCs, and / or XENs with mutation or deletion in one or more integrin or one or more laminin, respectively, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax, NEAA, P-mercaptoethanol, Knockout serum replacement, FGF2, Activin-A, and CHIR99021.

[0271] In some aspects, the in vitro system with engineered ESC, and / or XENs comprises ESCs, and / or XENs with mutation or deletion in one or more integrin or one or more laminin, respectively, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), P-mercaptoethanol (55mM), FGF2 (20 ng / mL), Activin-A (20 ng / mL), and CHIR99021 (3 pM).

[0272] In some aspects, the in vitro system with engineered ESC, and / or XENs comprises ESCs, and / or XENs with mutation or deletion in one or more integrin or one or more laminin, respectively, and a culture medium comprising DMEM / F-12 medium, neurobasal medium, N2, B27, Glutamax (1X), NEAA (1X), p-mercaptoethanol (55mM), 20% KnockOut Serum Replacement, FGF2 (6 ng / mL), Activin-A (25 ng / mL) and CHIR99021 (1.5 pM).

[0273] In some aspects, the system with derived engineered ESC, and / or XENs further comprises a growth restricting agent, wherein the growth restricting agent is selected from laminin, collagen, XENs, or any combination thereof. In some aspects, the disclosed system comprises a growth restricting agent laminin at a concentration of 1 - 200 pg / ml. In some aspects, the disclosed system comprises laminin at a concentration of 30 pg / ml. In some aspects, the disclosed system comprises a growth restriction agent collagen at a concentration of 1 -200 pg / ml. In some aspects, the disclosed system comprises collagen at a concentration of 30 pg / ml. In some aspects, the growth restriction agent in the disclosed system is XENs, wherein the XENs is present in the culture medium at an initial ESC to XEN ratio of about 1 :2 to about 8:1.VII. Applications

[0274] Any of the methods and / or in vitro culture systems involving derived ESCs, XENs and / or TSCs as described herein may be used to produce cells of any type. For example, derived ESCs, produced as described herein may be induced to differentiate into primordial germ cells, hematopoietic stem cells, muscle cells, cardiac muscle cells, liver cells, cartilage cells, epithelial cells, and urinary tract cells.

[0275] Any of the methods and / or in vitro culture systems involving derived ESCs, XENs, and / or TSCs as described herein may be used for non-clinical purposes, e.g., for research purposes. In some aspects, the methods and / or in vitro culture systems described herein may be used to study the behavior of stem cells. In some aspects, the methods and systems may be used for identifying novel biological pathways or process involved in pluripotency. In some aspects, the methods and / or in vitro culture systems described herein may be used to study embryo development. In some aspects, the methods and / or in vitro culture systems described herein may be used to study biological signaling pathways and processes involved in cell to cell communications. In some aspects, the methods and / or in vitro culture systems described herein may be used to study inter-species differences in biological signaling pathways and processes involved in cell to cell communications and embryo development.

[0276] In some aspects, the methods and / or in vitro culture systems involving derived ESCs, XENs, and / or TSCs as described herein may be used for producing embryo models. The embryo models may be obtained by differentiation of derived, co-cultured or multi-lineage cells produced by the methods and systems described herein. As a way non-limiting example, ESCs, TSCs, and XENs may be derived using culturing blastocysts in a disclosed culture medium comprising a FGF activator, a TGF- activator, and a WNT activator, and the derived ESCs, XENs, and / or TSCs can be further cultured in a differentiation medium, disclosed herein, to induce embryo development. The embryo models obtained using the methods and / or in vitro culture systems involving derived ESCs, XENs, and / or TSCs as described herein may be used for studying molecular mechanisms involved during development of tissues during embryo growth and evaluating the toxic effect of drugs, compounds, or microbial infection on the developing embryo.

[0277] In some embodiments, methods and / or in vitro culture systems involving ESCs, XENs, and / or TSCs as described herein may be used to evaluate whether a candidate such as a drug, compound or a microbe can alter the development, differentiation or growth of stem cells or embryo. For example, the candidate molecule can be added into the in vitro culture system as described herein. After being cultured under suitable conditions for a suitable period, the development, differentiation, or growth of the cells in the culture system can be compared with a control culture system that does not contain the candidate molecule. If the development, differentiation, or growth in the presence of the candidate molecule is altered ascompared to that in the absence of the candidate molecule, it indicates that the candidate molecule may affect development, differentiation, or growth of cells. Similarly, in further aspects, methods and / or in vitro culture systems involving ESCs, XENs, and / or TSCs as described herein can be used for toxicity assessments, cell growth assessments, cell proliferation assessments, cell differentiation assessments, tissue development assessments, and / or cell culture assessments.

[0278] In some aspects, methods and / or in vitro culture systems involving ESCs, XENs, and / or TSCs as described herein may be used to screen drugs or compounds that alter the development, differentiation or growth of stem cells or embryo. In some aspects, methods and / or in vitro culture systems involving ESCs, XENs, and / or TSCs as described herein may be used to monitor the effects of drugs, compounds or a microbial infection during the development, differentiation or growth of stem cells or embryo. Such methods or systems are useful to study the overall embryotoxic or cytotoxic potential of a drug or a compound formulation, specific side effects of the drug or compound on inducing birth defects, abnormal growth and development of the fetus, or cause implantation failure or abortions and the effect of various diseases, infections, microbial toxins on the developing embryo.

[0279] Further, the derived XENs and TSCs can used to form XEN-tumors and TSC- tumors, respectively. In some aspects, the ESCs, XENs, and / or TSCs as described herein may be implanted in vivo to form teratomas or tumors. Implantation of the disclosed cells can be done using any known method in the art, such as subcutaneous injection, and teratoma or tumor development can be examined after a period of 1-3 months. Such teratomas or tumors and may be used for evaluating, screening, or monitoring effects of drugs, or compounds or understanding biological mechanisms underlying tumor growth and development.

[0280] Additionally, the derived XENs and TSCs disclosed herein can be injected into blastocyst to form chimeric embryos, which can be used as a system for studying underlying developmental molecular mechanisms, or evaluating, screening, or monitoring drug or compounds affecting embryo development and growth.

[0281] In further aspects of the disclosure, the derived engineered ESCs and / or XENs can be used to study cell-cell interactions and mechanistic insights into the crosstalk among cocultured cells.VIII. Kits

[0282] The present disclosure also provides kits for use in producing the ESCs, XENs, and / or TSCs described herein. Such kits can include one or more containers comprising one or more activators described herein, e.g., FGF activator, a TGF- activator, and a WNT activator, and optionally, a population of cells (e.g., a population of reproductive cells or apopulation of PSCs). The kits may further comprise a cell culture medium as described herein, suitable for culturing the population of cells. Kits may further comprise a growth restriction agent such as laminin, collagen, or derived XSCs.

[0283] In some aspects, the kit can comprise instructions for use in accordance with any of the methods described herein. The included instructions can comprise a description of culturing a population of cells in a medium comprising a FGF activator, a TGF- |3 activator, and a WNT pathway activator as described herein. The kit may further comprise a description of obtaining a population of cells, e.g., obtaining a population of cells comprising blastocysts produced by natural mating, in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). In still other aspects, the instructions may comprise a description of culturing the derived ESCs, XENs, and / or TSCs, optionally in the presence of a growth restriction agent, thereby producing, growth restricted ESCs, co-culture ESCs, XENs, and / or TSCs and / or multilineage cells.

[0284] The instructions relating to the use of activators described herein generally include information as to dosage, and dosing schedule for the intended production of ESCs, XENs, and / or TSCs. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or subunit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine- readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0285] The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. A kit may have a sterile access port (e.g., the container may be a vial having a stopper pierceable by a hypodermic injection needle).

[0286] The cell culture media described herein may be used alone or as part of a kit and may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the invention provides articles of manufacture comprising contents of the kits described above.

[0287] Having described several aspects, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the present inventive concept. Additionally, a number of well-known processes and elements have not been described in order to avoid unnecessarily obscuring the present inventive concept. Accordingly, this description should not be taken as limiting the scope of the present inventive concept.

[0288] Those skilled in the art will appreciate that the presently disclosed aspects teach by way of example and not by limitation. Therefore, the matter contained in this description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the method and assemblies, which, as a matter of language, might be said to fall there between.EXAMPLES

[0289] The following examples are included to demonstrate preferred aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.MethodsMice

[0290] C57BL / 6 and CD-1 (ICR) female mice aged 8-10 weeks were purchased fromCharles River or Envigo (Harlen). NOD-SCID (NOD.CB17-Prkdcscid / J) female mice aged 8-10 weeks were purchased from the Jackson Lab. Mice were housed in 12-hr light / 12-hr dark cycle at 22.1°C -22.3°C and 33-44% humidity. All procedures related to animals were performed in accordance with the ethical guidelines of the University of Texas Southwestern Medical Center. The animal protocol was reviewed and approved by the UT Southwestern Institutional Animal Care and Use Committee (IACUC) [Protocol #2018-102430], All experiments followed the 2021 Guidelines for Stem Cell Research and Clinical Translation released by the International Society for Stem Cell Research (ISSCR). Human-mouse chimeric studies were reviewed and approved by the UT Southwestern Stem Cell Oversight Committee (SCRO) [Registration #14],Cvnomolgus Monkeys

[0291] All animals and experimental procedures were approved in 2021 by the ethical committee of the LPBR and Institute of Primate Translational Medicine and Kunming University of Science and Technology (IPTM, KUST), and performed by following the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) for the ethical treatment of non-human primates. 2 healthy femalecynomolgus monkeys (Macaca fascicularis), ranging in age from 5 to 8 years with body weights of 4 to 6 kg, were selected for use in this study. All animals were housed at the State Key Laboratory of Primate Biomedical Research (LPBR). All cynomolgus monkey embryos related work was conducted at the State Key Laboratory of Primate Biomedical Research.Harvesting and culture of mouse embryos

[0292] C57BL / 6 female mice (8-10 weeks old) were super ovulated by intraperitoneal (IP) injection with 5 IU of PMSG (Prospec), followed by IP injection with 5 IU of hCG (Sigma- Aldrich) 48 h later. After mating with C57BL / 6 male mice, embryos at 8-cell to morula stages were harvested at E2.75 [the presence of a virginal plug was defined as embryonic day 0.5 (E0.5)) in KSOM-HEPES by flushing oviducts and uterine horns. Embryos were cultured in the mKSOMaa overnight until blastocyst stage in a humidified atmosphere containing 5% (v / v) CO2 and 5% (v / v) O2 at 37 °C. CD-1 females (8 weeks old or older) in natural estrous cycles were mated with CD-1 males. Blastocysts were harvested at E3.5 by flushing uterine horns.Derivation and culture of FTW-mXENs, FTW-mTSCs and FTW-mESCs

[0293] Embryo manipulations were performed under a dissection microscope (Nikon SMZ800N). In brief, zona pellucidae (ZP) were removed from E3.5 blastocysts by brief treatment with acidic Tyrode’s solution (Millipore MR-004-D). After removing zona pellucidae, embryos were plated on MEFs in FTW medium [N2B27 basal medium supplemented with FGF2 (20 ng / mL, Peprotech), Activin-A (20 ng / mL, Peprotech) and CHIR99021 (3 pM, Selleckchem). After 6-8 days in culture, blastocyst outgrowths were passaged using TrypLE and re-seeded onto newly prepared MEFs. XEN, TSC, and ESC colonies were manually picked for further cultivation. Established mouse XEN, TSC, and ESC lines were cultured on MEFs plates pre-coated with 0.1% gelatin in FTW medium. The cells were cultured at 37 °C under 5% CO2, with daily media changes. For passaging, the cells were dissociated into single cells using TrypLE Express (GIBCO) and passaged onto new MEF-coated plates at a split ratio of 1 :20 (FTW-mESCs), 1 :10 (FTW-mTSCs), and 1 :50 (FTW-mXEN) every 3-4 days.Generation of fluorescent mouse FTW cells.

[0294] pCAG-IP-mKO or pCAG-IP-eGFP was used to label FTW-mXENs, FTW-mTSCs, and FTW-mESCs. In brief, 1-2 pg of pCAG-IP-mKO / eGFP plasmids were transfected into 1 x io6_ 2 x 106dissociated single cells using an electroporator (NEPA21 , Nepa Gene) following the protocol recommended by the manufacturer. Then, 0.5-1.0 pg ml’1of puromycin (Invitrogen) was added to the culture medium 2-3 days after transfection. Drug-resistant colonies were manually picked between 7 to 14 days and further expanded clonally.In vitro differentiation of FTW-mTSCs and FTW-mXENs

[0295] FTW-XENs were dissociated into single cells using TrypLE Express and seeded into a 6-well plate pre-coated with 5 g / mL laminin at a density of 2 x 105cells (mouse XEN) or 5 x 105cells (monkey and human XEN) per well in XEN differentiation medium for 9 days, with medium changes every other day. XEN differentiation medium was prepared using the following: 1 :1 (v / v) mixture of DMEM / F12 and neurobasal medium, 1X N2 supplement, 1X B27 minus insulin supplement, 1X GlutaMAX, 1X Nonessential amino acids, 0.1 mM - mercaptoethanol, 0.5% Penicillin-Streptomycin, and 10% FBS.Harvesting and culture of cynomolgus embryos

[0296] Cynomolgus monkey (Macaca fasicularis) ovarian stimulation, oocyte recovery and in vitro fertilization were performed as previously described. Briefly, healthy female cynomolgus monkeys were subjected to follicular stimulation by intramuscular injection of 20 III of recombinant human follitropin alpha (rhFSH, Gonal F, Merck Serono) for 8 days, then 1 ,000 IU recombinant human chorionic gonadotropin alpha (rhCG, OVIDREL, Merck Serono) was injected on day 9. Cumulus-oocyte complexes were collected by laparoscopic follicular aspiration 32-35 hours following rhCG administration. Follicular contents were placed in HEPES-buffered Tyrode’s albumin lactate pyruvate (TALP) medium containing 0.3% bovine serum albumin (BSA) (Sigma-Aldrich) at 37°C. Oocytes were stripped of cumulus cells by pipetting after a brief exposure (<1 min) to hyaluronidase (0.5 mg / mL) in TALP-HEPES to allow visual selection of nuclear maturity metaphase II (Mil; first polar body present) oocytes. The maturity oocytes were subjected to intracytoplasmic sperm injection (ICSI) immediately and then cultured in CMRL-1066 medium (Gibco, 11530037) containing 10% FBS at 37°C in 5% CO2. Fertilization was confirmed by the presence of the second polar body and two pronuclei. Zygotes were then cultured in the chemically defined hamster embryo culture medium-9 (HECM-9) containing 10% fetal bovine serum at 37 °C in 5% CO2 to allow embryo development. The blastocysts were collected at 7 days post fertilization (d.p.f.). The zona pellucida of blastocyst was removed by exposure to hyaluronidase from bovine testes (Sigma- Aldrich) about 30 seconds and embryos were cultured in vitro until 10 days post fertilization.Derivation and culture of FTW-cyXENs, FTW-cyTSCs, FTW-cyESCs

[0297] For the derivation of FTW-cyXENs and FTW-cyTSCs, 10 days post-fertilization (d.p.f.) cynomolgus monkey embryos were delicately dissected with a 31-gauge syringe needle and plated into wells of 4-well dish pre-coated with 0.1% gelatin and layered with mitotically inactivated MEF in FTW medium (done the same way as with mouse). To promote the proliferation of hypoblast, 1% KSR (Thermo Fisher Scientific, A3181502) and PDGF (10 ng / mL, R&D, 220-BB) were also added. After 8-10 days, XEN-like and TS-like colonies appeared. Single colonies were isolated and then dissociated by TrypLE (Thermo FisherScientific, 12604021) for 3 minutes at 37 °C, and passaged into a well of a 4-well dish. Established FTW-cyXENs and FTW-cyTSCs were passaged by TrypLE every 6-7 days (monkey) at a split ratio of 1 : 10 in FTW medium under 20% 02 and 5% CO2 at 37°C. T o derive FTW-cyESCs, blastocysts (7 days p.c.) were plated into a well of 4-well dish coated with 0.1% gelatin and MEFs in FTW cyESC medium [N2B27 basal medium supplemented with 20% KSR, FGF2 (6 ng / mL, Peprotech), Activin-A (25 ng / mL, Peprotech) and CHIR99021 (1.5 M, Selleckchem)] under 5% 02 and 5% CO2. After 8-10 days, the ES-like outgrowths appeared and were dissociated using TrypLE for 3 minutes at 37 °C. The established FTW-cyESCs were cultured in a 6-well plate pre-coated with 0.1 % gelatin MEFs in FTW cyESC medium under 20% 02 and 5% CO2 at 37°C.In vitro Differentiation of FTW-cyXENs

[0298] To induce differentiation, FTW-cyXENs were dissociated into single cells using TrypLE and seeded into a 6-well plate pre-coated with 5 pg / mL laminin at a density of 5 x 105 cells per well and kept in differentiation medium for 9 days with medium changes every other day. Differentiation medium was prepared as following: 1 :1 (v / v) mixture of DMEM / F12 and neurobasal medium, 0.5X N2 supplement, 0.5X B27 supplement, 1% GlutaMAX, 1 % Nonessential amino acids, 0.1 mM 0-Mercaptoethanol, 1% Penicillin-Streptomycin, and 10% FBS.In vitro Differentiation of FTW-cyTSCs

[0299] FTW-cyTSCs were differentiated into ST and EVT as the described with human cells. For the induction of EVT cells, FTW-cyTSCs were dissociated into single cells in a 6- well plate pre-coated with 1 mg / ml Collagen-IV at a density of 1 x 105 cells per well and cultured with 3 mL of EVT medium (DMEM / F12 supplemented with 1 % Penicillin- Streptomycin, 0.3% BSA, 4% KnockOut Serum Replacement ,0.1 mM 0-Mercaptoethanol ,1% ITS-X supplement, 100 ng / ml NRG1 , 2.5 mM Y27632, 7.5 mM A83-01). 2% Matrigel was added to the medium on the first day. At day 3, the medium was changed to EVT medium with 0.5% Matrigel, but without NRG1. At day 6, the medium was replaced with EVT medium with 0.5% Matrigel, but without NRG1 and KSR, and cells were cultured for an additional two days. For differentiation of ST (2D) cells, FTW-cyTSCs were dissociated in a 6-well plate pre-coated with 2.5 mg / ml Collagen-IV at a density of 2 x 105 cells per well and cultured in 3 mL of ST(2D) medium (DMEM / F12 supplemented with 0.1 mM p-Mercaptoethanol, 1% Penicillin- Streptomycin, 4% KSR, 0.3% BSA, 1% ITS-X supplement, 2.5 mM Y27632 and 2 mM Forskolin). The medium was replaced on day 3, and the cells were analyzed on day 6. For the differentiation of ST (3D) cells, 2 x 105 FTW-cyTSCs were seeded in a 3.5 cm Petri dish and cultured with 3 mL of ST (3D) medium (DMEM / F12 supplemented with 0.1 mM -Mercaptoethanol, 1% Penicillin-Streptomycin, 4% KSR, 0.3% BSA, 1 % ITS-X supplement, 2 mM Forskolin, 50 ng / ml EGF, and 2.5 mM Y27632). An equal amount of fresh ST (3D) medium was added at day 3. The cells were passed through a 40 m mesh filter to remove dead cells and debris at day 6.RT-PCR and qRT-PCR Analysis

[0300] Total RNA was isolated using the RNeasy Mini Kit (QIAGEN) following the manufacturer's instructions. Contaminating genomic DNA was removed by RNase-Free DNase Set (QIAGEN). RNA concentrations were measured on a spectrophotometer (DS-11+, DeNovix). cDNA was synthesized with iScript Reverse Transcription Supermix kit (BIO-RAD) and amplified with PrimeSTAR GXL DNA Polymerase (TaKaRa) or with SYBR Green PCR Master Mix (Thermo Fisher Scientific) on a Touch Thermal Cycler Real-Time PCR system (C1000, BIO-RAD). GAPDH was used as an internal normalization control. All primers used in this study are listed in Table 1 .Table 1 : Primers used in the examplesMOUSE qPCR / rt PCR PrimerMOUSE KO plasmid generationMONKEY rtPCR PrimerCell population doubling time analysis

[0301] The cell population doubling time was calculated using the doubling time online calculator ( h ftgffwww, .do b.Cell Growth Curve analysis

[0302] FTW-cyXENs, FTW-cyTSCs and FTW-cyESCs’s (1 x 105) were seeded onto 6- well plates coated with MEF in FTW medium, with media changes every day. Cells were harvested by TrypLE Express at the indicated time points, depleted of MEFs by plating the cell suspension onto 0.5% gelatin-coated plates for 30 minutes, and growth curves were generated by manual cell counting.Teratoma, XEN-tumor and TSC-tumor formation

[0303] For FTW-mTSC and FTW-mXEN teratomas, a total of 5 x 106cells were resuspended in 100 pL of DMEM-Matrigel solution (1 :1) and injected subcutaneously into 10- week-old immunodeficient NOD-SCID mice. After 2 weeks, FTW-mTSC teratomas were dissected and fixed with PBS containing 4% formaldehyde. After 3 months, FTW-mXEN teratomas were dissected and fixed with PBS containing 4% formaldehyde. For cyTSC teratomas, a total of 1 x 107cells were resuspended in 100 pL of DMEM-Matrigel solution (1 :1) and injected subcutaneously into 10-week-old immunodeficient NOD-SCID mice. After ? days, FTW-mTSC teratomas were dissected and fixed with PBS containing 4% formaldehyde. For co-culture experiments, the FTW-mESC only group was injected with 1 x 106FTW- mESCs, and the co-culture group was injected with 1 x 106FTW-mESCs and 2.5 x 105FTW- mXENs. After one month, teratomas were dissected, weighed, and then fixed. Paraffin- embedded teratomas were sliced and stained with hematoxylin and eosin. Teratomas were formed using the following cell numbers: 1 x 106FTW-mESCs only, or 1 x 106FTW-mESCs plus 1.25 x 105(8:1), 2.5 x 105(4:1), 5 x 105(2:1), 1 x 106(1 :1), 2 x 106(1 :2) FTW-mXENs. After one month teratomas were dissected, weighed, and fixed.Immunostaininq

[0304] Samples were fixed in 4% PFA for 15 min, washed three times with PBS and permeabilized with 0.5% Triton X-100 in PBS for 30 min at room temperature. Cells were then blocked with blocking buffer [5% (w / v) BSA; and 0.1 % (v / v) Tween 20 in PBS] for 1 h and incubated with the primary antibodies (Table 2) diluted in blocking buffer at room temperature for 2 h or at 4°C overnight. After three washes with PBST (PBS plus 0.1 % Tween 20), the cells were incubated with corresponding secondary antibodies (1 :300 diluted, Table 2) in blocking buffer at room temperature for 1 h. After additional three times PBST washes, cells were counterstained with 300 nM DAPI solution at room temperature for 20 min before mounting. Samples were imaged using a fluorescence (Echo Laboratories, CA) or a confocal microscope (A1 R, Nikon).Table 2: Antibodies used in examplesMouse Primary antibodyDilutionGene symbol Name _ Vendor Cat# RRID ratioHuman GATA-6 Affinity PurifiedGATA6 n R&o mD Sys 4t.ems A00F17 A g^B 210 1 , :d1n0n0 Polyclonal Ab antibodyHuman SOX17 Affinity Purified AR ^55SOX17 R&D Systems AF19 1 : 100 Polyclonal Ab antibody24060« , ab23 AB 778 ,EOMES Anti-TBR2 / Eomes antibody Abeam 1 :d1n0n0345 267Anti-CDX-2, Clone CDX2-8 MU3CDX2 8 antibody BioGenex 92A- AB_265 0 1 : 100 UC 531 ntibody Santa sc-SOX2 Sox-2 (E-4) a Cruz AB_108 Biotechnology 3658 1 : 50 23 42165POU5F1 Oct-3 / 4 (C-10) antibody Santa Cruz sc- AB_628 Biotechnology 5279 051 1 : 50GATA4 Anti-GATA4 antibody Abeam ab84 AB_106 1 : 100 593 70538AFP Human / Mouse alpha- etoprotein / AFP Antibody R&D MAB AB_357 F Systems 1368 658 1 : 100 SC-PAX6 Pax -6 (AD2.35) antibody Santa Cruz AB_630 Biotechnology 5310 1 : 50 8 089T Anti-Brachyury / Bry antibody Abeam ab20 AB_275 9665 092 1 : 100[EPR18113] 5FOXA1 FOXA1 antibody Abeam ab55 AB_941 178 631 1 : 100COL6A1 Rabbit Anti-Collagen VIPolyclonal Antibody Abeam ab65 AB_305 88 585 1 : 100Caspase 3 Cleaved Caspase-3 (Asp175) Cell signaling AB_234 Antibody technology 9661 1188 1 : 100DilutionGene symbol Name Vendor Cat# RRID ratio r .TARGATA-6 (D61 E4) XP® Rabbit Cell signaling AB_107 .mAb technology 05521:GATA4 H . u .m.. an , GATA-4 Biotiny ' lated R&D Sys *tems BAF2 AB 226 . .nr.Antibody ' 6 _0_6_ 3176 1 : 400Human SOX17 Affinity Pu AB_355SOX17 rified R&D Systems AF19 Polyclonal Ab antibody 1 : 400 24 060FOXA2 FoxA2 / HNF3-beta Antibody Cell Signaling AB_210Technology 3143 4878 1 : 400FOXA2 Anti-FOXA2 antibody Abeam ab60 AB_941 721 632 1 : 400SOX2 Anti-SOX2 antibody Abeam ab13 AB_281 7385 4892 1 : 400FOXA1 FOXA1 antibody Abeam ab55 AB_941 178 631 1 : 400COL6A1 Rabbit Anti-Collagen VI ab65 AB_305Polyclonal Antibody Abeam 88 585 1 : 400POU5F1 Oct-3 / 4 (C-10) antibody Santa Cruz sc- AB_628Biotechnology 5279 051 1 : 400GATA3 Recombinant Anti-GATA3 antibody Abeam ab19 AB_281 9428 9013 1 : 400 ab68 AB-CK7 Cytokeratin 7 - Cytoskeleton 459 113982 1 : 400Marker Abeam 4 sc-TFAP2C Santa Cruz 1276 AB_667 1 : 400AP-2y Antibody (6E4 / 4) Biotechnology 2 770 ab52 AB_880HLAG Anti-HLA G antibody [G233] Abeam 454 554 1 : 400PAS-HCGB Thermo Fisher 5859 AB_264 1 : 400 hCG beta Polyclonal Antibody Scientific 8 2336notIR50HCG Anti-Human Chorionic availabl 1 : 400 8DAKO eSecondary antibodyName Vendor Cat# RRIDDonkey anti AB_2 rabbit IgG (H+L) Invitrogen A31573 5361647 83Donkey anti AB_1 rabbit IgG (H+L) Invitrogen A21207 4163594 7Donkey anti AB_1 mouse IgG Invitrogen A21202 4160(H+L) 488 7Donkey anti AB_2 mouse IgG Invitrogen A32744 7628(H+L) 594 26Donkey anti AB_2 goat IgG (H+L) Invitrogen A32849 7628647 40Donkey anti AB_2 goat IgG (H+L) Invitrogen A11058 5341594 05 . .. , , AB 2Goat anti rabbit . ..IgG (H+L) 594lml,r0£|e"A1 1037“4° yoGoa +t an +t■i rabbuit- . .. AB 2IgG (H+L) 488l nvitr°gen A1 10345762Goat anti-Mouse AB_2IgG 1 , Alexa Invitrogen A21240 5358Fluor 647 09Goat anti-Mouse AB_2IgG 1 , Alexa Invitrogen A21121 5357Fluor 488 64Goat anti-Mouse AB_2 lgG2b, Alexa Invitrogen A21145 5357Fluor 594 81Goat anti-Mouse AB_2 lgG2b, Alexa Invitrogen A21141 5357Fluor 488 78Goat anti-Mouse AB_2 lgG2a, Alexa Invitrogen A21131 5357Fluor 488 71Goat anti-Mouse AB_2 lgG2a, Alexa Invitrogen A21135 5357Fluor 594 74Flow Cytometry

[0305] Cells were dissociated with T rypLE Express at 37 °C for 5 min. Then, the cells were fixed in 4% PFA at room temperature for 30 min and permeabilized with 0.5% (v / v) Triton X- 100 at room temperature for 30 min. The cells were then incubated in the primary antibody (Table 2) solution for 30 min and then the secondary antibody solution for 30 min at roomtemperature. Samples stained with only secondary antibodies were used as the negative controls. Between each step, the samples were washed twice with PBS containing 2% FBS. Finally, the stained cells were suspended in PBS containing 2% FBS and analyzed by flow cytometry (FACScalibur system, BD).Blastocyst injection of FTW-mXENs and mouse FTW-mTSCs

[0306] FTW-mXEN injection into mouse blastocysts was performed as described previously with slight modifications. Briefly, single cell suspensions of mouse and human FTW- mXENs were added to a 40 pL droplet of KSOM-HEPES containing the blastocysts and placed on an inverted microscope (Nikon) fitted with micromanipulators (Narishige). Individual cells were collected into a micropipette with 15-20 pm internal diameter (ID), and a Piezo Micro Manipulator (Prime Tech) was used to create a hole in the zona pellucida and trophectoderm layer of mouse blastocysts. 10-12 (FTW-mXEN / FTW-mTSCs) cells were introduced into the blastocoel. After microinjection, the blastocysts were cultured in mKSOMaa. For mouse embryo transfer, 8-12 weeks old ICR female mice were used as surrogates and were mated with vasectomized ICR male mice to induce pseudopregnancy. Ketamine (30 mg / ml) I Xylazine (4 mg / mL) and Buprenorphine (1 mg / mL) were used in surgery for maintaining anesthesia and relieving pain. Injected blastocysts were transferred to the surrogate uterine at E2.5. 14-30 blastocysts were transferred within 20-30 min per surrogate.Immunostaining and imaging of chimeric embryos

[0307] At E7.5 or E11.5, surrogates were euthanized, and embryos were isolated. Embryos were dissected and checked for fluorescence using zeiss Axio Zoom.V16 fluorescence stereo zoom microscope equipped with a Plan-Neofluar Z 1.0x / 0.25 (FWD 56 mm) objective and Axiocam 503 monochromatic camera. Embryos were fixed in 4% paraformaldehyde and incubated at 4°C for 30 min (E7.5 embryos) or overnight (E11.5 embryos). After overnight cryoprotection in 30% sucrose solution (Fisher), the embryos were embedded in Polyfreeze Tissue freezing medium (Polyscience, Inc) and frozen on dry ice. Sections (10 pm thick) of the different embryos were cut on a Leica cryostat (Leica CM 1950). For immunostaining, 10 mM citrate buffer (0.05% Tween 20 based) was used for antigen retrieval. The primary antibodies used were summarized in Table 2. After washing with TBST three times, the cells were incubated with corresponding secondary antibodies in blocking buffer at room temperature for 1 hour. Samples were counterstained with 300 nM DAPI solution at room temperature for 20 min and washed with PBST at least three times. Finally, slides were imaged using a fluorescence microscope (Echo Laboratories, CA).Mouse FTW stem cells co-culture assay

[0308] FTW-mESCs-eGFP, FTW-mTSCs-WT, and FTW-mXENs-mKO / WT were seeded onto MEF-coated plates either cultured separately or mixed at different ratios for co-cultures. The seeding ratio and density were empirically tested and decided on the basis of cell growth rate. A starting number of FTW-mESCs-eGFP (1.5 x 104cells), FTW-mTSCs-WT (3 x 104cells, and FTW-mXENs-mKO (7.5 x 103cells) were decided on for most of the cell-cell coculture assays. During co-culture experiments, cells were cultured in FTW medium on MEFs for 5 days and the following analyses were performed. For co-culture experiments with various different cell ratios, FTW-mESCs-eGFP were seeded at a fixed cell number (1.5 x 104cells per well) and with varying numbers of FTW-mTSCs-WT and / or FTW-mXENs-WT.

[0309] For 3D co-culture, AggreWell™800 was used to form Embryoid bodies (EBs). For separate culture experiments, FTW-mESCs-eGFP were seeded into an AggreWell™800 with 2.4 x 105cells per well (800 cells per microwell), and for co-culture experiments, 2.4 x 105cells FTW-mESCs-eGFP and 1.2 x 105FTW-mXENs-WT were added per well (400 cells per microwell). The next day, EBs were transferred into 6-well low-attachment plates for further culture. Media was changed every day for 4 days using FTW medium. For 3D co-culture experiments with different ratios, FTW-mESCs-eGFP were seeded at the fixed density of 2.4 x 105cells per well and with varying densities of FTW-mXENs-WT.

[0310] For the differentiation co-culture experiments, FTW-mESCs-eGFP and FTW- mXENs-mKO were seeded, and the medium was switched to differentiation medium containing DMEM / F12 supplemented with 10% fetal bovine serum (FBS) the next day. On day 5, the number of FTW-mESCs-eGFP cells was counted.Transwell culture assay

[0311] For transwell co-culture experiments, Millipore Transwell 0.4 pm PET hanging inserts (Millicell, MCH12H48) were used by placing them into 12-well plates. MEFs were coated on the top / bottom well. For separate culture groups, FTW-mESCs-eGFP (5000 cells) were seeded on the bottom wells, but not the top insert. For co-culture groups, FTW-mESCs- eGFP (5000 cells) and FTW-mXENs-WT (2500 cells) were seeded on the bottom and top insert of the wells, respectively. Half of the medium was changed every day and cell numbers were counted on day 5.Mouse ECM protein inhibition assay

[0312] Mouse FTW-mESC-GFP cells were passaged using TrypLE and seeded in a well of 6-well plate at 1.5 x 104cells in FTW cyESC medium under 20% O2 and 5% CO2 at 37°C. After 4 hours, the ECM protein mixtures (Matrigel_L: 0.5% (v / v), Matrigel_H: 2% (v / v), Laminin_L: 30 pg / ml, Laminin_H: 120 pg / ml, Collagen_L:15 pg / ml, Collagen_H:60 pg / ml,Vitronectin_L: 5 pg / ml, Vitronectin_H: 30 pg / ml) were directly added into the culture medium every day for 5 days. Medium and ECM proteins were changed every day.Mouse ECM protein inhibition assay

[0313] Mouse FTW-mESC-GFP cells or naive mESCs (cultured in 2iLIF and serum / LIF) and primed mEpiSCs were passaged using TrypLE and seeded in a well of 6-well plate at 1.5 X 104cells in FTW cyESC medium under 20% 02 and 5% CO2 at 37°_C. After 4 h, the ECM protein mixtures (Matrigel_L: 0.5% (v / v), Matrigel_H: 2% (v / v), Laminin_L: 30 mg / mL, Laminin_H: 120 mg / mL, Collagen_L: 15 mg / mL, Collagen_H: 60 mg / mL, Vitronectin_L: 5 mg / mL, Vitronectin_H: 30 mg / mL) were directly added into the culture medium every day for 5 days. Medium and ECM proteins were changed every day.Cell cycle assay

[0314] Cell cycle analysis was performed using the fluorescence single staining PI (Propyridine Iodide). Separate culture (FTW-mESCs-GFP [5 X 104) and Co-culture (FTW- mESCs-GFP [5 X 104] and FTW-mXEN-WT [1 X 104) cells were seeded on a 35mmdish. Each group has three experimental repeats. Cells were fixed with ice colded 70% ethanol for 30 min and staining PI for 30 min for subsequent experiments. Cells were analyzed on the BD flowcytometer and FlowJo software.Mouse FTW stem cell inhibitor tests

[0315] FTW-mESC cells, FTW-mXEN cells, conventional mESCs and mXENs were passaged using TrypLE and seeded in a well of 6-well plate at 1.5 X 104cells in FTW or conventinal medium under 20% 02 and5%CO2 at 37°_C. After 24 h, the inhibitors (PD173074: 1 mM, IWR-1 : 2.5 mM, LDN193189: 2 mM, EC359: 100 nM, SB431542: 2 mM) were directly added into the culture medium for 5 days. Medium with inhibitors was changed every day.Annexin V-PI assay

[0316] Cell death was analyzed by Annexin V-APC / PI fluorescence dual staining cell apoptosis detection kit (Procell, Cat# P-CA-207). Separate culture (FTW-mESCs-GFP [5 X 104]) and Co-culture (FTW-mESCs-GFP [5 X 104] and FTW-mXEN-WT [1 X 104]) cells were seeded on a 35mm dish. Cells were collected at day 3, day 4, and day 5 for further analysis. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Then, the Annexin V-APC / PI fluorescence double staining cell apoptosis detection kit was used for subsequent experiments as the kit described. Cells were analyzed on the BD flowcytometer and FlowJo software.Monkey FTW stem cells co-culture assay

[0317] FTW-cyESCs-GFP, FTW-cyTSCs-WT, and FTW-cyXENs-mKO or WT were seeded onto MEF-coated plates and mixed at different ratios for co-culture experiments. The seeding ratios and densities were empirically tested and decided on the basis of cell growth rate. These numbers were: FTW-cyESCs-GFP (5 x 104cells), FTW-cyTSCs-WT (30 x 104cells, and FTW-cyXENs-mKO / WT (35 x 104cells). During co-culture experiments, cells were cultured in MEF-coated plates in FTW cyESC medium for 5 days.Monkey ECM protein inhibition assay

[0318] Monkey FTW-cyESC-GFP cells were passaged by treatment with TrypLE and 5 x 104 cells were seeded into a well of 6-well plate in FTW medium under 20% O2 and 5% CO2 at 37°C. After 4 hours, ECM mixtures were added (Matrigel: 0.5%, 2%; laminin: 2.5pg / ml, 10pg / ml; CollagenlV:15 pg / ml, 60 pg / ml) directly added into the culture medium every day for 5 days. Medium and ECM proteins were changed every day.Quantification of FTW-EScs number

[0319] For all of the co-culture experiments, total ES cell numbers were quantified in the following manner. Cells were dissociated into single cells using TrypLE Express at 37°C for 4 min, and the total number of live cells were counted. Next, the relative percentage of eGFP+ FTW-ESCs-eGFP were determined using an LSR II Flow Cytometer (BD Bioscience). Total ES cell number (tN) for each group in both the co-cultured and separate culture conditions were determined by multiplying total cell volume (V) with cell concentration (CC) and percentage of eGFP+ cells (P). tN = V x CC x P. Cell density (cells cm-2) was calculated by dividing the total cell number by the surface area.Colony Size and fluorescence intensity analysis

[0320] All of the co-culture experiments involving colony size and intensity were analyzed in the following method. On day 5 of co-culture, fluorescently labeled cells were imaged with a Leica Microsystem DMi8 microscope using Leica Application Suite X software for analysis. The images were taken randomly by the software in a fixed area for each well and A Fiji pipeline was used to quantify the size and intensity of the ESCs colonies. Briefly, images are preprocessed with median filters to filter out noise and small debris, binary images are then created through Otsu thresholding, and watershed was applied to separate merged clones. The quantification was implemented with the Analyze Particles function of Fiji based on the binary images created earlier. The average ESC colony Pixels (P) for each group in cocultures and separate cultures were determined by multiplying colony Size (A) with colony intensity (I). P = A x |. For mouse cell experiments, there were three independent biological replicates for each group and 3 images for each sample. All of the colonies from 9 imageswere analyzed. For monkey cell experiments, 10 images were taken randomly and all of the colonies were analyzed in the 10 images.Ex vivo culture of E6.5-E6.75 epiblasts with or without visceral endoderm

[0321] To obtain embryos, ICR females were mated with males from ICR (Charles River Laboratories) in the afternoon, and the presence of vaginal plugs was checked the next morning. The day on which a plug was found was considered to be E0.5. Both male and female mice were used at ages between 6 to 25 weeks. All the animal experiments were performed under the ethical guidelines of the Kindai University, and animal protocols were reviewed and approved by the Kindai University Animal Care and Use Committee. The developmental stage of embryos is critical for adapting the cultivation of isolated epiblast to an STO-conditioned medium. The isolated epiblasts before the onset of gastrulation collapsed in culture, while those after at onset of gastrulation were stable for growth in an STO- conditioned medium and were applicable for further experiments. The embryos at E6.5-6.75 were surgically isolated in cold DM EM supplemented with 10% fetal calf serum (FCS, BioWest), and whole epiblasts were isolated by the mechanical removal of Reichert’s membrane, extra-embryonic ectoderm as well as visceral endoderm, depending on the cases, using fine forceps and a tungsten needle. The isolated epiblasts with or without visceral endoderm were plated into Nunclon Sphera-treated, 96-well U-shaped-bottom microplate (174925, Thermo Scientific) in STO-conditioned medium. The STO-conditioned medium was prepared as follows. Dulbecco's Modified Eagle's Medium (DMEM, SIGMA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1 % Penicillin-Streptomycin (10,000 U / mL, Gibco) was used to culture Mitomycin-C inactivated STO feeders for 2 days. After 48 hours, cultured epiblast outgrowths including visceral endoderm in the case were dissociated with TrypLE (Gibco, 12604013), and the total cell number was counted.Extended ex vivo culture of mouse blastocysts

[0322] Embryos at the 2-cell stage were incubated in 30 pL droplets of prewarmed potassium simplex optimization medium (KSOM) (MR-004-D, Sigma-Aldrich) under a layer of mineral oil (10029, Vitrolife). After a day, embryos at the 8-cell stage were shifted to fresh prewarmed KSOM droplets containing 0.5 / 1 mMPD0325901 (TOCRIS, 4192) for a 48-h treatment. Post- treatment, each blastocyst was injected with 8-10 FTW-mXENs (GFP+) and then placed in fresh KSOM droplets for about 3 h to allow the shrunken blastocyst to reexpand. Following the removal of the zona pellucida using Acid Tyrode’s Solution (T1788, Sigma- Aldrich) and a rinse in M2 medium (M7167, Sigma-Aldrich), 6-8 blastocysts were transferred to a well in a m-Slide 8-well plate (80826, I bidi). This plate was pre-coated with 100 pL of undiluted Matrigel and kept at 37°_C for 30 min in 250 pL of pre-equilibrated IVC1 medium(components see below). On day 1 , the medium was refreshed with prewarmed IVC1. By the morning of day 2, IVC1 was substituted with 200 pL of prewarmed IVC2 (component details provided below), and by evening, the embryos were fixed for immunofluorescence staining. IVC1 : CMRL 1066 (11530037, Gibco) supplemented with 20% (v / v) heat-inactivated fetal bovine serum (FBS) (35-076-CV, Corning), 2 mM L-glutamine (25030081 , Thermo Fisher Scientific), Penicillin (100 units / mL) / Streptomycin (100 mg / mL) (15140122, Thermo Fisher Scientific), Insulin-Transferrin-Selenium-X (ITS-X) (100 3) (51500056, Thermo Fisher Scientific), 8 nM P-Estradiol (E8875, Sigma-Aldrich), 200 ng / mL Progesterone (P0130, Sigma- Aldrich) and 25 pM N-acetyl-L-cysteine (A7250, Sigma-Aldrich), 4% (v / v) Matrigel. IVC2: CMRL 1066 supplemented with 25% (v / v) KnockOut Serum Replacement (10828028, Thermo Fisher Scientific), 5% rat serum (prepared in-house), 2 mM L-glutamine, Penicillin (100 units / mL) / Streptomycin (100 mg / mL), Insulin-Transferrin-Selenium-X (ITS-X), 4% (v / v) Matrigel.FTW-mXENs and FTW-cyXENs knockdown assay

[0323] FTW-mXENs and TFW-cyXENs were cultured until they reached 50-60% confluency. Afterward, 100pL of lentivirus (at a titer of 1 X 10A8 TU / ml) carrying shRNA targeting either PDGFRA, JAK2, or SRC was added to each individual well. Cells were then subjected to puromycin selection 72 h post-transfection. Subsequent to this, single cell clones were isolated for the Smart-seq2 protocol. Briefly, the reverse transcription reaction was conducted using SuperScript II (18064-071 , Invitrogen). This was followed by cDNA amplification using the KAPA HiFi HotStart Ready Mix (product number KK2602 from KAPA Biosystems) and PCR, set to 19 cycles. The concentration of cDNA was determined by Qubit 3 (Invitrogen) and was diluted to 10 ng / pL using nuclease-free water. Next, TB Green Premix Ex Taq II (RR820L, Takara) was used to establish a 25 pL real-time fluorescence PCR reaction (TB Green Premix Ex Taq II: 12.5 pL, upstream primer (10 pM): 0.5 pL, downstream primer (10 pM): 0.5 pL, cDNA: 10 ng, nuclease-free water: to 25 pL). Real-time PCR was performed with CFX ConnectTM Real Time System (Bio-Rad). The amount of target mRNA was determined using the AACt method with ACTB as the internal control.FTW-mESCs and FTW-mXENs knockout assay

[0324] For this study, we designed all single guide RNAs (sgRNAs) using the online software Benchling CRISPR. The sgRNA sequences are provided in Table 1. These sgRNAs were incorporated into the pSpCas9(BB)-2A-eGFP (PX458) plasmid from Addgene by ligating annealed oligonucleotides to a Bbsl-digested vector. The plasmid containing the specific sgRNA was subsequently transfected into either FTW-mESCs or FTW-mXENs using the NEPA2 electroporator (NEPA2, Nepa Gene 1). 48 h post- transfection, EGFP-positive cellswere isolated via flow cytometry, and 2,000 of these cells were seeded into a well of a 6-well plate. Individual clones were then selected and cultivated. Clones with homozygous knockouts were validated through Sanger sequencing.Monkey FTW-cyXENs knockout assay

[0325] For this study, all single guide RNAs (sgRNAs) were designed using the online software Benchling CRISPR. The sgRNA sequences are provided in Table 1. These sgRNAs were incorporated into the pSpCas9(BB)-2A-eGFP (PX458) plasmid from Addgene by ligating annealed oligonucleotides to a Bbsl-digested vector. Following the manufacturer’s guidelines, 2 x106FTW-cyXENs were transfected with 10 pg of the plasmid containing the specific sgRNA using an electroporator. After 48 h post-transfection, EGFP-positive cells were isolated using flow cytometry. 500 of these cells were then seeded in a 4-well plate. Individual clones were subsequently selected for further analysis. Their genome was extracted to analyze knockout clones using the Discover-sc Single Cell WGA Kit (Vazyme, N603-02). In brief, the cell lysate was prepared per the instructions, and monoclonal cells were aspirated into the lysate using a pipette. Following this, random primers and Phi29 enzyme facilitated the isothermal PCR amplification of the genome (conditions: 30° C for 2 h, followed by 65° C for 5 min), the extracted DNA was then diluted to a concentration of 10 ng / pL based on its concentration. We performed specific PCR amplification to verify knockout efficiency using Ex Premier DNA Polymerase (Takara, RR370A). The amplified products underwent sequencing on the Applied Biosystems 3730x1 platform (provided by Tsingke), and the sequencing results were analyzed using the SnapGene software (version 6.0.2).Plasmids

[0326] pSpCas9(BB)-2A-eGFP (PX458) plasmid was purchased from Addgene (plasmid #48138). pLKO.I-puro.shRNA was purchased from Sigma-Aldrich (Cat#SHC001). pCAG-IP- mKO and pCAG-IP-eGFP plasmids were obtained from T. Hishida. HBLV- JAK2 / PDGFRA / SRC shRNA-ZsGREEN-PURO plasmids were obtained from HANBIO.Bulk RNA-seguencing

[0327] RNA extraction was performed using a RNeasy Mini Kit (QIAGEN) using DNase treatment (QIAGEN). RNA was analyzed using a 2100 Bioanalyzer (Aglient Technologies). (Transcripts per Kilobase Million). For monkey cells, RNA was extracted with Trizol Reagent (15596026, Invitrogen). The RNA (~50 ng) reverse transcription reaction and amplification were performed using SuperScript II (18064-071 , Invitrogen), and KAPA HiFi HotStart Ready Mix (KK2602, KAPA). The cDNA was analyzed using a 2100 Bioanalyzer (Aglient Technologies). The RNA Library was generated using TruePrep DNA Library Prep Kit V2 forIllumina (TD501, Vazyme), then the library was adapted for sequencing on an Illumina NovaSeq 6000 platform (sequenced by Annoroad).Pre-processing of raw RNA-seg data

[0328] All reads were mapped to the mouse (GRCm38 / mm10), human (GRCh38 / hg38), and rhesus macaque genome (Mmul_10 / rheMac10) using hisat2 (version 2.2.1) with default settings. FeatureCoumt (version 2.0.1) was used to estimate read counts. Stringtie (version 2.1.4) was used to estimate fragments per kilobase of exon per million fragments mapped (FPKM) and transcripts per kilobase of exon model per million mapped reads (TPM) values, genes with an FPKM value >3 were considered as expressed.Comparison analysis with published available datasets

[0329] The previously published datasets including mouse (Bao et al., 2018; Cruz-Molina et al., 2017; Cui et al., 2019; Kubaczka et al., 2015; Mohammed et al., 2017; Wu et al., 2015; Wu et al., 2011; Ye et al., 2018; Zhao et al., 2015; Zhong et al., 2018), human(Linneberg- Agerholm et al., 2019; Xiang et al., 2020; Zhou et al., 2019) and monkey(Ma et al., 2019; Nakamura et al., 2016; Niu et al., 2019b) embryonic single-cell RNA sequencing and mouse cell lines (nEND) microarray datasets (Anderson et al., 2017) were obtained from GEO repository (NCBI) and incorporated into the analysis. The expression levels of scRNA-seq data were transformed into log2(TPM + 1), and those of microarray data were transformed into Iog2(intensity).Principal components analysis (PC A)

[0330] The principal component analysis (PCA) was performed using the prcomp function without scaling. The DEGs were defined as genes exhibiting more than twofold changes between the samples (P < 0.005) and the sum of the expression level of every gene was log2(TPM+1) > 0 with the variance > 0.Similarities inference between cells

[0331] A union of the top 2000 genes of highest variance was selected for experimental dataset and the published dataset and calculation of 20 canonical correlates (CCs) with diagonal CCA was done. After running CCA, the first 10 CCs were used for t-SNE visualization. The homology cell types were co-clustered in the same CCA cluster. Then, the correlation analysis was employed to detect the correspondence of cell subtype for the disclosed cells and the previously published embryonic cells by using expression matrix of 200 high variable genes that contributed to the first 10 CCs.Differential expression and GO and KEGG pathway analysis

[0332] Differentially expressed genes (DEGs) among clusters were detected by the Seurat function “Find All Markers”. Heatmaps showing the expression distribution of marker genes of cell clusters were created by pheatmap R package. The functions enrichKEGG and enrichGO in clusterProfiler R package were used to perform KEGG pathways and Gene Ontology (GO) biological processes enrichment analysis. A pathway or process with a P value 0.05 was considered to be significantly enriched. The enriched pathways and processes were visualized with the “ggplot” function in the ggplot2 package in R.Single cell samples preparation

[0333] Mouse blastocysts were collected at E3.5 and the zona pellucida removed by Tyler’s buffer. After removing the zona, blastocysts were washed in PBS / PVA two times. Digestion was performed in TrypLE Express for 10 minutes at 37°C, and cell clumps were cut up by mouth glass pipettes beginning with a larger size (50 urn diameter) to a smaller size (20 urn diameter). The totally digestion time was around 40-50 minutes. For the FTW-derive-day 8 outgrowth samples, blastocysts were collected and plated onto MEF-coated plates in FTW medium for 8 days, and then the outgrowth area were picked and dissociated. For the established FTW cell lines, all the cells were dissociated with TripLE Express for 3-5 minutes at 37 °C. scRNA-seq library preparing and sequencing

[0334] Single cell suspensions were diluted at a concentration of 1 ,100 cells / pL in 0.04% bovine serum albumin (BSA) / PBS for loading into 10X Chromium Single Cell G Chips (for mouse blastocysts, all the of the cells were loaded into the chip). Single cell libraries were prepared using the Chromium Single Cell 3’ Reagent Kit v3.1 (10X Genomics, Pleasanton, CA) following the manufacturer’s protocol. Briefly, single cells were partitioned into Gel beads in EMulsion (GEMs) in the GemCode instrument followed by cell lysis and barcoded reverse transcription of RNA, amplification, shearing and 5’ adaptor and sample index attachment. Libraries were sequenced on the Illumina NovaSeq 6000 platform. scRNA-seq data analysis

[0335] Single-cell gene expression count matrix was constructed with Cell Ranger (v6.0.0). The subsequent analyses were performed using the Seurat package (v4.6.0). For each dataset, transcription noise cells were removed by a series of criteria, including minimal expression of 200 genes per cell, mitochondrial read percentage < 20%, ribosomal read percentage > 5%, as well as specific minimal / maximal number of genes and maximal reads for each dataset. For the monkey, after stringent quality filtering, we generated 35987 single cell transcriptomes with a median unique molecular identifier (UM I) of 22277 and gene number of 5436. Cells passed quality control were merged and normalized using Seurat’s SCTmethod. Datasets from different sources were integrated using Seurat’s CCA method. The LIMAP dimensional reduction was performed using the first 30 PCs from the PCA analysis. Cells were then clustered using the FindClusters function in Seurat package. Cell identities were annotated based on the expression of cell marker genes. To examine the correlation among cell clusters, PCA analysis was first performed for each cell cluster and then correlation analysis was carried out using the PC1s extracted from the cell clusters.Cell clustering by nonlinear dimensional reduction

[0336] T o remove the influence of different experimental conditions on the samples, batch effect correction was carried out. Briefly, following the standard processing steps, including use of “SelectlntegrationFeatures” function to select 2,000 feature genes, “FindlntegrationAnchors” function to find a set of anchors between different samples, “IntegrateData” function to integrate data, “RunPCA” and “RunllMAP” function select 30 PCs dimensionality reduction, “DimPlot” function to graphs the output of a dimensional reduction technique on a 2D scatter plot. The Seurat package was applied to perform cell clustering analysis based on TPM expression values, and only the genes with an expression level greater than 5 were included for further analysis. Then uniform manifold approximation and projection (LIMAP) were applied for visualization. First, “FindlntegrationAnchors” function took these objects as input with parameters as “k.anchor = 5, anchor. features = 2000” and returned an anchor object. Then, “IntegrateData” function used the anchor object to integrate all datasets with the default parameter. Finally, the top-10 / 30 principal components (PCs) were used for clustering (with a resolution of 1) by Seurat.Pseudotime trajectory analysis

[0337] Slingshot was used to determine cell Pseudotime trajectories. Frist, the object that determines the cell type is passed into the “SingleCellExperiment” sim object. Then use a “Rtsne” function to dimensionality reduction (dims = 2, perplexity = 10, pea = TLIRE, pca_scale = FALSE, normalize = FALSE). Slingshot analysis was then performed on the diffusion map to determine per-cell pseudotime estimates and mapped back to the UMAP embedding. Monocle288 (v2.16.0) (available at http: / / cole-trapnell-lab.github.io / monocle-release) was further used for pseudotime trajectory analysis. The dimensionality of the cells was reduced using the DDRTree method. Subsequently, the cells were ordered in pseudotime. The results were visualized using the plot_cell_trajectory, pheatmap, and plot_genes_in_pseudotime functions.Gene regulatory networks

[0338] Single cell regulatory network inference and clustering (SCENIC) has been used to infer gene regulatory networks based on single cell expression profiles and identify cellstates, providing important biological insights into the mechanism of cell heterogeneity. To identify important transcriptional regulation during cell development, pySCENIC grn / ctx / aucell of the Python module tool pySCENIC was used to obtain core regulatory TF. The workflow first describes the input single-cell expression abundance profile matrix and applies a pertarget regression method (GRNBoost2) to infer co-expression modules from which indirect targets are pruned based on cis-regulatory motif discovery (cisTarget). Subsequently, the activity of these regulatory factors was quantified using aucells, and the regulatory factor activity score (RAS) was obtained by enrichment and scoring the target genes of the regulatory factors. The regulator specificity score (RSS) was calculated based on Jensen-Shannon divergence (JS) to determine the cluster-specific regulators, this process is implemented through R language “calcRSS” function and visualized through ggplot2.Gene set score

[0339] The “AddModuleScore” function within the Seurat package was employed to compute the scores for gene sets. The scores for the gene sets of FTW-cyTSC, FTW-cyCTB, and FTW-cySTB were derived using the differentially expressed genes (DEGs) from each subcluster identified in a previous study. These scores were subsequently visualized using ggplot2.Cell cycle analysis based on scRNA-seq data

[0340] The “CellCycleScoring” function within the Seurat package was employed to determine each cell’s cycle phase. This is accomplished by assessing the expression of hallmark marker genes for the G2 / M and S phases. For each cell, expression scores are derived for these classical G2 / M and S phase marker genes. Cells that lack expression of these markers are likely in the G1 phase.Analysis of cell-cell communication

[0341] To evaluate the differences in the cell-cell interactions among FTW-ESCs, FTW- TSCs and FTW-XENs under the co-culture and separate culture conditions, the CellChat package38 (v1.4.0) was utilized. Following the recommended protocol, the potential ligandreceptor interactions was determined among the three cell lineages in each condition and then the count and intensity of these interactions were compared between the co-culture and separate culture setups. Briefly, the normalized counts were input into CellChat and the standard preprocessing steps were applied, including the functions “identifyOverExpressedGenes”, “identifyOverExpressedlnteractions”, and “projectData” with a standard parameter set. Subsequently, the potential ligand-receptor interactions between infected and non-infected cells, were deduced by employing the functions“computeCommunProb”, “computeCommunProbPathway”, and “aggregateNet”. All procedures adopted default settings.

[0342] To annotate homologous genes, a list of human homologous genes were initially retrieved from Ensembl BioMart(https: / / www.ensembl.org / biomart / martview / 526377f4f1a12cc7650403f7c93d23bb) to use as a reference. The gene sequences were then examined to identify orthologous genes across humans, monkeys, and mice.14,818 expressed homologous genes were annotated from this process, of which 1 ,197 were identified as transcription factors (TFs). The list of TF genes was sourced from AnimalTFDB (http: / / bioinfo.life.hust.edu.en / AnimalTFDB / #l / ).CRISPR knockout

[0343] The online software Benchling CRISPR was used to design all single guide RNAs (sgRNAs) used in this study. The sequences of sgRNAs are included in Table 1. sgRNAs were cloned into the pSpCas9(BB)-2A-eGFP (Addgene, PX458) plasmid by ligating annealed oligonucleotides with Bbsl-digested vector. The plasmid carrying the specific sgRNA was then transfected into FTW-mESCs or FTW-mXENs by using an electroporator (NEPA2, Nepa Gene 1). EGFP positive cells were sorted by flow cytometry after 48 h transfection and 2000 cells were plated in one well of a 6 well plate. Single clones were picked and expanded. Homozygous knockout clones were confirmed by Sanger sequencing.Plasmids

[0344] pSpCas9(BB)-2A-eGFP (PX458) plasmid was purchased from Addgene (plasmid 48138). pCAG-IP-mKO, pCAG-IP-eGFP plasmids were obtained from T. Hishida.Statistical Analysis

[0345] All quantitative data is presented as the mean ± SD. Experiments were conducted at a minimum of three times with the repeat number indicated as “n” in figure legends. Differences between groups were evaluated by Student’s t-test (two-sided). P values are shown in the figures. Data visualization and analysis were done using GraphPad Prism version 7.0 and 8.0 (GraphPad Software, La Jolla, Ca) and Microsoft Excel (Microsoft 365).Example 1. A shared culture for all blastocyst lineages

[0346] It has been previously reported that de novo derivation of intermediate embryonic stem cells (ESCs) from mouse blastocysts can be performed by activating FGF, TGF-|3 and WNT pathways (FTW-mESCs). To enrich the FTW-mESC population, a MEK inhibitor (PD0325901) was transiently added to suppress the proliferation of extra-embryonic cells. Withdrawal of PD0325901 during FTW-mESCs derivation resulted in the co-appearance of epiblast-like cells (ELCs), trophoblast-like cells (TLCs), and extraembryonic endoderm-likecells (XLCs) in the blastocyst outgrowth and upon passaging (FIG. 2A). After manual picking, separate cultivation, and further passaging in the FTW condition, stable extraembryonic endoderm stem cells (designated as FTW-mXENs), trophoblast stem cells (designated as FTW-mTSCs), and FTW-mESCs could be derived from a single mouse blastocyst (FIG. 1A and FIG. 1 B). FTW-mXENs, FTW-mTSCs and FTW-mESCs expressed extraembryonic endoderm, trophoblast, and epiblast markers, respectively (FIG. 1C and FIGs. 2B-E). Next, FTW-mXENs and FTW-mTSCs were compared with mXENs and mTSCs in conventional conditions (FIGs. 2F-2I). FTW-mTSCs and FTW-mXENs expressed their respective lineage markers at comparable levels to mTSCs and mXENs, respectively (FIG. 2J). Although similar proliferation rates were observed for mTSCs and FTW-mTSCs, FTW-mXENs exhibited a significantly shorter doubling time than mXENs (FIG. 2K). In addition, pathway inhibitor treatments revealed differences in signaling dependencies between conventional and FTW- cultured mESCs and mXENs (FIG. 2L). Regarding conventional mTSCs and FTWmTSCs, we found little to no difference between the two upon signaling inhibitor treatments (data not shown). After random differentiation of FTW-mXENs in vitro, several parietal endoderm (PE) and visceral endoderm (VE) related genes were upregulated (FIG. 2M). Following subcutaneous injection into an immunodeficient NOD-SCID mouse, FTW-mXENs formed a teratoma-like tissue mass (designated as XEN-teratoma) after 3 months (FIG. 2N). Immunofluorescence (IF) analysis revealed that within the XEN-teratoma many cells expressed alpha-fetoprotein (AFP), some cells were GATA6+, and others stained positive for yolk sac markers FOXA1 and / or COL6A1 (FIGs. 2O-2P). T+ (mesoderm) or PAX6+ (ectoderm) cells were not detected in the XEN-teratoma (FIG. 20). For FTW-mTSCs, after random differentiation in vitro, multinucleated cells appeared (FIG. 2Q) and genes related to trophoblast differentiation were upregulated (FIG. 2R). FTW-mTSCs also formed a tissue mass (TSC-teratoma) containing multinucleated trophoblast giant-cell like cells two weeks after injection into a NOD-SCID mouse (FIG. 2S). Next, blastocyst injections were performed and found that GFP (green fluorescent protein) or mKO (monomeric Kusabira-Orange) labeled FTW-mXENs and FTW-mTSCs contributed to chimera formation in the yolk sac and placenta tissues of mouse conceptuses (E7.5 and E11.5), respectively (FIGs. 1D-1G, FIG. 2T and Table 3).

[0347] In summary, the results indicated that a shared culture condition could support de novo derivation and long-term culture of embryonic and extraembryonic stem cells from mouse blastocysts.Table 3: Chimera efficiency of the FTW-mXENs and FTW-mTSCsHost n- f # #aKr>+Cell lines Passages mouse|ssec ion Transferred Isolated , Chimeras s *t.rain stagae . b .last .ocyst .s em .bryos embry1os %FTW-mXENs P10-P15 ICR E11.5 51 6 11.8%(mKO)-Line A (6 / 51)FTW-mTSCs P1 14.3% (mKO)-Line A 0-P20 ICR E11.5 39 28 4 (4 / 28)C _e „ll lines r P,assages mouse Diss .ectionTTra . . . . . , # eGFP+ Chimeras st .ra .in stagae . b . n last .sferre ocyst .d Iso s em .lated bryos em .bry1os % c"7 c r oo o 9.0%Conventional-E7'5 65 22 2(2 / 22) mXENs (GFP)- P33-P36 B6(GFP)-Line B P33+3BbEH .5 16 9 44(44«Host Di # #Cell lines Passages mouse ssection # eGFP+ Chimeras st T ransferred Isolated strain age blastocysts embryos embryos %10.5%Conventional-(2 / 19)3.5-TSCs P20-P24 B6(GFP)-Line B 10%Example 2: Transcriptome profiling of mouse FTW stem cells

[0348] Bulk RNA-sequencing (RNA-seq) was performed to examine the global transcriptional profiles of FTW stem cells and compared them with published datasets from established mouse embryonic and extraembryonic stem cells (see methods). FTW-mXENs, FTW-mTSCs and FTW-mESCs were found to express their respective lineage markers and were clustered together with stem cells derived from the same tissue of origin (FIG. 3A and FIGs. 4A-4C). Based on comparison with in vivo datasets, it was found that FTW-mESCs, FTW-mTSCs and FTW-mXENs were transcriptionally most closely related to E5.5 epiblast(EPI), E5.25-E5.5 extraembryonic ectoderm (ExE) and E5.5 VE, respectively (FIG. 4D-4F). To resolve the transcriptional states of FTW stem cells, single-cell RNA-sequencing (scRNA- seq) was performed and revealed distinct FTW-mESCs, FTW-mTSCs and FTW-mXENs clusters and heterogeneity within each FTW stem cell type (FIG. 3G-3H, and FIG. 4F-4H). Consistently, by comparing with published single-cell transcriptomes derived from E3.5-E6.5 mouse embryos, FTW-mESCs, FTW-mTSCs, and FTW-mXENs also showed the highest correlation with E5.5 EPI, E5.5 TE (trophectoderm) and E5.5 PrE (primitive endoderm), respectively (FIG. 3B, FIG. 31). Uniform manifold approximation and projection (UMAP) analysis further revealed FTW stem cells clustered closely with cells from their respective lineages in E5.5 mouse conceptuses (FIG. 4E). To examine the temporal steps of FTW stem cells derivation, in addition to established FTW stem cell lines (passage 10), scRNA-seq of blastocysts (day 0) and blastocyst outgrowths (day 8) were performed. Analysis revealed a clear segregation of ELCs, TLCs and ELCs in day 8 blastocyst outgrowths (FIG. 3C, FIG. 3J). Monocle2 pseudotime analysis revealed differentiation trajectories from ICM / EPI to ELCs to FTW-mESCs, PrE to XLCs to FTW-mXENs, and TE to TLCs to FTW-mTSCs, respectively (FIG. 3K), and dynamic gene expression changes alongside the pseudotime trajectories (FIG. 3K). Slingshot pseudotime analysis was performed to delineate differentiation trajectories during derivation. Interestingly, in blastocyst outgrowths, ELCs have already acquired similar transcriptional states of stable FTW-mESCs while TLCs and ELCs clustered separately from established FTW-mTSCs and FTW-mXENs, respectively (FIG. 3C). Next, stage specific genes and enriched Gene Ontology (GO) terms were identified. Notably, several common stage-specific features across all lineages emerged in the analysis, e.g. blastocysts lineages (ICM / EPI, PrE, and TE) were enriched with terms related to the cellular response to hypoxia and lipid metabolic process, etc., suggesting these cells undergo a metabolic transition from in vivo to in vitro', cells in day 8 blastocyst outgrowths (ELCs, XLCs, and TLCs) shares terms related to actin dynamics and cell adhesion, etc., which implicates active cell movement and dynamic changes in cell shape; established FTW stem cells, on the other hand, are characterized by terms related to glycolysis and substrate adhesion-dependent cells spreading, etc., , which is indicative of their stabilization in the FTW condition (FIG. 3D-3F, FIG. 3L).

[0349] Taken together, these transcriptomic analyses confirmed lineage identities, and revealed temporal properties and derivation dynamics of FTW-mESCs, FTW-mTSCs and FTW-mXENs.Example 3: Cross-lineage FTW stem cell co-cultures

[0350] Having FTW-mESCs, FTW-mXENs and FTW-mTSCs derived and maintained in the same condition had enabled to establish co-cultures to study intercellular communicationsbetween embryonic and extraembryonic lineages (FIG. 5A). FTW-mESCs was labeled with GFP and were subjected to co-culture with mKO-labeled FTW-mXENs, in the presence or absence of FTW-mTSCs (unlabeled). After 5 days of co-culture in the FTW condition, many FTW-mESC colonies were surrounded by FTW-mXENs. These FTW-mESC colonies appeared smaller and more “domed” when compared to standalone colonies (not in contact with FTW-mXENs) and FTW-mESC colonies from separate culture (FIG. 5B). This phenomenon was not observed in FTW-mESCs co-cultured with either FTW-mTSCs or mouse embryonic fibroblasts (FIG. 5B). A significant reduction of the GFP signal "Area x Intensity / Colony” was found in FTW-mESCs co-cultured with FTW-mXENs and FTW- mXENs / FTW-mTSCs (FIG. 5C) but not with FTW-mTSCs and fibroblasts, when compared with separately cultured FTW-mESCs. The cell density (cell number per cm2) of FTW-mESCs was also calculated daily in each experimental group. On day 5, a significantly lower density of FTW-mESCs was found in FTW-ESCs / TSCs / XENs and FTW-ESCs / XENs groups than in control groups (FIG. 5D and FIG. 6A). The effects of different cell plating ratios were tested and a greater reduction in FTW-mESC densities were found when co-cultured with more FTW- mXENs in both 2D and 3D (FIG. 6B-6D). We next studied what led to the reduced FTW-mESC numbers in co-cultures. We noted that cocultured FTW-mESCs and FTW-mXENs still highly expressed sternness-related genes (Figure S3C) and showed levels of cell apoptosis comparable to separate-cultures (FIG. 6E-6F, FIG. 6M-6N), thereby ruling out cell differentiation and apoptosis as the cause. This decrease in FTW-mESC density in co-culture was not due to increased cell apoptosis (FIG. 6E-6F) or differentiation (FIG. 6G) and was dependent on direct contact with FTW-mXENs (FIGs. 6H-6I). Interestingly, significantly decreased and increased subpopulations of co-cultured FTW-mESCs in the G2 / M and G1 / S cell cycles were detected, respectively, suggesting the reduced FTW-mESC number in cocultures is due to cell cycle changes (FIG. 60). Moreover, it was found that the growth phenotype depended on direct contact between FTW-mESCs and FTW-mXENs (FIG. 6I and FIG. 6H).

[0351] Next, whether this growth inhibition phenotype was manifested during differentiation, was studied. The same number of FTW-mESCs either alone or together with FTW-mXENs were injected under the skin of NOD-SCID mice (FIG. 5E). Although in both conditions teratomas that contained tissues from all three germ lineages could be generated (FIG. 6 J), the average size and weight of teratomas generated by co-injection were smaller than FTW-mESCs alone (FIG. 5F-5H). Different FTW-ESC:FTW-XEN ratios was also tested and found that in general the more FTW-mXENs were injected, the smaller the teratomas (FIG. 6K). To study whether this growth restriction also exists between EPI and VE cells, E6.5- 6.75 mouse conceptuses were isolated, ExE and ectoplacental cone were removed, and EPI with or without VE (VE+A / E-) were used for ex vivo culture (FIG. 5I). Interestingly, the sizeand total cell number in the VE+ group were significantly smaller than those in the VE- group (FIG. 5J, FIG.5K and FIG. 6L), suggesting the proliferation of EPI is also limited by the VE. In addition, a PrE complementation assay was performed. Briefly, E2.5 mouse morulae were transiently treated with an ERK inhibitor, PD0325901 (PD), to inhibit PrE development, and subsequently, GFP-labeled FTW-mXENs were injected into the blastocysts to complement the extraembryonic endoderm lineages (FIG. 6P). The results showed that in PD-treated mouse embryos, following ex vivo culture, the number of EPI cells is significantly increased when compared to those in the mock-treated WT embryos. And the EPI cell number is restored to normal levels in the PD-treated embryos after blastocyst injection of FTW-mXEN cells (FIG. 6Q-6R).

[0352] Collectively, embryonic and extra-embryonic stem cell co-cultures were established and a contact-dependent growth restriction of FTW-mESCs by FTW-mXENs was identified, which may reflect an embryo size control mechanism by surrounding VE during mouse early post-implantation.Example 4: Mechanistic insights

[0353] To gain mechanistic insights into the crosstalk among co-cultured FTW stem cells, scRNA-seq analysis was performed (FIG. 7A). Pearson correlation analysis and LIMAP coembedding revealed transcriptional similarity between co-cultured and separately cultured cells (FIG. 7H, FIGs. 8A-8B, FIG. 8N). The CellChat toolkit was used to infer cell-cell communication. As expected, both the number and strength of cell-cell interactions increased in co-cultures when compared to separate cultures (FIG. 7B-7C, FIG. 7H). Interestingly, a significant proportion of these interactions originated from FTW-mXENs towards the other two cell types, especially FTW-mESCs (FIG. 7C, FIG. 8B). Interestingly, a significant proportion of these interactions originated from FTW-mXENs toward the other two cell types (FIG. 7C- 7D). CellChat analysis also predicted several signaling pathways mediating these interactions, which included signaling through extracellular matrix (ECM) proteins such as LAMININ and COLLAGEN (FIG. 7D). LAMININ and COLLAGEN are known components of the basement membrane (BM) lining the basal side of the post-implantation EPI, which are in part produced by the VE in mice. Next, it was determined whether ECM protein(s) could phenocopy the growth restriction of co-cultured FTW-mESCs. Supplementation of separately cultured FTW- mESCs with Matrigel (-60% LAMININ, -30% COLLAGEN IV), LAMININ, or COLLAGEN IV, or a combination of LAMININ and COLLAGEN IV, but not VITRONECTIN could inhibit FTW- mESC growth in a dosage dependent manner (FIG. 7E, and FIG. 8C-8E, FIG. 8O-8P). Interestingly, this growth inhibition effect of ECMs was more pronounced for FTW-mESCs than for naive mESCs and primed EpiSCs (FIG. 8P). Consistently, knockout of Laminin y1 (Lamcl) in FTW-mXENs could partially rescue the growth phenotype of co-cultured FTW-mESCs (FIG. 7F, FIG. 8E-8F, FIG. 8O-8P). As INTEGRIN-01 plays an important role in ECM protein signaling and is a cell surface receptor for LAMININ-yl integrin [ 1 (Itgbl) knockout FTW-mESCs was generated. It was observed that loss-of-function of Itgbl could also mitigate the retarded growth of co-cultured FTW-mESCs (FIG. 8G and FIG. 8H).

[0354] In addition, the transcriptomic differences between separately cultured and cocultured FTW-mESCs were studied by bulk RNA-seq. Through comparative analysis, it was identified 502 differentially expressed genes (DEGs) shared between FTW-mESCs in XENs / ESCs and XENs / TSCs / ESCs co-cultures, when compared to separately cultured FTW- mESCs (FIG. 8I and Table 4). Interestingly, the majority of DEGs (492) were down-regulated genes in co-cultured FTW-mESCs. Consistent with their decreased proliferation, the down- regulated DEGs included genes related to cell proliferation and embryo size (e.g., several members of activator protein 1 [AP-1] transcription factor complex such as Fos, Fosl1, Fosb, Batf among others (FIG. 8J). GO and Bioplanet 2019 analyses of the down- regulated DEGs revealed top enriched terms including “extracellular matrix and structure organization”, “lnterleukin-1 and TGF-beta regulation of extracellular matrix”, and p1 integrin cell surface interactions (FIGs. 8K-8L). In addition, it was found that many matrix metalloproteinases (MMPs) (e.g., Mmps 2, 3, 9, 10, 12 etc.) were down-regulated in co-cultured FTW-mESCs (FIG. 8M). MMPs have been reported to modulate cell proliferation, migration, and morphogenesis by degrading ECM proteins, and inhibition of MMPs also led to reduced growth of EPI in vivo and ESCs in vitro, which are consistent with the findings.

[0355] In summary, these analyses helped gain insights into the crosstalk among co- cultured embryonic and extraembryonic stem cells and identified ECM signaling as one of the mechanisms mediating the reduced proliferation of FTW-mESCs by FTW-mXENs (FIG. 7G).Table 4: Differentially expressed genes between co-cultured and separately cultured mouse FTW stem cells.Example 5: FTW Stem cells from monkey blastocysts

[0356] By using the FTW condition, several stable extra-embryonic (FTW-cyXENs and FTW-cyTSCs) and embryonic (FTW-cyESCs, 20% KSR [knockout serum replacement] was needed) stem cell lines from 7-10 d.p.f. (days post-fertilization) cynomolgus monkey blastocysts were successfully derived (FIG. 9A-9D, and Table 5). FTW-cyESCs could also be directly converted from naive-like ESCs through culture adaptation (FIG. 10M). Once established, FTW-cyXENs, FTW-cyTSCs and FTW-cyESCs proliferated well (FIG. 10A, FIG. 10F, and FIG. 10L), maintained stable colony morphology (FIG. 9B) and normal karyotypes (FIG.10B and FIG. 10G) after long-term culture, expressed hypoblast (HYP), trophoblast, and EPI related genes, respectively (FIG. 9E and FIG. 10A-10H, FIGs. 10L-10M, FIG. FIG. 9C, FIG. 10C, FIG. 10D, FIG. 10H, FIG. 101, and FIG. 10M). Upon random differentiation in vitro, FTW-cyXENs could generate a XEN-tumor in vivo (FIG. 10E) and visceral- and yolk-sac- endoderm- (VE / YE) (FOXA1+GATA4-) like cells and extra-embryonic-mesenchyme-cell- (EXMC-) (COL6AVGATA4-) like cells in vitro (FIG. 9F, FIG. 101, FIG. 9D). FTW-cyTSCs were capable of differentiating into extravillous trophoblast (EVT)- like cells and multinucleated syncytiotrophoblast (SCT)-like cells in vitro (FIG. 9G-9H, FIG. 9E), and of generating a TSC- tumor in vivo (FIG. 10J and FIG. 10K). FTW-cyESCs could form teratomas that contained all three germ layer tissues (FIG. 10J-10K, FIG. 10O).Table 5: Summary of cell lines derivation from monkey embryosNo. Stage of DerivationCell lines embryo for No. cell lines Efficiency derivation (%)FTW-cyESCs 2 (E7) 2 100FTW-cyXENs 11 (E7 / E8 / E19) 8 72.73FTW-cyTSCs 11 (E7 / E8 / E10) 4 36.36

[0357] Next, transcriptomic profiling of cynomolgus monkey FTW stem cells was performed. Based on marker gene expressions, it was found that although the majority of FTW cells maintained sternness (FTW-cyESC [SOX2 and NAGOG], FTW-cyXEN [SOX17 and GATA6], and FTW-cyTSC [NR2F2 and LRP2] subclusters), small subpopulations (FTW-cyPS [MIXL1 , LHX1 , and GSC], FTW-cyVE / YE [IHH and MIXL1], FTW-cyEXMC [COL6A1 and HAND2], FTW-cyCTB [C1QBP and COMMD6], and FTW-cySTB [CGA and CRH] subclusters) expressed genes related to early differentiation (FIG. 5I). The lineage identity of each subcluster was further revealed by correlation analysis with monkey in vivo reference datasets and gene set score analysis with human and monkey embryo datasets (FIG. 9H and FIG. 10P). In addition, by using SCENIC analysis, the regulon activities of each subcluster were determined (FIG. 10Q). scRNA-seq analysis revealed that FTW-cyESCs, FTW-cyXENs and FTW-cyTSCs segregated into three distinct clusters on LIMAP and expressed their respective lineage marker genes (FIG. 11A-11B). Bulk RNA-seq analysis helped identify DEGs among FTW-cyESCs, FTW-cyXENs and FTW-cyTSCs, which were enriched in GO terms including: “Signaling pathways regulating pluripotency of stem cells” and “VEGF signaling pathway” (FTW-cyESCs); “ECM-receptor interaction” and “TGF-beta signaling” (FTW-cyXENs); “ECM- receptor interaction” and “Hippo signaling pathway” (FTW-cyTSCs) (FIG. 11C). The result of the Pearson correlation analysis showed that FTW-cyESCs, FTW-cyXENs and FTW-cyTSCs shared the highest correlation coefficients with their in vivo counterparts (FIG. 11D). The derivation of FTW-cyXENs offers an opportunity to study species-specific TF (transcription factor) regulatory networks that regulate primate hypoblast cells. To this end, SCENIC analysis was applied to scRNA-seq datasets of monkey and mouse FTW-XENs to identify speciesspecific TF regulons (FIG. 9K). For functional validations, a few monkey- specific TFs (HMGA1 , SALL1 , and NFE2L1) were selected and performed genetic perturbations in FTW- cyXENs using CRISPR-Cas9. Noticeably, all knockout clones (4 / 4 HMGA1 , 4 / 4 SALL1 , and 9 / 9 NFE2L1) showed stalled growth (FIG. 9L). In addition, the expressions of HYP-related genes (e.g., GATA6 and GATA4) were significantly impaired in these knockout clones (FIG. 9M-9N). These results demonstrated the indispensable roles of HMGA1 , SALL1 , and NFE2L1 in FTW-cyXENs’ self-renewal and sternness.Example 6: Cross-lineage FTW stem cell co-cultures with monkey cells

[0358] Next, it was investigated whether the growth restriction of FTW-ESCs by FTW- XENs was conserved in monkeys. To this end, co-culture of FTW-cyESCs with FTW-cyXENs and / or FTW-cyTSCs was performed. Consistent with mouse findings, it was found that growth of FTW-cyESCs was greatly inhibited by FTW-cyXENs but marginally affected by FTW- cyTSCs or fibroblasts (FIGs. 12A-12B and FIG. 13A-13C, FIGs. 13H-13I). To providemechanistic insights into embryonic and extra-embryonic lineage crosstalk in monkeys, scRNA-seq analysis of co-cultures of FTW-cyESCs / FTW-cyXENs (2 lines) and FTW- cyESCs / FTW-cyXENs / FTW-cyTSCs (3 lines) were performed using 10x Genomics Chromium and compared with single cell transcriptomes derived from separate cultures (FIG. 12E, FIG. 13J, Table 6 and Table 7). Consistent with mouse findings, reduced and increased subpopulations of FTWcyESCs in G2 / M and G1 cell cycles, respectively, were observed in co-cultures when compared to separate cultures (FIG. 13K). Through DEG and GO analyses, several terms, e.g., “Focal adhesion”, “Cadherin binding”, and “Cell-cell adhesion”, were identified as enriched in co-cultured FTW-cyESCs, FTW-cyXENs and FTW-cyTSCs, respectively, suggesting more active cell-cell interactions (FIG. 13B-13D, FIG. 13H-J). Consistent with mouse results, CellChat analysis also predicted signaling through ECM proteins, e.g., LAMININ and COLLAGEN, mediated crosstalk between FTW-cyXENs and FTW-cyESCs (FIG. 12C, FIG. 12E, FIG. 13E, FIG. 13K Table 8). To confirm the effect(s) of ECM protein(s), Matrigel, LAMININ, or COLLAGEN IV were supplemented to separately cultured FTW-cyESCs, and found that each could phenocopy FTW-cyXENs’ inhibitory effect on the growth of FTW-cyESCs in a dosage dependent manner (FIG. 12D and FIGs. 13F-13H).

[0359] Taken together, these results extended the findings in mice to monkeys and revealed a conserved mechanism underlying growth inhibition of FTW-ESCs by FTW-XENs.Table 6: Summary of scRNA-seq samples from MouseA total of 37,987 cells were used for further analyses after stringent filtration„ . Numbers of Average numbers of genes Average numbers ofRampies cell expressed UMIsrephcatelFTW-dayO 145 16,348 158,245FTW-derevation-6 34318,716 11 ,2038daysNote: Remove cells with less than 200 genes and genes expressed less than 3 cells• Remove cells with mitochondria proportion greater than 25%• Remove cells with ribosome proportion less than 5%• Remove cells with abnormally high / low counts & features (based on QC plots) mitochondria in one cellTable 7: Summary of scRNA-seq samples from MonkeyA total of 37,987 cells were used for further analyses after stringent filtration Numbers Average numbers of Averagegenes expressed numbers of UMIsFTW-cyESCs5 3626,155 22,792 rephcatelFTW- .c. yX. E.Ns 4.,36c„cc„ „corephcatel 6 5,355 22,358FTW-cyXENs2,814 5,208 23,046 rephcate2FTW-cyTSCs4,879 20,842 rephcatelFTW-cyTSCs2 8464,912 22,480 rephcate2F™-cy2line rephcatelFTW-cy2hne4 8684 ,868 13,992 rephcate2FTW-cy3hne5 3696,135 24,840 rephcatelFTW-cy3hne3 5247,014 33,758 rephcate2 Note: The Filter condition is less than 500 genes, less than 10,000 and not more than 60,000 UMIs or not more than 15 percent UMIs mapping on mitochondria in one cellTable 8: Cell signaling pathways involved in cell-cell communications during co-culture of FTW-cyXENs and FTW-cyESCsExample 7: Cross species comparisons

[0360] Embryonic and extraembryonic stem cells from both mice and monkeys derived and cultured in the same condition gave a unique opportunity to examine species-conserved and -divergent features not influenced by different culture parameters. Using the single-cell regulatory network inference and clustering (SCENIC) pipeline, the regulatory networks underlying the maintenance of FTW-ESCs, FTW-XENs and FTW-TSCs was studied in both species. Analysis identified several conserved and species-specific transcription factor (TF)- driven regulons in each stem cell type (FIG. 14A-14C). SCENIC analysis also revealed common and distinct targets of well-known lineage TFs such as NANOG, SOX17 and GATA3 (FIG. 14A-14C). Next, cross-species comparison of intercommunications between lineages was performed, which uncovered both species-conserved (e.g., MK, LAMININ, COLLAGEN and FN1) and -divergent (e.g., CDH, AGRN and HSPG [mouse]; FGF, NCAM and BMP [monkey]) signaling crosstalk (FIG. 14D).

[0361] Collectively, these results reveal interspecies transcriptomic similarities and differences in early embryonic and extraembryonic cells and their intercommunications, which provide insights into evolutionary convergent and divergent processes underlying epiblast, hypoblast and trophoblast development across different species in vivo.Example 8: Generation and characterization of FTW-hXENs from human naive ESCs.

[0362] In addition to mice and monkeys, it was found that human FTW-XENs (FTW- hXENs) could also be generated from naive ESCs and extended pluripotent stem cells (EPSCs) by culture adaptation (FIG. 15A-15B, and FIG. 16A-16C). FTW-hXENs expressed HYP markers, e.g., GATA4, GATA6, FOXA2, and SOX17, but not pluripotency markers (FIG. 15C, FIG. 16C-16D). Although FTW-hXENs could be passaged multiple times (naive ESC- derived: >10 passages; EPSC-derived: >25 passages) and maintained stable HYP marker gene expression, it was found that they grew noticeably more slowly than FTW-mXENs and FTW-cyXENs. RNAseq for FTW-hXENs was performed and compared with HYP cells from human embryos cultured ex vivo. Principal-component analysis (PCA) revealed that on the PC1 axis FTW-hXENs aligned with human E8HYP cells (FIG. 16E). To determine the differentiation potential of FTW-hXENs, FTW-hXENs was cultured on laminin-coated plates in basal medium supplemented with 10% fetal bovine serum (FBS) for 9 days. Through IF analyses it was found that both EXMC-like (COL6A1+ GATA6_) and VE / YE-like (FOXA1 + GATA6_) cells were present in the differentiation culture (FIG. 16F).

[0363] Across-species comparisons of mouse, monkey, and human FTW-XENs was performed. Hierarchical clustering and correlation analysis showed that FTW-cyXENs clustered closer with FTW-hXENs than FTW-mXENs (FIG. 16G). It was found that 2,608 genes were commonly shared among mouse, monkey, and human FTW-XENs, which were enriched in GO terms, including regulation of cellular PH, negative regulation of cell-matrix adhesion, and positive regulation of vasculogenesis, among others (FIG 15D). In addition, 2,132, 2,667, and 1 ,767 genes were identified specifically expressed in human, monkey, and mouse FTW-XENs, respectively (FIG. 15D). Top enriched species-specific GO terms include: (1) human: nervous system development and axon guidance; (2) monkey: mitochondria translation and negative regulation ubiquitin-protein ligase activity; and (3) mouse: inflammatory response and neutrophil activation involved in immune response (FIG. 16H). The expression patterns of both divergent and conserved TFs identified through cross-species comparisons among mouse, monkey, and human FTW-XENs (FIG. 161). To delineate evolutionarily conserved genes regulating the FTW-XEN state, pairwise transcriptomic comparisons between mouse, monkey, and human FTW-XENs and EPls from corresponding developmental stages (mouse, E5.5 EPI55; human, d.p.f. 8 and 9 EPIs53,54; monkey, d.p.f. 8 and 9 EPIs50) were carried out. Based on this analysis, 735 DEGs (XEN vs. EPI) were identified that were shared by all three species (FIG. 16J), and hub-gene-network analysis of these 735 genes predicted several core FTW-XENs regulators, including several well-known hypoblast-related genes (e.g., GATA4, GATA6, SOX17, and PDGFA), as well as genes with unknown functions in hypoblast development (e.g., SRC, JAK2, PIK3R1 , and CCL2) (FIG.15E).

[0364] Next, we selected several candidate genes including SRC, PDGFRA, and JAK2 for functional validation via shRNA-based knockdown experiments in both mouse and monkey FTWXENs (FIG. 16K). It was found that individual knockdown of SRC, PDGFRA, and JAK2 disturbed the expression patterns of several PrE / HYP-related genes in both monkey and mouse FTW-XENs. Interestingly, while knockdown of SRC and JAK2 significantly up- regulated the expression levels of FOXA2 and LAMC1 in both species (FIG. 15F), PDGFRA knockdown resulted in an opposite change in GATA4 and GATA6 expression between mouse and monkey FTW-XENs (FIG. 15F). These results revealed conserved and divergent functions of FTW-XEN regulators between rodent and primate species.

[0365] The transcriptomic differences between separately cultured and co-cultured FTW- hXENs, differentiated FTW-hXEN at day 9 (FTW-hXENs-dif) and published in vivo datasets of hypoblasts were studied by RNA-seq. The comparative analysis identified differentially expressed genes. Further, correlation analysis of gene clusters in FTW-hXEN-LCs with epiblast, cytotrophoblast, syncytiotrophoblast and the sub-clusters of hypoblast cells revealedthat FTW-hXEN-LCs exhibited high expression levels of genes in the sub-clusters of hypoblast cells (FIG. 16L). Next, mouse blastocyst were injected with human FTW-XEN-LCs. Injected blastocysts were able to form twin embryos and chimeric tissues (FIG. 16M-16N).

[0366] In summary, the results indicate that a culture medium with FGF activator, TGF- activator and WNT activator, supported de novo derivation and long-term culture of hXENs from human naive ESCs.Summary of Examples

[0367] Most stem cell cultures include pathway inhibitors to suppress differentiation in favor of self-renewal, e.g., ground state mouse ESC culture, which often necessitate the design of new conditions for different stem cell types and different species. By activating multiple developmental signaling pathways at once, it was found that several embryonic (mouse, cynomolgus monkey, and horse) and extraembryonic stem cells (mouse and cynomolgus monkey) from multiple species could be derived and stably maintained in the same condition. This signaling “excited” state can potentially be attained in a variety of stem cell types by striking a balance between differentiation and self-renewal via switching on a combination of signaling pathways including but not limited to FGF, TGF-|3 and WNT pathways used in this study. Further the culture medium supported de novo derivation and long-term culture of hXENs from human naive ESCs.

[0368] The ability to grow embryonic and extraembryonic stem cells in the same culture environment opens new avenues to dissect the molecular and cellular mechanisms underlying lineage crosstalk during early mammalian development. Based on FTW stem cell co-cultures, a previously unrecognized non-cell autonomous control of pluripotent cell proliferation by extraembryonic endoderm cells was discovered, which is in part mediated by the ECM signaling, in both mice and monkeys (FIG. 17). It was further revealed that a similar overgrowth phenotype in mouse epiblast in the absence of visceral endoderm, providing evidence supporting the in vivo relevance of the observed proliferation restriction.

[0369] The disclosed study identified that laminin-integrin signaling contributed to the growth restriction of FTW-mESCs by FTW-mXENs (FIG. 7D-7F and FIGs. 8C-8F). This is likely due to irregularly distributed matrix materials surrounding integrin |31 -deficient FTW- mESCs during co-culture with FTW-mXENs as opposed to a continuous basement membrane layer in WT FTW-mESCs. A previous study demonstrated that integrin pi-deficient mESCs underwent massive cell death during transition from naive pluripotency toward the formative pluripotency. This contrasts with integrin pi-deficient formative FTW-mESCs (FIG. 8G-8H). Further, FTW-mESCs exhibited functional formative features including the ability to form blastocyst chimeras and direct responsiveness to primordial germ cell specification, retainingthe expression of many naive pluripotency genes and are recognized as more naive-like formative PSCs as opposed to more primed-like formative stem (FS) cells.

[0370] Advancements in both extended primate (human and cynomolgus monkey) embryo cultures and stem cell embryo models provided an unprecedented opportunity to study primate early post-implantation development. However, the dynamic nature of a developing embryo / embryo model and its cellular complexity make it difficult to dissect the key cell-cell interactions. Here, the establishment of cynomolgus monkey FTW-ESCs, XENs and TSCs under the same condition enabled, for the first time, to delineate cell-cell interactions in a tractable and accessible manner in primates. In summary, the FTW embryonic and extraembryonic stem cells and the co-culture strategy developed in this disclosure provide superior starting cells for generating more robust integrated stem cell embryo models and developing more faithful differentiation protocols for regenerative medicine. A unified culture condition for all three primary tissues of mouse and cynomolgus monkey blastocysts was therefore established, facilitating stem cell coculture experiments and uncovering intricate interactions between embryonic and extraembryonic lineages.

Claims

CLAIMSWhat is claimed is:

1. A method for derivation of embryonic stem cells (ESCs), extraembryonic endoderm stem cells (XENs), and / or trophoblast stem cells (TSCs) in vitro, the method comprising: i. obtaining reproductive cells or naive pluripotent stem cells (PSCs); ii. culturing the reproductive cells or naive human PSCs in a culture medium that comprises a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF- ) activator, and a WNT activator; iii. simultaneously deriving stable ESCs, XENs, and TSCs.

2. The method of claim 1, wherein the method further comprises: iv. isolating the epiblast-like, endoderm-like, and / or trophoblast-like cells from the cell outgrowth; and v. culturing and passaging the isolated cells to obtain stable ESCs, XENs, or TSCs.

3. The method of any one of claims 1 to 3, wherein the ESCs, XENs, and TSCs are produced from reproductive cells.

4. The method of claim 4, wherein the reproductive cells comprise blastocyst.

5. The method of any one of claims 1 to 3, wherein the derivation is from a single or multiple blastocysts.

6. The method of claim 4, wherein the reproductive cells are obtained from a mammal.

7. The method of claim 7, wherein the reproductive cells are obtained from a human, a rodent, or a non-human primate.

8. A method of restricting growth of ESCs in a cell culture, the method comprising: i. obtaining a population of ESCs derived from reproductive cells or PSCs; ii. culturing ESCs in a culture medium that comprises a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF- ) activator, and a WNT activator;iii. adding a growth restricting agent to the culture medium, wherein the agent is selected from laminin, collagen, XENs, or any combination thereof; and iv. maintaining the cells in the culture for at least 5 days to obtain a population of growth restricted ESCs.

9. The method of claim 8, wherein ESCs and XENs are obtained using a method of claim 1.

10. The method of claim 8 or 9, wherein the growth restricting agent is laminin and the laminin is at a concentration of 1 - 200 pg / ml in the culture medium.

11. The method of claim 8 or 9, wherein the growth restricting agent is collagen and the collagen is at a concentration of 1 - 200 pg / ml in the culture medium.

12. The method of claim 8 or 9, wherein the growth restricting agent is XENs and the XENs is added at an initial ESC to XEN ratio of about 1 :2 to about 8: 1 to the culture medium.

13. The method of claim 8, wherein growth restricted ESCs comprise ESCs with reduced cell density, and / or reduced expression of one or more Lyz2, Gpnmb, Saa3, B2m, H2- D1 , Thbsl , Fbln2, Ctsl, Bgn, Plau, Marco, Col1a2, Ctss, Timp2, C1qa, Lilrb4a, Csf1 , Col1a1 , Tnfaip2, Den, Lox, Thbs2, Il1 rn, Plek, Adgrel , Mpegl , Msr1 , Serpinel Adam8, H2-M2, Cybb, C1qc, Mmp3, Ccl3, Anxal , Postn, Cd274, Vim, Xist, Mmp9, Lgals3bp, CxcH , Wfdc17, Sdc3, lfi202b, Clec4e, Acssl , Vcaml, C1qb, Ccl2, Col5a3, Egr1 , Fcgr3, C3, C3ar1 , 111a, Fcgr2b, Lilr4b, Ccl6, Plk2, Clec4n, Ccl9, Pf4, Csf2rb, Axl, Atp6v0d2, Ms4a6d, Cd44, App, Zeb2, Ms4a7, Greml , Ptgs2, Tnc, Slfn2, Itgb2, Col6a1 , Aebpl , Mmp12, Col5a1 , Slc7a2, Mmp2, Tyrobp, Mmp13, Cxcl2, Trem2, S100a6, Id2, Ecm1 , Ltbp2, Cd53, Bmp1 , Serpinfl , Ptges, Plxndl , Feerig, Fxyd5, Cd36, Arhgdib, Ccl5, Cyr61 , Sirpa, Rgs1 , Slc15a3, Rac2, AI607873, Apbbl ip, Xdh, Clec4d, Ccl7, Slc11 a1 , Bst1 , Alcam, Tgfbi, Col5a2, Glrx, Tnfrsf23, Slfn5, Gas7, Gas6, Tnfaip3, Ctla2a, 111 rl1 , Atf3, Itpripl2, Ddr2, 6720427l07Rik, Cd84, Ptprc, Itgb3, Prrxl , Fabp4, Pmp22, Cav1 , Mical2, Ctgf, Col6a2, Inhba, Igfbp7, Tm4sf1 , Lum, Hk3, Fam46c, Cd33, Adamtsl , Clec7a, Lcn2, Serpinb2, Mir17hg, Col8a1 , Il4ra, Csf2rb2, Adssll , Irg1 , Gsap, Ccrl2, Cd300lf, Tmem176b, Egr2, Rgs16, Rab7b, Rhob, Grem2, Col12a1 , Col6a3, Lgals3, lfi204, Rassf4, Rnd3, Fam198b, Cd14, Pcdh7, Marchl , Cp, Ncf4, lfi203, Ptger4, Csfl r, Smpdl3a, Havcr2, H2-T23, Hclsl , Sigled , Naip2, Emp1 , WIs, Ier5, Rps2-ps13, Cd93, Serpina3g, Penk, Serpina3h, Layn, Cryab, Thbd, Olr1 , Nlrc5, Acta2, Col16a1 , Pik3ap1 , Thy1 , Ncf1 , AF251705, Mmp10, Synpo, Igf1 , Ly9, Ptx3, Npy, II33, Tns1 , Gda, Mafb, Casp4, Tfpi2, Cd180, Mr1 , Lmxlb, Ets1 , Ccdc80, Ugt1a7c, Gm15487, Gm8995, Rnasel, Cav2, Il7r, Mrc2, Itpr2, C5ar1 , Tgfbr2, Cyp1b1 , P2ry6, Gbp9, Dusp8, Acpp, Ifi44, Antxr2, Cxcl3, Ly6a, Nrp1 , Rgl1 , Oaf, Ccl8, Clec2d,Ptpre, Pdgfra, Arhgap25, Arsb, S1 pr1 , Adgrg6, Fam20c, Sh3kbp1 , Runxl , Kitl, Gab2, Col3a1 , Rspo4, 111 Ora, 111 b, Arhgap24, Ikbke, Emilin2, Cd34, Itga4, Hacd4, Nptxl , Steap4, Ccbel , Mmp8, Caspl , Entpdl , Sqrdl, Efnb3, Cd302, Gem, Dkk3, Rnase4, DuspIO, Mnda, Psmb8, Cd82, Sod3, Osbpl5, Lats2, Fgd3, Arhgap31 , Cd86, Ms4a14, Mcoln2, Pik3cg, Cyp4v3, Slc25a24, Tagln, Cebpa, Rsph4a, Fas, Ereg, Klhl6, Col4a5, 1810011O10Rik, Itgam, Arg2, Tmem176a, TI , Flrt2, Efempl , Pla2g4a, C1s1 , B430306N03Rik, Mn1 , Procr, Stabl , Plat, Cd200, Nos2, Rasgefl b, Wnt5a, Ccr1 , Sept5, Gap43, Mef2c, Creb5, Ifitl , Negri , Parva, Gpr176, Orai2, S100a8, Pappa, 111 r1 , Prl2c3, Raetld, Hspalb, Adamts5, Evi2a, 01 ra, Zfp703, Filipll, Fgf7, Dclkl , Nov, Heph, Sema3a, Pcdh19, Pgm5, Slc2a9, Irf2, Vgll3, Fcgr4, Cyth4, Mgstl , Gbp8, Emp3, H20rb, Fhl2, Casp12, Dkk2, Fill , 4930523C07Rik, Ngf, Maspl , Lrrc15, Cxcl5, Scarf2, Lrrc18, Nbl1 , Gm10143, Pdzrn3, Pdlim4, Osmr, Slc22a4, Mmp19, Myo18b, Ctso, Angpt4, Mxd4, Fads3, Adgrg2, Fam78b, Hgf, Cpq, Serpina3n, Prl2c2, Rbms3, Pcdh9, Tfec, Ldlrad4, Rgs2, Cd40, Fbxo32, Mir22hg, Sparcl 1 , Tgfb2, Pde4b, Cspg4, Cd200r1 , Abca8b, Ctsk, Tap2, Ehd2, Svepl , Rcan2, Vax2os, Dchsl , Gm14453, Zfpm2, Slc22a13b-ps, Gm42531 , Gng11 , Pla2g4c, Isg15, Serpinb6b, Cdh13, Gm33280, Milrl , Slc8a1 , Maf, Cnripl , Slpi, Fam13a, Runx2, Bdkrb2, Till , Itga11 , Gbp4, Gm15559, H10rb, Il15ra, Rab32, Mctpl , Sspn, Dachl , Pamrl , Lat2, Mcpt8, Msrb3, Serpinb9, Cd52, Rgma, Tbxasl , Itga2, Osr2, Nedd9, Gdnf, Nol4, list, Eya4, Slc16a2, C920025E04Rik, Lcelg, Nr2f2, Foxd , II7, Pcp4l1 , Ednra, 9330175E14Rik, Lama2, Plxna4, Serpinbla, Nipall , Mfap4, Irx1 , Rspo3, Prss23, Nsg1 , Dnm3os, Osginl , Pxdd , Fat4, Fat3, Snai2, Thsd7a, Gm26809, Tgfb3, Cxxc4, Meis3, Rbm24, Cplx2, Vax2, Areg, Gabrbl , Lancl3, PI15, Lrrc32, Fgf8, RP24-439I22.8, Scara3, Fgf2, Kcnn3, 4933427D06Rik, Rnf150, or any combination thereof, when compared to derived ESCs.

14. A method for obtaining multi-lineage stem cells in a shared cell culture condition, the method comprising: i. obtaining a population of ESCs derived from reproductive cells or PSCs; ii. obtaining a population of XENs and / or TSCs derived from reproductive cells or PSCs; iii. co-culturing ESCs, XENs, and / or TSCs in a culture medium that comprises a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF-P) activator, and a WNT activator; iv. maintaining the mixed cells in the culture medium for at least 5 days to obtain a population of the multi-lineage stem cells.

15. The method of claim 14, wherein the multi-lineage stem cells comprise ESCs and XENs cells.

16. The method of claim 14, wherein the multi-lineage stem cells comprise ESCs and TSCs cells.

17. The method of claim 14, wherein the multi-lineage stem cells comprise ESCs, XENs, and TSCs cells.

18. The method of any one of claims 14 to 17, wherein the method further comprises adding laminin at a concentration of 1 - 200 pg / ml in the culture medium.

19. The method of any one of claims 14 to 17, wherein the method further comprises adding collagen at a concentration of 1 - 200 pg / ml in the culture medium.

20. The method of any one of claims 14 to 19, wherein ESCs, XENs, and / or TSCs are obtained using a method of claim 1.

21. The method of claim 14, wherein step (iii) comprises combining ESCs and XENs at an initial ESC to XEN ratio of about 1 :2 to about 8:1.

22. The method of claim 14, wherein step (iii) comprises combining ESCs and TSCs at an initial ESC to TSC ratio of about 1 :2 to about 8:1.

23. The method of claim 14, wherein step (iii) comprises combining ESCs, XENs, and TSCs at an initial ESC to XEN to TSC ratio of about 1 :2:2 to about 8:1 :1.

24. The method of any one of the preceding claims, wherein the culture medium further comprises neurobasal medium.

25. The method of any one of the preceding claims, wherein the FGF activator is FGF2.

26. The method of any one of the preceding claims, wherein the TGF-|3 activator is Activin- A.

27. The method of any one of the preceding claims, wherein the WNT activator is a GSK3 inhibitor.

28. The method of claim 27, wherein the GSK3 inhibitor is CHIR99021.

29. The method of any one of the preceding claims, wherein the XENs and / or TSCs express extraembryonic endoderm or trophoblast markers, respectively.

30. The method of any one of the preceding claims, wherein the XENs and TSCs can form XEN-tumors and TSC-tumors, respectively.

31. An in vitro co-culture system comprising: i. ESCs derived from reproductive cells or PSCs; ii. XENs, and / or TSCs derived from reproductive cells or PSCs; iii. a growth restricting agent for the culture medium, wherein the agent is selected from laminin, collagen, or any combination thereof; and iv. a culture medium comprising a FGF activator, a TGF-£ activator, and a WNT activator.

32. The system of claim 31 , wherein the mixed population of ESCs, XENs, and / or TSCs form multi-lineage stem cells.

33. The system of any one of claims 31 or 32, comprising ESCs and XENs cells.

34. The system of claim 33, wherein ESCs and XENs are present at an initial ESC to XEN ratio of about 1 :2 to about 8: 1.

35. The system of any one of claims 31 to 32, comprising ESCs and TSCs cells.

36. The system of claim 35, wherein ESCs and TSCs are present at an initial ESC to TSC ratio of about 1 :2 to about 8: 1.

37. The system of any one of claims 31 to 32, comprising ESCs, XENs, and TSCs cells.

38. The system of claim 37, wherein ESCs, XENs, and TSCs are present at an initial ESC to XEN to TSC ratio of about 1 :2:2 to about 8:1 :1.

39. The system of any one of claims 31 to 38, comprising laminin.

40. The system of claim 39, wherein the laminin is at a concentration of 1 - 200 pg / ml in the culture medium.

41. The system of any one of claims 31 to 38, comprising collagen.

42. The system of claim 41 , wherein the collagen is at a concentration of 1 - 200 pg / ml in the culture medium.

43. The system of any one of claims 31 to 40, wherein ESCs, XENs, and / or TSCs are obtained using a method of claim 1.

44. The system of any one of claims 31 to 41 , wherein the culture medium further comprises neurobasal medium.

45. The system of any one of claims 31 to 42, wherein the FGF activator is FGF2.

46. The system of any one of claims 31 to 43, wherein the TGF-|3 activator is Activin-A.

47. The system of any one of claims 31 to 33, wherein the WNT activator is a GSK3 inhibitor.

48. The system of claim 45, wherein the GSK3 inhibitor is CHIR99021.

49. The system of any one of claims 31 to 46, wherein the XENs and / or TSCs express extraembryonic endoderm or trophoblast markers, respectively.

50. The system of any one of claims 31 to 47, wherein the XENs and TSCs can form XEN- tumors and TSC-tumors, respectively.

51. An in vitro co-culture system comprising: i. ESCs derived from blastocysts, wherein the ESCs have a mutation or deletion in one or more integrin gene and / or; XENs derived from blastocysts, wherein the XENs have a mutation or deletion in one or more laminin gene; ii. a culture medium comprising a fibroblast growth factor (FGF) activator, a transforming growth factor beta (TGF-|3) activator, and a WNT activator.

52. An embryo model produced by differentiation of cells produced by the method of any one of the claims 1 to 30 or the system of any one of claims 31 to 48.

53. A method for cellular analysis, the method comprising using cells produced by the method of any one of the claims 1 to 30 or the system of any one of claims 31 to 48.

54. The system of claim 51 , wherein the cellular analysis comprise drug assessments, toxicity assessments, cell growth assessments, cell proliferation assessments, celldifferentiation assessments, tissue development assessments, cell culture assessments or any combination thereof.

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