Extraembryonic mesoderm cells derived from human embryonic stem cells rely on Wnt pathway activation

Abstract

Extraembryonic mesoderm cells (EXMCs) are involved in the development of multiple embryonic lineages and umbilical cord formation, where they subsequently develop into mesenchymal stem cells (MSCs). Although EXMCs can be generated from human naïve embryonic stem cells (ESCs), it is unclear whether human primed ESCs (hpESCs) can differentiate into EXMCs that subsequently produce MSCs. The present report described a three-dimensional differentiation protocol to induce hpESCs into EXMCs by activating the Wnt pathway using CHIR99021. Single-cell transcriptome and immunostaining analyses revealed that the EXMC characteristics were similar to those of post-implantation embryonic EXMCs. Cell sorting was used to purify and expand the EXMCs. Importantly, these EXMCs secreted extracellular matrix proteins, including COL3A1 and differentiated into MSCs. Inconsistent with other MSC types, these MSCs exhibited a strong differentiation potential for chondrogenic and osteogenic cells and lacked adipocyte differentiation. Together, these findings provided a protocol to generate EXMCs and subsequent MSCs from hpESCs.

References

[1] RossC, Boroviak TE. Origin and function of the yolk sac in primate embryogenesis. Nat Commun. 2020;11(1):3760.

[2] O’RahillyR, Müller F. Developmental stages in human embryos: revised and new measurements. Cells Tissues Organs. 2010;192(2):73-84.

[3] SaykaliB, Mathiah N, NahabooW, et al. Distinct mesoderm migration phenotypes in extra-embryonic and embryonic regions of the early mouse embryo. elife. 2019;8:8.

[4] BernardoAS, FaialT, GardnerL, et al. BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages. Cell Stem Cell. 2011;9(2):144-155.

[5] XiangL, YinY, ZhengY, et al. A developmental landscape of 3D-cultured human pre-gastrulation embryos. Nature. 2020;577(7791):537-542.

[6] AiZ, NiuB, YinY, et al. Dissecting peri-implantation development using cultured human embryos and embryo-like assembloids. Cell Res. 2023;33(9):661-678.

[7] YangR, GoedelA, KangY, et al. Amnion signals are essential for mesoderm formation in primates. Nat Commun. 2021;12(1):5126.

[8] Castillo-VenzorA, Penfold CA, MorganMD, et al. Origin and segregation of the human germline. Life Sci Alliance. 2023;6(8):e202201706.

[9] TamPP, Beddington RS. The formation of mesodermal tissues in the mouse embryo during gastrulation and early organogenesis. Development. 1987;99(1):109-126.

[10] MarkouliC, de Deckersberg EC, DziedzickaD, et al. Sustained intrinsic WNT and BMP4 activation impairs hESC differentiation to definitive endoderm and drives the cells towards extra-embryonic mesoderm. Sci Rep. 2021;11(1):8242.

[11] RostovskayaM, Andrews S, ReikW, Rugg-GunnPJ. Amniogenesis occurs in two independent waves in primates. Cell Stem Cell. 2022;29(5):744-759.

[12] IoS, KabataM, IemuraY, et al. Capturing human trophoblast development with naive pluripotent stem cells in vitro. Cell Stem Cell. 2021;28(6):1023-1039.

[13] SimunovicM, SiggiaED, BrivanlouAH. In vitro attachment and symmetry breaking of a human embryo model assembled from primed embryonic stem cells. Cell Stem Cell. 2022;29(6):962-972.

[14] PhamTXA, PandaA, KagawaH, et al. Modeling human extraembryonic mesoderm cells using naive pluripotent stem cells. Cell Stem Cell. 2022;29(9):1346-1365.

[15] ArnoldSJ, Robertson EJ. Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo. Nat Rev Mol Cell Biol. 2009;10(2):91-103.

[16] MartynI, KannoTY, RuzoA, Siggia ED, BrivanlouAH. Self-organization of a human organizer by combined Wnt and Nodal signalling. Nature. 2018;558(7708):132-135.

[17] Ben-HaimN, LuC, Guzman-AyalaM, et al. The nodal precursor acting via activin receptors induces mesoderm by maintaining a source of its convertases and BMP4. Dev Cell. 2006;11(3):313-323.

[18] CreaseDJ, DysonS, GurdonJB. Cooperation between the activin and Wnt pathways in the spatial control of organizer gene expression. Proc Natl Acad Sci USA. 1998;95(8):4398-4403.

[19] BaronMH. Early patterning of the mouse embryo: implications for hematopoietic commitment and differentiation. Exp Hematol. 2005;33(9):1015-1020.

[20] DownsKM. The mouse allantois: new insights at the embryonic-extraembryonic interface. Philos Trans R Soc Lond Ser B Biol Sci. 2022;377(1865):20210251.

[21] DownsKM, Rodriguez AM. The mouse fetal-placental arterial connection: A paradigm involving the primitive streak and visceral endoderm with implications for human development. Wiley Interdiscip Rev Dev Biol. 2020;9(2):e362.

[22] EndersAC, KingBF. Formation and differentiation of extraembryonic mesoderm in the rhesus monkey. Am J Anat. 1988;181(4):327-340.

[23] KaragiannisED, BoydenES. Expansion microscopy: development and neuroscience applications. Curr Opin Neurobiol. 2018;50:56-63.

[24] GomesA, CoelhoP, SoaresR, Costa R. Human umbilical cord mesenchymal stem cells in type 2 diabetes mellitus: the emerging therapeutic approach. Cell Tissue Res. 2021;385(3):497-518.

[25] BeeravoluN, McKeeC, AlamriA, et al. Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta. J Vis Exp. 2017;122:e55224.

[26] AiZ, NiuB, DuanK, et al. Modulation of Wnt and Activin/Nodal supports efficient derivation, cloning and suspension expansion of human pluripotent stem cells. Biomaterials. 2020;249:120015.

[27] HaoJ, CaoJ, WangL, et al. Requirements for human embryonic stem cells. Cell Prolif. 2020;53(12):e12925.

[28] LiJ, LiN, WeiJ, et al. Genetically engineered mesenchymal stem cells with dopamine synthesis for Parkinson’s disease in animal models. NPJ Parkinsons Dis. 2022;8(1):175.

[29] ChenX, HuangJ, WuJ, et al. Human mesenchymal stem cells. Cell Prolif. 2022;55(4):e13141.

[30] McDermaidA, MonierB, ZhaoJ, Liu B, MaQ. Interpretation of differential gene expression results of RNA-seq data: review and integration. Brief Bioinform. 2019;20(6):2044-2054.

[31] ShiDL. Wnt/planar cell polarity signaling controls morphogenetic movements of gastrulation and neural tube closure. Cell Mol Life Sci. 2022;79(12):586.

[32] SrivatsanSR, RegierMC, BarkanE, et al. Embryo-scale, single-cell spatial transcriptomics. Science. 2021;373(6550):111-117.

[33] ChoudhuryJ, PandeyD, ChaturvediPK, GuptaS. Epigenetic regulation of epithelial to mesenchymal transition: a trophoblast perspective. Mol Hum Reprod. 2022;28(5):gaac013.

[34] TyserRCV, Mahammadov E, NakanohS, VallierL, Scialdone A, SrinivasS. Single-cell transcriptomic characterization of a gastrulating human embryo. Nature. 2021;600(7888):285-289.

[35] GemayelJ, ChakerD, el HachemG, et al. Mesenchymal stem cells-derived secretome and extracellular vesicles: perspective and challenges in cancer therapy and clinical applications. Clin Transl Oncol. 2023;25(7):2056-2068.

[36] HwangNS, ZhangC, HwangYS, Varghese S. Mesenchymal stem cell differentiation and roles in regenerative medicine. Wiley Interdiscip Rev Syst Biol Med. 2009;1(1):97-106.

[37] KinderSJ, TsangTE, QuinlanGA, Hadjantonakis AK, NagyA, TamPPL. The orderly allocation of mesodermal cells to the extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo. Development. 1999;126(21):4691-4701.

[38] ParameswaranM, TamPP. Regionalisation of cell fate and morphogenetic movement of the mesoderm during mouse gastrulation. Dev Genet. 1995;17(1):16-28.

[39] Maddox-HyttelP, Alexopoulos NI, VajtaG, et al. Immunohistochemical and ultrastructural characterization of the initial post-hatching development of bovine embryos. Reproduction. 2003;125(4):607-623.

[40] TamPP, LoebelDA, TanakaSS. Building the mouse gastrula: signals, asymmetry and lineages. Curr Opin Genet Dev. 2006;16(4):419-425.

[41] LanT, LuoM, WeiX. Mesenchymal stem/stromal cells in cancer therapy. J Hematol Oncol. 2021;14(1):195.

[42] KramperaM, Le Blanc K. Mesenchymal stromal cells: Putative microenvironmental modulators become cell therapy. Cell Stem Cell. 2021;28(10):1708-1725.

[43] FuJ, WangY, JiangY, du J, XuJ, LiuY. Systemic therapy of MSCs in bone regeneration: a systematic review and meta-analysis. Stem Cell Res Ther. 2021;12(1):377.

[44] ZhengW, LiH, HuK, LiL, BeiM. Chondromalacia patellae: current options and emerging cell therapies. Stem Cell Res Ther. 2021;12(1):412.

[45] FarkasK, Ferretti E. Derivation of Human Extraembryonic Mesoderm-like Cells from Primitive Endoderm. Int J Mol Sci. 2023;24(14):11366.

[46] GuoG, von Meyenn F, RostovskayaM, et al. Epigenetic resetting of human pluripotency. Development. 2017;144(15):2748-2763.

[47] AbasR, Masrudin SS, HarunAM, OmarNS. Gastrulation and Body Axes Formation: A Molecular Concept and Its Clinical Correlates. Malays J Med Sci. 2022;29(6):6-14.

[48] van den BrinkSC, Alemany A, van BatenburgV, et al. Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids. Nature. 2020;582(7812):405-409.

[49] OlmstedZT, Paredes-Espinosa MB, PaluhJL. Generation of human elongating multi-lineage organized cardiac gastruloids. STAR Protoc. 2022;3(4):101898.

[50] KnöflerM, HaiderS, SalehL, Pollheimer J, GamageTKJB, JamesJ. Human placenta and trophoblast development: key molecular mechanisms and model systems. Cell Mol Life Sci. 2019;76(18):3479-3496.

[51] OldakB, Wildschutz E, BondarenkoV, et al. Complete human day 14 post-implantation embryo models from naive ES cells. Nature. 2023;622(7983):562-573.

[52] MorisN, AnlasK, van den BrinkSC, et al. An in vitro model of early anteroposterior organization during human development. Nature. 2020;582(7812):410-415.

[53] ZhengG, HuangR, QiuG, et al. Mesenchymal stromal cell-derived extracellular vesicles: regenerative and immunomodulatory effects and potential applications in sepsis. Cell Tissue Res. 2018;374(1):1-15.

RIGHTS & PERMISSIONS

2024 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.