Self‐organization of human iPS cells into trophectoderm mimicking cysts induced by adhesion restriction using microstructured mesh scaffolds

Abstract

Cellular dynamics leading to the formation of the trophectoderm in humans remain poorly understood owing to limited accessibility to human embryos for research into early human embryogenesis. Compared to animal models, organoids formed by self-organization of stem cells in vitro may provide better insights into differentiation and complex morphogenetic processes occurring during early human embryogenesis. Here we demonstrate that modulating the cell culture microenvironment alone can trigger self-organization of human induced pluripotent stem cells (hiPSCs) to yield trophectoderm-mimicking cysts without chemical induction. To modulate the adhesion microenvironment, we used the mesh culture technique recently developed by our group, which involves culturing hiPSCs on suspended micro-structured meshes with limited surface area for cell adhesion. We show that this adhesion-restriction strategy can trigger a two-stage self-organization of hiPSCs; first into stem cell sheets, which express pluripotency signatures until around day 8-10, then into spherical cysts following differentiation and self-organization of the sheet-forming cells. Detailed morphological analysis using immunofluorescence microscopy with both confocal and two-photon microscopes revealed the anatomy of the cysts as consisting of a squamous epithelial wall richly expressing E-cadherin and CDX2. We also confirmed that the cysts exhibit a polarized morphology with basal protrusions, which show migratory behavior when anchored. Together, our results point to the formation of cysts which morphologically resemble the trophectoderm at the late-stage blastocyst. Thus, the mesh culture microenvironment can initiate self-organization of hiPSCs into trophectoderm-mimicking cysts as organoids with potential application in the study of early embryogenesis and also in drug development.

Establishment of human trophoblast stem cells from human induced pluripotent stem cell-derived cystic cells under micromesh culture

“…Micromesh scaffolds have been used for hiPSC differentiation into trophectoderm-like cysts [19, 20]. Consistent with previously reported data, our data showed that cystic structures emerged on day 16 when using a 4-mm nickel micromesh (Fig.…”

“…Notably, micromesh-cultured hepatic cells have been shown to exhibit increased expression of hepatic marker genes and enhanced cell maturation [18]. Furthermore, Okeyo et al [19] reported that micromesh culture induces spontaneous differentiation of hiPSCs into trophoblast cyst structures without BMP4 treatment.…”

A Novel Human Placental Barrier Model Based on Trophoblast Stem Cells Derived from Human Induced Pluripotent Stem Cells

“…40 Previous reports by Okeyo et al have shown that the hiPSCs (clone TIG1-4F hiPSC from lung fibroblasts, provided by Dr. Takashi Tada of Kyoto University, Kyoto, Japan) can differentiate into TSC-like cells. 19,20 In this study, we used the TSCs derived from the hiPSC line ChiPSC22, which is more ordinary and commercially available than the above hiPSC. Furthermore, we have been tried to obtain TSCs using the other common hiPSC lines 201B7 and 253G1, however, these hiPSC lines did not effectively differentiate into trophectoderm.…”

“…Hori and Kurosawa reported that micromesh-cultured hepatic cells have been shown to exhibit increased expression of hepatic marker genes and enhanced cell maturation. 18 Previous reports by Okeyo et al 19,20 have shown that limited culture on micromesh can induce hiPSCs to differentiate into trophoblast cysts. Since the cells are suspended in the culture medium in a cell sheet state maintained by cell-cell adhesion, nutrition and oxygen can be supplied directly from both sides of the cell sheet.…”

“…The apertures of these micromeshes are much larger than the size of single cells, hence the cells in these openings bind each other only by cell-cell adhesion. [18][19][20] Additionally, micromesh cell culture has several features not found in other 2D culture methods. Hori and Kurosawa reported that micromesh-cultured hepatic cells have been shown to exhibit increased expression of hepatic marker genes and enhanced cell maturation.…”