Micropattern differentiation of mouse pluripotent stem cells recapitulates embryo regionalized cell fate patterning

Abstract

During gastrulation epiblast cells exit pluripotency as they specify and spatially arrange the three germ layers of the embryo. Similarly, human pluripotent stem cells (PSCs) undergo spatially organized fate specification on micropatterned surfaces. Since in vivo validation is not possible for the human, we developed a mouse PSC micropattern system and, with direct comparisons to mouse embryos, reveal the robust specification of distinct regional identities. BMP, WNT, ACTIVIN and FGF directed mouse epiblast-like cells to undergo an epithelial-to-mesenchymal transition and radially pattern posterior mesoderm fates. Conversely, WNT, ACTIVIN and FGF patterned anterior identities, including definitive endoderm. By contrast, epiblast stem cells, a developmentally advanced state, only specified anterior identities, but without patterning. The mouse micropattern system offers a robust scalable method to generate regionalized cell types present in vivo, resolve how signals promote distinct identities and generate patterns, and compare mechanisms operating in vivo and in vitro and across species.

Dissecting the dynamics of signaling events in the BMP, WNT, and NODAL cascade during self-organized fate patterning in human gastruloids

“…In a previous study, we showed that spatially confined human embryonic stem cells (hESCs), treated with BMP4 (gastruloids) self-organize to form radial patterns of distinct germ layers: an outer ring of extra-embryonic cells, followed by endodermal and mesodermal rings, and an ectodermal center, thus recapitulating some aspects of gastrulation in vitro (5). These findings have since been reproduced in other labs (6), and a comparable system for mouse ESCs has been developed (7). Three-dimensional models have also been developed that recapitulate aspects of early mammalian development (8)(9)(10)(11), and in some cases, even morphologically resemble embryos (12)(13)(14).…”

“…Inside of the BRA+ ring, center cells co-express the pluripotency markers NANOG and SOX2, with the centermost cells showing lower NANOG levels ( Figure S1B), a possible sign of the onset of ectodermal differentiation (20). Compared to the MEF-CM protocol in which NANOG expression was completely lost in the center cells (5), the mTeSR1 protocol recapitulates an earlier time point in gastrulation when primitive streak formation has begun, but the remainder of the epiblast remains pluripotent with only shallow gradients in pluripotency markers such as NANOG (1,7).…”

Bioengineered microenvironment to culture early embryos

“…Early embryonic development is accompanied by the maintenance of pluripotency, differentiation and the order of various pluripotent stem cells. [34][35][36] However, natural embryos are small in size, bringing difficulties to conducting analytical studies, especially after the implantation. Embryoid bodies derived from in vitro cultured stem cells can help in understanding the specific history of the early embryonic development.…”

“…Stem cells can self-assemble into endoderm, mesoderm and ectoderm tissues according to a certain geometric pattern under natural conditions. 35,57,96 After the reported stress-adjusted neuroectoderm developmental model, 47 Chan et al 97 TSCs are designed to develop into early embryos in vitro, the following challenges require particular attention: as the expression profiles F I G U R E 6 From a single cell to 3D models, embryonic development is regulated by mechanics and geometry. 1 The mechanical properties such as the stiffness of the hydrogels around the cells can regulate the development of the cells.…”

“…95 Using 2D models, researchers can use micropatterning to control the self-assembly of stem cells. 35,57,96 Furthermore, the researchers applied stress to regulate embryonic spatial patterning. 47 (Figure 7), incomplete in structure and lack of extraembryonic tissue.…”