Complexity in the spatial organization of human embryonic stem cell (hESC) cultures creates heterogeneous microenvironments (niches) that influence hESC fate. This study demonstrates that the rate and trajectory of hESC differentiation can be controlled by engineering hESC niche properties. Niche size and composition regulate the balance between differentiation‐inducing and ‐inhibiting factors. Mechanistically, a niche size‐dependent spatial gradient of Smad1 signaling is generated as a result of antagonistic interactions between hESCs and hESC‐derived extra‐embryonic endoderm (ExE). These interactions are mediated by the localized secretion of bone morphogenetic protein‐2 (BMP2) by ExE and its antagonist, growth differentiation factor‐3 (GDF3) by hESCs. Micropatterning of hESCs treated with small interfering (si) RNA against GDF3, BMP2 and Smad1, as well treatments with a Rho‐associated kinase (ROCK) inhibitor demonstrate that independent control of Smad1 activation can rescue the colony size‐dependent differentiation of hESCs. Our results illustrate, for the first time, a role for Smad1 in the integration of spatial information and in the niche‐size‐dependent control of hESC self‐renewal and differentiation.

The trophectoderm layer of the blastocyst-stage embryo is the precursor for all trophoblast cells in the placenta. Human trophoblast stem (TS) cells have emerged as an attractive tool for studies on early trophoblast development. However, the use of TS cell models is constrained by the limited genetic diversity of existing TS cell lines, and restrictions on using human fetal tissue or embryos needed to generate additional lines. Here we report the derivation of two distinct stem cell types of the trophectoderm lineage from human pluripotent stem cells. Analogous to villous cytotrophoblasts in vivo, the first is a CDX2- stem cell comparable to placenta-derived TS cells - they both exhibit identical expression of key markers, are maintained in culture and differentiate under similar conditions, and share high transcriptome similarity. The second is a CDX2+ stem cell with distinct cell culture requirements, and differences in gene expression and differentiation, relative to CDX2- stem cells. Derivation of TS cells from pluripotent stem cells will significantly enable construction of in vitro models for normal and pathological placental development.