During early development, extrinsic triggers prompt pluripotent cells to begin the process of differentiation. When and how human embryonic stem cells (hESCs) irreversibly commit to differentiation is a fundamental yet unanswered question. By combining single-cell imaging, genomic approaches, and mathematical modeling, we find that hESCs commit to exiting pluripotency unexpectedly early. We show that bone morphogenetic protein 4 (BMP4), an important differentiation trigger, induces a subset of early genes to mirror the sustained, bistable dynamics of upstream signaling. Induction of one of these genes, GATA3, drives differentiation in the absence of BMP4. Conversely, GATA3 knockout delays differentiation and prevents fast commitment to differentiation. We show that positive feedback at the level of the GATA3-BMP4 axis induces fast, irreversible commitment to differentiation. We propose that early commitment may be a feature of BMP-driven fate choices and that interlinked feedback is the molecular basis for an irreversible transition from pluripotency to differentiation.

Pluripotent stem cells (PSCs) could provide a powerful system to model development of the human esophagus, whose distinct tissue organization compared to rodent esophagus suggests that developmental mechanisms may not be conserved between species. We therefore established an efficient protocol for generating esophageal progenitor cells (EPCs) from human PSCs. We found that inhibition of TGF-ß and BMP signaling is required for sequential specification of EPCs, which can be further purified using cell-surface markers. These EPCs resemble their human fetal counterparts and can recapitulate normal development of esophageal stratified squamous epithelium during in vitro 3D cultures and in vivo. Importantly, combining hPSC differentiation strategies with mouse genetics elucidated a critical role for Notch signaling in the formation of this epithelium. These studies therefore not only provide an efficient approach to generate EPCs, but also offer a model system to study the regulatory mechanisms underlying development of the human esophagus.

Hematopoietic stem cell (HSC) self-renewal and aging are tightly regulated by paracrine factors from the bone marrow niche. However, whether HSC rejuvenation could be achieved by engineering a bone marrow niche ex vivo remains unknown. Here, we show that matrix stiffness fine-tunes HSC niche factor expression by bone marrow stromal cells (BMSCs). Increased stiffness activates Yap/Taz signaling to promote BMSC expansion upon 2D culture, which is largely reversed by 3D culture in soft gelatin methacrylate hydrogels. Notably, 3D co-culture with BMSCs promotes HSC maintenance and lymphopoiesis, reverses aging hallmarks of HSCs, and restores their long-term multilineage reconstitution capacity. In situ atomic force microscopy analysis reveals that mouse bone marrow stiffens with age, which correlates with a compromised HSC niche. Taken together, this study highlights the biomechanical regulation of the HSC niche by BMSCs, which could be harnessed to engineer a soft bone marrow niche for HSC rejuvenation.