Hydroxyapatite describes both the natural mineral phase of bone as well as the widely used calcium-phosphate implant substitute. Given that hydroxyapatite is a major component of the in vivo surface with which osteoblasts interact, it is surprising that most studies examining the regulation of osteoblast growth and differentiation utilize plastic surfaces. Here we demonstrate that the phenotype of mouse MC3T3-E1 osteoblasts is significantly altered on hydroxyapatite compared with plastic surfaces. Specifically, alkaline phosphatase activity and messenger RNA levels, markers of early stages of osteoblast differentiation, are increased in osteoblasts cultured on hydroxyapatite. The precocious appearance of alkaline phosphatase activity on the hydroxyapatite surface suggests that osteoblast differentiation is activated earlier compared with plastic surfaces. Osteocalcin expression, a marker of late-stage differentiation, is also increased on hydroxyapatite and further demonstrates enhanced differentiation. Cell counts indicate that fewer osteoblasts are present on hydroxyapatite versus plastic surfaces 24 h after plating. Measurement of osteoblast attachment, apoptosis, and necrosis indicated no differences between surfaces. In contrast, the number of bromodeoxyuridine-incorporating cells was significantly decreased on hydroxyapatite compared with plastic surfaces. Taken together, our findings indicate that hydroxyapatite enhances osteoblast differentiation while also suppressing growth.

Generation of functional beta cells for regenerative medicine would benefit from an increased understanding of pancreatic endocrine cell differentiation. During pancreatic development, expression of Ngn3 defines the progenitor cells that will differentiate into one of five endocrine lineages, including the beta cell. Here, we identify and further characterize a distinct endocrine progenitor population defined by differential expression of a transcription factor named Fev in murine and human endocrine cell development. Using single-cell RNA-sequencing and genetic lineage tracing, we build a transcriptomic profile of murine Fev+ progenitors that is distinct from that of Ngn3+ progenitors and reconstruct lineage relationships among Ngn3+, Fev+, and differentiated, hormone+ cells. We further identify candidate transcriptional regulators along the differentiation trajectory of this Fev+ progenitor population toward the alpha or beta cell lineages. Translating our findings in murine pancreatic development to that of human, we identify an analogous FEV+ population and putative gene regulators of endocrine cell fate in human fetal pancreas. In a stepwise differentiation platform designed to generate beta cells from human embryonic stem cells (hESCs), we also uncover robust FEV expression in endocrine progenitors prior to their differentiation into insulin-producing beta cells. Single-cell RNA-sequencing and lineage reconstruction of hESC-derived cells at the differentiated beta cell stage reveal FEV+ cells that may represent endocrine progenitors that have failed to acquire a beta cell identity; this failure of FEV+ endocrine cell differentiation may facilitate in uncovering the required molecular signals and genetic circuitry that push endocrine progenitors toward a beta cell fate. This work establishes a new model of pancreatic endocrine cell differentiation and identifies a Fev+ endocrine progenitor as a new cell stage in endocrine lineage determination. Disclosure D.M. Wong: None. L. Byrnes: None. J.O. Bouza: None. G. Peixoto: None. R. McMullen: None. J.B. Sneddon: Advisory Panel; Spouse/Partner; Akouos. Advisory Panel; Self; Encellin. Advisory Panel; Spouse/Partner; Encellin. Consultant; Self; Semma Therapeutics, Inc. Funding Nora Eccles Treadwell Foundation; University of California, San Francisco