SummaryEmbryonic stem cells (ESCs), which are derived from a peri-implantation embryo, are routinely cultured in medium containing diabetic glucose (Glc) concentrations. While pregnancy in women with pre-existing diabetes may result in small embryos, whether such high Glc levels affect ESC growth remains uncovered. We show here that long-term exposure of ESCs to diabetic Glc inhibits their proliferation, thereby mimicking in vivo findings. Molecularly, Glc exposure increased oxidative stress and activated Forkhead box O3a (FOXO3a), promoting increased expression and activity of the ROS-removal enzymes superoxide dismutase and catalase and the cell-cycle inhibitors p21cip1 and p27kip1. Diabetic Glc also promoted β-catenin nuclear localization and the formation of a complex with FOXO3a that localized to the promoters of Sod2, p21cip1, and potentially p27kip1. Our results demonstrate an adaptive response to increases in oxidative stress induced by diabetic Glc conditions that promote ROS removal, but also result in a decrease in proliferation.

SUMMARY Human pluripotent stem cell-derived osteoblasts possess great potential for utilization in bone disorder elucidation and repair; yet, while the general ability of human pluripotent stem cells to differentiate into osteoblasts and lay down bone-specific matrix has been shown, previous studies lack the complete characterization of the process whereby such osteoblasts are derived as well as a comparison between the osteogenic efficiency of multiple cell lines. Here, we compared the osteogenic potential of two human induced pluripotent stem cell lines (RIV9 and RIV4) to human H9 embryonic stem cells. Generally capable of osteogenic differentiation, the overall osteogenic yield was lower in the RIV9 and RIV4 lines and correlated with differential expression of osteocalcin (OCN) in mature cultures and PAX7 and TWIST1 during early differentiation. In the undifferentiated cells the promoters of the latter two genes were differentially methylated potentially explaining the variation in differentiation efficiency. Furthermore, the expression signatures of selected neural crest and mesodermal genes and proteins suggested that H9 cells preferentially gave rise to neural crest-derived osteoblasts, whereas the osteoblasts in the RIV9 cultures were generated both through a mesodermal and a neural crest route albeit each at a lower rate. These data suggest that epigenetic dissimilarities between multiple PSC lines may lead to differences in lineage derivation and mineralization. Since osteoblast progenitors from one origin inadequately repair a defect in the other, these data underscore the importance of screening human pluripotent stem cells lines for the identity of the osteoprogenitors they lay down.

The specification of pluripotent stem cells into the bone-forming osteoblasts has been explored in a number of studies. However, the current body of literature has yet to adequately address the role of Wnt glycoproteins in the differentiation of pluripotent stem cells along the osteogenic lineage. During mouse embryonic stem cell (ESC) in vitro osteogenesis, the noncanonical WNT5a is expressed early on. Cells either sorted by their positive WNT5a expression or when supplemented with recombinant WNT5a (rWNT5a) during a 2-day window showed significantly enhanced osteogenic yield. Mechanistically, rWNT5a supplementation upregulated protein kinase C (PKC), calcium/calmodulin-dependent kinase II (CamKII) and c-Jun N-terminal kinase (JNK) activity while antagonizing the key effector of canonical Wnt signaling: β-catenin. Conversely, when recombinant WNT3a (rWNT3a) or other positive regulators of β-catenin were employed during this same time window there was a decrease in osteogenic marker expression. However, if rWNT3a was supplemented during a time window following rWNT5a treatment, osteogenic differentiation was enhanced both in murine and human ESCs. Elucidating the role of these WNT ligands in directing the early stages of osteogenesis has the potential to considerably improve tissue engineering protocols and applications for regenerative medicine.

Introduction Tobacco smoking has been implicated in an array of adverse health outcomes, including those that affect adult bone. However, little is known about the impact of tobacco products on developing bone tissue as it develops in the embryo. Methods Here, human embryonic stem cells were differentiated into osteoblasts in vitro and concomitantly exposed to various concentrations of smoke solutions from two conventional, one additive-free and two harm reduction brands of cigarettes. Differentiation inhibition was determined by calcium assays that quantified matrix mineralization and compared to the cytotoxicity of the tobacco product. Results Exposure to mainstream smoke from conventional and additive-free cigarettes caused no inhibition of cell viability or mineralization, while sidestream smoke concentration-dependently produced cell death. In contrast, mineralization was inhibited only by the highest mainstream concentration of harm-reduction smoke solution. Additionally, sidestream smoke solution from the harm-reduction cigarettes impeded calcification at concentrations lower than those determined to be cytotoxic for conventional products. Conclusions Sidestream smoke impaired in vitro osteogenesis at sub-toxic concentrations. In addition, though often perceived as safer, smoke from harm reduction cigarettes was more potent in inhibiting in vitro osteogenesis than smoke from conventional cigarettes. Implications This study adds to a growing list of adverse outcomes associated with prenatal tobacco exposure. Specifically, in vitro exposure to tobacco products interfered with osteogenic differentiation of human embryonic stem cells, a well-established surrogate model for human embryonic bone development. Contrasting a diverse array of tobacco products unveiled that sidestream smoke was generally more developmentally osteotoxic than mainstream smoke and that harm-reduction products may not be less harmful than conventional products, adverse effects that were seemingly independent of nicotine.

To prevent congenital defects arising from maternal exposure, safety regulations require pre-market developmental toxicity screens for industrial chemicals and pharmaceuticals. Traditional embryotoxicity approaches depend heavily on the use of low-throughput animal models which may not adequately predict human risk. The validated embryonic stem cell test (EST) developed in murine embryonic stem cells addressed the former problem over 15 years ago. Here, we present a proof-of-concept study to address the latter challenge by updating all three endpoints of the classic mouse EST with endpoints derived from human induced pluripotent stem cells (hiPSCs) and human fibroblasts. Exposure of hiPSCs to selected test chemicals inhibited differentiation at lower concentrations than observed in the mouse EST. The hiPSC-EST also discerned adverse developmental outcomes driven by novel environmental toxicants. Evaluation of the early cardiac gene TBX5 yielded similar toxicity patterns as the full-length hiPSC-EST. Together, these findings support the further development of hiPSCs and early molecular endpoints as a biologically relevant embryotoxicity screening approach for individual chemicals and mixtures.