SummaryNicotinamide, the amide form of vitamin B3, is widely used in disease treatments and stem cell applications. However, nicotinamide's impact often cannot be attributed to its nutritional functions. In a vitamin screen, we find that nicotinamide promotes cell survival and differentiation in human pluripotent stem cells. Nicotinamide inhibits the phosphorylation of myosin light chain, suppresses actomyosin contraction, and leads to improved cell survival after individualization. Further analysis demonstrates that nicotinamide is an inhibitor of multiple kinases, including ROCK and casein kinase 1. We demonstrate that nicotinamide affects human embryonic stem cell pluripotency and differentiation as a selective kinase inhibitor. The findings in this report may help researchers design better strategies to develop nicotinamide-related stem cell applications and disease treatments.

Summary Pyruvate is a key metabolite in glycolysis and the tricarboxylic acid (TCA) cycle. Exogenous pyruvate modulates metabolism, provides cellular protection, and is essential for the maintenance of human preimplantation embryos and human embryonic stem cells (hESCs). However, little is known about how pyruvate contributes to cell-fate determination during epiblast stage. In this study, we used hESCs as a model to demonstrate that elevated exogenous pyruvate shifts metabolic balance toward oxidative phosphorylation in both maintenance and differentiation conditions. During differentiation, pyruvate potentiates mesoderm and endoderm lineage specification. Pyruvate production and its mitochondrial metabolism are required in BMP4-induced mesoderm differentiation. However, the TCA-cycle metabolites do not have the same effect as pyruvate on differentiation. Further study shows that pyruvate increases AMP/ATP ratio, activates AMPK, and modulates the mTOR pathway to enhance mesoderm differentiation. This study reveals that exogenous pyruvate not only controls metabolism but also modulates signaling pathways in hESC differentiation.

Cell density has profound impacts on the cell culture practices of human pluripotent stem cells. The regulation of cell growth, cell death, pluripotency and differentiation converge at high density, but it is largely unknown how different regulatory mechanisms act at this stage. We use a chemically defined medium to systemically examine cellular activities and the impact of medium components in high-density culture. We show that medium acidosis is the main factor that alters cell cycle, gene expression and cellular metabolism at high cell density. The low medium pH leads to inhibition of glucose consumption, cell cycle arrest, and subsequent cell death. At high cell density, the suppression of medium acidosis with sodium bicarbonate (NaHCO3) significantly increases culture capacity for stem cell survival, derivation, maintenance and differentiation. Our study provides a simple and effective tool to improve stem cell maintenance and applications.

Pluripotent stem cells (PSCs) can be maintained in a continuum of cellular states with distinct features. Exogenous lipid supplements can relieve the dependence on de novo lipogenesis and shift global metabolism. However, it is largely unexplored how specific lipid components regulate metabolism and subsequently the pluripotency state. In this study, we report that the metabolic landscape of human PSCs (hPSCs) is shifted by signaling lipid lysophosphatidic acid (LPA), which naturally exists. LPA leads to a distinctive transcriptome profile that is not associated with de novo lipogenesis. Although exogenous lipids such as cholesterol, common free fatty acids, and LPA can affect cellular metabolism, they are not necessary for maintaining primed pluripotency. Instead, LPA induces distinct and reversible phenotypes in cell cycle, morphology, and mitochondria. This study reveals a distinct primed state that could be used to alter cell physiology in hPSCs for basic research and stem cell applications.

Graphical Abstract Highlights d Exogenous insulin/IGF signal promotes heterogeneity in early mesoderm differentiation d Endogenous IGF induced during mesoderm differentiation suppresses cardiomyocyte fate d Inhibition of IGF pathway by LY294002 leads to effective cardiomyocyte differentiation d LY294002 inhibits the CK2 pathway to promote cardiomyocyte cell fate SUMMARYDuring embryogenesis, various cell types emerge simultaneously from their common progenitors under the influence of intrinsic signals. Human embryonic stem cells can differentiate to diverse cell types of three embryonic lineages, making them an excellent system for understanding the regulatory mechanism that maintains the balance of different cell types in embryogenesis. In this report, we demonstrate that insulin-like growth factor (IGF) proteins are endogenously expressed during differentiation, and their temporal expression contributes to the cell fate diversity in mesoderm differentiation. Small molecule LY294002 inhibits the IGF pathway to promote cardiomyocyte differentiation while suppressing epicardial and noncardiac cell fates. LY294002induced cardiomyocytes demonstrate characteristic cardiomyocyte features and provide insights into the molecular mechanisms underlying cardiac differentiation. We further show that LY294002 induces cardiomyocytes through CK2 pathway inhibition. This study elucidates the crucial roles of endogenous IGF in mesoderm differentiation and shows that the inhibition of the IGF pathway is an effective approach for generating cardiomyocytes.