One of the key characteristics of stem cells is their capacity to divide for long periods of time in an environment where most of the cells are quiescent. Therefore, a critical question in stem cell biology is how stem cells escape cell division stop signals. Here, we report the necessity of the microRNA (miRNA) pathway for proper control of germline stem cell (GSC) division in Drosophila melanogaster. Analysis of GSCs mutant for dicer-1 (dcr-1), the double-stranded RNaseIII essential for miRNA biogenesis, revealed a marked reduction in the rate of germline cyst production. These dcr-1 mutant GSCs exhibit normal identity but are defective in cell cycle control. On the basis of cell cycle markers and genetic interactions, we conclude that dcr-1 mutant GSCs are delayed in the G1 to S transition, which is dependent on the cyclin-dependent kinase inhibitor Dacapo, suggesting that miRNAs are required for stem cells to bypass the normal G1/S checkpoint. Hence, the miRNA pathway might be part of a mechanism that makes stem cells insensitive to environmental signals that normally stop the cell cycle at the G1/S transition.

For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that embryonic stem cells derived from both mice and humans display two stable yet epigenetically distinct states of pluripotency, naïve and primed. We now show that nicotinamide-N-methyl transferase (NNMT) and metabolic state regulate pluripotency in hESCs. Specifically, in naïve hESCs NNMT and its enzymatic product 1-methylnicotinamide (1-MNA) are highly upregulated, and NNMT is required for low SAM levels and H3K27me3 repressive state. NNMT consumes SAM in naïve cells, making it unavailable for histone methylation that represses Wnt and activates HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development.

Background Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) have great potential as a cell source for therapeutic applications such as regenerative medicine, disease modeling, drug screening, and toxicity testing. This potential is limited, however, by the immature state of the cardiomyocytes acquired using current protocols. Tri-iodo-L-thyronine (T3) is a growth hormone that is essential for optimal heart growth. In this study, we investigated the effect of T3 on hiPSC-CM maturation. Methods and Results A one-week treatment with T3 increased cardiomyocyte size, anisotropy, and sarcomere length. T3 treatment was associated with reduced cell cycle activity, manifest as reduced DNA synthesis and increased expression of the cyclin-dependent kinase inhibitor p21. Contractile force analyses were performed on individual cardiomyocytes using arrays of microposts, revealing an almost two-fold higher force per-beat after T3 treatment and also an enhancement in contractile kinetics. This improvement in force generation was accompanied by an increase in rates of calcium release and reuptake, along with a significant increase in sarcoendoplasmic reticulum ATPase expression. Finally, although mitochondrial genomes were not numerically increased, extracellular flux analysis showed a significant increase in maximal mitochondrial respiratory capacity and respiratory reserve capability after T3 treatment. Conclusions Using a broad spectrum of morphological, molecular, and functional parameters, we conclude that T3 is a driver for hiPSC-CM maturation. T3 treatment may enhance the utility of hiPSC-CMs for therapy, disease modeling, or drug/toxicity screens.

In metazoans, transition from fetal to adult heart is accompanied by a switch in energy metabolism-glycolysis to fatty acid oxidation. The molecular factors regulating this metabolic switch remain largely unexplored. We first demonstrate that the molecular signatures in 1-year (y) matured human embryonic stem cell-derived cardiomyocytes (hESC-CMs) are similar to those seen in in vivo-derived mature cardiac tissues, thus making them an excellent model to study human cardiac maturation. We further show that let-7 is the most highly up-regulated microRNA (miRNA) family during in vitro human cardiac maturation. Gain-and loss-of-function analyses of let-7g in hESC-CMs demonstrate it is both required and sufficient for maturation, but not for early differentiation of CMs. Overexpression of let-7 family members in hESC-CMs enhances cell size, sarcomere length, force of contraction, and respiratory capacity. Interestingly, large-scale expression data, target analysis, and metabolic flux assays suggest this let-7-driven CM maturation could be a result of down-regulation of the phosphoinositide 3 kinase (PI3K)/AKT protein kinase/insulin pathway and an up-regulation of fatty acid metabolism. These results indicate let-7 is an important mediator in augmenting metabolic energetics in maturing CMs. Promoting maturation of hESC-CMs with let-7 overexpression will be highly significant for basic and applied research.everal coronary heart diseases (CHDs) are characterized by cardiac dysfunctions predominantly manifested during cardiac maturation (1, 2). Dramatic changes in energy metabolism occur during this postnatal cardiac maturation (3). At early embryonic development, glycolysis is a major source of energy for cardiomyocytes (CMs) (4, 5). However, as the cardiomyocytes mature, mitochondrial oxidative metabolism increases with fatty acid oxidation, providing 90% of the heart's energy demands (6-8). This switch in cardiac metabolism has been shown to have important implications during in vivo cardiac maturation (9). In contrast to the relatively advanced knowledge of the genetic network that contributes to heart development during embryogenesis (10, 11), molecular factors that regulate peri-and postnatal cardiac maturation, particularly in relation to the metabolic switch, remain largely unclear. So far, studies to understand the transition of the glycolysisdependent fetal heart to oxidative metabolism in the adult heart have been mostly related to the peroxisome proliferatoractivated receptor (PPAR)/estrogen-related receptor/PPARγ coactivator-1α circuit (7,8,12). However, it is currently unknown what other factors act upstream or in synergy with this pathway in controlling cardiac energetics.miRNAs have emerged as key factors in controlling the complex regulatory network in a developing heart (13). Genetic studies that enrich or deplete miRNAs in specific cardiac tissue types and large-scale gene expression studies have demonstrated that they achieve such complex control at the level of cardiac gene expression (14-16). We sou...

SummaryAlthough human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as a novel platform for heart regeneration, disease modeling, and drug screening, their immaturity significantly hinders their application. A hallmark of postnatal cardiomyocyte maturation is the metabolic substrate switch from glucose to fatty acids. We hypothesized that fatty acid supplementation would enhance hPSC-CM maturation. Fatty acid treatment induces cardiomyocyte hypertrophy and significantly increases cardiomyocyte force production. The improvement in force generation is accompanied by enhanced calcium transient peak height and kinetics, and by increased action potential upstroke velocity and membrane capacitance. Fatty acids also enhance mitochondrial respiratory reserve capacity. RNA sequencing showed that fatty acid treatment upregulates genes involved in fatty acid β-oxidation and downregulates genes in lipid synthesis. Signal pathway analyses reveal that fatty acid treatment results in phosphorylation and activation of multiple intracellular kinases. Thus, fatty acids increase human cardiomyocyte hypertrophy, force generation, calcium dynamics, action potential upstroke velocity, and oxidative capacity. This enhanced maturation should facilitate hPSC-CM usage for cell therapy, disease modeling, and drug/toxicity screens.

Stem cells are maintained and retain their capacity to continue dividing because of the influence of a niche. Although niches are important to maintain "stemness" in a wide variety of tissues, control of these niches is poorly understood. The Drosophila germline stem cells (GSCs) reside in a somatic cell niche. We show that Notch activation can induce the expression of niche-cell markers even in an adult fly; overexpression of Delta in the germline, or activated Notch in the somatic cells, results in extra niche cells, up to 10-fold over the normal number. In turn, these ectopic niche cells induce ectopic GSCs. Conversely, when GCSs do not produce functional Notch ligands, Delta and Serrate, the TGF-beta pathway is not activated in the GSCs, and they differentiate and subsequently leave the niche. Importantly, clonal analysis reveals that the receiving end of the Notch pathway is required in the somatic cells. These data show that a feedback loop exists between the stem cells and niche cells. Demonstration that stem cells can contribute to niche function has far-reaching consequences for stem cell therapies and may provide insight into how cancer can spread throughout an organism via populations of cancer stem cells.

In both mice and humans, pluripotent stem cells (PSCs) exist in at least two distinct states of pluripotency, known as the naïve and primed states. Our understanding of the intrinsic and extrinsic factors that enable PSCs to self-renew and to transition between different pluripotent states is important for understanding early development. In mouse embryonic stem cells (mESCs), Wnt proteins stimulate mESC self-renewal and support the naïve state. In human embryonic stem cells (hESCs), Wnt/β-catenin signaling is active in naïve-state hESCs and is reduced or absent in primed-state hESCs. However, the role of Wnt/β-catenin signaling in naïve hESCs remains largely unknown. Here, we demonstrate that inhibition of the secretion of Wnts or inhibition of the stabilization of β-catenin in naïve hESCs reduces cell proliferation and colony formation. Moreover, we show that addition of recombinant Wnt3a partially rescues cell proliferation in naïve hESCs caused by inhibition of Wnt secretion. Notably, inhibition of Wnt/β-catenin signaling in naïve hESCs did not cause differentiation. Instead, it induced primed hESC-like proteomic and metabolic profiles. Thus, our results suggest that naïve hESCs secrete Wnts that activate autocrine or paracrine Wnt/β-catenin signaling to promote efficient self-renewal and inhibit the transition to the primed state.Wnt/β-catenin signaling | naïve pluripotency | naïve-to-primed transition | human embryonic stem cells | self-renewal

BackgroundPresently, there is no effective treatment for the lethal muscle wasting disease Duchenne muscular dystrophy (DMD). Here we show that increased sphingosine-1-phoshate (S1P) through direct injection or via the administration of the small molecule 2-acetyl-4(5)-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, has beneficial effects in acutely injured dystrophic muscles of mdx mice.MethodsWe treated mdx mice with and without acute injury and characterized the histopathological and functional effects of increasing S1P levels. We also tested exogenous and direct administration of S1P on mdx muscles to examine the molecular pathways under which S1P promotes regeneration in dystrophic muscles.ResultsShort-term treatment with THI significantly increased muscle fiber size and extensor digitorum longus (EDL) muscle specific force in acutely injured mdx limb muscles. In addition, the accumulation of fibrosis and fat deposition, hallmarks of DMD pathology and impaired muscle regeneration, were lower in the injured muscles of THI-treated mdx mice. Furthermore, increased muscle force was observed in uninjured EDL muscles with a longer-term treatment of THI. Such regenerative effects were linked to the response of myogenic cells, since intramuscular injection of S1P increased the number of Myf5nlacz/+ positive myogenic cells and newly regenerated myofibers in injured mdx muscles. Intramuscular injection of biotinylated-S1P localized to muscle fibers, including newly regenerated fibers, which also stained positive for S1P receptor 1 (S1PR1). Importantly, plasma membrane and perinuclear localization of phosphorylated S1PR1 was observed in regenerating muscle fibers of mdx muscles. Intramuscular increases of S1P levels, S1PR1 and phosphorylated ribosomal protein S6 (P-rpS6), and elevated EDL muscle specific force, suggest S1P promoted the upregulation of anabolic pathways that mediate skeletal muscle mass and function.ConclusionsThese data show that S1P is beneficial for muscle regeneration and functional gain in dystrophic mice, and that THI, or other pharmacological agents that raise S1P levels systemically, may be developed into an effective treatment for improving muscle function and reducing the pathology of DMD.