There has been considerable progress in identifying signaling pathways directing the differentiation of human pluripotent stem cells (hPSCs) into specialized cell types including neurons. However, extrinsic factor-based differentiation of hPSCs is a slow, step-wise process mimicking the protracted timing of normal human development. Using a small molecule screen we identified a combination of five small molecule pathway inhibitors sufficient to yield hPSC-derived neurons at >75% efficiency within 10 days of differentiation. The resulting neurons express canonical markers and functional properties of human nociceptors including TTX-resistant, SCN10A-dependent sodium currents and response to nociceptive stimuli including ATP and capsaicin. Neuronal fate acquisition occurs three-fold faster than during in vivo1 development suggesting that use of small molecule pathway inhibitors could develop into a general strategy for accelerating developmental timing in vitro. The quick and high efficiency derivation of nociceptors offers unprecedented access to this medically relevant cell type for studies of human pain.

Adipogenesis involves cell proliferation and differentiation, both of which have been shown to be regulated by micro (mi)RNA. During mouse preadipocyte 3T3L1 cell differentiation, we found that miR-17-92, a miRNA cluster that promotes cell proliferation in various cancers, was significantly up-regulated at the clonal expansion stage of adipocyte differentiation. Stable transfection of 3T3L1 cells with miR-17-92 resulted in accelerated differentiation and increased triglyceride accumulation after hormonal stimulation. By using a luciferase reporter assay, we demonstrated that miR-17-92 directly targeted the 3 UTR region of Rb2/p130, accounting for subsequently reduced Rb2/p130 mRNA and protein quantities at the stage of clonal expansion. siRNA-mediated knock-down of Rb2/p130 at the same stage of clonal expansion recapitulated the phenotype of overexpression of miR-17-92 in the stably transfected 3T3L1 cells. These data indicate that miR-17-92 promotes adipocyte differentiation by targeting and negatively regulating Rb2/p130. G iven the global obesity epidemic, deciphering the mechanisms underlying adipocyte differentiation in mammals poses an important challenge. The mouse 3T3L1 preadipocyte cell line has been an ideal in vitro system for unraveling the molecular events of adipocyte differentiation (1, 2). The entire process of 3T3L1 preadipocyte cell differentiation involves several cellular stages. First, proliferating preadipocytes become growth-arrested by contact inhibition, which can be reversed by hormonal induction and reentry into the cell cycle. After several rounds of clonal expansion, the cells again become quiescent with the initiation of coordinate transcriptional activation of adipocyte genes such as C/Ebp␣, Ap2, Lpl, Srebp, and Ppar␥. Expression of these genes leads to terminal adipocyte differentiation (3).Among the above cellular programs, clonal expansion is one of the key events taking place in early adipogenesis (4, 5). Blocking cell cycle reentry with a DNA synthesis inhibitor prevents adipocyte differentiation, suggesting that an active clonal expansion is required for the differentiation process (4, 6). Clonal expansion is critically regulated by retinoblastoma family (pRB) genes through interactions with the E2F transcription factors. A significant role of two members of the pRB family p130 and p107 in early adipocyte differentiation is suggested by the dramatic change in their expression within the first 24 h (i.e., clonal expansion) of hormonal induction. In growth-arrested preadipocytes, there are significant levels of p130 with little or no detectable p107. However, this trend is completely reversed at 24 h, with a significant decrease in p130 accompanied by a marked increase in p107. This dramatic regulation has been designated as the p130:p107 switch (6). The functional significance of the p130:p107 switch has now been demonstrated by disrupting the p130 expression pattern during the mitotic clonal expansion phase, resulting in complete inhibition of adipocyte differentiation (7).In...

Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into functional neurons. However, the protracted timing of human neuron specification and functional maturation remains a key challenge that hampers the routine application of hPSC-derived lineages in disease modeling and regenerative medicine. Using a combinatorial small-molecule screen, we previously identified conditions for the rapid differentiation of hPSCs into peripheral sensory neurons. Here we generalize the approach to central nervous system (CNS) fates by developing a small-molecule approach for accelerated induction of early-born cortical neurons. Combinatorial application of 6 pathway inhibitors induces post-mitotic cortical neurons with functional electrophysiological properties by day 16 of differentiation, in the absence of glial cell co-culture. The resulting neurons, transplanted at 8 days of differentiation into the postnatal mouse cortex, are functional and establish long-distance projections, as shown using iDISCO whole brain imaging. Accelerated differentiation into cortical neuron fates should facilitate hPSC-based strategies for disease modeling and cell therapy in CNS disorders.