Our analysis revealed that mTOR inhibitors added alone to

Our analysis revealed that mTOR inhibitors added alone to SF-defined medium can induce mesendoderm, and our follow-up studies confirmed that rapamycin enhances the formation of HE and blood progenitors. Ten-point dose curves revealed a bimodal effect where rapamycin at low concentrations mildly enhances the purity and number of OCT4+SOX2+ PSCs, and higher concentrations reduce the OCT4+SOX2+ population and enhance mesendoderm differentiation. These results reconcile two previous studies: in line with Easley et al. (2010) we observe no negative effect on pluripotency of rapamycin at low concentrations, and in line with Zhou et al. (2009) we observe loss of pluripotency and induction of mesendoderm at higher concentrations. In contrast to Zhou et al., we found that rapamycin strongly inhibits DE induction. This difference may be serum mediated or cell-line dependent. Additionally, emerging evidence suggests that separate induction mechanisms may confer distinct mesoderm/endoderm subtype potentials (Mendjan et al., 2014). Although both GSK3β and mTOR inhibition appear to enhance mesendoderm, rapamycin-treated hPSCs appear to generate significantly higher frequencies of CD34+VECAD+CD73− HE phenotype Cyanine3.5 carboxylic acid cost compared with CHIR99021 (GSK3β inhibitor)-treated conditions. GSK3β inhibition has been reported to direct hPSC differentiation to blood progenitor (Sturgeon et al., 2014) and cardiac cells (Lian et al., 2012), supporting its role as a driver of lateral plate-derived tissues. Our results indicate that mTOR inhibition is an efficient driver of hPSC differentiation to blood progenitor competent mesoderm. Comparing the mesendoderm subtypes that emerge in GSK3β- and mTOR-inhibited differentiation conditions is an important future direction of our study. The observation that subnanomolar concentrations of rapamycin have a moderate enhancing effect on pluripotency corresponds with findings of He et al. (2012) showing that rapamycin at 0.01 and 0.03 nM enhances mouse somatic cell reprogramming efficiency nearly 2-fold while concentrations of 0.05 nM or greater have no effect or reduce reprogramming efficiency. Autophagy regulates homeostasis of pluripotency-associated transcription factors including OCT4 in hPSCs, with autophagy inhibition increasing transcription factor levels yet leading to differentiation (Cho et al., 2014). Accordingly, low levels of mTOR inhibition may inhibit autophagy and enhance OCT4 levels in hPSCs sufficiently to enhance pluripotency, with higher levels of mTOR inhibition increasing OCT4 levels beyond a threshold to initiate differentiation. Alternatively, given that differentiated cell types such as hPSC-derived extra-embryonic endoderm are known to inhibit pluripotency (Peerani et al., 2007), it may be that rapamycin selectively inhibits these cell types, leading to increased hPSC numbers. Another possibility is that the cell-fate effects of discrete mTOR inhibition levels may be directly mediated by differential SMAD activation, as mTORC1 inhibition activates SMAD1 and SMAD5 in human prostate cancer cells (Wahdan-Alaswad et al., 2012), and mTORC2 inhibition can enhance SMAD2 and SMAD3 activity via regulation of the SMAD2/3-T220/T179 linker residue (Yu et al., 2015). Understanding the complex interplay between discrete levels of mTOR signaling, autophagy, pluripotency transcription factor homeostasis, signaling pathway activation, and effects on cell-fate choice and subpopulation dynamics remains an important direction of future studies. The methods we present here are applicable to systems with heterogeneous subpopulations and complex micro-environmental regulation, such as in vitro stem cell and cancer models, and can be applied iteratively. Changes in micro-environmental cues have been attributed to cell-fate transitions (Bonfanti et al., 2010), and heterogeneity in micro-environmental context (such as population size, local cell density, and relative cell position) can deterministically explain phenotypic variance (Snijder et al., 2009). Improved techniques to control the cellular micro-environment in conjunction with improved single-cell analytics will further develop our understanding of molecular cell-fate regulation.