br Acknowledgments The study is supported by funds to

Acknowledgments The study is supported by funds to W-Q.G. from the Chinese Ministry of Science and Technology (2012CB966800, 2013CB945600, and 2012CB967903), the National Natural Science Foundation of China (81130038 and 81372189), the Science and Technology Commission of Shanghai Municipality (Pujiang program), the Shanghai Education Committee Key Discipline and Specialty Foundation (J50208), the Shanghai Health Bureau Key Discipline and Specialty Foundation, and the KC Wong foundation.
Introduction Human pluripotent stem clofibrate (hPSCs) and their differentiated derivatives offer the exciting opportunity to develop tools to study and treat human diseases. However, robust and reproducible control of hPSC fate remains challenging. Small molecules offer one approach to control hPSC fate, and the discovery and characterization of these compounds can be facilitated by cell-based phenotypic high-throughput screening (HTS). Emerging data from hPSC assays has revealed variable and contradictory observations, even with matched cell lines and protocols (Haibe-Kains et al., 2013). Although the factors underlying this variability are not completely known, population context has been identified as a main contributor to assay inconsistency (Snijder et al., 2012). Spatially heterogeneous (Peerani et al., 2007) micro-environmental factors such as endogenous ligands, extra-cellular matrix proteins (ECMPs), and cell subpopulations are strong regulators of hPSC fate. Specifically, spatial cell distribution has been shown to affect hPSC self-renewal (Maherali and Hochedlinger, 2008), differentiation trajectories in both normal and patient-derived cells (Cai et al., 2009; Chambers et al., 2009), and disease phenotypes (Sun et al., 2012). Consequently, robust assays that combine defined culture conditions with comprehensive analysis of cell responses to exogenous cues are needed. To this end, we developed a chemically defined cell patterning-based high-throughput (HTP) assay, engineering colony size, local cell density, medium composition, and substrate for rapid and robust measurement of hPSC fate responses to exogenous cues (Nazareth et al., 2013). We applied the assay to screen a library of kinase inhibitors for effects on four early hPSC fates. For each compound, the change in yield and purity in the resulting pluripotent, neuroectoderm (NE), mesendoderm, and extra-embryonic populations were simultaneously tracked, allowing for estimation of selection and induction events. Our analysis identified mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin, as having a strong mesendoderm-inducing effect on hPSCs. Rapamycin was subsequently shown to synergize with bone morphogenetic protein 4 (BMP4) and activin A to enhance BRACHYURY induction more than 3-fold, an effect that propagated to equivalent enhancements of hemogenic endothelium and blood progenitor cells. This study demonstrates the advantages of controlling micro-environmental parameters and measuring multiple subpopulation outputs in parallel on PSC fate screening assays. This strategy should enhance discovery in more complex and predictive multi-cell population drug-screening assays.
Results
Discussion We have applied an engineered micro-environment based HTS platform to screen a kinase inhibitor library simultaneously for regulators of PSCs, NE, and mesendoderm. Small-molecule control of cell fate is attractive for scale-up purposes, and a focused library could be instrumental in elucidating endogenous regulators of hPSC fate decisions. In line with a previous screen (Desbordes et al., 2008), we found no single compound able to maintain long-term pluripotency, indicating that exogenous pathway agonists or possibly combinations of small-molecule inhibitors may be required for hPSC maintenance. Our multi-lineage readout enabled the discovery of specific pathways endogenously activated in hPSCs amenable to small-molecule control of lineage-specific differentiation. Patterning enabled a more rapid (48 hr versus 7 days) assay and robust response than non-pattern-based screens (Desbordes et al., 2008). Additionally, the short assay duration limits the number of emergent subpopulations, allowing simultaneous detection of these subpopulations to be tracked by OCT4 and SOX2 costaining. In conjunction with cell-number readouts, selection and induction events can be reasonably discriminated.