High-throughput Analysis and Control of the Pluripotent Stem Cell Microenvironment to Interrogate and Direct Cell Fate

Human pluripotent stem cells (hPSCs) are heralded to transform regenerative medicine and drug discovery and development, as they can potentially supply a scalable source of clinically relevant cell populations. Numerous exogenous and endogenous factors interplay to control stem cell fate; however, these are often unrecognized and unreported, undermining reproducibility and precluding the systematic analysis of hPSC response to molecular and chemical perturbations. We have developed a high-throughput (HTP) platform to screen hPSCs in configurable micro-environments, in which we optimized parameters including colony size and cell density to achieve rapid and robust cell fate responses to cues. We used this platform to perform single-cell protein expression profiling, revealing that Oct4 and Sox2 co-staining discriminate pluripotent, neuroectoderm, primitive streak, and extraembryonic cell fates. We applied this code to analyze dose responses of 27 developmental factors to delineate lineage-specific concentration optima and to quantify cell-line endogenous signaling pathway activation and differentiation bias. We additionally demonstrate that response profiles can classify cell lines by similarity, are stable through passages, predict definitive endoderm induction efficiency and can be used to rescue differentiation of cell-lines reticent to cardiac induction. We next applied this platform to screen 400 small molecule kinase inhibitors, measuring resultant yield and purity of multiple lineages on a per colony basis. Enrichment analysis revealed mTOR inhibitors as strong inducers of mesendoderm cells. This mesendoderm enhancing effect of mTOR inhibitors is blocked by TGFβ or BMP receptor inhibition, and knockdown of Raptor, a mTORC1 component, via siRNA phenocopied the effects of small molecule mTOR inhibitors such as rapamycin. When rapamycin was added to a blood differentiation protocol, CD34+VECAD+ hemogenic endothelium purity and yield were increased 3 fold, with a concomitant enhancement of blood colony forming cells (CFCs). Lastly, to aid translation of these approaches, we validate a protocol of our 96-well hPSC assay in multiple international laboratories via the International Stem Cell Initiative (ISCI) consortium and provide open-source GUI-based software for analysis of spatial context in HTP studies. Ultimately, these technologies and applications enable a more comprehensive elucidation of the regulatory networks linking cell-extrinsic factors, signal transduction, transcription factor regulation, and cell fate decisions.Ph.D.2017-02-12 00:00:0