Selective targeting of cancer stem cells (CSCs) offers promise for a new generation of therapeutics. However, assays for both human CSCs and normal stem cells that are amenable to robust biological screens are limited. Using a discovery platform that reveals differences between neoplastic and normal human pluripotent stem cells (hPSC), we identify small molecules from libraries of known compounds that induce differentiation to overcome neoplastic self-renewal. Surprisingly, thioridazine, an antipsychotic drug, selectively targets the neoplastic cells, and impairs human somatic CSCs capable of in vivo leukemic disease initiation while having no effect on normal blood SCs. The drug antagonizes dopamine receptors that are expressed on CSCs and on breast cancer cells as well. These results suggest that dopamine receptors may serve as a biomarker for diverse malignancies, demonstrate the utility of using neoplastic hPSCs for identifying CSC-targeting drugs, and provide support for the use of differentiation as a therapeutic strategy.

Human embryonic stem cells (hESC) have potential applications as tools for drug screening to identify small molecule regulators of self-renewal or differentiation. Elucidating the mechanisms governing lineage commitment in hESC will allow for efficient derivation of specified cell types for clinical use. Recognizing the early steps in loss of pluripotency is key to achieving both goals of drug screening and derivation of therapeutically relevant cell types. Here we report the use of a real time cell cycle fluorescent reporter for the first time in hESC that indicates onset of differentiation in a lineage unbiased manner. Pluripotent hESC possess a short cell cycle length, due primarily to a truncated G1 phase. G1 lengthens concomitant with differentiation. Stable hESC lines expressing the live cell cycle reporter exhibit fluorescence only during G1. Due to the short length of pluripotent G1 phase, G1 fluorescence is only weakly and transiently detected, however it is quickly increased to easily detectable levels upon onset of differentiation. We hypothesize that lengthened G1 phase can be used as an indicator of differentiation status of individual human embryonic stem cells. Cells with lengthened G1 are typically negative for pluripotency markers OCT4, Tra-1-60 and SSEA-3 following differentiation. Differentiated cells with lengthened G1 also demonstrate increased levels of lineage-specific differentiation markers at both the protein and mRNA level. Automated image analysis of hESC indicates this mutually exclusive relationship between lengthened G1 and pluripotency exists both on the cellular level and in colonies as a whole. Here we have shown that lengthened G1 indicates both loss of pluripotency and gain of lineage markers.

The clinical applicability of direct cell fate conversion depends on obtaining tissue from patients that is easy to harvest, store, and manipulate for reprogramming. Here, we generate induced neural progenitor cells (iNPCs) from neonatal and adult peripheral blood using single-factor OCT4 reprogramming. Unlike fibroblasts that share molecular hallmarks of neural crest, OCT4 reprogramming of blood was facilitated by SMAD+GSK-3 inhibition to overcome restrictions on neural fate conversion. Blood-derived (BD) iNPCs differentiate in vivo and respond to guided differentiation in vitro, producing glia (astrocytes and oligodendrocytes) and multiple neuronal subtypes, including dopaminergic (CNS related) and nociceptive neurons (peripheral nervous system [PNS]). Furthermore, nociceptive neurons phenocopy chemotherapy-induced neurotoxicity in a system suitable for high-throughput drug screening. Our findings provide an easily accessible approach for generating human NPCs that harbor extensive developmental potential, enabling the study of clinically relevant neural diseases directly from patient cohorts.

Human embryonic stem cells (hESCs) expressing pluripotency markers are assumed to possess equipotent developmental potential. However, disparate responses to differentiation stimuli functionally illustrate that hESCs generate a spectrum of differentiated cell types, suggestive of lineage bias. Here, we reveal specific cell surface markers that allow subfractionation of hESCs expressing hallmark markers of pluripotency. By direct de novo isolation of these subsets, we demonstrate that propensities for lineage differentiation are balanced with reduced clonogenic self-renewal. Histone modification marks of gene loci associated with pluripotency versus lineage specificity predicted cell fate potential of these subfractions, thereby supporting the absence of uniform bivalency as a molecular paradigm to describe cell fate determination of pluripotent cells. Our study reveals that cell fate potential is encoded within cells comprising hESC cultures, highlighting them as a means to understand the mechanisms of lineage specification of pluripotent cells.

Human pluripotent stem cells (hPSCs) have been reported in naive and primed states. However, the ability to generate mature cell types remains the imperative property for utility of hPSCs. Here, we reveal that the naive state enhances self-renewal while restricting lineage differentiation in vitro to neural default fate. Molecular analyses indicate expression of multiple lineage-associated transcripts in naive hPSCs that failed to predict biased functional differentiation capacity. Naive hPSCs can be converted to primed state over long-term serial passage that permits recovery of multi-germ layer differentiation. Suppression of OCT4 but not NANOG allows immediate recovery directly from naive state. To this end, we identified chemical inhibitors of OCT4 that restore naive hPSC differentiation. Our study reveals unique cell-fate restrictions in human pluripotent states and provides an approach to overcome these barriers that harness both efficient naive hPSC growth while maintaining in vitro differentiation essential for hPSC applications.