Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors

Abstract

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.

Substratum-induced differentiation of human pluripotent stem cells reveals the coactivator YAP is a potent regulator of neuronal specification

“…6). YAP activity is decreased during mammalian neurogenesis (19,36,37) in conjunction with Sonic Hedgehog (36,37) and Smad (38) signaling pathways, which are ubiquitous targets of soluble factors used to promote neuronal differentiation of hES cells (39)(40)(41)(42)(43)(44)(45)(46).…”

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

“…This was done by applying critical morphogens which are important for the specification of these neuron types during in vivo development. Other protocols have also been developed to promote the differentiation of hESCs into neurons using either additional factors [18][19][20] such as small molecules or by co-culturing with other cell types to help promote differentiation 21 .…”

Differentiation of Human Embryonic Stem Cells towards a Sensory Neural Phenotype

“…They obtained however less than 2% peripherin positive cells from cultures with bFGF. Our protocol did however not reach the high levels of 40-75% peripherin positive neurons found in hESC culture induced with small molecules (Lee et al, 2010, Chambers et al, 2012. Other reported protocols yielded about 10% peripheral neurons when neural progenitor cells, as starting cell populations, were cultured on PA6 stromal cell feeders, and less than 1% of peripheral neurons when starting from hESC (Pomp et al, 2005, Brokhman et al, 2008.…”

“…However, very few reports focus on peripheral nervous system (PNS) neurons. (Erceg et al, 2009, Chambers et al, 2012, Brokhman et al, 2008, Pomp et al, 2005, Shi et al, 2007.…”