Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes

Abstract

Specification of cell identity during development depends on exposure of cells to sequences of extrinsic cues delivered at precise times and concentrations. Identification of combinations of patterning molecules that control cell fate is essential for the effective use of human pluripotent stem cells (hPSCs) for basic and translational studies. Here we describe a scalable, automated approach to systematically test the combinatorial actions of small molecules for the targeted differentiation of hPSCs. Applied to the generation of neuronal subtypes, this analysis revealed an unappreciated role for canonical Wnt signaling in specifying motor neuron diversity from hPSCs and allowed us to define rapid (14 days), efficient procedures to generate spinal and cranial motor neurons as well as spinal interneurons and sensory neurons. Our systematic approach to improving hPSC-targeted differentiation should facilitate disease modeling studies and drug screening assays.

SMN deficiency in severe models of spinal muscular atrophy causes widespread intron retention and DNA damage

“…The pluripotency of the hiPSCs was confirmed by staining for pluripotency markers and in vitro differentiation into three germ layers. Differentiation of hiPSCs into motor neuron cultures was carried out as described previously (72), with slight modifications. In brief, hiPSC colonies were dissociated with EDTA and placed in suspension in low-adhesion flasks (Corning) to form embryoid bodies in medium consisting of DMEM/F12/ Neurobasal medium (Life Technologies), N2 supplement (Life Technologies), B27 supplement (Life Technologies), ascorbic acid (20 μM), and β-mercaptoethanol (0.055 mM).…”

Functional characterization of human pluripotent stem cell-derived arterial endothelial cells

“…Cell fate is determined by the temporal exposure to combinatorial developmental cues (21). Therefore, we examined the combinatorial effect of these factors to further improve AEC differentiation.…”

Is this a brain which I see before me? Modeling human neural development with pluripotent stem cells

“…Similarly, spinal cord identity can be induced through the combinatorial addition of the 'caudal morphogens' RA, Wnts and FGFs (Amoroso et al, 2013;Boulting et al, 2011;Dimos et al, 2008;Li et al, 2005;Maury et al, 2015;Takazawa et al, 2012;Wichterle et al, 2002), again similar to the mechanisms operating in vivo ( appear to be derived from a common class of transient precursors called neuromesodermal progenitors (NMPs) (Brown and Storey, 2000;Gouti et al, 2015;Tzouanacou et al, 2009), which can be specified into neural or mesodermal fates (Chapman et al, 1996;Chapman and Papaioannou, 1998;Takemoto et al, 2011). This in vivo developmental scheme can be observed in PSC-derived models, where NMP-like cells can be derived from PSCs using Wnt and FGF ligands (Gouti et al, 2014;Turner et al, 2014).…”