Human embryonic (ESC) and induced pluripotent stem cells (iPSC) present exciting opportunities for studying development and in vitro disease modeling. However, reported variability in iPSC behavior has called their utility into question. We therefore constituted a test set of 16 iPSCs lines from 7 individuals of varying gender and health status, characterized them extensively for pluripotency, and evaluated their ability to terminally differentiate. Using standardized procedures in two independent laboratories, 13 of the iPSC lines gave rise to functional motor neurons with a range of efficiencies similar to ESCs. Although three iPSC lines were resistant to neural differentiation, early neuralization rescued their performance. Therefore, all lines in the test set passed a stringent test of differentiation capacity despite variations in expression of early pluripotency markers, transgenes and karyotype. This novel iPSC/ESC test set is a robust resource for those interested in the basic biology of stem cells and their applications.

Human pluripotent stem cells are a promising source of differentiated cells for developmental studies, cell transplantation, disease modeling, and drug testing. However, their widespread use even for intensely studied cell types like spinal motor neurons is hindered by the long duration and low yields of existing protocols for in vitro differentiation and by the molecular heterogeneity of the populations generated. We report a combination of small molecules that within 3 weeks induce motor neurons at up to 50% abundance and with defined subtype identities of relevance to neurodegenerative disease. Despite their accelerated differentiation, motor neurons expressed combinations of HB9, ISL1 and column-specific markers that mirror those observed in vivo in human fetal spinal cord. They also exhibited spontaneous and induced activity, and projected axons towards muscles when grafted into developing chick spinal cord. Strikingly, this novel protocol preferentially generates motor neurons expressing markers of limb-innervating lateral motor column motor neurons (FOXP1+/LHX3−). Access to high-yield cultures of human limb-innervating motor neuron subtypes will facilitate in-depth study of motor neuron subtype-specific properties, disease modeling, and development of large-scale cell-based screening assays.

Efficient transcriptional programming promises to open new frontiers in regenerative medicine. However, mechanisms by which programming factors transform cell fate are unknown, preventing more rational selection of factors to generate desirable cell types. Three transcription factors, Ngn2, Isl1 and Lhx3, were sufficient to program rapidly and efficiently spinal motor neuron identity when expressed in differentiating mouse embryonic stem cells. Replacement of Lhx3 by Phox2a led to specification of cranial, rather than spinal, motor neurons. Chromatin immunoprecipitation–sequencing analysis of Isl1, Lhx3 and Phox2a binding sites revealed that the two cell fates were programmed by the recruitment of Isl1-Lhx3 and Isl1-Phox2a complexes to distinct genomic locations characterized by a unique grammar of homeodomain binding motifs. Our findings suggest that synergistic interactions among transcription factors determine the specificity of their recruitment to cell type–specific binding sites and illustrate how a single transcription factor can be repurposed to program different cell types.

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Neuronal maturation requires dramatic morphological and functional changes, but the molecular mechanisms governing this process are not well understood. Here, we studied the role of Rbfox1, Rbfox2, and Rbfox3 proteins, a family of tissue-specific splicing regulators mutated in multiple neurodevelopmental disorders. We generated Rbfox triple knockout (tKO) ventral spinal neurons to define a comprehensive network of alternative exons under Rbfox regulation and to investigate their functional importance in the developing neurons. Rbfox tKO neurons exhibit defects in alternative splicing of many cytoskeletal, membrane, and synaptic proteins, and display immature electrophysiological activity. The axon initial segment (AIS), a subcellular structure important for action potential initiation, is diminished upon Rbfox depletion. We identified an Rbfox-regulated splicing switch in ankyrin G, the AIS "interaction hub" protein, that regulates ankyrin G-beta spectrin affinity and AIS assembly. Our data show that the Rbfox-regulated splicing program plays a crucial role in structural and functional maturation of postmitotic neurons.