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Human pluripotent stem cells (hPSCs) are a powerful tool for disease modeling of hard-to-access tissues (such as the brain). Current protocols either direct neuronal differentiation with small molecules or use transcription-factor-mediated programming. In this study, we couple overexpression of transcription fac-tor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced motor neurons (liMoNes/liMNs). This approach induces canonical MN markers including MN-specific Hb9/ MNX1 in more than 95% of cells. liMNs resemble bona fide hPSC-derived MN, exhibit spontaneous elec-trical activity, express synaptic markers, and can contact muscle cells in vitro. Pooled, multiplexed sin-gle-cell RNA sequencing on 50 hPSC lines reveals reproducible populations of distinct subtypes of cer-vical and brachial MNs that resemble their in vivo, embryonic counterparts. Combining small molecule patterning with Ngn2 overexpression facilitates high-yield, reproducible production of disease-relevant MN subtypes, which is fundamental in propelling our knowledge of MN biology and its disruption in dis-ease.

Recent genetic discoveries in schizophrenia have highlighted neuronal genes with functions in the synapse. Although emblematic of neurons, the development of synapses and neuronal maturation relies on interactions with glial cells including astrocytes. To study the role of glia-neuron interactions in schizophrenia, we generated RNA sequence data from human pluripotent stem cell (hPSC) derived neurons that were cocultured with glial cells. We found that expression of genes characteristic of astrocytes induced the expression of post-synaptic genetic programs in neurons, consistent with advanced neuronal maturation. We further found that the astrocyte-induced genes in neurons were associated with risk for schizophrenia. To understand how glial cells promoted neuronal maturation, we studied the association of transcript abundances in glial cells to gene expression in neurons. We found that expression of synaptic cell adhesion molecules in glial cells corresponded to induced synaptic transcripts in neurons and were associated with genetic risk for schizophrenia. These included 11 genes in significant GWAS loci and three with direct genetic evidence for the disorder (MAGI2, NRXN1, LRRC4B, and MSI2). Our results suggest that astrocyte-expressed genes with functions in the synapse are associated with schizophrenia and promote synaptic genetic programs in neurons, and further highlight the potential role for astrocyte-neuron interactions in schizophrenia.

Direct neuronal conversion describes the process of generating induced neurons from somatic cells such as fibroblasts by overexpressing cell type-specific transcription factors, microRNAs or by culturing in the presence of small molecules. This was first achieved by expressing Brn2, Ascl1 and Myt1L in mouse fibroblasts, and was later achieved in human cells by the inclusion of additional factors such as NeuroD1. Here, we present the first protocol for directly converting porcine fibroblasts into induced neurons. We used lentivirus-mediated delivery of previously identified neuron-specifying transcription factors and microRNAs and evaluated morphology and neuron marker expression after ten days of conversion. We found that Ascl1 and microRNAs, miR-9/9* and miR-124 together generated more neuronal cells than other conditions tested. The porcine induced neurons expressed common mature markers such as MAP2 and Synaptophysin after four weeks of conversion. Transcriptomic analysis revealed that fibroblast-specific signatures were silenced early in the conversion process, while the neuron-specific genes became more abundant during conversion. We generated a heterogeneous population of glutamatergic and GABAergic neurons.