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Stem cell-based approach to identify regulatory TFs during mammalian cell differentiation

ABSTRACT: Cell differentiation is controlled by transcription factors (TFs). However, we still do not fully understand which TFs are needed for even one mammalian differentiation pathway. To address this, we developed a detailed and integrated method. This method combines pluripotent stem cell differentiation in the lab, single-cell transcriptomics, and a CRISPR-based TF loss-of-function screen. Using this approach, we identified all the TFs required for spinal motor neuron (MN) differentiation in mice and their requirement in human MN differentiation. Our transcriptomic analysis revealed that while over 1,300 TFs are transcribed during ventral spinal cord differentiation, only 55 have annotated functions in the literature. The screen identified 232 TFs and other transcription-regulating genes essential for the progression of ventral spinal cord differentiation, including 116 TFs specifically required for MN differentiation. This approach led to three critical observations. First, the motor neuron progenitor (pMN) stage is a regulatory hub that correlates with a reduction in cell potential. Second, a secondary screen in human ventral spinal cord differentiation revealed a greater degree of conservation j between mouse pMNs and the human-specific ventral MN progenitors (vpMNs). This finding was unexpected, as the conservation was anticipated to be with the canonical human pMN stage. Third, the screen underscored the importance of the numerous and yet understudied ZF TFs controlling mammalian cell differentiation. In summary, we present a universal, unbiased strategy for studying TFs in mammalian differentiation, comparable to methodologies used in simpler organisms. This approach enhances our understanding of MN differentiation and provides a practical framework to identify all TFs required for any differentiation trajectory that can be recapitulated in vitro, offering many applications in various fields of developmental biology.

ORGANISM(S): Mus musculus

PROVIDER: GSE285057 | GEO | 2025/09/01

REPOSITORIES: GEO

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Project description:SMARD1 is an infantile autosomal recessive motor neuron (MN) disease, caused by mutations in the Immunoglobulin mu binding protein 2 (IGHMBP2). We investigated the potential of a spinal cord neural stem cell population isolated on the basis of aldehyde dehydrogenase activity (ALDH) to modify disease progression of nmd mice, an animal model of SMARD1. ALDHhiSSClo stem cells are self-renewing and multipotent and when intratechally transplanted in nmd mice generate motor neurons properly localized in the spinal cord ventral horns. Transplanted nmd animals presented delayed disease progression, sparing of motor neurons and ventral root axons and increased life-span. To further investigate the molecular events responsible for these differences, microarray and Real time RT-PCR analysis of wild-type, mutated and transplanted nmd spinal cord were undertaken. We demonstrated a down-regulation of genes involved in excitatory amino acid toxicity and oxidative stress handling, as well as an up-regulation of genes related to the chromatin organization in nmd compared to wild-type mice, suggesting that they may play a role in SMARD1 pathogenesis. Spinal cord of nmd transplanted mice expressed high transcript levels for genes related to neurogenesis such as Doublecortin (DCX), LIS1 and drebrin. The presence of DCX-expressing cells in adult nmd spinal cord suggests that both exogenous and endogenous neurogenesis may contribute to the observed nmd phenotype amelioration. Experiment Overall Design: We intratechally transplanted a spinal cord neural stem cell population isolated on the basis of aldehyde dehydrogenase activity (ALDH) in nmd mice, an animal model of SMARD1. Experiment Overall Design: We analyzed the lumbar spinal cord tract of transplanted nmd mice (n=3), untransplanted nmd mice (n=3) and wild-type mice (n=3) (all mice are of male gender). The latter groups underwent surgical procedure with vehicle. The surgical procedure and cell transplantation were done at P1 and the animals were sacrificed at 6 weeks of age.

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Stem cell-based approach to identify regulatory TFs during mammalian cell differentiation

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