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Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder caused by mutations in genes encoding components of the primary cilium and is characterized by hyperphagic obesity. To investigate the molecular basis of obesity in human BBS, we developed a cellular model of BBS using induced pluripotent stem cell-derived (iPSC-derived) hypothalamic arcuate-like neurons. BBS mutations [BBS1.sup.M390R] and BBS[10.sup.C91fsX95] did not affect neuronal differentiation efficiency but caused morphological defects, including impaired neurite outgrowth and longer primary cilia. Single-cell RNA sequencing of [BBS1.sup.M390R] hypothalamic neurons identified several downregulated pathways, including insulin and cAMP signaling and axon guidance. Additional studies demonstrated that [BBS1.sup.M390R] and BBS[10.sup.C91fsX95] mutations impaired insulin signaling in both human fibroblasts and iPSC-derived neurons. Overexpression of intact BBS10 fully restored insulin signaling by restoring insulin receptor tyrosine phosphorylation in BBS[10.sup.C91fsX95] neurons. Moreover, mutations in BBS1 and BBS10 impaired leptin-mediated p-STAT3 activation in iPSC-derived hypothalamic neurons. Correction of the BBS mutation by CRISPR rescued leptin signaling. POMC expression and neuropeptide production were decreased in [BBS1.sup.M390R] and BBS[10.sup.C91fsX95] iPSC-derived hypothalamic neurons. In the aggregate, these data provide insights into the anatomic and functional mechanisms by which components of the BBSome in CNS primary cilia mediate effects on energy homeostasis., Introduction Bardet-Biedl syndrome (BBS) is an autosomal recessive disorder characterized by severe, early-onset obesity, as well as an array of other diagnostic features typical of ciliopathies including polydactyly, retinal degeneration, [...]

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder caused by mutations in genes encoding components of the primary cilium and is characterized by hyperphagic obesity. To investigate the molecular basis of obesity in human BBS, we developed a cellular model of BBS using induced pluripotent stem cell-derived (iPSC-derived) hypothalamic arcuate-like neurons. BBS mutations BBS1M390R and BBS10C91fsX95 did not affect neuronal differentiation efficiency but caused morphological defects, including impaired neurite outgrowth and longer primary cilia. Single-cell RNA sequencing of BBS1M390R hypothalamic neurons identified several downregulated pathways, including insulin and cAMP signaling and axon guidance. Additional studies demonstrated that BBS1M390R and BBS10C91fsX95 mutations impaired insulin signaling in both human fibroblasts and iPSC-derived neurons. Overexpression of intact BBS10 fully restored insulin signaling by restoring insulin receptor tyrosine phosphorylation in BBS10C91fsX95 neurons. Moreover, mutations in BBS1 and BBS10 impaired leptin-mediated p-STAT3 activation in iPSC-derived hypothalamic neurons. Correction of the BBS mutation by CRISPR rescued leptin signaling. POMC expression and neuropeptide production were decreased in BBS1M390R and BBS10C91fsX95 iPSC-derived hypothalamic neurons. In the aggregate, these data provide insights into the anatomic and functional mechanisms by which components of the BBSome in CNS primary cilia mediate effects on energy homeostasis. [ABSTRACT FROM AUTHOR]

The hypothalamus, composed of several nuclei, is essential for maintaining our body's homeostasis. The arcuate nucleus (ARC), located in the mediobasal hypothalamus, contains neuronal populations with eminent roles in energy and glucose homeostasis as well as reproduction. These neuronal populations are of great interest for translational research. To fulfill this promise, we used a robotic cell culture platform to provide a scalable and chemically defined approach for differentiating human pluripotent stem cells (hPSCs) into pro-opiomelanocortin (POMC), somatostatin (SST), tyrosine hydroxylase (TH) and gonadotropin-releasing hormone (GnRH) neuronal subpopulations with an ARC-like signature. This robust approach is reproducible across several distinct hPSC lines and exhibits a stepwise induction of key ventral diencephalon and ARC markers in transcriptomic profiling experiments. This is further corroborated by direct comparison to human fetal hypothalamus, and the enriched expression of genes implicated in obesity and type 2 diabetes (T2D). Genome-wide chromatin accessibility profiling by ATAC-seq identified accessible regulatory regions that can be utilized to predict candidate enhancers related to metabolic disorders and hypothalamic development. In depth molecular, cellular, and functional experiments unveiled the responsiveness of the hPSC-derived hypothalamic neurons to hormonal stimuli, such as insulin, neuropeptides including kisspeptin, and incretin mimetic drugs such as Exendin-4, highlighting their potential utility as physiologically relevant cellular models for disease studies. In addition, differential glucose and insulin treatments uncovered adaptability within the generated ARC neurons in the dynamic regulation of POMC and insulin receptors. In summary, the establishment of this model represents a novel, chemically defined, and scalable platform for manufacturing large numbers of hypothalamic arcuate neurons and serves as a valuable resource for modeling metabolic and reproductive disorders.

Mouse embryonic stem cells (mESCs) are pluripotent cells capable of differentiating in vitro to form the ~200 types of cells of the developing embryo and adult, including cells of the nervous system. This makes mESCs a useful tool for studying the molecular mechanisms of mammalian embryonic development. Many protocols involving the use of growth factors and small molecules to differentiate mESCs into neural progenitors and neurons currently exist. However, there is a paucity of protocols available that recapitulate the developmental process. Our laboratory has developed a protocol to recapitulate mammalian neural lineage development by differentiating mESCs to mature neurons via intermediate cell populations observed during in vivo embryo development. This protocol uses the amino acid L-proline to direct the differentiation of mESCs, grown as embryoid bodies, into Sox1 + neurectoderm, followed by differentiation to form Nestin + , BLBP + , and NeuN + neural cell types.