Investigations of the neuro-molecular physiology of obesity using hypothalamic neurons derived from human pluripotent stem cells

My thesis research focused on establishing an in vitro model for understanding the molecular neurophysiology of obesity using, as "proof-of-principle", neurons derived from human pluripotent stem cells (hPSCs) derived from individuals with monogenic forms of obesity. Three related projects are described in details:;I. Differentiation of hypothalamic-like neurons from human pluripotent stem cells (Chapter 2) .;This project was designed to establish an in vitro model for studying hypothalamic cell-molecular physiology in neurons derived from hPSCs. After screening several morphogens and other molecules affecting neuronal differentiation, we developed a protocol that combined early activation of sonic hedgehog signaling followed by timed NOTCH inhibition resulting in the generation of hypothalamic arcuate nucleus (ARC)-like neurons. Neuronal cells expressing pro-opiomelanocortin (POMC), neuropeptide-Y/agouti-related protein (NPY/AgRP) were generated from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) obtained from patients with monogenic forms of obesity. These hypothalamic-like neurons accounted for over 90% of differentiated cells and exhibited transcriptional profiles characteristic of specific hypothalamic neurons (and explicitly lacking pituitary markers). Importantly, these cells displayed hypothalamic neuronal characteristics, including production and secretion of neuropeptides and responsiveness to metabolic hormones such as insulin and leptin.;II. Using stem cell-derived hypothalamic neurons to investigate the neurophysiology of obesity caused by prohormone convertase 1/3 deficiency (Chapter 3)..;My second project investigated the use the hPSC-differentiated hypothalamic neurons to assess the cellular physiology of hESC-derived hypothalamic neurons with induced knockdown or mutations of proprotein convertase subtilisin/kexin type 1 (PCSK1, encodes prohormone covertase 1/3 (PC1/3)). To understand the molecular neurophysiology of the obesity in PC1/3-deficient subjects, we generated PCSK1 deficient hESC lines with CRISPR or by knocking down PCSK1 with shRNA, and assessed the POMC processing in the hypothalamic ARC-like neurons made from these lines. The molecular mechanisms underlying the invocation of these possibly compensatory processes are under study. The findings provide confidence that the hypothalamic neurons generated by the techniques described in Chapter 2 display molecular phenotypes consistent with a mutation in one of the important neuropeptide processing pathways.;III. Using iPSC-derived neurons to investigate the molecular pathogenesis of obesity in Bardet-Biedl Syndrome (Chapter 4)..;To further investigate the use of iPSC-derived neurons in the study of the neurobiology of obesity, I analyzed structural and molecular physiologic phenotypes cells derived from patients with Bardet-Biedl Syndrome (BBS). BBS is a rare autosomal recessive disease characterized by multiorgan dysfunction, including polydactyly, hyperphagic obesity, retinal degeneration, renal cysts and cognitive impairments. Eighteen discrete genes have been implicated in specific instances of BBS, and all cognate proteins that have been identified encode constituents of the basal body of the primary cilium. The primary cilium has also been implicated in other clinical obesities, including the Alstrom syndrome, and the effects of a highly prevalent FTO allele on body weight. We found that ciliogenesis and neurite outgrowth were affected in both BBS1 and BBS10 mutant iPSC-derived neurons as reflected by longer primary cilia, shorter neurite length, and fewer processes. Furthermore, insulin-induced AKT phosphorylation at Thr308 was greatly reduced in both BBS1 and BBS10 mutant neurons compared to controls. These data demonstrate that BBS proteins are essential for insulin and leptin signaling in neurons and fibroblasts, in a cellular context independent of the effects of obesity. (Abstract shortened by UMI.).