The hypothalamus coordinates various physiological processes, including food intake and energy expenditure, that are essential for energy homeostasis. Alterations in hypothalamic pathways disrupt energy balance, resulting in extreme body weight alterations. A lack of access to human hypothalamic neurons has hampered in depth evaluation of the neuro-molecular mechanisms that lead to metabolic phenotypes, such as obesity. Liheng Wang and colleagues at Columbia University developed a protocol for efficient generation of human hypothalamic-like neurons from embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) obtained from both healthy individuals and patients with monogenic forms of obesity. Human ESCs were successfully converted into NKX2.1+ hypothalamic progenitors through early activation of sonic hedgehog signaling plus dual inhibition of SMADs, followed by inhibition of NOTCH signaling. RNA-seq analysis confirmed that the differentiated cells express a hypothalamic transcriptional profile. Importantly, biochemical analysis revealed that these hypothalamic-like neurons were able to sense and respond to the metabolic hormones such as insulin and leptin. The results of this study provide a resource for further evaluation of hypothalamic-pathways in the control of human body weight. The accompanying image shows hypothalamic neurons that were differentiated from human pluripotent stem cells. These cells express neuropeptides that are relevant for body weight homeostasis, including POMC (red) and NPY (green). Nuclei are stained blue with hoechst.
The hypothalamus is the central regulator of systemic energy homeostasis, and its dysfunction can result in extreme body weight alterations. Insights into the complex cellular physiology of this region are critical to the understanding of obesity pathogenesis; however, human hypothalamic cells are largely inaccessible for direct study. Here, we developed a protocol for efficient generation of hypothalamic neurons from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) obtained from patients with monogenetic forms of obesity. Combined early activation of sonic hedgehog signaling followed by timed NOTCH inhibition in human ESCs/iPSCs resulted in efficient conversion into hypothalamic NKX2.1+ precursors. Application of a NOTCH inhibitor and brain-derived neurotrophic factor (BDNF) further directed the cells into arcuate nucleus hypothalamic-like neurons that express hypothalamic neuron markers proopiomelanocortin (POMC), neuropeptide Y (NPY), agouti-related peptide (AGRP), somatostatin, and dopamine. These hypothalamic-like neurons accounted for over 90% of differentiated cells and exhibited transcriptional profiles defined by a hypothalamic-specific gene expression signature that lacked pituitary markers. Importantly, these cells displayed hypothalamic neuron characteristics, including production and secretion of neuropeptides and increased p-AKT and p-STAT3 in response to insulin and leptin. Our results suggest that these hypothalamic-like neurons have potential for further investigation of the neurophysiology of body weight regulation and evaluation of therapeutic targets for obesity.
Liheng Wang, Kana Meece, Damian J. Williams, Kinyui Alice Lo, Matthew Zimmer, Garrett Heinrich, Jayne Martin Carli, Charles A. Leduc, Lei Sun, Lori M. Zeltser, Matthew Freeby, Robin Goland, Stephen H. Tsang, Sharon L. Wardlaw, Dieter Egli, Rudolph L. Leibel