Differentiation of hypothalamic-like neurons from human pluripotent stem cells
Liheng Wang, … , Dieter Egli, Rudolph L. Leibel
Liheng Wang, … , Dieter Egli, Rudolph L. Leibel
Published January 2, 2015
Citation Information: J Clin Invest. 2015;125(2):796-808. https://doi.org/10.1172/JCI79220.
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Technical Advance Stem cells

Differentiation of hypothalamic-like neurons from human pluripotent stem cells

Abstract

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.

Authors

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

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Figure 6

Efficient generation of hypothalamic ARC-like neurons from iPSCs generated from BBS obese subjects.

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Efficient generation of hypothalamic ARC-like neurons from iPSCs generat...
(A) Quantification of the percentage of NKX2.1+ cells in day-12 progenitors derived from 2 control iPSC lines (control 1, control 2) and 3 BBS iPSC lines (BBS1A, BBS1B, BBS10B). n = 5 wells for each bar. (B) Immunostaining of day-25 and day-43 iPSC-derived neurons. Scale bars: 100 μm. (C) Quantification of the percentage of POMC neurons (n = 3 to 5 wells). (D) Heat map of the expression of hypothalamic TFs and neuronal markers in day-27 hESC-derived and day-45 iPSC-derived neurons based on the FPKM value from RNA-seq. Red color indicates high expression; green color indicates low expression. Action potentials in day-30 control iPS–derived and day-35 BBS iPS–derived neurons. (E and F) Quantification of secreted neuropeptides as indicated on day 45 of differentiation. The volume of each sample was 1 ml. n = 3 for each bar. (G) Western blot analysis of insulin signaling in day-34 hESC-derived and in iPSC-derived neurons.