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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

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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 3

In vitro hESC–differentiated neurons show hypothalamic-specific gene signatures.

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In vitro hESC–differentiated neurons show hypothalamic-specific gene sig...
(A) Heat map of 1 matrix clustering of hypothalamic- and pituitary-specific transcripts in undifferentiated hESCs on indicated days of differentiation. Green and red indicate low and high expression, respectively. (B) qPCR analysis of indicated factors in hESCs and of day-12– and day-24–differentiated cells (n = 3) and adult human hypothalamus. (C) Immunohistochemistry for prolactin and BEP in day-33 hESC-derived neurons. (D) Gene expression of hypothalamic and pituitary TFs from RNA-seq of hESCs (n = 1) and day-12 progenitors (n = 3).
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