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 5

hESC-derived hypothalamic ARC-like neurons sense metabolic signals insulin and leptin.

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hESC-derived hypothalamic ARC-like neurons sense metabolic signals insul...
(A) Western blot analysis of insulin-treated (1 μg/ml) day-33 hESC-derived hypothalamic neurons. Antibodies for p-AKT (Thr308) and p-AKT (Ser473) were probed. (B) Inhibition of insulin signaling by AKTi decreases the production of neuropeptides in day-38 hESC-derived POMC neurons. Total neuropeptide is the sum of the quantities of neuropeptides in the medium and neural lysates. n = 3 for each bar. (C) Transcript levels of LEPR in hESCs and 12, 18, 24, 36, and 40 days of differentiation (n = 3). (D) Western blot analysis of leptin-treated (1 μg/ml) day-33 hESC-derived hypothalamic neurons. p-STAT3 and STAT3 were probed. (E) Immunocytochemistry for p-STAT3 and αMSH in day-53 hESC-derived hypothalamic neurons cocultured with mouse cortical astrocytes. Rectangle indicates 2 subtypes of POMC neurons: leptin sensing and nonsensing. Scale bars: 200 μm; 25 μm (small panels). (F) Action potential firing of neurons with or without mouse astrocyte coculture. Dashed lines indicate –60 mV. (G) Quantification of the maximal number of action potentials in neurons cocultured with mouse cortical astrocytes (n = 6) and neurons cultured without astrocytes (n = 10). (H) Quantification of the ratios for POMC+ NPY+ in day-35 neurons with or without astrocyte coculture. The ratio is calculated with the following equation: (number of POMC+ NPY+)/(total number of POMC neurons). n = 3–5. **P < 0.01, 2-tailed Student’s t test.