Efficient Generation of Hypothalamic Neurons from Human Pluripotent Stem Cells

Liheng Wang1, Dieter Egli2, Rudolph L. Leibel1

1 Division of Molecular Genetics, Department of Pediatrics, Columbia University College of Physicians and Surgeons, New York, New York, 2 New York Stem Cell Foundation Research Institute, New York, New York
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 21.5
DOI:  10.1002/cphg.3
Online Posting Date:  July, 2016
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Abstract

The hypothalamus comprises neuronal clusters that are essential for body weight regulation and other physiological functions. Insights into the complex cellular physiology of this region of the brain are critical to understanding the pathogenesis of obesity, but human hypothalamic cells are largely inaccessible for direct study. Here we describe a technique for generation of arcuate‐like hypothalamic neurons from human pluripotent stem (hPS) cells. Early activation of SHH signaling and inhibition of BMP and TGFβ signaling, followed by timed inhibition of NOTCH, can efficiently differentiate hPS cells into NKX2.1+ hypothalamic progenitors. Subsequent incubation with BDNF induces the differentiation and maturation of pro‐opiomelanocortin and neuropeptide Y neurons, which are major cell types in the arcuate hypothalamus. These neurons have molecular and cellular characteristics consistent with arcuate neurons. © 2016 by John Wiley & Sons, Inc.

Keywords: ARC neurons; feeder‐free; hypothalamic neurons; human pluripotent stem cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of hES or iPS Cells for Neuron Differentiation
  • Basic Protocol 2: Neuron Induction: Generation of Hypothalamic Neuron Progenitors from hES or iPS Cells
  • Basic Protocol 3: Neuron Differentiation and Maturation from NKX2.1+ Progenitors
  • Support Protocol 1: Cryopreservation of Day 12 Neuron Progenitors
  • Support Protocol 2: Thawing Frozen Day 12 Neuron Progenitors
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of hES or iPS Cells for Neuron Differentiation

  Materials
  • hES or iPS cell line
  • Matrigel (BD Biosciences, cat. no. 354277)
  • DMEM/F12 medium (Thermo Fisher Scientific, cat. no. 11320‐082)
  • hES medium (see recipe)
  • Mouse embryonic fibroblasts (MEFs)
  • TrypLE Express Enzyme (ThermoFisher, cat. no. 12605‐036)
  • mTeSR1 complete kit (Stemcell Technologies, cat. no. 05850)
  • Y‐27632, ROCK inhibitor (Selleckchem, cat. no. S1049)
  • 0.4% (w/v) Trypan blue (Thermo Fisher Scientific, cat. no. T10282)
  • 6‐well cell culture plates (Thermo Scientific, cat. no. 14‐832‐11)
  • 15‐ml and 50‐ml conical tubes
  • 5‐ml round‐bottom polystyrene tube with 35‐μm mesh cell strainer cap (Thermo Fisher Scientific)
  • Eppendorf 5702 centrifuge with A‐4‐38 rotor
  • 1.5‐ml microcentrifuge tubes
  • Inverted microscope
  • Countess automated cell counter and cell counting chambers (Thermo Fisher Scientific)
  • 37°C, 5% CO 2 humidified incubator

Basic Protocol 2: Neuron Induction: Generation of Hypothalamic Neuron Progenitors from hES or iPS Cells

  Materials (also see protocol 1)
  • hES or iPS cell cultures, 95% to 100% confluent (from protocol 1)
  • KSR medium (see recipe)
  • 100 μg/ml SHH, recombinant human sonic hedgehog N‐terminus (see recipe)
  • 5 mM purmorphamine (see recipe)
  • 10 mM SB431542 (see recipe)
  • 2.5 mM LDN‐193189 (see recipe)
  • N2 medium (see recipe)
  • 10 mM DAPT (see recipe)
  • 50× B‐27 (Thermo Fisher Scientific, cat. no. 12587‐010)

Basic Protocol 3: Neuron Differentiation and Maturation from NKX2.1+ Progenitors

  Materials (also see Basic Protocols protocol 11 and protocol 22)
  • 0.01% poly‐L‐ornithine (Sigma, cat. no. P4957)
  • 1 mg/ml laminin, natural mouse protein (Thermo Fisher Scientific, cat. no. 23017‐015)
  • Hypothalamic neuron progenitor cells (from protocol 2)
  • TrypLE express enzyme (ThermoFisher, cat. no. 12605‐036)
  • N2 medium (see recipe)
  • B‐27
  • Y‐27632, ROCK inhibitor
  • 10 mM DAPT (see recipe)
  • 0.4% Trypan blue
  • 40 μg/ml BDNF (see recipe)
  • 6‐well, 12‐well, and 24‐well plates
  • 15‐ml conical tubes
  • 5‐ml round‐bottom tubes with 35‐µm mesh cell‐strainer caps
  • Countess automated cell counter and counting chambers
  • Inverted microscope
  • 37°C water bath
  • 37°C, 5% CO 2 humidified incubator

Support Protocol 1: Cryopreservation of Day 12 Neuron Progenitors

  Materials
  • Day 12 hypothalamic neuron progenitor cells (harvested and counted in protocol 3, steps 4 to 14)
  • Cell freezing medium (see recipe)
  • 1.8‐ml cryo tubes (Thermo Fisher Scientific, cat. no. 377267)
  • Mr. Frosty freezing container (Thermo Fisher Scientific, cat. no. 5100‐0001)
  • −80°C freezer
  • Liquid‐nitrogen storage

Support Protocol 2: Thawing Frozen Day 12 Neuron Progenitors

  Materials (also see Basic Protocols protocol 11 and protocol 22)
  • Frozen day 12 neuron progenitors
  • N2 medium
  • B‐27
  • Y‐27632, ROCK inhibitor
  • 0.01% poly‐L‐ornithine
  • 1 mg/ml laminin
  • 6‐well, 12‐well, 24‐well, or 4‐well plates, cell‐culture treated
  • 15‐ml conical tubes
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Figures

Videos

Literature Cited

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