Generation and Characterization of Functional Human Hypothalamic Neurons

Peter Kirwan1, Magdalena Jura1, Florian T. Merkle1

1 The Anne McLaren Laboratory, Wellcome Trust–Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.33
DOI:  10.1002/cpns.40
Online Posting Date:  October, 2017
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Abstract

Neurons in the hypothalamus orchestrate homeostatic physiological processes and behaviors essential for life. Defects in the function of hypothalamic neurons cause a spectrum of human diseases, including obesity, infertility, growth defects, sleep disorders, social disorders, and stress disorders. These diseases have been studied in animal models such as mice, but the rarity and relative inaccessibility of mouse hypothalamic neurons and species‐specific differences between mice and humans highlight the need for human cellular models of hypothalamic diseases. We and others have developed methods to differentiate human pluripotent stem cells (hPSCs) into hypothalamic neurons and related cell types, such as astrocytes. This protocol builds on published studies by providing detailed step‐by‐step instructions for neuronal differentiation, quality control, long‐term neuronal maintenance, and the functional interrogation of hypothalamic cells by calcium imaging. Together, these protocols should enable any group with appropriate facilities to generate and study human hypothalamic cells. © 2017 by John Wiley & Sons, Inc.

Keywords: hypothalamus; protocol; human; differentiation; pluripotent stem cell; neuron; POMC; obesity

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: hPSC Maintenance
  • Basic Protocol 2: Hypothalamic Differentiation
  • Support Protocol 1: Cortical Neuron (Control) Differentiation
  • Basic Protocol 3: Neuronal Maturation
  • Support Protocol 2: Quality Control: Confirmation of Hypothalamic Patterning and Neurogenesis
  • Support Protocol 3: Fura‐2 Imaging of Hypothalamic Cultures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: hPSC Maintenance

  Materials
  • Geltrex LDEV‐Free Reduced Growth Factor Basement Membrane Matrix (Thermo Fisher Scientific, cat. no. A1413202)
  • DMEM/F12 without phenol red (Thermo Fisher Scientific, cat. no. 21041025)
  • Dulbecco's phosphate‐buffered saline, calcium‐ and magnesium‐free (DPBS––), pH range 7.0 to 7.3 (Thermo Fisher Scientific, cat. no. 14190250)
  • hiPSC or hESC cell line confirmed to be free of pathogens and unwanted mutations (e.g., WA09 cell line; WiCell, cat. no. RRID:CVCL_9773)
  • mTeSR1 medium (see recipe)
  • 0.5 mM ethylenediaminetetraacetic acid [EDTA; dilute 0.5 M EDTA (Sigma‐Aldrich, cat. no. 03690) 1:1000 in DPBS–– (see above); filter sterilize and store up to 1 year at 4°C]
  • mTeSR1 medium (see recipe) containing 10 µM Y‐27632 dihydrochloride (ROCK inhibitor; see recipe)
  • 6‐well plate, 12‐well plate, 24‐well plate, or 10‐cm plate;
  • Inverted phase‐contrast microscope
  • 15‐ and 50‐ml V‐bottom polypropylene tubes (e.g., Corning Falcon)
  • 37°C, 20% O 2, 5% CO 2 humidified incubator
NOTE: To ensure cultures are kept sterile, cells should be handled in a Class II biosafety cabinet using standard sterile technique at all times (Mather & Roberts, ).

Basic Protocol 2: Hypothalamic Differentiation

  Materials
  • Nearly confluent culture of hESCs or hiPSCs (generated in protocol 1)
  • Dulbecco's phosphate‐buffered saline, calcium‐ and magnesium‐free (DPBS––), pH range 7.0 to 7.3 (Thermo Fisher Scientific, cat. no. 14190250)
  • 1× TrypLE Express, no phenol red (Life Technologies, cat. no. 12604021)
  • hPSC wash medium (see recipe)
  • mTeSR1 medium (see recipe)
  • mTeSR1 medium (see recipe) containing 10 µM Y‐27632 dihydrochloride (ROCK inhibitor; see recipe)
  • 0.4% trypan blue (Invitrogen, cat. no. T10282)
  • N2B27 medium (see recipe)
  • XAV939 (see recipe)
  • LDN‐193189 (see recipe)
  • SB431542 (see recipe)
  • Smoothened agonist (SAG; see recipe)
  • Purmorphamine (see recipe)
  • DAPT (see recipe)
  • Maturation medium (see recipe)
  • 6‐well plate, 12‐well plate, 24‐well plate, or 10‐cm plate;
  • 15‐ and 50‐ml V‐bottom polypropylene tubes (e.g., Corning Falcon)
  • Laboratory centrifuge (Eppendorf 5804, rotor A‐4‐44, or similar)
  • 0.5‐ and 1.5‐ml polypropylene tubes (e.g., Eppendorf)
  • Automated cell counter and associated consumables (e.g., Life Technologies, Countess II Automated Cell Counter, cat no. AMQAX1000)
  • Cell counting slides (Countess Cell Counting Chamber Slides, Life Technologies, cat. no. C10228)
  • Inverted phase‐contrast microscope;
  • Additional reagents and equipment for Geltrex coating of plates and culture of hPSCs (see protocol 1)

Support Protocol 1: Cortical Neuron (Control) Differentiation

  Additional Materials (also see protocol 2)
  • FGF2 (see recipe)
At step 2 of protocol 1, if cells plated for differentiation are evenly distributed over the plate and at a density of approximately 75%, start the differentiation by washing cultures once with DPBS–– and adding Day 0 (D0) medium (see below). Every second day, make full medium changes (5 to 6 ml per 6‐well plate, 10 to 15 ml per 10‐cm plate) using media with the following compositions corresponding to the day of differention:
  • Day 0 (D0): N2B27 + 2 µM XAV939 + 100 nM LDN‐193189 + 10 µM SB431542
  • Day 2 (D2): N2B27 + 2 µM XAV939 + 100 nM LDN‐193189 + 10 µM SB431542
  • Day 4 (D4): N2B27 + 1.5 µM XAV939 + 75 nM LDN‐193189 + 7.5 µM SB431542
  • Day 6 (D6): N2B27 + 1 µM XAV939 + 50 nM LDN‐193189 + 5 µM SB431542
  • Day 8 (D8): N2B27 + 0.5 µM XAV939 + 25 nM LDN‐193189 + 2.5 µM SB431542
  • Day 10 (D10): N2B27
  • Day 12 (D12): N2B27 + 20 ng/ml FGF2
  • Day 14 (D14): dissociation and replating in maturation medium + 20 ng/ml FGF2 + 10 µM Y‐27632 (see protocol 4).

Basic Protocol 3: Neuronal Maturation

  Materials
  • Dulbecco's phosphate‐buffered saline, calcium‐ and magnesium‐free (DPBS––), pH range 7.0 to 7.3 (Thermo Fisher Scientific, cat. no. 14190250)
  • Neural progenitor cells after differentiation ( protocol 2)
  • 1× TrypLE Express, no phenol red (Life Technologies, cat. no. 12604021)
  • Trituration medium (see recipe)
  • Maturation medium (see recipe)
  • Maturation medium (see recipe) containing 10 µM Y‐27632 dihydrochloride (ROCK inhibitor; see recipe)
  • BDNF (see recipe)
  • 0.4% trypan blue (Invitrogen, cat. no. AM7962)
  • Papain (Worthington Biochemical Corporation, cat. no. LK003176)
  • 6‐well plate, 12‐well plate, 24‐well plate, or 10‐cm plate;
  • Inverted phase‐contrast microscope
  • 15‐ml and 50‐ml V‐bottom polypropylene tubes (e.g., Corning Falcon)
  • Laboratory centrifuge (Eppendorf 5804, rotor A‐4‐44, or similar)
  • 0.5‐ and 1.5‐ml polypropylene tubes (e.g., Eppendorf)
  • Automated cell counter and associated consumables (e.g., Life Technologies, Countess II Automated Cell Counter, cat no. AMQAX1000)
  • Cell counting slide (Countess Cell Counting Chamber Slides, cat. no. C10228)
  • Additional reagents and equipment for coating plates with Geltrex ( protocol 1) and differentiation of neurons ( protocol 2 or protocol 3)

Support Protocol 2: Quality Control: Confirmation of Hypothalamic Patterning and Neurogenesis

  Materials
  • 5 M hydrochloric acid (HCl; Thermo Fisher, cat. no. MFCD00011324–2.5 liters];
  • 70% ethanol
  • Differentiated neuronal culture ( protocol 4, step 2, substeps a to l)
  • Dulbecco's phosphate‐buffered saline, calcium‐ and magnesium‐free (DPBS––), pH range 7.0 to 7.3 (Thermo Fisher Scientific, cat. no. 14190250)
  • 4% paraformaldehyde in PBS (Santa Cruz, cat. no. 30525‐89‐4 or Alfa Aesar, cat. no. J61899)
  • Primary antibodies (Table 3.33.2)
  • Tris‐buffered saline (TBS; see recipe)
  • Normal donkey serum (Jackson Immunoresearch Labs, cat. no. 017‐000‐121)
  • Triton X‐100 (Sigma‐Aldrich, cat. no. T8787)
  • Alexa Fluor‐conjugated secondary antibody (Thermo Fisher Scientific; conjugated to Alexa Fluor 488, 555, 594, or 647)
  • 4′,6‐diamidine‐2′‐phenylindole dihydrochloride (DAPI; see recipe)
  • Sodium azide (Sigma‐Aldrich, cat. no. 769320)
  • ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, cat. no. P36965)
  • RNA isolation kit (RNeasy Mini Kit; Qiagen, cat. no. 74104)
  • Human brain total RNA (Thermo Fisher Scientific, cat. no. AM7962)
  • Glass coverslips (Thermo Scientific, cat. no. MENZCB00120RAC20)
  • 65°C rolling or shaking incubator
  • Glass beaker
  • 6‐well plate, 12‐well plate, 24‐well plate, or 10‐cm plate
  • Forceps
  • Glass microscope slides
  • Additional reagents and equipment for Geltrex coating of plates ( protocol 1, step 1) and RT‐qPCR (Toyohara et al., )

Support Protocol 3: Fura‐2 Imaging of Hypothalamic Cultures

  Materials
  • hPSC‐derived hypothalamic neurons, Day 30+ (preferably Day 40 to Day 60; see protocol 4)
  • 1× TrypLE Express, no phenol red (Life Technologies, cat. no. 12604021)
  • Papain (Worthington Biochemical Corporation, cat. no. LK003176)
  • Fura‐2AM loading solution (see recipe)
  • Artificial cerebrospinal fluid (ACSF; see recipe)
  • ACSF with 30 mM KCl (see recipe)
  • 35‐mm Ibidi dishes (Thistle Scientific, cat. no. 80136), coated with Geltrex (see protocol 1. step 1)
  • Perfusion system; we use a custom‐built gravity perfusion system; however commercial systems are available, e.g., MPS‐2 (World Precision Instruments)
  • Imaging system:
  • Inverted microscope, e.g., Eclipse TE2000U (Nikon)
  • Epifluorescence illuminator and monochromator to provide excitation wavelengths at 340 nm, 360 nm, 380 nm (e.g., Optoscan; Cairn Research Ltd.)
  • Emission filter: 510 nm
  • Objective: 40× Nikon oil immersion, N.A. 1.3
  • EMCCD camera, e.g., Orca‐ER‐1394 (Hamamatsu)
  • Imaging software: e.g., Metafluor (Molecular Devices, cat. no. RRID: SCR_014294)
  • Additional reagents and equipment for Geltrex coating ( protocol 1, step 1)
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Figures

Videos

Literature Cited

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