Neural Stem Cell or Human Induced Pluripotent Stem Cell–Derived GABA‐ergic Progenitor Cell Grafting in an Animal Model of Chronic Temporal Lobe Epilepsy

Dinesh Upadhya1, Bharathi Hattiangady1, Geetha A. Shetty1, Gabriele Zanirati1, Maheedhar Kodali1, Ashok K. Shetty1

1 Department of Molecular and Cellular Medicine, Texas A&M University Health Science Center College of Medicine, College Station, Texas
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2D.7
DOI:  10.1002/cpsc.9
Online Posting Date:  August, 2016
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Grafting of neural stem cells (NSCs) or GABA‐ergic progenitor cells (GPCs) into the hippocampus could offer an alternative therapy to hippocampal resection in patients with drug‐resistant chronic epilepsy, which afflicts >30% of temporal lobe epilepsy (TLE) cases. Multipotent, self‐renewing NSCs could be expanded from multiple regions of the developing and adult brain, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). On the other hand, GPCs could be generated from the medial and lateral ganglionic eminences of the embryonic brain and from hESCs and hiPSCs. To provide comprehensive methodologies involved in testing the efficacy of transplantation of NSCs and GPCs in a rat model of chronic TLE, NSCs derived from the rat medial ganglionic eminence (MGE) and MGE‐like GPCs derived from hiPSCs are taken as examples in this unit. The topics comprise description of the required materials, reagents and equipment, methods for obtaining rat MGE‐NSCs and hiPSC‐derived MGE‐like GPCs in culture, generation of chronically epileptic rats, intrahippocampal grafting procedure, post‐grafting evaluation of the effects of grafts on spontaneous recurrent seizures and cognitive and mood impairments, analyses of the yield and the fate of graft‐derived cells, and the effects of grafts on the host hippocampus. © 2016 by John Wiley & Sons, Inc.

Keywords: cell transplantation; chronic temporal lobe epilepsy; cognitive and mood impairments; GABA‐ergic progenitors; glial‐cell line derived neurotrophic factor; hippocampal neurogenesis; human induced pluripotent stem cells; medial ganglionic eminence; phenotypic differentiation of graft‐derived cells; spontaneous recurrent seizures

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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation of Rats Exhibiting Chronic TLE: Induction of Status Epilepticus (SE) in Adult Male F344 Rats
  • Basic Protocol 2: Selecting Rats Exhibiting Chronic TLE
  • Basic Protocol 3: Analyzing Cognitive Function in CERs Chosen for Grafting Studies
  • Basic Protocol 4: Harvesting and Preparation of Rat MGE‐NSC Suspension for Grafting
  • Basic Protocol 5: Preparation of MGE‐LIKE GABA‐ERGIC Progenitor Cells from Human‐Induced Pluripotent Stem Cells
  • Basic Protocol 6: Grafting of NSCs OR GPCs into the Hippocampi of Chronically Epileptic Rats
  • Basic Protocol 7: Analyses of Rat MGE‐NSC or Human GPC Grafting‐Mediated Changes in Seizures
  • Basic Protocol 8: Analyses of NSC or GPC Grafting Mediated Effects on Cognitive and mood Function
  • Basic Protocol 9: Analyses of the Yield and Differentiation of Graft‐Derived Cells
  • Basic Protocol 10: Analyses of Effect of NSC or GPC Grafting on the Host Hippocampus
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Generation of Rats Exhibiting Chronic TLE: Induction of Status Epilepticus (SE) in Adult Male F344 Rats

  • Experimental animals: 4‐ to 5‐month‐old male Fischer 344 (F344) rats
  • Kainic acid (KA; Milestone PharmTech)
  • Saline (0.9% NaCl)
  • Diazepam
  • Ringer's lactate solution, sterile
  • Regular rat chow soaked in water (soft pellets) and transgel
  • Additional reagents and equipment for intraperitoneal and subcutaneous injections of drugs to rats (Donovan and Brown, )

Basic Protocol 2: Selecting Rats Exhibiting Chronic TLE

  • Chronically epileptic rats (CERs; see Basic Protocols protocol 11 and protocol 22)
  • Open field apparatus, suitable for rats
  • Noldus‐Ethovision or ANY‐maze video tracking system (Stoelting; http://www.any‐

Basic Protocol 3: Analyzing Cognitive Function in CERs Chosen for Grafting Studies

  • Human‐induced pluripotent stem cell lines of interest
  • TeSR‐E8 medium (see recipe)
  • Matrigel‐coated 6‐well plates (see recipe)
  • Dispase solution (see recipe)
  • TeSR‐E6 medium (see recipe)
  • TGF‐β3, human recombinant (Sigma‐Aldrich, cat. no. SRP3171)
  • Neural induction medium (NIM; see recipe)
  • Fetal bovine serum (FBS; Gibco, cat. no. 10437)
  • Recombinant human sonic hedgehog (C24II) N‐terminus, CF (SHH; see recipe)
  • Purmorphamine (see recipe)
  • B27 supplement, without retinoic acid, 50× (Gibco, cat. no. 12587‐010)
  • TrypLE Express Enzyme, 1× (Invitrogen, cat. no. 12604‐021)
  • Neuronal differentiation medium (NDM; see recipe)
  • Cyclic AMP (cAMP; see recipe)
  • Brain‐derived neurotrophic factor (BDNF; see recipe)
  • Glial cell line–derived neurotrophic factor (GDNF; see recipe)
  • Insulin‐like growth factor (IGF1; see recipe
  • Natural mouse laminin, 1 mg ml−1 (laminin; Invitrogen, cat. no. 23017‐015)
  • Poly‐L‐ornithine solution (PLO; Sigma‐Aldrich, cat. no. P4957)
  • Tissue culture plates (6‐well and 24‐well)
  • 37°C, 5% CO 2 incubator
  • 15‐ and 50‐ml centrifuge tubes
  • Disposable serological pipets, 5, 10 and 25 ml
  • 1‐ml pipets
  • Phase‐contrast inverted microscope
  • Benchtop centrifuge
NOTE: We tried a few hiPSC lines for generating GABA‐ergic progenitors. However, IMR90‐4 (Wisconsin International Stem Cell Bank, cat. no. iPS (IMR90)‐4 line gave us consistent and reproducible results.

Basic Protocol 4: Harvesting and Preparation of Rat MGE‐NSC Suspension for Grafting

  • Chronically epileptic rats (CERs; see Basic Protocols protocol 11 and protocol 22)
  • Anesthetic cocktail (see recipe)
  • Artificial tears
  • 70% ethanol
  • Betadine
  • Cyclosporine
  • Isopropyl alcohol
  • 3% hydrogen peroxide
  • NSC suspension, BrdU‐labeled ( protocol 4)
  • Bone wax
  • Buprenorphine
  • Ringer's lactate or sterile saline (0.9% NaCl; Hospira)
  • Soft food/hydrogel/transgel
  • Animal cages
  • Surgical shaver
  • Sterile surgical drape
  • Stereotaxic equipment: any stereotaxic device made for rat neurosurgery can be used; in our laboratory, we use digital stereotaxic equipment purchased from MyNeurolab (
  • Sterile applicators with cotton tips
  • Autoclaved surgical instruments:
  • Scalpel blade holders
  • Skin retractors
  • Blunt forceps of different sizes
  • Autoclipping device loaded with 9‐mm wound clips (for stapling the skin incision after the grafting surgery)
  • Autoclip remover
  • Sterile scalpel blades
  • Hand‐ or foot controlled electrical driller with different sizes of drill bits to make small burr holes in the skull using the desired stereotaxic coordinates
  • 10‐µl Hamilton syringes
  • Heating pads to maintain the body temperature of the rat during and after the surgery
  • Additional reagents and equipment for injection of the rat (Donovan and Brown, )
NOTE: Personnel protective equipment including sterile surgical latex gloves, disposable laboratory coats, head cover, booties, surgical masks and approved respirators (for handling human GPCs) should be worn.NOTE: A glass bead sterilizer should be available to sterilize the stainless steel surgical instruments during the surgery of multiple animals in one surgery session.

Basic Protocol 5: Preparation of MGE‐LIKE GABA‐ERGIC Progenitor Cells from Human‐Induced Pluripotent Stem Cells

  • NSC or GPC grafted chronically epileptic rats ( protocol 6)
  • Isoflurane
  • 1% (w/v) heparin in 0.9% NaCl, sterile
  • 4% paraformaldehyde (see recipe)
  • 0.1 M sodium phosphate buffer, pH 7.4 (Fisher Scientific, cat. no. NC9552713)
  • 10%, 20%, and 30% (w/v) sucrose in sodium phosphate buffer, pH 7.4
  • Cryo‐embedding gel (Fisher Scientific cat. no. SH75‐125D)
  • 0.1 M sodium phosphate buffer, pH 7.4, containing 0.01% sodium azide
  • 0.1 M Tris‐buffered saline (TBS; see recipe) containing 20% (v/v) methanol and 2% (v/v) hydrogen peroxide
  • 0.1 M Tris‐buffered saline (TBS; see recipe)
  • 50% (v/v) formamide/2× SSC (see SSC recipe in Ariizumi et al., )
  • 2× SSC (see Ariizumi et al. )
  • 2 N HCl
  • 0.1 M borate buffer, pH 8.5 (see recipe)
  • Blocking solution: TBS containing 10% (v/v) normal horse serum and 0.01% Triton X‐100
  • Primary antibody solution: mouse anti‐BrdU (BD Biosciences, 1:500) or mouse‐human nuclear antigen (HNA; Millipore, 1: 200)
  • Secondary antibody solution: biotinylated anti–mouse IgG (Vector Labs) diluted 1:200 in TBS containing 2% normal horse serum
  • ABC reagent (from Elite ABC kit; Vector Labs)
  • DAB kit (Vector Labs)
  • Phosphate‐buffered saline (PBS; (Sigma, cat. no. P3813‐10PAK))
  • Vaporizer
  • Flat fixation board (e.g., rectangular Thermocol board covered with thin plastic sheet)
  • Pushpins
  • Dissection instruments (heavy‐duty scissors, tissue forceps, microdissecting scissors, bone cutter, tissue retractors and clamps, scalpel, scalpel blades, and spatula) to open the thoracic cavity and to remove the perfusion‐fixed brain from the skull
  • Perfusion pump and perfusion tube (World Precision Instruments, cat. no. 500328; http://www.wpi‐
  • 20‐ to 25‐G needles
  • Cryostat (Leica)
  • 24‐well plates
  • 37°C and 65°C oven
  • Light and fluorescent microscopes and a confocal microscope (Nikon, Olympus)
  • Imaging system for stereological counting of graft‐derived cells e.g., the StereoInvestigator system; MicroBrightField (
  • Additional reagents and equipment for immunohistochemistry (Hofman, )
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