Neural Stem Cell Transplantation in Mouse Brain

Jean‐Pyo Lee1,2, Scott McKercher1, Franz‐Josef Muller1,3, Evan Y. Snyder1,2

1 Stem Cell and Regeneration Program, Center for Neuroscience and Aging Research, Burnham Institute for Medical Research, La Jolla, California, 2 Department of Pediatrics, University of California San Diego, School of Medicine, La Jolla, California, 3 Zentrum für Integrative Psychiatrie, Kiel, Germany
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.10
DOI:  10.1002/0471142301.ns0310s42
Online Posting Date:  January, 2008
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Abstract

Neural stem cells (NSCs) are the most primordial, least committed cells of the nervous system, and transplantation of these multipotent cells holds the promise of regenerative therapy for many central nervous system (CNS) diseases. This unit describes methods for NSC transplantation into neonatal mouse pups, embryonic mouse brain, and adult mouse brain. A description of options for detection of labeled donor cells in engrafted mouse brain is provided along with an example protocol for detecting lacZ-expressing cells in situ. Also included is a protocol for preparing NSCs for transplantation. Curr. Protoc. Neurosci. 42:3.10.1-3.10.23. © 2008 by John Wiley & Sons, Inc.

Keywords: neural stem cell; CNS; transplant

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Neural Stem Cell Transplantation into Neonatal Mice
  • Basic Protocol 2: Neural Stem Cell Transplantation into Fetal Mouse Brain In Utero
  • Basic Protocol 3: Transplanting Neural Stem Cells into Adult Mouse Brain
  • Support Protocol 1: Preparing Neural Stem Cells for Transplantation
  • Basic Protocol 4: Detecting lacZ-Expressing Donor Cells in Neural Stem Cell-Engrafted Mouse Brain
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Neural Stem Cell Transplantation into Neonatal Mice

 Materials
  • P0 to P3 mouse pups (less than 3-days-old)
  • NSCs resuspended in PBS with trypan blue (see Support Protocol 1)
  • Fiber optic light source for transillumination (Dolan-Jenner Industries)
  • Micropipet puller (Sutter Instrument Co., Model P-87)
  • Borosilicate glass (Sutter Instrument, no. B100–75-15)
  • Warming pad
  • Aspirator tube assemblies for calibrated microcapillary pipets (Sigma Aldrich, no. A5177–5EA)
  • Additional reagents and equipment for preparing NSCs resuspended in PBS with trypan blue (Support Protocol 1) and preparing glass micropipets (unit 6.3)

Basic Protocol 2: Neural Stem Cell Transplantation into Fetal Mouse Brain In Utero

 Materials
  • Mice of gestational age E13.5
  • Depilatory (e.g., Nair hair-removal lotion)
  • 70% ethanol prep pad (PDI, B603)
  • Sterile Ringer's lactate with 5% dextrose (Hospira, Inc.)
  • NSCs resuspended in PBS with trypan blue (Support Protocol 1)
  • Iodine (antiseptic germicide, Clinipad)
  • Neosporin antibiotic ointment
  • Buprenorphine
  • Bupivacaine
  • Sterile surgical instruments:
  • Two fine scissors,
  • Scalpel with #11 surgical blade (Bard–Parker)
  • Forceps
  • Surgical gloves
  • UV light
  • Operational stage (Fig. 3.10.2) with Styrofoam platforms and 18-G needles for positioning mice
  • Operating microscope, includes bright-field objectives with foot pedals (Carl Zeiss, Opmi-6Fc)
  • Anesthesia machine with isoflurane vaporizer and oxygen and nitrous oxide tanks (MDS Matrix)
  • Isoflurane, isoflurane reservoir, and gas tanks
  • Fiber optic light source (Dolan-Jenner Industries)
  • Sterile foil
  • Plastic wrap (e.g., Saran wrap)
  • 4 metal hooks (bent to hold the abdomen open)
  • Sterile gauze and wet cotton swabs
  • Glass capillaries
  • Micropipet puller (Sutter Instrument Co., Model P-87)
  • Suture (# 6 black braided silk with Taper C-1, Ethicon)
  • 37°C warming pad
  • Additional reagents and equipment for preparing NSCs resuspended in PBS with trypan blue (Support Protocol 1) and preparing glass micropipets (unit 6.3)
     FigureFigure 3.10.2 Illustration of operation stage for in utero transplantation. S-shaped metal hooks are used to hold the skin after incision to expose the fetus during surgery.

Basic Protocol 3: Transplanting Neural Stem Cells into Adult Mouse Brain

 Materials
  • Adult mouse
  • Betadine solution
  • 70% ethanol prep pad (PDI, B603)
  • NSCs resuspended in PBS with trypan blue (Support Protocol 1)
  • Bone wax (Fine Science Tools)
  • Sterile surgical instruments including:
  • Scalpel with #11 surgical blade (Bard–Parker)
  • Fine scissors
  • Forceps
  • UV light
  • Operating microscope
  • Anesthesia machine with isoflurane vaporizer and oxygen and nitrous oxide tanks (MDS Matrix)
  • Isoflurane, isoflurane reservoir, and gas tanks
  • Fiber optic light source (Dolan-Jenner Industries)
  • Sterilized foil
  • Electric shaver
  • Small animal stereotaxic system (David Kopf Instruments, model 900)
  • Ear bars (David Kopf Instruments)
  • Dry cotton swab
  • Mouse brain atlas (Paxinos and Watson, 1986)
  • 30-G, 2-in. needle (Hamilton)
  • Bone drill with 0.5-mm drill burrs (Fine Science Tools)
  • Ruler
  • 5-µl syringe (Hamilton Co., 7633-01)
  • Sterile gauze and wet cotton swabs
  • Suture (# 6 black braided silk with Taper C-1, Ethicon)
  • Warming pad
  • Additional reagents and equipment for preparing NSCs resuspended in PBS with trypan blue (Support Protocol 1)

Support Protocol 1: Preparing Neural Stem Cells for Transplantation

 Materials
  • NSC cultures in 10-cm tissue culture dishes (Corning)
  • Phosphate buffered saline (PBS, without calcium or magnesium, HyClone)
  • Trypsin/EDTA (HyClone)
  • Dulbecco's modified Eagle medium (DMEM)/F12 (Invitrogen)
  • Fetal bovine serum (FBS, HyClone)
  • 0.4% trypan blue dye (Sigma)
  • 100× penicillin/streptomycin (Irvine Scientific)
  • Inverted microscope
  • 15-ml Falcon centrifuge tubes
  • Additional reagents and equipment for counting cells with a hemacytometer and trypan blue staining (appendix 3B)

Basic Protocol 4: Detecting lacZ-Expressing Donor Cells in Neural Stem Cell-Engrafted Mouse Brain

 Materials
  • Injected experimental animals (Basic Protocols 1 to 3)
  • Pentobarbital
  • 4% paraformaldehyde in PBS (see recipe)
  • 10% and 30% sucrose in PBS
  • OCT embedding medium
  • 1× PBS, pH 7.6 (HyClone)
  • Xgal solutions A, B, and C (see recipes)
  • Slides (charged, e.g., Fisher Superfrost Plus)
  • Moist chamber: plastic box containing wet paper towels
  • Bright-field microscope
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Figures

  •  FigureFigure 3.10.1 Transillumination of anesthetized newborn mouse pup head immediately following bilateral intracerebroventricular injection of NSCs mixed with trypan blue. (A) Orientation of fiber optic light source, mouse pup, and investigator. (B) Close-up view of transilluminated mouse pup head showing location of injected cells mixed with trypan blue. The injectate often fills as far rostrally as the olfactory bulbs and as far caudally as the third ventricle and cisterna (allowing the cells access to brainstem and high cervical spinal cord).
    View Image
  •  FigureFigure 3.10.2 Illustration of operation stage for in utero transplantation. S-shaped metal hooks are used to hold the skin after incision to expose the fetus during surgery.
    View Image
  •  FigureFigure 3.10.3 Incision site for transplantation of neural stem cells into adult mouse brain. Nude mouse is shown here for better visualization.
    View Image
  •  FigureFigure 3.10.4 NSCs transplanted into newborn pups and analyzed at adulthood. (A) Representative thick coronal sections through the forebrain showing widely disseminated integration of blue Xgal+ donor-derived cells following transplantation at birth of lacZ-expressing mNSCs into the cerebral ventricles. Similar distributions were obtained whether the NSCs were from mouse clone C17.2 (A), ROSA mouse neurospheres (B,C), or human neuroectoderm (D). Donor NSC-derived cells are recognized by Xgal (A,B), immunoreactivity to a -gal-specific antibody (C, red) or human mitochondria-specific antibody (hMito; D, red; Adapted from Lee et al., 2007).
    View Image
  •  FigureFigure 3.10.5 In utero transplantation of NSCs into cerebroventricles. (A) Immediately after transplantation, injected donor cells mixed with trypan blue are visualized by transillumination of the embryo. The injectate often fills as far rostrally as the olfactory bulbs and as far caudally as the third ventricle. (B through E) Blue Xgal+ donor-derived cells are distributed in semiserial coronal sections at adulthood following unilateral ventricular injection of lacZ-expressing NSCs.
    View Image

Videos

Literature Cited

Literature Cited
    Ahmed, S., Reynolds, B.A., and Weiss, S. 1995. BDNF enhances the differentiation but not the survival of CNS stem cell-derived neuronal precursors. J. Neurosci. 15:5765-5778.
    Auerbach, J.M., Eiden, M.V., and McKay, R.D. 2000. Transplanted CNS stem cells form functional synapses in vivo. Eur. J. Neurosci. 12:1696-1704.
    Flax, J.D., Aurora, S., Yang, C., Simonin, C., Wills, A.M., Billinghurst, L.L., Jendoubi, M., Sidman, R.L., Wolfe, J.H., Kim, S.U., and Snyder, E.Y. 1998. Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes. Nat. Biotechnol. 16:1033-1039.
    Imitola, J., Raddassi, K., Park, K.I., Mueller, F.J., Nieto, M., Teng, Y.D., Frenkel, D., Li, J., Sidman, R.L., Walsh, C.A., Snyder, E.Y., and Khoury, S.J. 2004. Directed migration of neural stem cells to sites of CNS injury by the stromal cell-derived factor 1alpha/CXC chemokine receptor 4 pathway. Proc. Natl. Acad. Sci. U.S.A. 101:18117-18122.
    Jeyakumar, M., Thomas, R., Elliot-Smith, E., Smith, D.A., van der Spoel, A.C., d'Azzo, A., Perry, V.H., Butters, T.D., Dwek, R.A., and Platt, F.M. 2003. Central nervous system inflammation is a hallmark of pathogenesis in mouse models of GM1 and GM2 gangliosidosis. Brain 126:974-987.
    Lacorazza, H.D., Flax, J.D., Snyder, E.Y., and Jendoubi, M. 1996. Expression of human beta-hexosaminidase alpha-subunit gene (the gene defect of Tay-Sachs disease) in mouse brains upon engraftment of transduced progenitor cells. Nat. Med. 2:424-429.
    Lee, J.P., Jeyakumar, M., Gonzalez, R., Takahashi, H., Lee, P.J., Baek, R.C., Clark, D., Rose, H., Fu, G., Clarke, J., McKercher, S., Meerloo, J., Muller, F.J., Park, K.I., Butters, T.D., Dwek, R.A., Schwartz, P., Tong, G., Wenger, D., Lipton, S.A., Seyfriend, T.N., Platt, F.M., and Snyder, E.Y. 2007. Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease. Nat. Med. 13:439-447.
    Lu, P., Jones, L.L., Snyder, E.Y., and Tuszynski, M.H. 2003. Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Exp. Neurol. 181:115-129.
    Myerowitz, R., Lawson, D., Mizukami, H., Mi, Y., Tifft, C.J., and Proia, R.L. 2002. Molecular pathophysiology in Tay-Sachs and Sandhoff diseases as revealed by gene expression profiling. Hum. Mol. Genet. 11:1343-1350.
    Neufeld, E.F. 1991. Lysosomal storage diseases. Annu. Rev. Biochem. 60:257-280.
    Park, K.I., Teng, Y.D., and Snyder, E.Y. 2002. The injured brain interacts reciprocally with neural stem cells supported by scaffolds to reconstitute lost tissue. Nat. Biotechnol. 20:1111-1117.
    Parker, M.A., Anderson, J.K., Corliss, D.A., Abraria, V.E., Sidman, R.L., Park, K.I., Teng, Y.D., Cotanche, D.A., and Snyder, E.Y. 2005. Expression profile of an operationally-defined neural stem cell clone. Exp. Neurol. 194:320-332.
    Paxinos, G. and Watson, C. 1986. The Rat Brain in Stereotaxic Coordinates, 2nd ed. Academic Press, London.
    Snyder, E.Y., Taylor, R.M., and Wolfe, J.H. 1995. Neural progenitor cell engraftment corrects lysosomal storage throughout the MPS VII mouse brain. Nature 374:367-370.
    Taylor, R.M., Lee, J.P., Palacino, J.J., Bower, K.A., Li, J., Vanier, M.T., Wenger, D.A., Sidman, R.L., and Snyder, E.Y. 2006. Intrinsic resistance of neural stem cells to toxic metabolites may make them well suited for cell non-autonomous disorders: Evidence from a mouse model of Krabbe leukodystrophy. J. Neurochem. 97:1585-1599.
    Teng, Y.D., Lavik, E.B., Qu, X., Park, K.I., Ourednik, J., Zurakowski, D., Langer, R., and Snyder, E.Y. 2002. Functional recovery following traumatic spinal cord injury mediated by a unique polymer scaffold seeded with neural stem cells. Proc. Natl. Acad. Sci. U.S.A. 99:3024-3029.
    Wada, R., Tifft, C.J., and Proia, R.L. 2000. Microglial activation precedes acute neurodegeneration in Sandhoff disease and is suppressed by bone marrow transplantation. Proc. Natl. Acad. Sci. U.S.A. 97:10954-10959.
    Windrem, M.S., Nunes, M.C., Rashbaum, W.K., Schwartz, T.H., Goodman, R.A., McKhann, G., 2nd, Roy, N.S., and Goldman, S.A. 2004. Fetal and adult human oligodendrocyte progenitor cell isolates myelinate the congenitally dysmyelinated brain. Nat. Med. 10:93-97.
    Yandava, B.D., Billinghurst, L.L., and Snyder, E.Y. 1999. “Global” cell replacement is feasible via neural stem cell transplantation: Evidence from the dysmyelinated shiverer mouse brain. Proc. Natl. Acad. Sci. U.S.A. 96:7029-7034.
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