Chicken embryonic brain: an in vivo model for verifying neural stem cell potency

Laboratory investigation

Restricted access

Object

The multipotency of neural stem cells (NSCs) can be assessed in vitro by detection of stage-specific markers in response to a suitable differentiation signal. This test is frequently used because it is fast and affordable. However, it is not clear how the in vitro potential for multilineage differentiation and stem cell marker expression would reflect the ability of NSCs to engraft into the brain following transplantation. The authors undertook this study to directly compare the in vitro potency and in vivo migration of human NSCs (hNSCs) expanded under conditions of gradually increased concentration of fetal bovine serum (FBS) as a maturation factor.

Methods

Human NSCs isolated from fetal brain were propagated in serum free media (SF-hNSCs) and in media containing 0.1% and 0.2% serum. At Passage 4 in tissue culture the NSCs were harvested and either differentiated in vitro or transplanted into the lateral ventricle of chicken embryonic brain at the late stage of its development (Hamburger and Hamilton Stage 26). The in vitro differentiation was evaluated by immunostaining with neural or glial specific markers, and the in vivo migration was assessed using immunohistology.

Results

The authors found that SF-hNSCs successfully engrafted into the chicken embryonic brain, which correlated with their ability to differentiate in vitro. NSCs grown at as low as 0.1% concentration of FBS failed to demonstrate the robust in vivo migration pattern but still preserved the capability to differentiate in vitro. Furthermore, NSCs generated in media containing a higher concentration of FBS (0.2%) lost both the in vivo engraftment and in vitro differentiation potential.

Conclusions

The present study suggests that marker expression and in vitro differentiation assays might not provide adequate information regarding the behavior of NSCs following their transplantation. The in vivo migration following injection into chicken embryonic brain may provide an important assay of the potency of NSCs.

Abbreviations used in this paper:FBS = fetal bovine serum; GFAP = glial fibrillary acidic protein; HH26 = Hamburger and Hamilton Stage 26; hNSC = human NSC; NSC = neural stem cell; OCT = optimal cutting temperature; PBS = phosphate-buffered saline; SF-hNSC = hNSC grown in a serum-free environment; TUJ1 = tubulin β-III (neuronal Class III β-tubulin).
Article Information

Contributor Notes

Address correspondence to: Michael L. Levy, M.D., Ph.D., Division of Pediatric Neurosurgery, 8010 Frost Street, Suite 502, San Diego, California 92123. email: mlevy@rchsd.org.Please include this information when citing this paper: published online March 1, 2013; DOI: 10.3171/2013.1.JNS12698.
Headings
References
  • 1

    Alvarado-Mallart RMMartinez SLance-Jones CC: Pluripotentiality of the 2-day-old avian germinative neuroepithelium. Dev Biol 139:75881990

    • Search Google Scholar
    • Export Citation
  • 2

    Aroca PLorente-Cánovas BMateos FRPuelles L: Locus coeruleus neurons originate in alar rhombomere 1 and migrate into the basal plate: Studies in chick and mouse embryos. J Comp Neurol 496:8028182006

    • Search Google Scholar
    • Export Citation
  • 3

    Arthur ARychkov GShi SKoblar SAGronthos S: Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues. Stem Cells 26:178717952008

    • Search Google Scholar
    • Export Citation
  • 4

    Breunig JJSarkisian MRArellano JIMorozov YMAyoub AESojitra S: Primary cilia regulate hippocampal neurogenesis by mediating sonic hedgehog signaling. Proc Natl Acad Sci U S A 105:13127131322008

    • Search Google Scholar
    • Export Citation
  • 5

    Brüstle OChoudhary KKarram KHüttner AMurray KDubois-Dalcq M: Chimeric brains generated by intraventricular transplantation of fetal human brain cells into embryonic rats. Nat Biotechnol 16:104010441998

    • Search Google Scholar
    • Export Citation
  • 6

    Brüstle OMaskos UMcKay RD: Host-guided migration allows targeted introduction of neurons into the embryonic brain. Neuron 15:127512851995

    • Search Google Scholar
    • Export Citation
  • 7

    Campbell KOlsson MBjörklund A: Regional incorporation and site-specific differentiation of striatal precursors transplanted to the embryonic forebrain ventricle. Neuron 15:125912731995

    • Search Google Scholar
    • Export Citation
  • 8

    Demeter KHerberth BDuda EDomonkos AJaffredo THerman JP: Fate of cloned embryonic neuroectodermal cells implanted into the adult, newborn and embryonic forebrain. Exp Neurol 188:2542672004

    • Search Google Scholar
    • Export Citation
  • 9

    Elias LAWang DDKriegstein AR: Gap junction adhesion is necessary for radial migration in the neocortex. Nature 448:9019072007

  • 10

    Fishell G: Striatal precursors adopt cortical identities in response to local cues. Development 121:8038121995

  • 11

    Gage FH: Mammalian neural stem cells. Science 287:143314382000

  • 12

    Gaiano NFishell G: Transplantation as a tool to study progenitors within the vertebrate nervous system. J Neurobiol 36:1521611998

    • Search Google Scholar
    • Export Citation
  • 13

    Gonzalez-Perez ORomero-Rodriguez RSoriano-Navarro MGarcia-Verdugo JMAlvarez-Buylla A: Epidermal growth factor induces the progeny of subventricular zone type B cells to migrate and differentiate into oligodendrocytes. Stem Cells 27:203220432009

    • Search Google Scholar
    • Export Citation
  • 14

    Graham VKhudyakov JEllis PPevny L: SOX2 functions to maintain neural progenitor identity. Neuron 39:7497652003

  • 15

    Gritti AParati EACova LFrolichsthal PGalli RWanke E: Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor. J Neurosci 16:109111001996

    • Search Google Scholar
    • Export Citation
  • 16

    Hamburger VHamilton HL: A series of normal stages in the development of the chick embryo. J Morphol 88:49921951

  • 17

    Kim MHabiba ADoherty JMMills JCMercer RWHuettner JE: Regulation of mouse embryonic stem cell neural differentiation by retinoic acid. Dev Biol 328:4564712009

    • Search Google Scholar
    • Export Citation
  • 18

    Kuwabara THsieh JMuotri AYeo GWarashina MLie DC: Wnt-mediated activation of NeuroD1 and retro-elements during adult neurogenesis. Nat Neurosci 12:109711052009

    • Search Google Scholar
    • Export Citation
  • 19

    LaVaute TMYoo YDPankratz MTWeick JPGerstner JRZhang SC: Regulation of neural specification from human embryonic stem cells by BMP and FGF. Stem Cells 27:174117492009

    • Search Google Scholar
    • Export Citation
  • 20

    Lendahl UZimmerman LBMcKay RD: CNS stem cells express a new class of intermediate filament protein. Cell 60:5855951990

  • 21

    Liapi APritchett JJones OFujii NParnavelas JGNadarajah B: Stromal-derived factor 1 signalling regulates radial and tangential migration in the developing cerebral cortex. Dev Neurosci 30:1171312008

    • Search Google Scholar
    • Export Citation
  • 22

    Morrison SJSpradling AC: Stem cells and niches: mechanisms that promote stem cell maintenance throughout life. Cell 132:5986112008

    • Search Google Scholar
    • Export Citation
  • 23

    Olsson MBjerregaard KWinkler CGates MBjörklund ACampbell K: Incorporation of mouse neural progenitors transplanted into the rat embryonic forebrain is developmentally regulated and dependent on regional and adhesive properties. Eur J Neurosci 10:71851998

    • Search Google Scholar
    • Export Citation
  • 24

    Renfranz PJCunningham MGMcKay RD: Region-specific differentiation of the hippocampal stem cell line HiB5 upon implantation into the developing mammalian brain. Cell 66:7137291991

    • Search Google Scholar
    • Export Citation
  • 25

    Rex MOrme AUwanogho DTointon KWigmore PMSharpe PT: Dynamic expression of chicken Sox2 and Sox3 genes in ectoderm induced to form neural tissue. Dev Dyn 209:3233321997

    • Search Google Scholar
    • Export Citation
  • 26

    Schmidt MHBicker FNikolic IMeister JBabuke TPicuric S: Epidermal growth factor-like domain 7 (EGFL7) modulates Notch signalling and affects neural stem cell renewal. Nat Cell Biol 11:8738802009

    • Search Google Scholar
    • Export Citation
  • 27

    Shen QWang YKokovay ELin GChuang SMGoderie SK: Adult SVZ stem cells lie in a vascular niche: a quantitative analysis of niche cell-cell interactions. Cell Stem Cell 3:2893002008

    • Search Google Scholar
    • Export Citation
  • 28

    Sigurjonsson OEPerreault MCEgeland TGlover JC: Adult human hematopoietic stem cells produce neurons efficiently in the regenerating chicken embryo spinal cord. Proc Natl Acad Sci U S A 102:522752322005

    • Search Google Scholar
    • Export Citation
  • 29

    Snyder EYDeitcher DLWalsh CArnold-Aldea SHartwieg EACepko CL: Multipotent neural cell lines can engraft and participate in development of mouse cerebellum. Cell 68:33511992

    • Search Google Scholar
    • Export Citation
  • 30

    Striedter GFBeydler S: Distribution of radial glia in the developing telencephalon of chicks. J Comp Neurol 387:3994201997

  • 31

    Vicario-Abejón CJohe KKHazel TGCollazo DMcKay RD: Functions of basic fibroblast growth factor and neurotrophins in the differentiation of hippocampal neurons. Neuron 15:1051141995

    • Search Google Scholar
    • Export Citation
  • 32

    White PMAnderson DJ: In vivo transplantation of mammalian neural crest cells into chick hosts reveals a new autonomic sublineage restriction. Development 126:435143631999

    • Search Google Scholar
    • Export Citation
  • 33

    Wilson SWHouart C: Early steps in the development of the forebrain. Dev Cell 6:1671812004

  • 34

    Zhao CDeng WGage FH: Mechanisms and functional implications of adult neurogenesis. Cell 132:6456602008

TrendMD
Metrics

Metrics

All Time Past Year Past 30 Days
Abstract Views 322 273 19
Full Text Views 107 60 1
PDF Downloads 218 62 2
EPUB Downloads 0 0 0
PubMed
Google Scholar