Combined extrinsic and intrinsic manipulations exert complementary neuronal enrichment in embryonic rat neural precursor cultures: An in vitro and in vivo analysis

Orion Furmanski, Shyam Gajavelli, Jeung Woon Lee, Maria E. Collado, Stanislava Jergova, Jacqueline Sagen

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

Numerous central nervous system (CNS) disorders share a common pathology in dysregulation of γ-aminobutyric acid (GABA) inhibitory signaling. Transplantation of GABA-releasing cells at the site of disinhibition holds promise for alleviating disease symptoms with fewer side effects than traditional drug therapies. We manipulated fibroblast growth factor (FGF)-2 deprivation and mammalian achaete-scute homolog (MASH)1 transcription factor levels in an attempt to amplify the default GABAergic neuronal fate in cultured rat embryonic neural precursor cells (NPCs) for use in transplantation studies. Naïve and MASH1 lentivirus-transduced NPCs were maintained in FGF-2 or deprived of FGF-2 for varying lengths of time. Immunostaining and quantitative analysis showed that GABA- and β-III-tubulin-immunoreactive cells generally decreased through successive passages, suggesting a loss of neurogenic potential in rat neurospheres expanded in vitro. However, FGF-2 deprivation resulted in a small, but significantly increased population of GABAergic cells derived from passaged neurospheres. In contrast to naive and GFP lentivirus-transduced clones, MASH1 transduction resulted in increased bromodeoxyuridine (BrdU) incorporation and clonal colony size. Western blotting showed that MASH1 overexpression and FGF-2 deprivation additively increased β-III-tubulin and decreased cyclic nucleotide phosphodiesterase (CNPase) expression, whereas FGF-2 deprivation alone attenuated glial fibrillary acidic protein (GFAP) expression. These results suggest that low FGF-2 signaling and MASH1 activity can operate in concert to enrich NPC cultures for a GABA neuronal phenotype. When transplanted into the adult rat spinal cord, this combination also yielded GABAergic neurons. These findings indicate that, even for successful utilization of the default GABAergic neuronal precursor fate, a combination of both extrinsic and intrinsic manipulations will likely be necessary to realize the full potential of NSC grafts in restoring function.

Original languageEnglish (US)
Pages (from-to)56-71
Number of pages16
JournalJournal of Comparative Neurology
Volume515
Issue number1
DOIs
StatePublished - Jul 1 2009

Keywords

  • Clonal analysis
  • Differentiation
  • FGF-2
  • GABA
  • MASH1
  • Transplantation

ASJC Scopus subject areas

  • Neuroscience(all)

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