title: Combined extrinsic and intrinsic manipulations exert complementary neuronal enrichment in embryonic rat neural precursor cultures: an in vitro and in vivo analysis.
abstract: Numerous central nervous system (CNS) disorders share a common pathology in dysregulation of gamma-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 beta-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 naïve 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 beta-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.
authors.affiliation: The Miami Project to Cure Paralysis, University of Miami, Miller School of Medicine, Miami, Florida 33136, USA.
authors.forename: Orion
authors.initials: O
authors.lastname: Furmanski
authors.forename: Shyam
authors.initials: S
authors.lastname: Gajavelli
authors.forename: Jeung Woon
authors.initials: JW
authors.lastname: Lee
authors.forename: Maria E
authors.initials: ME
authors.lastname: Collado
authors.forename: Stanislava
authors.initials: S
authors.lastname: Jergova
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Sagen
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics*; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D016380:Brain Tissue Transplantation / Q000379:methods*; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D019070:Cell Lineage / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1 / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism / Q000494:pharmacology*; D007395:Interneurons / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D008297:Male; D009433:Neural Inhibition / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism / Q000601:surgery; D033581:Stem Cell Transplantation / Q000379:methods*; D014161:Transduction, Genetic / Q000379:methods; D014404:Tubulin / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D015415:Biomarkers; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D016222:Fibroblast Growth Factor 2; D014404:Tubulin; D005680:gamma-Aminobutyric Acid; C499318:Ascl1 protein, rat