title: Sonic hedgehog and retinoic Acid induce bone marrow-derived stem cells to differentiate into glutamatergic neural cells.
abstract: Studies have showed that transplanted stem cells in the inner ear won't regenerate to replace the damaged sensory hair cells. They can spontaneously differentiate into mesenchymal cells and fibrocytes in the damaged inner ear. Only mature sensory cells of MSCs-derived possess the great potency for cell transplantation in the treatment of sensorineural hearing loss. So, we try to establish an efficient generation of the glutamatergic sensory neural phenotype for the cell transplantation of the hearing loss. We isolated MSCs from femoral and tibial bones according to their adherence to culture dishes. After purification, proliferation, and passaged, cells became homogeneous in appearance, showing more uniformity and grew in a monolayer with a typical spindle-shape morphology. The cell surface markers were assessed using FACS to characterize the isolated cells. For neural induction to harvest the glutamatergic sensory neurons, passage 3 MSCs were incubated with preinduced medium for 24 hr, and neural-induced medium for an additional 14 days. The cells exhibit a typical neural shape. RT-PCR analysis indicated that the mRNA levels of the neural cell marker nestin, Tau, MAP-2, β-tubulin III, GluR-3, and GluR-4 were higher compared with primary MSCs. Immunohistochemistry and western-blotting proofed that nestin, MAP-2, β-tubulin III, and GluR-4 proteins indeed exhibit their expression difference in the induced cells compared to the MSCs. We show an efficient protocol by the combined applications of Sonic Hedgehog (Shh) and Retinoic Acid (RA) to induce MSCs to differentiate into the glutamatergic sensory neuron which were identified from the morphological, biochemical, and molecular characteristics.
authors.affiliation: a Department of Human Anatomy, College of Basic Medical Sciences , Second Military Medical University , Shanghai , PR China.
authors.forename: Zhenhai
authors.initials: Z
authors.lastname: Yu
authors.forename: Shixing
authors.initials: S
authors.lastname: Wu
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liu
authors.forename: Haiyan
authors.initials: H
authors.lastname: Lin
authors.forename: Lei
authors.initials: L
authors.lastname: Chen
authors.forename: Xinli
authors.initials: X
authors.lastname: Yuan
authors.forename: Zhiying
authors.initials: Z
authors.lastname: Zhang
authors.forename: Fang
authors.initials: F
authors.lastname: Liu
authors.forename: Chuansen
authors.initials: C
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000276:immunology; D053823:Hedgehog Proteins / Q000276:immunology*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D058953:Neural Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013329:Structure-Activity Relationship; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: D053823:Hedgehog Proteins; C546888:Shh protein, rat; D014212:Tretinoin
keywords: Retinoic Acid; Sonic Hedgehog; glutamatergic sensory neurons; induced; mesenchymal stem cell