Estimulación trófica de células madre embrionarias de cuerpo estriado de rata en un bioensayo 3D

ANA MARIA CRUZ GAITAN; MIRTA REYNALDO; CARRI, NÉSTOR GABRIEL

Ibague, Colombia

Encuentro; SdN; 2010

IBRO-Sociedad de Neurosciencias, Ibague, Colombia.

Trophic stimulation of embryonic stem cells from rat corpus striatum in 3D bioassay  Few years ago were discovered progenitors cells in the Corpus Striatum (CS) from the sub ventricular zone of mammalian brain, but little is known about their proliferation and differentiation that have become essential for neural regeneration research. The purpose of this study was to analyze CS cells morpho-immune profiles using 3D bioassays under basal conditions (DMEM-F12, bFGF, B27, 37°C, humidity 100% and CO2 5%) and subsequent trophic stimulation (NGF, NT-3, or NTN) on collagen gel. Primary cells obtained from CS of rat embryos at E13-14, were cultivated in floating for the forming neurospheres, after 48h an aliquot was placed inside gelifield collagen I under trophic factors action, after 72h one passage was realized from first culture using the initial conditions until forming new neurospheres (48h), these were cultivated in collagen gel I with trophic stimulation. The expression of proliferation markers (PCNA, Ki67) and neural progenitor markers (GFAP, Nestin, Vimentin, O4, A2B5, Pax6, S100, TubIII, and NeuN) were analyzed by means inmunocitochemistry. Optimum generation and growth of neurites after 24 and 48h was obtained in all stimulated cultures, especially with NT-3. Also, we showed glial cells grew out. Antibodys staining was strongly positive opposite NeuN and O4 that were faint. We can conclude that the bioassay system allowed us to conclude that CS cells can generate neural progenitors useful for research on proliferation and differentiation, NSC culture inside gelifield collagen I under NT-3 support is an excellent culture medium for this cells type and their neurites. Finally this bioassay showed high reproducibility, and optimum resolution to  visualize neurons and glial cells outgrowth.  Key words: Neural stem cells, trophic support, collagen gel I, morpho-inmune profile. BIBLIOGRAFÍA   1. ALTMAN, J.; DAS, G. D. Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J. Comp. Neurol. 124: 319–336; 1965. 2. ARGIBAY, P. Medicina regenerativa y stem cells. De la terapia celular a la ingeniería de tejidos. Universidad Nacional de Quilmes Editorial. 2005. 3. BALDWIN, S.P., KREWSON, C.E., & SALTZMAN, W. M. (1996). PC12 cell aggreration and neurite growth in gels of collagen, laminin and fibronection, Int J Dev Neurosci, 14, 351-364. 4. CARRI, NG, SN VILLEGAS y FA POLETTA. Preparación de cultivos neurales en microensayos de baja densidad. Capítulo 17, en: Neurogénesis, Aspectos moleculares del desarrollo neural. Protocolos de Laboratorio. Anales del Tercer Taller sobre Neurogénesis. Impreso en Barza SRL. 1999.