Direct cell-cell contact required for neurotrophic effect of chromaffin cells on neural progenitor cells.
Previous studies showed that neural progenitor cultures could be maintained without exogenously added FGF-2 when co-cultured with chromaffin cells. In addition, progenitor cells displayed dramatically increased neuronal differentiation in the presence of chromaffin cells. These findings suggested an approach to improved neural progenitor transplant outcomes using co-transplantation or administration of chromaffin cell-derived factors. The aim of this study was to determine whether the observed survival and differentiation effects were due to diffusible factors or required direct cell-cell contact (DC). Rat neural progenitors were cultured under six different conditions: (1) Standard N2 media with FGF-2; (2) N2 without FGF-2; (3) N2 with FGF+conditioned media (CM) from chromaffin cultures; (4) N2 without FGF-2+CM; (5) Transwells (TW), progenitor+chromaffin cells grown together but separated by a membrane allowing movement of diffusible agents but preventing direct contact; (6) direct contact co-cultures of progenitors and chromaffin cells. Cultures were evaluated for survival, proliferation, and differentiation. Cultures with FGF-2 proliferated and formed floating neurospheres while those grown in N2 without FGF-2 failed to thrive. Those grown either with CM or in transwells showed significantly improved survival. Survival was comparable to the exogenous FGF groups when progenitors were allowed direct contact with chromaffin cells. Proliferation was low in all cultures except those receiving exogenous FGF-2. Direct contact co-cultures exhibited a marked increase in beta-tubulin III+ processes compared to all other groups, indicating differentiation towards a neuronal phenotype. The results of this study suggest that diffusible agents produced by chromaffin cells can sustain viable progenitor cells in vitro even in the absence of added FGF-2 but that the effects on progenitor cell neuronal differentiation require direct cell-cell contact.
Schumm
Michael A
MA
The Miami Project to Cure Paralysis, School of Medicine, University of Miami, Lois Pope Life Center, 1095 NW 14th Terrace (R-48), Miami, FL 33136, USA.
Castellanos
Daniel A
DA
Frydel
Beata R
BR
Sagen
Jacqueline
J
D000313:Adrenal Medulla / Q000166:cytology; D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism; D002417:Cattle; D002450:Cell Communication / Q000502:physiology*; D002452:Cell Count; D002454:Cell Differentiation; D002470:Cell Survival; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000502:physiology*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D018920:Coculture Techniques / Q000379:methods; D003114:Colony-Forming Units Assay; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004622:Embryo, Mammalian; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D007150:Immunohistochemistry; D007211:Indoles / Q000378:metabolism; D007381:Intermediate Filament Proteins / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D009419:Nerve Tissue Proteins; D064231:Nestin; D009474:Neurons; D051381:Rats; D018100:Receptors, Histamine H3 / Q000378:metabolism; D013234:Stem Cells / Q000502:physiology*; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D017077:Culture Media, Conditioned; D007211:Indoles; D007381:Intermediate Filament Proteins; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; C577486:Nes protein, rat; D064231:Nestin; D018100:Receptors, Histamine H3; D014404:Tubulin; C462941:beta3 tubulin, mouse; D016222:Fibroblast Growth Factor 2; C007293:DAPI; D014446:Tyrosine 3-Monooxygenase; D001973:Bromodeoxyuridine