Rapid differentiation of NT2 cells in Sertoli-NT2 cell tissue constructs grown in the rotating wall bioreactor.
Cell replacement therapy is of great interest as a long-term treatment of neurodegenerative diseases such as Parkinson's disease (PD). We have previously shown that Sertoli cells (SC) provide neurotrophic support to transplants of dopaminergic fetal neurons and NT2N neurons, derived from the human clonal precursors cell line NTera2/D1 (NT2), which differentiate into dopaminergic NT2N neurons when exposed to retinoic acid. We have created SC-NT2 cell tissue constructs cultured in the high aspect ratio vessel (HARV) rotating wall bioreactor. Sertoli cells, NT2, and SC plus NT2 cells combined in starting ratios of 1:1, 1:2, 1:4 and 1:8 were cultured in the HARV in DMEM with 10% fetal bovine serum and 1% growth factor reduced Matrigel for 3 days, without retinoic acid. Conventional, non-HARV, cultures grown in the same culture medium were used as controls. The presence of tyrosine hydroxylase (TH) was assessed in all culture conditions. Sertoli-neuron-aggregated-cell (SNAC) tissue constructs grown at starting ratios of 1:1 to 1:4 contained a significant amount of TH after 3 days of culture in the HARV. No TH was detected in SC HARV cultures, or SC, NT2 or SC-NT2 conventional co-cultures. Quantitative stereology of immunolabled 1:4 SNAC revealed that approximately 9% of NT2 cells differentiate into TH-positive (TH+) NT2N neurons after 3 days of culture in the HARV, without retinoic acid. SNAC tissue constructs also released dopamine (DA) when stimulated with KCl, suggesting that TH-positive NT2N neurons in the SNAC adopted a functional dopaminergic phenotype. SNAC tissue constructs may be an important source of dopaminergic neurons for neuronal transplantation.
Saporta
Samuel
S
Department of Anatomy, College of Medicine, Health Sciences Center, University of South Florida, Tampa, FL 33612, USA. ssaporta@hsc.usf.edu
Willing
Alison E
AE
Shamekh
Rania
R
Bickford
Paula
P
Paredes
Daniel
D
Cameron
Don F
DF
D000818:Animals; D000831:Animals, Newborn; D019149:Bioreactors; D015153:Blotting, Western / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007211:Indoles / Q000378:metabolism; D008297:Male; D009474:Neurons / Q000166:cytology*; D051381:Rats; D012708:Sertoli Cells / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D007211:Indoles; C007293:DAPI; D014446:Tyrosine 3-Monooxygenase