Growth hormone promotes proliferation of adult neurosphere cultures.
OBJECTIVE
Growth hormone (GH) and its receptor (GHR) are widely expressed in the CNS. During development, GH signaling regulates both proliferation of neural progenitor cells as well as their differentiation into neurons and glia. Here we have examined the effect of GH signaling on adult subventricular zone derived neural progenitor cells cultured as neurospheres.


METHODS
GH was added to adult wild-type (WT) neurosphere cultures and neurosphere growth measured using the MTT cell proliferation assay. To examine the influence of endogenous GH production on neural progenitors, neurospheres derived from GH receptor knockout (GHRKO) mice were examined by measuring neurosphere sizes and Ki67 and TUNEL immunoreactivity. In addition, neurosphere growth curves were compared following long term culture. Finally, the differentiation of WT vs. GHRKO neurospheres was compared using immunocytochemistry for betaIII-tubulin and GFAP.


RESULTS
While GH alone was insufficient to support neurosphere formation, it enhanced neurosphere growth by 20% in the presence of epidermal growth factor and fibroblast growth factor-2. Compared to wildtype neurospheres, GHRKO neurospheres were smaller, contained fewer proliferating cells and exhibited reduced self-renewal in long term culture. Addition of GH increased STAT5 phosphorylation levels in neurosphere cells. Upon differentiation, GHRKO neurospheres showed accelerated neurogenesis, although over time similar numbers of betaIII-tubulin positive neurons were generated by cells of both genotypes.


CONCLUSIONS
GH functions as an autocrine mitogen in adult neurosphere cultures and promotes proliferation of neural progenitor cells as well as self-renewal of neurosphere cultures. In addition, signaling through the GHR appeared to delay neuronal differentiation in adult neurospheres.
McLenachan
S
S
Neural Regeneration Laboratory, Centre for Neuroscience, University of Melbourne, Parkville, Melbourne, Victoria 3010, Australia.
Lum
M-G
MG
Waters
M J
MJ
Turnley
A M
AM
D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D013006:Growth Hormone / Q000494:pharmacology*; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D055495:Neurogenesis; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D010766:Phosphorylation; D017978:Receptors, Growth Factor / Q000502:physiology*; D050799:STAT5 Transcription Factor; D018874:Spheroids, Cellular / Q000378:metabolism; D013234:Stem Cells / Q000502:physiology*; D014404:Tubulin / Q000378:metabolism
D017978:Receptors, Growth Factor; D050799:STAT5 Transcription Factor; D014404:Tubulin; D013006:Growth Hormone