Abstract
Background
Mammalian spermatogonial stem cells (SSC) are able to differentiate into different
cell types in vitro, which are valuable sources for regenerative medicine and gene
transfer studies. We investigated the differentiation potential of chicken SSC into
osteoblasts, neuron-like cells and adipocytes in vitro.
Methods
Chicken SSC from the testes of 18- and 20-day-old chicken embryos were cultured in
different induction media for three passages in vitro. For differentiation into osteoblasts,
SSC were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 1 × 10−4 µmol/mL desamethasone, 10 µmol/mL (β-sodium glycerophosphate and 0.05 mg/mL vitamin C, and examined by microscopy after Von Kossa's, cytochemical and immunohistochemical
staining. For differentiation into neuron-like cells, SSC were cultured in DMEM supplemented
with 1 × 10−3 µmol/mL retinoic acid (RA), 5.0 µmol/mL 3-isobutyl-1-methylxanthine (IBMX) and examined by microscopy after toluidine
blue or immunohistochemical staining. For differentiation into adipocytes, SSC were
cultured in DMEM supplemented with 1 × 10−3 µmol/mL dexamethasone, 0.01 mg/mL insulin, 0.5 µmol/mL IBMX and examined by microscopy after Oil red O staining and reverse transcriptase-polymerase
chain reaction (RT-PCR) for gene expression of peroxisome proliferation activation
receptor-γ (PPAR-γ).
Results
After 15 and 21 days of culture in the induction medium for osteoblast differentiation,
75% and 80% chicken SSC differentiated into osteoblasts, as confirmed by Von Kossa's,
calcium-cobalt and collagen I antibody staining. After 3 and 7 days of culture in
the induction medium for neuron-like cell differentiation, 78% and 85% SSC became
neuron-like cells, as confirmed by staining with toluidine blue and the monoclonal
antibody against neuron-specific enolase, nestin and glial fibrillary acidic protein.
After 7 days of culture in the induction for adipocyte differentiation, 85% SSC differentiated
into adipocytes, as confirmed by Oil red O staining and RT-PCT for PPAR-γ gene expression.
Discussion
Our results show that chicken SSC can differentiate into osteoblasts, neuron-like
cells and adipocytes under similar conditions as for directional differentiation of
mammalian SSC in vitro. The findings show the feasibility of using SSC-derived cells
for developmental biology and gene transfer studies in chickens.
Key Words
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Article info
Publication history
Accepted:
October 21,
2009
Received:
August 13,
2009
Identification
Copyright
© 2010 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
