Induction of Stem Cell Differentiation
Mesenchymal Differentiation Standard published protocols were used to promote differentiation
of passage 3-6 MSCs along mesenchymal lineages, including osteogenic [2], adipogenic [1], and chondrogenic lineages [3,4].
Briefly,
to induce osteogenic differentiation, confluent MSCs were incubated in osteogenic medium (control medium, supplemented with 10 nM
dexamethasone, 50 ug/ml L-ascorbic acid phosphate, and 5 mM beta-glycerophosphate [all from Sigma-Aldrich]) for 12 days, before being
stained for alkaline phosphatase activity and mineralization using the von Kossa stain. Adipogenic differentiation was stimulated
by culturing confluent MSCs in adipogenic medium (control medium with 10 nM dexamethasone, 50 ug/ml L-ascorbic acid phosphate, 500
uM isobutylmethyxanthine [IBMX; Sigma-Aldrich] and 60 uM indomethacin [Sigma-Aldrich]) for 12 days, before identifying adipocytes
by oil red O staining. MSCs were grown in micromass cultures to induce chondrogenic differentiation. 2 x105 cells in chondrogenic
medium (DMEM containing 110 ug/ml sodium pyruvate, 100 U/ml penicillin, 100 ug/ml streptomycin, 1% human recombinant insulin, human
transferrin, and selenous acid [ITS] + Premix [BD Biosciences, Oxford, U.K., http://www.bd.com], 50 ug/ml L-ascorbic acid phosphate,
100 nM dexamethasone, 50 ug/ml proline [Sigma-Aldrich], and 10 ng/ml recombinant human transforming growth factor?beta3 [TGF-beta3;
R&D Systems, Oxon, UK, http://www.rndsystems.com]) were placed in universals and pelleted by centrifugation at 800 rpm for 5 minutes,
before incubation for 15 days.
References:
1. Pittenger MF, Mackay AM,
2. Jaiswal N, Haynesworth SE, Caplan AI et al. Osteogenic differentiation of purified, culture-expanded
human mesenchymal stem cells in vitro. J Cell Biochem 1997;64:295?312.
3. Yoo JU, Barthel TS, Nishimura K et al. The chondrogenic
potential of human bone-marrow-derived mesenchymal progenitor cells. J Bone Joint Surg Am 1998;80:1745?1757.
4. Mackay AM,
Protocols
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