Multi-ponent differentiation of mesenchymal stem cells

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, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284:143?147.
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, Beck SC, Murphy JM et al. Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng 1998;4:415?428.