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Differentiation Methods for MSCs In Vitro
Adipogenic Differentiation. To induce adipogenic differentiation, MSCs are cultured as
monolayers in DMEM and allowed to become confluent. The cells are cultured for 3-7 days
more, and then the medium is changed to adipogenic induction medium (MDI + I medium)
containing 0.5 mM methyl-isobutylxanthine, 1 µM dexamethasone and 10 µg/mL insulin,
100 µM indomethacin, and 10% FBS in low glucose (1 g/L) DMEM. The MSCs are incubated
in this medium for 48-72 hours, and the medium is changed to adipogenic maintenance
(AM) medium containing 10 µg/mL insulin and 10% FBS in DMEM for 24 hours. The cells
are then re-treated with (MDI + I) for second and third treatment rounds. The cultures are
maintained in AM medium for about 1 week and then assayed. Nile red is a fluorescent
vital dye, and staining is used to quantify lipid vacuoles using a UV plate reader and
counterstaining DNA with DAPI (4′,6-diamidino-2-phenylindole) to label DNA content as
described (Pittenger et al. 1999). The adipogenic MSCs can then be fixed and stained with
oil red O for nonquantitative histological evaluation.
Chondrogenic Differentiation. Chondrogenic differentiation is induced by placing 2.5 × 10 5
human MSCs into defined chondrogenic medium and subjecting them to gentle
centrifugation (800 g for 5 minutes) in a 15-mL conical polypropylene tube, where they
consolidate into a pellet within 24 hours. Chondrogenic media consists of high-glucose (4.5
g/L) DMEM supplemented with 6.25 µg/mL insulin, 6.25 µg/mL transferrin, 6.25 µg/mL
selenous acid, 5.33 µg/mL linoleic acid, 1.25 mg/mL bovine serum albumin (ITS+,
Collaborative Research, Cambridge, MA), 0.1 µM dexamethasone, 10 ng/mL TGF-β3, 50
µg/mL ascorbate 2-phosphate, 2 mM pyruvate, and antibiotics. [Note: (1) The TGF-β3 is
stored in small aliquots at −80°C to avoid freeze-thawing damage. (2) For rat MSCs, 10 ng/
mL BMP2 is also included for efficient chondrogenic differentiation.] The chondrogenic
differentiation medium is changed every day due to the labile TGF. After 2-3 weeks, the
pellets are fixed in 4% formaldehyde in phosphate-buffered saline (PBS), paraffin embedded
for histology, sectioned and analyzed by immunostaining for collagen II expression,
collagen I, and so on. Sections are also stained with safranin O for detection of
proteoglycans and toluidine blue for metachromasia. For rat MSCs, chondrogenic
differentiation is enhanced by the addition of 1 µg/mL BMP2.
Osteogenic Differentiation. To promote osteogenic differentiation of MSCs, approximately 3
× 10 4 cells are seeded onto 35-mm dishes in low-glucose DMEM with 10% FBS. After 24
hours, this medium is replaced with osteogenic differentiation medium composed of the
same medium supplemented with 50 µM ascorbate 2-phosphate, 10 mM β-glycerol
phosphate, and 100 nM dexamethasone. The medium is changed about every 3 days, and
after 10-14 days the cells are stained with alizarin red and compared to MSCs maintained in
normal culture medium. In separate cultures, mineralization is quantified by measuring
calcium deposition (Kit 587-M, Sigma Chemical Co.).
2000; Pittenger et al. 1999). The surface molecules also indicate which direct
cell-cell interactions can occur between MSCs and other cells. The undif-
ferentiated MSCs can serve as a stromal cell feeder layer for the propaga-
tion of HSCs and embryonic stem cells, providing an all-human system that
avoids the concerns of xenogeneic feeder layers. The addition of other assays
will strengthen the evaluation of MSC differentiation, and data suggest that
MSCs may differentiate to endothelial cells as they form vessel-like struc-
tures in vivo that express smooth muscle actin, and increased blood low
is seen in regions where MSCs have been implanted (Pittenger and Martin
2004, Shake et al. 2000, Quevedo et al. 2000). However, these vessel-like
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