Fig 5. MSCs decrease oligodendrocyte death.

(A) Oligodendrocyte progenitor cells (OPCs) were isolated from neonatal rat brains and cultured in PDGF-supplemented Sato medium. For preparation of MSC-conditioned media (MSC-CM), MSCs were plated in Mouse Mesencult medium (MMM) alone or supplemented with interferon-gamma (IFNγ). After 24–48 hr, media was discarded, MSCs were washed with PBS, and subsequently received fresh MMM without additional factors. Conditioned media from these MSCs were harvested after 24 hours and filtered. After 3–4 days of culture in PDGF-supplemented Sato medium, OPCs then received 1:1 ratio of Sato medium: MMM (-MSC-CM, conditioned-medium), 1:1 of Sato: un-stimulated MSC-CM (+MSC-CM), or 1:1 of Sato: IFNγ-stimulated MSC-CM, for a period of 7–9 days. Cells were then harvested and MBP/Annexin V-stained for downstream flow cytometric analysis. Cells were gated on MBP+ cells for Annexin V analysis. Shown are a representative histogram and a bar graph consisting of data from three independent experiments. (B) To evaluate the effect of MSC action on oligodendrocyte death in vivo, female C57BL/6 mice were cuprizone-fed and treated with MSCs or phosphate-buffered saline vehicle as previously described (n = 5/treatment group). After 14 days of treatment, corpora callosa were harvested from brains and manually minced. Single cell suspensions were prepared, stained for the oligodendrocyte marker galactocerebroside (GalC) and for Annexin V and 7-AAD to evaluate cell survival (Annexin V^neg/7-AAD^neg cells). Cells were analyzed by flow cytometry. Shown are compiled bar graph data from two independent experiments. Significance was measured by Student’s t-test, with *p<0.05.