Murine MSCs are heterogeneous for CD105L expression.

<p>(A) Adipose tissue (subcutaneous and epididymal) from Balb/c mice were digested with collagenase type I for 30 minutes and the resulting cell suspension was stained for several cell surface markers before (day 0), 1, 2 and 6 days after plating on tissue-culture treated plastic and analyzed by flow cytometry. (B) ASC cultures were harvested with EDTA (2mM in PBS), TrypLE, or EDTA followed by treatment with collagenase type I or TrypLE followed by treatment with collagenase type 1 and analyzed by flow cytometry. The expression level (MFI) of each marker was normalized to the corresponding staining on EDTA-harvested cells. (C) Passage 4 mASCs were stained for a panel of surface antigens and analyzed by flow cytometry. Percentages of positive cells are represented as means (SEM) of 4 independent experiments. (D) ASCs from Balb/c and C57Bl/6 mice and BM-MSC from Balb/c mice were stained for CD105 and analyzed by flow cytometry. (E) Total RNA was purified from three independent mASC-cell preparations (passage 4-5), reverse transcribed and the expression of CD105L, CD105S and β-actin were analyzed by qPCR. (F) Total mASCs were cultured at either 5% O₂ or 21% O₂ for five passages and CD105 expression was analyzed for each passage by flow cytometry. (G) ASCs were cultured with or without TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) for 24 hours after which the cells were harvested and analyzed by flow cytometry for a panel of MSC markers. Results in A, B and F are shown as mean (SEM) of 3 independent experiments; in C and D a representative experiment is shown out of at least 4 independent experiments; data in G is represented as mean (SEM) of 2 independent experiments.