CD105 defines two distinct MSC subpopulations in mouse.

<p>(A) Balb/c ASCs were double-stained for CD105 in combination with CD29, CD44, CD45, CD49f, sca-1 and MHC class I and analyzed by flow cytometry. One representative experiment out of 3 independent experiments is shown. (B) MACS-purified CD105^- and CD105⁺ mASCs were cultured separately for 9 passages and CD105 expression was analyzed at each passage by flow cytometry. (C) CD105^- and CD105⁺ mASCs were starved for 24 hours and then stimulated with 10 ng/ml recombinant human TGF-β1 for 0, 2, 6 and 24 hours. At each time point total RNA was purified and reverse transcribed and the expression of CD105L was measured using qPCR. (D) ASC^tot, CD105^- and CD105⁺ cells were plated at 2500 cells/cm in T25 cell culture flasks and cultured at 5% O₂ until reaching 70-80% confluency. Cells were harvested, counted and reseeded at the same density for 10 passages. (E) ASC^tot, CD105^- and CD105⁺ cells were cultured in 10 cm petri dishes (2000 cells/plate) for 14 days for CFU-F assay as described in materials and methods. Cells were subsequently fixed with PFA and stained with cresyl violet to visualize CFU-F. (F) Total RNA was extracted from ASC^tot, and MACS-separated CD105^- and CD105⁺ mASCs and reverse transcribed into cDNA. qPCR analysis was performed on genes involved in ASC multipotency (nanog), adipogenesis (LPL, PPAR-γ), osteogenesis (osteocalcin, ALP) and chondrogenesis (Sox-9). Results are shown as mean (SEM) of three independent experiments.