Cell culture of freshly isolated +/+ BMSC and −/− BMSC was performed as previously described30 (link),51 (link). Prospectively isolated BMSCs were seeded in BMSC medium, which consisted of low-glucose α-MEM (1 g glucose/L, M4526, Sigma-Aldrich), supplied with 2 mM L-glutamine, 10 U/L heparin (preservative-free, Merck, Darmstadt, Germany) and 20 U/ml penicillin–streptomycin. 10% (v/v) pooled human platelet lysate (prepared as described in31 (link)) was added freshly to the cell culture. Seeding density was on average 1000 cells/cm2 for healthy donor samples, or 2500 cells/cm2 for MDS and AML samples, respectively. Cells were propagated in a humidified incubator at 37 °C and 5% CO2. Two-thirds of the medium were changed twice a week.
Colony-forming efficiency (CFE) of fibroblast-like colonies was observed every second day for up to four weeks or until colonies grew confluent52 (link). Colonies ≥ 25 cells were counted as being derived from CFU-F. −/− BMSC did not form fibroblast like colonies. CFU-F frequency was calculated by dividing the number of colonies observed by the number of input cells seeded, followed by normalization of colony number per 104 input cells.
Colony-forming efficiency (CFE) of fibroblast-like colonies was observed every second day for up to four weeks or until colonies grew confluent52 (link). Colonies ≥ 25 cells were counted as being derived from CFU-F. −/− BMSC did not form fibroblast like colonies. CFU-F frequency was calculated by dividing the number of colonies observed by the number of input cells seeded, followed by normalization of colony number per 104 input cells.
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