Fig. 1
Single and combined treatment effect of cyclic mechanical stretch (CMS) and hyperoxia (HOX) on human umbilical cord derived MSC (UC-MSC) from newborn infants resulting in reduced proliferative capacity and cell viability. (A) Characterization of CD surface markers of MSC isolated from umbilical cords of newborn infants. Multicolor flow cytometry revealed expression of MSC-characteristic surface markers CD146, CD90, CD73 and CD105 whereas the hematopoietic marker CD45 was absent, and CD165, CD34 and CD11b only weakly positive. Positive fraction of stained cells versus isotype. Representative data of n = 7 independent UC-MSC cultures experiments. Red = unstained control, blue = specific staining. PE-Cy7 = Phycoerythrin-Cyanine 7; APC =; Allophycocyanin; BV421 = Brilliant Violet 421; PerCP = Peridinin-Chlorophyll-Protein; FITC = Fluorescein isothiocyanate; APC-Cy7 = APC-Cyanine 7; BV605 = Brilliant Violet 605. (B) Quantitative FACS data (MFI ratio log10 of all n = 7 independent UC-MSC cultures included into the FACS analyses). (C) Bright-field microscopy of proliferation after 72 h of selective and combined exposure to CMS and/or hyperoxia (HOX 80%). Scale bar 250 μm. Representative data of n = 3 independent UC-MSC cultures experiments. (D) Change in cell expansion index (CEI) was calculated as the quotient of [cell count at the end of the experiment/ cell count at the start of the experiment] using manual cell counting of image recordings. CMS and/or hyperoxic exposure (HOX 80%) led to a dose-dependent inhibition of MSC proliferation. n = 3 independent MSC cultures. (E) Representative flow cytometry analysis of n = 12 independent UC-MSC cultures experiments with exposure to CMS and/or HOX (HOX 80%). Live/dead staining using Annexin V/SYTOX Blue double staining and flow cytometry analysis. Presented is the absolute cell death (%) = dead cells / frequency of single cells. (F) Percentage increase of absolute apoptosis induced by selective and combined treatment with CMS and/or HOX (HOX 80%) compared to unexposed UC-MSC. n = 12 independent MSC cultures. Data are expressed as box plot with median and interquartile range, *p < 0.05, *** p < 0.001, by paired t-test, comparing exposures to the control situation.
