All other authors declare that they have no competing interests.. and luciferase expression, respectively, in differentiated hBM-MSCs. Scale bars: 100 m (GFP); 5 mm (luciferase). Abbreviations: GFP: green fluorescent protein; hBM-MSCs: human bone marrow mesenchymal stem cells. NIHMS1054963-supplement-Supp_FigS1-3.pdf (660K) GUID:?0C009DEC-EAE9-48B5-AB9D-DA6626185839 Supp FigS4-9: Figure S4 Differentiation potential of CD264-sorted hBM-MSCs. CD264+ and CD264? populations of hBM-MSCs from both donors were cultured for 21 days in either osteo-, adipo-, or chondrogenic medium. Control cultures were maintained in growth medium (a, d, k, n, u, x). To visualize differentiation, Alizarin Red S was used to detect calcified extracellular matrix during osteogenesis (a-f); AdipoRed, lipid accumulation during adipogenesis (k-p); and Alcian Blue, sulfated glycosaminoglycans during chondrogenesis in micromass cultures (u-z). To quantify osteo-, adipo- and chondrogenic differentiation, Alizarin Red S was extracted and absorbance was read at 562 nm (g-j); AdipoRed fluorescence was excited at 485 nm and emission was measured at 572 nm (q-t); and 1,9-dimethyl-methylene blue absorbance of digested pellet cultures was measured at 656 nm after dye decomplexation and compared against a standard curve from known chondroitin sulfate concentrations (aa-ad). Relative differentiation values are reported per microgram DNA and relative to the control cultures. Data are expressed as the mean SEM for = 4 biological replicates. *< 0.05 and **< 0.01 vs donor-matched CD264? hBM-MSCs. Scale bars = 200 m. Abbreviations: GAG: glycosaminoglycans; hBM-MSCs: human bone marrow mesenchymal stem cells; SEM: standard error of the mean.Figure S5 Visualizing hBM-MSC attachment to HA/TCP granules and scaffold aggregation. (a-c) Fluorescence images of transduced hBM-MSCs that were cultured on 40 mg porous HA/TCP granules for 6 hours at the stated inoculum. Scale bars = 200 m. Arrows indicate diameter of inner pore (125 m, a) and outer shell (500 m, b). (d, e) Scaffold architecture before and after aggregation with mouse fibrinogen and thrombin. Scale bars = 1 cm. Abbreviations: HA/TCP: 15% hydroxyapatite/85% -tricalcium phosphate; hBM-MSCs: human bone marrow mesenchymal stem cells. Figure S6 Dependence of PTEN1 implant bioluminescence on hBM-MSC seeding density. Constructs were prepared with 40 mg HA/TCP granules that Collagen proline hydroxylase inhibitor Collagen proline hydroxylase inhibitor were seeded with eGFP-FLuc hBM-MSCs. Representative images and background-corrected bioluminescence for the following seeding conditions: (a, b) CD264? hBM-MSCs at three different seeding densities per mouse (= 3 mice), and (c, d) CD264+ (black) and CD264? (white) hBM-MSCs at the same seeding density per mouse for both 1105 and 1106 cells/40 mg granules (= 3 mice per seeding density). Each mouse is denoted by a different symbol (, , ). Abbreviations: eGFP: enhanced green fluorescent protein; FLuc: firefly luciferase; HA/TCP: 15% hydroxyapatite/85% -tricalcium phosphate; hBM-MSCs: human bone marrow mesenchymal stem cells. Figure S7 Sample calculation of survival metrics from bioluminescence imaging data. Collagen proline hydroxylase inhibitor An exponential regression of the bioluminescence Collagen proline hydroxylase inhibitor signals over 31 days was performed, and Collagen proline hydroxylase inhibitor the rate of decay was obtained from the exponential coefficient. The implant half-life was calculated from the rate of decay. The week 4: week 1 luminescence signal was calculated from the ratio of the final luminescence (sum of day 28 and day 31) to the initial luminescence (sum of day 0 and day 4). Abbreviations: s: seconds; wk: week. Figure S8 NG2 surface expression on hBM-MSCs. (a) Representative histogram and (b) mean fluorescence intensity ratio (mean SEM, = 3 biological replicates) from flow cytometric analysis of NG2 surface expression for both donor 1 (blue) and donor 2 (red) relative to isotype (white). The NG2 MFI ratio for donor 2 hBM-MSCs was on average > 1.5 times the value for donor 1 hBM-MSCs. *< 0.05 vs donor 1. Abbreviations: APC: allophycocyanin; hBM-MSCs: human bone marrow mesenchymal stem cells; MFI: mean fluorescence intensity; NG2: neuron-glial antigen 2; SEM: standard error of the mean. Figure S9 survival of CD264+ and CD264? hBM-MSCs. CD264-sorted eGFP-FLuc hBM-MSCs were seeded on 40 mg HA/TCP granules, aggregated with mouse fibrinogen and thrombin, and cultured for two months. (a) Initial and final bioluminescence images and (b) temporal profile of background-corrected bioluminescence from a representative culture of donor 1 hBM-MSCs. Abbreviations: eGFP: enhanced green fluorescent protein; FLuc: firefly luciferase; HA/TCP: 15% hydroxyapatite/85% -tricalcium phosphate; hBM-MSCs: human bone marrow mesenchymal stem cells. NIHMS1054963-supplement-Supp_FigS4-9.pdf (765K) GUID:?C506A2DE-9863-4F3F-A6B5-F9E9473F198F Abstract mesenchymal stem cell (MSC) survival is relevant to therapeutic applications requiring engraftment and potentially to non-engraftment applications as well. MSCs are a mixture of progenitors at different stages of cellular aging, but the contribution of this heterogeneity to the.