Human Bone Marrow Contains Mesenchymal Stromal Stem Cells That Differentiate In Vitro into Contractile Myofibroblasts Controlling T Lymphocyte Proliferation

Figure 4

Biological properties of MSC cultured in 3D collagen membranes. (a) Regulation of T lymphocyte proliferation by MSC-laden membranes. T lymphocytes were labeled with CFSE prior to culture, and proliferation was assessed as CSFE fluorescence decrease. Left panel: T lymphocyte proliferation in the absence of a membrane and MSC: white profile: quiescent T lymphocytes; gray profile: T cells stimulated with a3-28. Right panel: proliferation of T cells stimulated with a3-28 in the presence of membranes: gray profile: empty membrane; white profile: membrane containing MSC. These data are representative of 5 experiments. (b) In vitro differentiation in collagen membranes: left panels: control cultures in the presence of HPL only; upper right: adipocytic (oil Red O stain); median right: chondrocytic (Goldner’s stain); lower right: osteoblastic (Alizarin R stain) differentiation. Original magnifications are 10x, 20x, and 20x for adipocytic, chondrocytic, and osteoblastic differentiation, respectively. These data are representative of 3 experiments. (c) Biological activities of MSC-laden membranes after storage at 4°C for 0–5 days. Left: XTT reduction: an empty membrane (top) and a MSC-laden membrane (bottom) stored for 5 days at 4°C and subsequently exposed to XTT for 2 hours at 37°C are shown. The petri grid consists in 2 × 2 mm squares. Right: effect of MSC cocultured with activated T lymphocytes upon tryp degradation and T lymphocyte proliferation: “Tryp”: residual tryp concentration in various supernatants, “Prolif”: T lymphocyte proliferation. “No MSC”: cultures of T cells stimulated with a3-28 in the absence of MSC; “MBR 37°C” and “MBR 4°C”: cocultures of T lymphocytes and membranes seeded with MSC, without or with a previous storage of the latter at 4°C prior to the assay, respectively. The vertical axis refers either to the tryp concentration expressed in micromolar, or to the T cell proliferation expressed as the inverse of the median CFSE fluorescence. These data are representative of 2 experiments. (d) Flow cytometry analyses of surface molecules on MSC after membrane dissolution with collagenase II. The analyses are gated on 7AAD-negative live cells. Flow cytometry data are from one experiment, representative of 6.

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