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Chondrogenic differentiation capability of C3-MSCs and C3 MSCs RUNX2 knock-down (Aa) Macroscopic views of 5 chondroid spheres made from treated C3 MSCs (shRUNX2), and 5 identically processed control pellets generated from mock treated C3 MSCs (ctrl). (b) Chondrospheres from mock cells generate satisfactory cartilage. In contrast, treated cells regularly failed to form good cartilage differentiation. Hematoxylin/Eosin (H/E); Alcian blue/Hematoxylin (Alcian blue/H); Alcian blue stain. Scale bar = 200 µm, 100 µm. (c) Macroscopic views of 3 chondroid spheres generated from C3 MSCs, and 3 pellets generated from C3 MSCs shRUNX2 are shown. Hematoxylin and eosin staining of C3-MSCs (left) pellets induced by culture with chondrogenic medium (Cambrex) for 3 weeks. Immunostaining of C3-MSCs (left) pellets with anti-human type II collagen (COL II), anti-human type X collagen (COL X) and anti-human Sox-9 (Sox9) and counterstained with hematoxylin. In contrast, RUNX2 knock-down (right) pellets regularly failed to form good cartilage differentiation (Alcian blue/Hematoxylin). (d) Western Blot analysis of C3 MSCs infected with control lentivirus or shRUNX2 lentivirus. RUNX2 protein is effectively knocked-down. (B) RUNX2 knock-down in human C3 MSCs supporting hematopoietic cells and progenitors in LTCs. Cord blood CD34+ cells were plated onto C3-MSC layers. Weekly cultures were halved to be reFed, harvested cells were counted (shown in a) and replated in semisolid hematopoietic secondary cultures to evaluate their progenitor cell content (b). Graphs derive from one of three different experiments performed in triplicates. C3 MSCs represent cells infected with missense control viral particles. BM6-MSCs are included as standard hematopoietic supportive cells. The treatment did not affect the overall survival of hematopoietic cells (a), but selectively decreased the hematopoietic progenitors output (b). At wk 5 the culture was terminated harvesting both the cells in suspension (s) and the ones entrapped in the adherent layer (a), which were cumulated to generate the points wk5 (s + a).

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