BM-derived MSCs exhibit superior chondrogenic and osteogenic potential. (A) BM-MSCs were differentiated in vitro on Transwell membranes for 28 days in chondrogenic induction medium and then fixed, paraffin-embedded, and analyzed. Macroscopic view (left) of a representative cartilage tissue piece generated by 5 × 105 MSCs. Safranin O (middle) and toluidine blue staining (right) was used for visualization of glycosaminoglycans produced by differentiated hypertrophic chondrocytes. (B) Gene expression analysis of collagen 2a (COL2A), collagen 10a (COL10A), parathyroid hormone receptor 1 (PTHR1), aggrecan (ACAN), and distal-less homeobox 6 (DLX6) by quantitative reverse transcription–polymerase chain reaction after 28 days of chondrogenic induction comparing BM (black bars) to non-BM-derived cells (dark gray, light gray, and white bars). Bar graphs depict fold expression (mean ± SD) of differentiated MSCs over the respective cells in the uninduced state. (C) Endochondral ossification of BM-MSCs precedes marrow development in vivo. Time course analysis (2, 4, and 6 weeks posttranspant) of BM-MSCs transplanted in NSG mice (noninductive protocol), removed and sectioned for histology. Representative images show hematoxylin and eosin (H&E; upper row), pentachrome (second row), and alcian blue staining (third row). Immunohistochemical staining for human vimentin (huVimentin, bottom row) delineates human origin of the developed tissue. (D) Top: quantification of cartilage, bone, and hematopoietic tissue at 2-, 4-, and 6-week time points following transplant (noninductive protocol). Bottom: pentachrome-stained tissue sections (n = 4 ossicles per time point from 4 different human donors) were evaluated for relative percentage of respective tissue using the area calculation tool in ImageJ software. Representative pseudocolored images illustrating areas used for quantification are shown. (E) Bone formation in vivo is restricted to BM-MSCs. Two protocols (noninductive and osteoinductive) for testing bone and marrow niche formation capacity of MSCs are illustrated. Cells (2 × 106) were either directly injected subcutaneously in ECM (matrigel, noninductive protocol) or, to achieve maximal osteogenic induction, were cultured for 72 hours in a medium inducing osteogenic differentiation before being seeded onto (TCP-HA) particles. Thereafter, TCP-HA with attached MSCs was surgically implanted. Transplanted NSG mice received a daily anabolic dose of parathyroid hormone (PTH) for the first 21 days of the experiment to further enhance osteogenesis in vivo (osteoinductive protocol). After 4 weeks, in vivo imaging was performed to evaluate bone formation in both protocols used. Active bone growth and resorption was semiquantitatively evaluated in vivo using the bisphosphonate imaging agent OsteoSense on a Maestro imaging instrument. (F) Top: bar graphs represent bone scores from transplants containing BM-MSCs and WAT-, UC-, and skin-derived MSCs or control implants without cells (n = 3 per source). Cells were applied following either a noninductive (left) or an osteoinductive (right) transplantation protocol. Eight weeks posttransplant, mice were euthanized, and explants were analyzed to confirm in vivo imaging results. Bottom: representative photographs of H&E-stained tissues sections derived from all 4 different MSC-sources are shown as indicated. Student t test *P < .05, **P < .00001.
