(1) In vitro, bone marrow-derived stromal cells (BMSCs) demonstrate inter-donor phenotypic variability, which presents challenges for the development of regenerative therapies. Here, we investigated whether the frequency of putative BMSC sub-populations within the freshly isolated mononuclear cell fraction of bone marrow is phenotypically predictive for the in vitro derived stromal cell culture. (2) Vertebral body, iliac crest, and femoral head bone marrow were acquired from 33 patients (10 female and 23 male, age range 14–91). BMSC sub-populations were identified within freshly isolated mononuclear cell fractions based on cell-surface marker profiles. Stromal cells were expanded in monolayer on tissue culture plastic. Phenotypic assessment of in vitro derived cell cultures was performed by examining growth kinetics, chondrogenic, osteogenic, and adipogenic differentiation. (3) Gender, donor age, and anatomical site were neither predictive for the total yield nor the population doubling time of in vitro derived BMSC cultures. The abundance of freshly isolated progenitor sub-populations (CD45−CD34−CD73+, CD45−CD34−CD146+, NG2+CD146+) was not phenotypically predictive of derived stromal cell cultures in terms of growth kinetics nor plasticity. BMSCs derived from iliac crest and vertebral body bone marrow were more responsive to chondrogenic induction, forming superior cartilaginous tissue in vitro, compared to those isolated from femoral head. (4) The identification of discrete progenitor populations in bone marrow by current cell-surface marker profiling is not predictive for subsequently derived in vitro BMSC cultures. Overall, the iliac crest and the vertebral body offer a more reliable tissue source of stromal progenitor cells for cartilage repair strategies compared to femoral head.

Introduction: Mesenchymal stem cells (MSCs) are considered a promising cell source for cartilage repair strategies due to their chondrogenic differentiation potential. However, their in-vitro tendency to progress towards hypertrophy limits their clinical use. This unfavourable result may be due to the fact that MSCs used in tissue engineering approaches are all at the same developmental stage, and have lost crucial spatial and temporal signalling cues. In this study, we sought to investigate the effect of a spatial parathyroid hormone-related protein (PTHrP) signalling gradient on the chondrogenic differentiation of MSCs and progression to hypertrophy. Methods: Human bone marrow-derived MSCs were transduced with adenoviral vectors overexpressing PTHrP and seeded into fibrin-poly(ester-urethane) scaffolds. To investigate the effect of a spatial PTHrP signalling gradient, scaffolds were seeded with PTHrP-overexpressing MSCs positioned on top of the scaffold, with untransduced MSCs seeded evenly within. Scaffolds were cultured with or without 2 ng/ml TGF-β1 for 28 days. Results: PTHrP overexpression increased GAG production by MSCs irrespective of TGF-β1 treatment, and exerted differential effects on chondrogenic and hypetrophic gene expression when MSCs were cultured in the presence of a PTHrP signalling gradient. Furthermore, PTHrP-overexpressing MSCs were associated with an increase of endogenous TGF-β1 production and reduced total MMP-13 secretion compared to controls. Conclusion: The presence of a spatial PTHrP signalling gradient may support chondrogenic differentiation of MSCs and promote the formation of a more stable cartilage phenotype in tissue engineering applications.