Premature induction of hypertrophy during in vitro chondrogenesis of human mesenchymal stem cells correlates with calcification and vascular invasion after ectopic transplantation in SCID mice

Abstract

Objective. Functional suitability and phenotypic stability of ectopic transplants are crucial factors in the clinical application of mesenchymal stem cells (MSCs) for articular cartilage repair, and might require a stringent control of chondrogenic differentiation. This study evaluated whether human bone marrow-derived MSCs adopt natural differentiation stages during induction of chondrogenesis in vitro, and whether they can form ectopic stable cartilage that is resistant to vascular invasion and calcification in vivo.Methods. During in vitro chondrogenesis of MSCs, the expression of 44 cartilage-, stem cell-, and bone-related genes and the deposition of aggrecan and types II and X collagen were determined. Similarly treated, expanded articular chondrocytes served as controls. MSC pellets were allowed to differentiate in chondrogenic medium for 3-7 weeks, after which the chondrocytes were implanted subcutaneously into SCID mice; after 4 weeks in vivo, samples were evaluated by histology.Results. The 3-stage chondrogenic differentiation cascade initiated in MSCs was primarily characterized by sequential up-regulation of common cartilage genes. Premature induction of hypertrophy-related molecules (type X collagen and matrix metalloproteinase 13) occurred before production of type II collagen and was followed by up-regulation of alkaline phosphatase activity. In contrast, hypertrophy-associated genes were not induced in chondrocyte controls. Whereas control chondrocyte pellets resisted calcification and vascular invasion in vivo, most MSC pellets mineralized, in spite of persisting proteoglycan and type II collagen content.Conclusion. An unnatural pathway of differentiation to chondrocyte-like cells was induced in MSCs by common in vitro protocols. MSC pellets transplanted to ectopic sites in SCID mice underwent alterations related to endochondral ossification rather than adopting a stable chondrogenic phenotype. Further studies are needed to evaluate whether a more stringent control of MSC differentiation to chondrocytes can be achieved during cartilage repair in a natural joint environment.

Chondrogenic differentiation of adipose tissue‐derived mesenchymal stem cells: Greater doses of growth factor are necessary

“…When a fivefold combination dose of TGFb 2 and IGF-I was treated to ATMSCs, gene expressions comparable to BMMSCs were demonstrated not only for chondrogenic genes but also for COL1A1. This concurrent increase of type I collagen gene expression was reported from chondrogenesis from BMMSCs by previous studies 25,26 and underscores a problem intrinsic to MSCs. Future studies should address solutions to mitigate this limitations that MSCs have for neochondrogenesis.…”

Cartilage fragments from osteoarthritic knee promote chondrogenesis of mesenchymal stem cells without exogenous growth factor induction

“…This implied that cartilage fragments might drive cells toward chondrogenesis. As for type X collagen, one of the characteristic components of hypertrophic chondrocyte, [16][17][18] its gene expression was high in our study. This might be due to the use of immortalized MSC cell line as the cell source.…”

Skeletal tissue engineering using embryonic stem cells

“…Instability of engineered cartilage was also observed when MSCs, differentiated into the chondrogenic lineage for 3 weeks, were implanted into immuno-deficient mice. The cartilage either degraded, or fibrous or fibrocartilagelike implants were retrieved [210,238]. It appeared that longer in vitro incubation resulted in fewer degraded cartilage samples after implantation [238].…”

“…The cartilage either degraded, or fibrous or fibrocartilagelike implants were retrieved [210,238]. It appeared that longer in vitro incubation resulted in fewer degraded cartilage samples after implantation [238]. Thus, the stability of ESC-derived cartilage in vivo might be improved by changing the in vitro differentiation protocol.…”