Donor variation and loss of multipotency during in vitro expansion of human mesenchymal stem cells for bone tissue engineering

Abstract

The use of multipotent human mesenchymal stem cells (hMSCs) for tissue engineering has been a subject of extensive research. The donor variation in growth, differentiation and in vivo bone forming ability of hMSCs is a bottleneck for standardization of therapeutic protocols. In this study, we isolated and characterized hMSCs from 19 independent donors, aged between 27 and 85 years, and investigated the extent of heterogeneity of the cells and the extent to which hMSCs can be expanded without loosing multipotency. Dexamethasone-induced ALP expression varied between 1.2-and 3.7-fold, but no correlation was found with age, gender, or source of isolation. The cells from donors with a higher percentage of ALP-positive cells in control and dexamethasone-induced groups showed more calcium deposition than cells with lower percentage of ALP positive cells. Despite the variability in osteogenic gene expression among the donors tested, ALP, Collagen type 1, osteocalcin, and S100A4 showed similar trends during the course of osteogenic differentiation. In vitro expansion studies showed that hMSCs can be effectively expanded up to four passages (approximately 10-12 population doublings from a P0 culture) while retaining their multipotency. Our in vivo studies suggest a correlation between in vitro ALP expression and in vivo bone formation. In conclusion, irrespective of age, gender, and source of isolation, cells from all donors showed osteogenic potential. The variability in ALP expression appears to be a result of sampling method and cellular heterogeneity among the donor population. ß

The Effect of Donor Variation and Senescence on Endothelial Differentiation of Human Mesenchymal Stromal Cells

“…Mesenchymal stromal cells (MSC), a much used source of autologous cells, can be isolated relatively easily from multiple sources including adipose tissue, tibia, femur, lumbar spine and trabecular bone [1][2][3][4]. They can be then expanded in vitro and are characterized by their multipotency.…”

“…We have recently described a robust protocol to induce endothelial-like cells from bone marrow-derived MSCs in vitro and demonstrated their ability to contribute to the vasculature upon implantation in a mouse model. What remains to be addressed in order to bring endothelial-like MSCs to the clinic is both the large inter-donor variation in multilineage potential [1,21] and the phenomenon of loss of multipotency upon culture expansion of MSCs. We and others have observed striking differences between hMSCs of different donors with regard to growth rate, expression of both lineage-specific and non-specific markers such as ALP and STRO-1 and their response to in vitro differentiation and ectopic bone formation [1,[21][22][23].…”

“…What remains to be addressed in order to bring endothelial-like MSCs to the clinic is both the large inter-donor variation in multilineage potential [1,21] and the phenomenon of loss of multipotency upon culture expansion of MSCs. We and others have observed striking differences between hMSCs of different donors with regard to growth rate, expression of both lineage-specific and non-specific markers such as ALP and STRO-1 and their response to in vitro differentiation and ectopic bone formation [1,[21][22][23]. Similar situation can occur upon endothelial differentiation which can explain the controversy concerning the ability of MSCs to acquire endothelial characteristics.…”

Skeletal tissue engineering using embryonic stem cells

“…Similar observations have been reported by many groups. The mechanism is still poorly understood, but donor variation, as also observed for osteogenic differentiation of human MSCs [229], and the heterogeneity of the MSC population are factors known to play a part. Similarly, we observed varying efficiencies of cartilage formation when we analyzed several mouse ESC lines.…”

“…Even though MSCs from most human donors show osteogenic potential, there is large variation in bone forming capacity by human MSCs, and multipotency is gradually lost upon expansion [229]. Most importantly, bone formation by MSCs is currently insufficient for successful tissue engineering [230].…”