Abstract
Stromal vascular fraction (SVF) cells of human adipose tissue have the capacity to generate osteogenic grafts with intrinsic vasculogenic properties. However, adipose-derived stromal/stem cells (ASC), even after minimal monolayer expansion, display poor osteogenic capacity in vivo. We investigated whether ASC bone-forming capacity may be maintained by culture within a self-produced extracellular matrix (ECM) that recapitulates the native environment. SVF cells expanded without passaging up to 28 days (Unpass-ASC) deposited a fibronectin-rich extracellular matrix and displayed greater clonogenicity and differentiation potential in vitro compared to ASC expanded only for 6 days (P0-ASC) or for 28 days with regular passaging (Pass-ASC). When implanted subcutaneously, Unpass-ASC produced bone tissue similarly to SVF cells, in contrast to P0- and Pass-ASC, which mainly formed fibrous tissue. Interestingly, clonogenic progenitors from native SVF and Unpass-ASC expressed low levels of the fibronectin receptor α5 integrin (CD49e), which was instead upregulated in P0- and Pass-ASC. Mechanistically, induced activation of α5β1 integrin in Unpass-ASC led to a significant loss of bone formation in vivo. This study shows that ECM and regulation of α5β1-integrin signaling preserve ASC progenitor properties, including bone tissue-forming capacity, during in vitro expansion.
Highlights
Stromal vascular fraction (SVF) cells of human adipose tissue have the capacity to generate osteogenic grafts with intrinsic vasculogenic properties
Unpass, P0 and Pass-adipose-derived stromal/stem cells (ASC) were generated from stromal vascular fraction (SVF) cells (Fig. 1A) plated on tissue plastic dishes and grown either for 6 or 28 days without passaging at cell confluence (P0- and Unpass-ASC respectively) or, when reaching 80% of confluence, detached and reseeded into new dishes (Pass-ASC) for the same time (28 days)
Characterization of the composition of the extracellular matrix (ECM) deposited after 28 days of culture showed that Unpass-ASC produced a matrix that stained negatively for collagen type I and was strongly positive for Fibronectin (FN) (Fig. 1E and F respectively)
Summary
Stromal vascular fraction (SVF) cells of human adipose tissue have the capacity to generate osteogenic grafts with intrinsic vasculogenic properties. Adipose-derived stromal/stem cells (ASC), even after minimal monolayer expansion, display poor osteogenic capacity in vivo. Even minimally monolayer-expanded SVF cells lose their osteogenic capacity[3], unless they are either pre-differentiated into osteoblasts[5,6] or chondrocytes[7,8] in vitro, or exposed to bone morphogenetic protein-2 (BMP-2)[9]. These data suggest that SVF cells cannot maintain their osteoprogenitor properties during monolayer expansion on tissue usb.ch) www.nature.com/scientificreports/.
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