Our unpublished data has indicated that the perivascular stem cells (PSCs) have increased chondrogenic potential compared to mesenchymal stem cells (MSCs) derived in culture. There has been a recent change in the theory that stem cells work by a paracrine effect rather than differentiation. There are minimal data demonstrating the persistence of implanted stem cells when used for engraftment. This study aimed to develop an autologous large animal model for perivascular stem cells as well as to determine if cells were retained in the articular cartilage defects. The reactivity of anti-human and anti-ovine antibodies was ascertained using immunohistochemistry and fluorescence-activated cell sorting (FACS). A panel of antibodies were combined and used to identify and purify pericytes (CD34-CD45-CD146+) and adventitial cells (CD34+CD45-CD146-) using FACS. The purified cells were cultured and their identity checked using FACS. These cultured cells demonstrated osteogenic, adipogenic and chondrogenic potential. Autologous ovine PSCs (oPSCs) were isolated, cultured and transfected using a GFP virus. The transfection rate was 88%. The cells were implanted into an articular cartilage defect on the medial femoral condyle using a hydrogel, four weeks following implantation the condyle was explanted and confocal laser scanning microscopy demonstrated the presence of oPSCs in the defect. Histology did not demonstrate any repair tissue at this early time point. These data have confirmed the viability our large animal model and that the implanted stem cells were retained in the defect four weeks following implantation.
Mesenchymal stem cells (MSCs) reside around blood vessels in all organs. This reservoir of progenitors can be ‘recruited’ in response to injury. The ability to manipulate stem cells therapeutically within injured tissue provides an attractive alternative to transplantation. Stem cells are regulated by neighbouring cells. We hypothesized that endothelial cells (ECs) influence MSC differentiation into bone and fat. MSCs were sorted from fat using fluorescent activated sorting. Their capacity to differentiate into bone, fat and cartilage was used to confirm MSC phenotype. MSCs and ECs were cultured in two-dimensions (standard culture dishes) and three-dimensions (vascular networks suspended in gel). Cocultures were exposed to osteogenic and adipogenic media. The role of EC-released factors on MSC differentiation was determined using a system in which cells share media but do not contact. Wnt pathway modulators were used to investigate the role of Wnt signalling. MSCs differentiated into bone, fat and cartilage. MSCs and ECs integrated in two- and three-dimensions. MSCs and ECs formed vessel-like structures in three-dimensions. When cultured with ECs, MSC differentiation to bone was accelerated while differentiation to fat was inhibited. This effect on osteogenesis was maintained when cells shared media but did not contact. Coculture with Wnt modulators confirmed that this effect is in part, mediated through Wnt signalling. Our data suggest that ECs influence MSC differentiation. Therapeutic targeting of EC-MSCs signalling may enable manipulation of MSCs in vivo avoiding the need for cell transplantation. This could enable trauma and orthopaedic patients who have healthy resident stem cells to self-repair.
Perivascular stem cells (PSCs) from lipoaspirate demonstrate increased purity and immaturity with greater engraftment potential than standard mesenchymal stem cells (MSCs). MSCs from the infra-patellar fat pad (IFP) have previously demonstrated increased chondrogenic potential. This study investigated the availability and potential of PSCs harvested from the infra-patellar fat pad of the human knee for musculoskeletal regeneration. Tissue sections of IFP were stained with markers for PSCs, MSCs and endothelial cells to confirm their presence and location. Samples were obtained from patients undergoing TKR (n=13) or ACL reconstructions (n=10). Pericytes and adventitial cells made up 3.8% and 21.2% respectively of the stromal vascular fraction. The total number of pericytes and adventitial cells were 4.6±2.2×104 and 16.2±3.2×104 respectively. Cells were cultured both separately and combined. Cell identity was ascertained using fluorescence-activated cell sorting, immunocytochemistry and PCR. Cultured PSCs were differentiated using chondrogneic, osteogenic, adipogenic and myogenic medias. Differentiation was determined using Alcian Blue, Alizarin red, Oil Red O and myosin staining. This study demonstrates that the IPFP is a viable source of PSCs that can be harvested either arthroscopically or through an arthrotomy by orthopaedic surgeons for cell-based musculoskeletal regeneration. Their potential now needs to be compared to conventional MSCs.
Perivascular stem cells (PSCs) from lipoaspirate demonstrate increased purity and immaturity with greater engraftment potential than standard mesenchymal stem cells (MSCs). MSCs from the infra-patellar fat pad (IFP) have previously demonstrated increased chondrogenic potential. This study investigated the availability and potential of PSCs harvested from the infra-patellar fat pad of the human knee for musculoskeletal regeneration. Sections of IFP were stained with markers for PSCs, MSCs and endothelial cells to confirm their presence and location. Samples were obtained from patients undergoing TKR (n=13) or ACL reconstructions (n=10). Pericytes and adventitial cells made up 3.8% and 21.2% respectively of the stromal vascular fraction. The total number of pericytes and adventitial cells were 4.6±2.2×104 and 16.2±3.2×104 respectively. Cells were cultured both separately and combined. Cell identity was ascertained using fluorescence-activated cell sorting and immunocytochemistry. Cultured PSCs were differentiated using chondrogneic, osteogenic, adipogenic and myogenic medias. Differentiation was determined using Alcian Blue, Alizarin red, Oil Red O and mysosin staining. This study demonstrates that the IFP is a viable source of PSCs that can be harvested either arthroscopically or through an arthrotomy by orthopaedic surgeons for cell-based musculoskeletal regeneration. Their potential now needs to be compared to conventional MSCs.
