Introduction: The existence of peripheral blood (PB) derived mesenchynal stem cells (PBMSCs) have been documented in several species including human. The circulating skeletal stem cells may provide a new source of stem cells that may be used for skeletal and other tissue engineering applications. The objective of this study is to further investigate and compare the biological characteristics of the PBMSCs with bone marrow derived MSCs in the GFP rats. Methods: The peripheral blood (PB) from the GFP rats was harvested by cardiac puncture using syringes containing sodium heparin. Mononuclear cells were isolated by density gradient centrifugation method and plated at a density of 1–3~105/cm2 in flasks with D-MEM medium containing 15% FCS. The bone marrow (BM) was also collected for obtaining BMMSCs, the bone chips for osteoblastic cells, and the skin for skin fibroblasts. The phenotypes of the cells were characterized by immunocytochemistry (ICC), and flow cytometry methods. Gene expression profiles of 3-paired PBMSCs and BMMSCs cDNA samples were examined by Affymetrix gene chips microarray analysis. The multipotent differentiation potentials of PBMSCs into osteoblasts, chondrocytes, and adipocytes were examined under specific inductive conditions and checked with lineage specific markers. Finally, the osteogenic potential of the PBMSCs was examined by an in vivo implantation model in which the PBMSCs were seeded with HA-TCP powder complexes, and implanted subcutaneously in the severe compromised immunodeficiency (SCID) mice for 12 weeks, whereas the bone-derived osteoblasts and skin fibroblasts were used as controls. Results: Compared with the BMMSCs, the PBMSCs shared some but not all common surface markers as demonstrated by (ICC) and flow cytometry examinations. The osteogenic differentiation of PBMSCs was defined with positive staining of type I collagen and osteocalcin; positive staining for alkaline phosphatase and Von Kossa staining for mineralized bone nodules. Adipogenic differentiation was evidenced by positive Oil red-O staining for accumulated lipids, and chondrogenic differentiation by positive type II collagen and Saferinin O positive staining. For gene expression profiles, in the Affymetrix chip general analysis, 83 genes were up regulated and 84 genes down regulated in the PBMSCs (vs BMMSCs, > 2 fold, E-B/B-E> 100, p< 0.05). Most of which genes are related to cell proliferation, differentiation, cytoskeleton, and calcium/iron homeostasis. After 12 weeks implantation in SCID mice, newly formed lamellar bone was clearly evident in the groups with PBMSCs implants, so as in the groups with osteoblasts implants, but only fibrous tissue was found in the group implanted with skin fibroblasts. Discussion: This study demonstrated that the multi-potent PBMSCs in the GFP rats resemble BMMSCs in many aspects, but they are distinguishable from the BMMSCs in some biological characteristics and gene profiles. Our study has confirmed that these PBMSCs possess osteogenic potential in vitro and in vivo, suggesting that these circulating stem cells could serve as an alternative source as bone marrow derived MSCs for tissue engineering purposes

Objectives

In order to ensure safety of the cell-based therapy for bone regeneration, we examined in vivo biodistribution of locally or systemically transplanted osteoblast-like cells generated from bone marrow (BM) derived mononuclear cells.

Mesenchymal stem cells (MSCs) are immunosuppressive and have been used to facilitate tissue repair in the context of allogeneic implantation. However, xenogeneic cell transplantation has not been fully explored. The present study investigated the feasibility of xenogeneic MSCs implantation in mice. MSCs were harvested from the bone marrow of GFP rats (Green Fluorescent Protein transgenic rats), and cultured as previously described. 1 million GFP MSCs were loaded onto the synthetic HA/TCP porous Skelite blocks and implanted intramuscularly into the quadriceps of the MF1 and SCID mice. After 11 weeks, the implants were harvested and processed for histology examination. Upon termination, the mononuclear cells from the peripheral blood of each animal were also collected for mixed lymphocyte culture to examine lymphocyte proliferation potential and T-cell mediated cell lysis (cytotoxic) assays. In the SCID mice, there was sparse osteoid tissue formation in the implants, whereas only dense connective tissues were seen in the implants of the MF1 mice. Osteocalcin mRNA expression was confirmed in the osteoid tissues in the implants from the SCID mice, but it was not detected in the MF1 mice by RT in situ PCR examination. Cells of GFP-rat origin were observed in both the MF1 and SCID mice (more so in the SCID mice) after 11 weeks implantation, which were confirmed by positive immunostaining of anti-GFP antibody. In the MF1 mice after 11 weeks xenogeneic MSCs implantation, the rate of lymphocyte proliferation was significantly increased when mixed with the GFP-MSCs compared to that of mixed lymphocyte culture assays in the SCID or MF1 mice without xenogeneic MSCs implantation, suggesting that implantation of xenoge-neic MSCs has promoted host anti-graft immunogenic responses towards to otherwise immunosuppressive MSCs. In conclusion, xenogeneic rat MSCs transplanted in immunocompetent mice has survived for prolonged period, but their function was comprised to certain extent and this may be due to the increased host anti-graft immune sensitization after exposed to the xenogeneic MSCs

Introduction: MSCs were demonstrated to exist within peripheral blood (PB) of several mammalian species including human, guinea pig, mice, rat, and rabbit. We have found increased numbers of circulating MSCs in human peripheral blood after fracture and in patients with cancers. We have also compared the difference between circulating MSCs and bone marrow MSCs and evaluated their potential clinical applications in tissue engineering and cell therapy. Methods and findings: Using culture conditions similar to those defined for bone marrow derived mesenchymal stromal cells (BMMSCs), we have isolated and expanded multi-colony and single colony derived PBMSCs strains from the GFP transgenic rats. Aspects of molecular, cellular and developmental properties of this poorly characterized peripheral blood subpopulation were examined. PBMSCs share some common phenotypic characteristics with BMMSCs, but are distinguishable in gene expression profile by cDNA microarray analysis, with 84 up-regulated and 83 down-regulated genes (> 2 fold, E-B/B-E> 100, P< 0.05). Most of these genes are related to cell proliferation, differentiation, cyto-skeleton, and calcium/iron homeostasis. Differentially expressed genes with fold change ≥10 were further confirmed with quantitative real time RT-PCR, and these genes are: retinol-binding protein 1 (CRBP1), cadherin 2, bone morphogenetic protein 6 (BMP6), SRY-box containing gene 11 (Sox11), the aquaporin 1 (AQP1), and so on, and they can be potential targets for further investigations. We have demonstrated that single colony derived PBMSCs strains possess extensive proliferation and multipotent differentiation potentials into osteoblasts, adipocytes, chondrocytes, endothelial cells and neuronal cells. In terms of potential clinical implications of PBMSCs, we have demonstrated that allogenic PB-MSCs enhance bone regeneration in rabbit ulna critical-sized bone defect model. We also demonstrated that BM-MSCs can be recruited towards to the sites of bone fracture and participate fracture healing. We are now working on using MSCs as a gene delivery vehicle for management of would healing or cancer therapy, and ways of enhancing the homing and recruitment MSCs towards to specific sites after their systemic delivery. Conclusion: Taken the above data together, PB-MSCs may be a new cell source for cell therapy, tissue engineering and gene therapy strategies