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Mesenchymal Stem Cells and Osteoblast-Chondroblast Differentiation Jane E

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Mesenchymal Stem Cells and Osteoblast-Chondroblast Differentiation Jane E European Cells and Materials Vol. 16. Suppl. 4, 2008 (page 1) ISSN 1473-2262 Mesenchymal Stem Cells and Osteoblast-Chondroblast Differentiation Jane E. Aubin Dept. of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8 of simultaneous marker expression for multiple lineages INTRODUCTION: The fact that bone formation takes was seen in morphologically indistinguishable CFU-F, place not only during development but throughout life suggesting that gene priming may contribute to suggests that there is a large reservoir of cells in the mesenchymal cell fate selection. Mesenchymal stem and body capable of osteogenesis. The nature of these cells progenitor cell fate choice can also be altered by over the lifetime of the animal, contributions from stem cytokines and hormones, e.g., leukemia inhibitory factor cell versus committed progenitor pools, and (LIF) and 1,25(OH) D 2. identification of developmental transition steps remain 2 3 subjects of intense study. Osteoprogenitors (colony forming unit-osteoblast (CFU-O)) arise from multipotential mesenchymal stem cells (often designated CFU-fibroblast or CFU-F) that also give rise QuickTime™ and a TIFF (Uncompressed) decompressor to chondroblasts, adipocytes and myoblasts and in are needed to see this picture. which fate choice may be both stochastic and driven by environmental cues.. METHODS: Primary cultures of fetal (E21) rat or neonatal (P1-2) mouse calvaria and young adult mouse Fig. 1: Hormones, e.g. 1,25(OH)2D3 alter the fate or rat bone marrow stromal cells were prepared and 2 cultured as described1. Cells were cultured at high choice of mesenchymal stem and progenitor cells. (5X103 cells/cm2) or low (5 cells/cm2) or a limiting dilution series, under conditions for osteogenic, DISCUSSION & CONCLUSIONS: Our data support chondrogenic, adipocytic or myogenic conditions as both a deterministic/hierarchical and non-deterministic appropriate. In some experiments, cells were analyzed (environmental cues and modifiers) model of and sorted by flow cytometry or magnetic beads. At mesenchymal stem cells in which gene priming and various times, RNA was extracted for PCR or cells were non-reciprocal regulation of fate selection play roles, fixed and stained by histochemistry or and offer new strategies for enriching for multipotential immunohistochemistry for marker expression. cells for regenerative medicine applications. 1 RESULTS: CFU-O comprise only a proportion of the REFERENCES: J.E. Aubin and J. Triffitt (2002) CFU-F, express limited self-renewal and occur at Mesenchymal stem cells and the osteoblast lineage. frequencies of ~1/105 to 1/102 cells in bone marrow or Principles of Bone Biology, 2nd ed. (eds J.P. Bilezikian, L.G. Raisz, and G.A. Rodan) Academic Press, pp 59- stromal and calvaria-derived populations respectively. 2 Fractionation based on Hoechst dye efflux (side 81. S. Zhang, S. Uchida, T. Inoue, M. Chan, E. population or SP) or alkaline phosphatase (ALP) and/or Mockler and J.E. Aubin (2006) Side population (SP) parathyroid hormone/parathyroid hormone related cells isolated from fetal rat calvaria are enriched for protein receptor (PTH1R) expression enriches bone, cartilage, adipose tissue and neural progenitors. significantly for CFU-F and CFU-O, and reveals that Bone 38:662-670. both cell autonomous and non-autonomous ACKNOWLEDGEMENTS: This work is supported developmental events occur, that the majority of by the Canadian Institutes of Health Research (FRN immature progenitors reside in the ALP/PTH1R 483033). negative fraction and that cell autonomous clonogenic osteoprogenitors reside only in the latter pool. By other novel fractionation strategies based on positive and negative selection with lineage markers, we achieved >150-fold enrichment for a multipotential mesenchymal population with robust chondrogenic and osteogenic differentiation capacity in vitro and in vivo. Replica plating, global amplification poly(A)PCR, BrdU labelling and immunocytochemistry revealed at least seven transitional stages in osteoblast differentiation. Statistical analysis of the gene expression profiles uncovered multiple potential developmental pathways in osteoblast differentiation. A hierarchical distribution European Cells and Materials Vol. 16. Suppl. 4, 2008 (page 2) ISSN 1473-2262 Xenogenic Transplantation of Human Mesenchymal Stem Cells for Treatment of Critical Size Bone Defects in Sheep Philipp Niemeyer1, Thomas Schönberger1, Joachim Hahn2, Norbert P. Südkamp1, Erich Schneider2, Simon Pearce2 and Stefan Milz2 1Department of Orthopedic Surgery and Traumatology, Freiburg University Hospital, GER 2 AO Research Institute, AO Foundation, Davos, CH INTRODUCTION: Mesenchymal stem cells level however, there was no statistically (MSC) from bone marrow represent an attractive significant difference between the two groups. cell source for tissue engineering purposes such as the regeneration of bone. Due to a lack of expression of immunologically relevant surface antigens, this cell type might even be available for non-autologous cell transplantation. Although immunosuppressive properties have been demonstrated in vitro and xenogenic MSC show an engraftment after transplantation in immunocompetent mice, it remains unclear if HLA-mismatched MSC have a regeneration Fig.1: Sufficient bridging of the defect was found in 3 potential equal to autologous MSC. animals of the autologous MSC group (radiography at 24 weeks (A). Giemsa-Eosin staining of conventional METHODS: After isolation and cultivation on histology (B). In vivo fluorescent sequence labeling mineralized collagen as described earlier3, revealed that bone formation in these defects occurred as late as 20 weeks after surgery (C). xenogenic human and autologous ovine MSC were transplanted into a 3.0 cm diaphyseal tibia defect in Swiss Alpine sheep. Animals were sacrificed after 3 and 6 months. Unloaded mineralized collagen scaffolds served as a control. Radiography was performed every 2 weeks, in addition histological evaluation was performed Fig.2: Human-specific in-situ hybridization revealed after euthanasia (including in-situ hybridization presence of human MSC after xenogenic transplantation in 3 of 7 animals for detection of human MSC). Bone regeneration was analyzed using semi DISCUSSION & CONCLUSIONS: Although quantitative scoring systems on radiographic and xenogenic MSC could be detected in a significant histological levels. Furthermore, the amount of number of animals after transplantation, no severe newly formed bone was quantified using the immune response was detected. Nevertheless digital image software analysis program GIMP. xenogenic transplantation of MSC seems to lead to a lesser rate of bone formation compared to RESULTS: Autologous MSC lead to significant autologous ovine MSC which performed best. increase in radiological bone density in the defect Identification of the biological principle beyond after 6 weeks compared to unloaded controls (p < this observation will be part of further studies. 0.05). This difference could also be confirmed by histological evaluation after euthanasia (p < 0.01). REFERENCES: Compared to the autologous MSC group, the 1 Le Blanc K, et al. (2003) HLA expression and transplantation of xenogenic MSC leads to a immunologic properties of MSC … Exp Hematol 31:890- lower rate of bone formation (p < 0.05). 896. Nevertheless, an engraftment of xenogenic MSC 2 Niemeyer P, et al. (2007) Comparison of immunological was detected in 3 out of 7 animals using human properties of bone marrow stroma cells and adipose tissue- specific in situ hybridization, while no severe derived stem cells …. Tissue Eng 13:111-121. systemic or histological immune response could be detected. Bone formation in the xenogenic ACKNOWLEDGEMENTS: group was higher than in the unloaded control The Project was supported by the AO Research group with statistical significance in radiological Fund (No. 04-N94) of the AO Foundation, CH and data evaluation. On semi quantitative histological by the Albert-Ludwigs-University Freiburg, Germany. European Cells and Materials Vol. 16. Suppl. 4, 2008 (page 3) ISSN 1473-2262 Syndecan-4 Deficiency Leads to an Osteoporotic Bone Structure in vivo and an Impaired Osteoblast Functionality in vitro R. Stange +, M. Timmen +, T. Stoltenberg +, H. Hidding +, F. Echtermeyer *, K. Neugebauer *, T. Pap *, M. Raschke + +Dept. of Trauma, Hand and Reconstructive Surgery, University Hospital Muenster *Division of Molecular Medicine of Musculoskeletal Tissue, Dept. of Orthopaedics, University Hospital Muenster, Germany 100 INTRODUCTION: Members of the syndecan family of 90 18 80 heparan sulfate proteoglycans play important roles in cell 46 70 adhesion and cell communication by serving as co- 60 50 receptors for both extracellular matrix molecules and 40 82 whole% area growth factors together with integrins. Syndecan-4 is 30 54 20 ubiquitously expressed and appears to be involved in cell 10 proliferation, differentiation, adhesion and migration. It c 0 a b wild type syndecan-4 -/- has been demonstrated that syndecan-4 is upregulated in Fig. 1: Histological analysis (v. the dermis after injury, and syndecan-4 knockout mice Kossa) of a lumbar vertebra of wild type (a) and show a delay in wound healing1. During embryogenesis, syndecan-4 knockout mice (b); c Quantification of syndecan-4 is important for regulation of chondrocyte mineralized bone area differentiation and enchondral ossification. To determine the influence of syndecan-4 on bone morphology and In vitro analysis of osteoblast
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