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Association between in vivo bone formation and ex vivo migration capacity of human bone marrow stromal cells

ABSTRACT: Introduction: There is a clinical need for developing systemic transplantation protocols for use of human skeletal stem cells (also known bone marrow stromal stem cells) (hBMSC) in tissue regeneration. In systemic transplantation studies, only a limited number of hBMSC home to injured tissues suggesting that only a subpopulation of hBMSC possess âhomingâ capacity. Thus, we tested the hypothesis that a subpopulation of hBMSC defined by ability to form heterotopic bone in vivo, is capable of homing to injured bone. Methods: We tested ex vivo and in vivo homing capacity of a number of clonal cell populations derived from telomerized hBMSC (hBMSC-TERT) with variable ability to form heterotopic bone when implanted subcutaneously in immune deficient mice. In vitro transwell migration assay was used and in vivo homing ability to bone fractures in mice was visualized by bioluminescence imaging (BLI). In order to identify the molecular phenotype associated with enhanced migration, we carried out comparative DNA microarray analysis of gene expression of hBMSC-derived high bone forming (HBF) clones versus low bone forming (LBF) clones. Results: In this study, clonal cell populations forming in vivo bone were shown to exhibit higher ex vivo transwell migration and following intravenous infusion, enhanced in vivo homing ability to bone fractures. Comparative microarray analysis of LBF versus HBF clones identified a significant enrichment of gene categories of cell chemo-attraction, adhesion and migration. Among these genes, platelet-derived growth factor receptor (PDGF-R) Î± and Î² were highly expressed in HBF clones. Further studies showed that the chemoattractive effects of PDGF in vitro was more enhanced in HBF clones compared to LBF clones and this effect was abolished in presence of a PDGFR-Î²-specific inhibitor: SU-16f. Sorted PDGFR-Î²+ cells from LBF clones showed that the PDGFR-Î²+ enriched cell population exhibited strong chemoattractance towards PDGF. Conclusion: Our data demonstrate phenotypic and molecular association between in vivo bone formation and migratory capacity of hBMSC. PDGFR-Î² can be used as a potential marker for the prospective selection of hBMSC populations with high migration and bone formation capacities suitable for clinical trials for enhancing bone regeneration. Total RNA obtained from telomerized human bone marrow derived mesenchymal stromal cells (T4P38a) with high in vivo bone forming capacity and (T4P74a) with low bone forming capacity and three single cell clones with high bone forming capacities (DD8, AD10 and BB10) and three clones with low bone forming capacities (CF1, CB4 and CD4). All cells were cultured under standard conditions and RNAs were isolated at baseline with no induction. each sample was represented in doublicate as a and b.

ORGANISM(S): Homo sapiens

SUBMITTER: Wasco Wruck

PROVIDER: E-GEOD-69358 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Association between in vivo bone formation and ex vivo migratory capacity of human bone marrow stromal cells.

Andersen Rikke K RK Zaher Walid W Larsen Kenneth H KH Ditzel Nicholas N Drews Katharina K Wruck Wasco W Adjaye James J Abdallah Basem M BM Kassem Moustapha M

Stem cell research & therapy 20151008

<h4>Introduction</h4>There is a clinical need for developing systemic transplantation protocols for use of human skeletal stem cells (also known bone marrow stromal stem cells) (hBMSC) in tissue regeneration. In systemic transplantation studies, only a limited number of hBMSC home to injured tissues suggesting that only a subpopulation of hBMSC possesses "homing" capacity. Thus, we tested the hypothesis that a subpopulation of hBMSC defined by ability to form heterotopic bone in vivo, is capable ...[more]

PMID: 26450135

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Project description:Introduction: There is a clinical need for developing systemic transplantation protocols for use of human skeletal stem cells (also known bone marrow stromal stem cells) (hBMSC) in tissue regeneration. In systemic transplantation studies, only a limited number of hBMSC home to injured tissues suggesting that only a subpopulation of hBMSC possess “homing” capacity. Thus, we tested the hypothesis that a subpopulation of hBMSC defined by ability to form heterotopic bone in vivo, is capable of homing to injured bone. Methods: We tested ex vivo and in vivo homing capacity of a number of clonal cell populations derived from telomerized hBMSC (hBMSC-TERT) with variable ability to form heterotopic bone when implanted subcutaneously in immune deficient mice. In vitro transwell migration assay was used and in vivo homing ability to bone fractures in mice was visualized by bioluminescence imaging (BLI). In order to identify the molecular phenotype associated with enhanced migration, we carried out comparative DNA microarray analysis of gene expression of hBMSC-derived high bone forming (HBF) clones versus low bone forming (LBF) clones. Results: In this study, clonal cell populations forming in vivo bone were shown to exhibit higher ex vivo transwell migration and following intravenous infusion, enhanced in vivo homing ability to bone fractures. Comparative microarray analysis of LBF versus HBF clones identified a significant enrichment of gene categories of cell chemo-attraction, adhesion and migration. Among these genes, platelet-derived growth factor receptor (PDGF-R) α and β were highly expressed in HBF clones. Further studies showed that the chemoattractive effects of PDGF in vitro was more enhanced in HBF clones compared to LBF clones and this effect was abolished in presence of a PDGFR-β-specific inhibitor: SU-16f. Sorted PDGFR-β+ cells from LBF clones showed that the PDGFR-β+ enriched cell population exhibited strong chemoattractance towards PDGF. Conclusion: Our data demonstrate phenotypic and molecular association between in vivo bone formation and migratory capacity of hBMSC. PDGFR-β can be used as a potential marker for the prospective selection of hBMSC populations with high migration and bone formation capacities suitable for clinical trials for enhancing bone regeneration.

Project description:The unique properties of the bone marrow allow for migration and proliferation of multipl myeloma (MM) cells, while also providing the perfect environment for development of quiescent, drug-resistant MM cell clones. Bone marrow adipocytes (BMAds), which have recently been identified as important contributors to systemic adipokine levels, bone strength, hematopoiesis, and progression of metastatic and primary bone marrow cancers, such as MM. Recent studies in myeloma suggest that BMAds can be reprogrammed by tumor cells to contribute to myeloma-induced bone disease, and reciprocally, BMAds support MM cells in vitro. Importantly, most data investigating BMAds have been generated using adipocytes derived by differentiating bone marrow-derived mesenchymal stromal cells (MSCs) into adipocytes in vitro using adipogenic media, due to the extreme technical challenges associated with isolating and culturing primary adipocytes. However, if studies could be performed with primary adipocytes, they likely will recapitulate in vivo biology better than MSC-derived adipocyte, as the differentiation process is artificial and differs from in vivo differentiation, and progenitor cell(s) of the primary BMAd may not be the same as the MSCs precursors used for adipogenic differentiation in vitro. Therefore, we developed and refined three methods for culturing primary BMAds (pBMAds): 2D coverslips, 2D transwells, and 3D silk scaffolds, all of which can be cultured alone or with MM cells to investigate bidirectional tumor-host signaling. To develop an in vitro model with a tissue-like structure to mimic the bone marrow microenvironment, we developed the first 3D, tissue engineered model utilizing pBMAds derived from human bone marrow. We found that pBMAds, which are extremely fragile, can be isolated and stably cultured in 2D for 10 days and in 3D for short term (~2 weeks) or long term (1 month) in vitro. To investigate the relationship between pBMAds and myeloma, MM cells can be added to investigate physical relationships through confocal imaging and soluble signaling molecules via mass spectrometry. In sum, we developed three in vitro cell culture systems to study primary bone marrow adipocytes and myeloma cells, which could be adapted to investigate many diseases and biological processes involving the bone marrow, including other bone-homing tumor types.

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Association between in vivo bone formation and ex vivo migration capacity of human bone marrow stromal cells

Publications

Association between in vivo bone formation and ex vivo migratory capacity of human bone marrow stromal cells.

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