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Development of an ex vivo xenogeneic bone environment producing human platelet-like cells.

ABSTRACT: The efficiency of in vitro platelet production is considerably low compared with physiological activity due to the lack of pivotal factors that are essential in vivo. We developed an ex vivo platelet production system, introducing human megakaryocytes into an isolated porcine thighbone and culturing in closed circuit. The efficiency of the ex vivo platelet production system was compared to those in vivo and in vitro. CD61+ platelet-like cells were counted by immunostaining and flow cytometry. Results showed that 4.41 ± 0.27 × 103 CD61+ platelet-like cells were produced by 1 × 103 megakaryocytes in the ex vivo system, while 3.80 ± 0.87 × 103 and 0.12 ± 0.02 × 103 were produced in the in vivo and in vitro systems, respectively. Notably, ex vivo and in vitro production systems generated cells that responded well to thrombin stimulation and expressed functional molecules, such as CD62P. Overall, our ex vivo production system was comparable to in vivo production system and produced platelet-like cells that were functionally superior to those produced in vitro. In future, the present ex vivo production system implementing xenogeneic bone marrow would offer a promising alternative for industrial-scale production of platelet-like cells.

SUBMITTER: Fujiyama S

PROVIDER: S-EPMC7138292 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Development of an ex vivo xenogeneic bone environment producing human platelet-like cells.

Fujiyama Shingo S Hori Nobuyasu N Sato Toshiyuki T Enosawa Shin S Murata Mitsuru M Kobayashi Eiji E

PloS one 20200407 4

The efficiency of in vitro platelet production is considerably low compared with physiological activity due to the lack of pivotal factors that are essential in vivo. We developed an ex vivo platelet production system, introducing human megakaryocytes into an isolated porcine thighbone and culturing in closed circuit. The efficiency of the ex vivo platelet production system was compared to those in vivo and in vitro. CD61+ platelet-like cells were counted by immunostaining and flow cytometry. Re ...[more]

PMID: 32255777

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Project description:IntroductionThere 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 of homing to injured bone.MethodsWe 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 the in vivo homing ability of transplanted hBMSC 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.ResultsHBF clones were exhibited higher ex vivo transwell migration and following intravenous injection, better in vivo homing ability to bone fracture when compared to LBF clones. Comparative microarray analysis of HBF versus LBF clones identified enrichment of gene categories of chemo-attraction, adhesion and migration associated genes. Among these, platelet-derived growth factor receptor (PDGFR) α and β were highly expressed in HBF clones. Follow up studies showed that the chemoattractant effects of PDGF in vitro was more enhanced in HBF compared to LBF clones and this effect was reduced in presence of a PDGFRβ-specific inhibitor: SU-16 f. Also, PDGF exerted greater chemoattractant effect on PDGFRβ(+) cells sorted from LBF clones compared to PDGFRβ(-) cells.ConclusionOur data demonstrate phenotypic and molecular association between in vivo bone forming ability and migratory capacity of hBMSC. PDGFRβ 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: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.

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Development of an ex vivo xenogeneic bone environment producing human platelet-like cells.

Publications

Development of an ex vivo xenogeneic bone environment producing human platelet-like cells.

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