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Heparin Anticoagulant for Human Bone Marrow Does Not Influence In Vitro Performance of Human Mesenchymal Stromal Cells.

ABSTRACT: Mesenchymal stromal cells (MSCs) are a promising cell source for tissue engineering and regenerative medicine. In our lab, we found that MSC preparations from bone marrow of many different donors had a limited capacity of in vitro differentiation into osteogenic and chondrogenic lineages-a capacity claimed to be inherent to MSCs. The current study was designed to test the hypothesis that the amount of heparin used as anticoagulant during bone marrow harvest had an inhibitory influence on the in vitro differentiation capacity of isolated MSCs. Bone marrow was obtained from the femoral cavity of twelve donors during total hip arthroplasty in the absence or presence of heparin. No coagulation was observed in the absence of heparin. The number of mononuclear cells was independent of heparin addition. Isolated MSCs were characterized by morphology, population doubling times, expression of cell surface antigens and in vitro differentiation. Results of these analyses were independent of the amount of heparin. Transcriptome analyses of cells from three randomly chosen donors and quantitative realtime PCR (qRT-PCR) analysis from cells of all donors demonstrated no clear effect of heparin on the transcriptome of the cells. This excludes heparin as a potential source of disparate results.

SUBMITTER: Roger Y

PROVIDER: S-EPMC7408646 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Heparin Anticoagulant for Human Bone Marrow Does Not Influence In Vitro Performance of Human Mesenchymal Stromal Cells.

Roger Yvonne Y Burmeister Laura L Hamm Anika A Elger Kirsten K Dittrich-Breiholz Oliver O Flörkemeier Thilo T Hoffmann Andrea A

Cells 20200629 7

Mesenchymal stromal cells (MSCs) are a promising cell source for tissue engineering and regenerative medicine. In our lab, we found that MSC preparations from bone marrow of many different donors had a limited capacity of in vitro differentiation into osteogenic and chondrogenic lineages-a capacity claimed to be inherent to MSCs. The current study was designed to test the hypothesis that the amount of heparin used as anticoagulant during bone marrow harvest had an inhibitory influence on the in ...[more]

PMID: 32610653

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Project description:Background: Mesenchymal stromal cells (MSCs) are a promising cell source for tissue engineering and regenerative medicine. In our lab, we found that cell preparations from bone marrow of many donors had a limited capacity for in vitro-differentiation into the osteogenic and chondrogenic lineages although this is a capacity claimed to be inherent to this kind of cells. Therefore the current study was designed to test the hypothesis whether the amount of heparin used as anti-coagulant during bone marrow harvest has inhibitory influence on the in vitro-differentiation capacity of the isolated MSCs. Methods: Bone marrow was obtained from twelve donors with good physical condition during preparation of the femoral cavity for the implantation of the femoral stem for a total hip arthroplasty in the absence or presence of heparin. Isolated MSCs were characterized by plastic adhesion, morphology, population doubling times, expression of cell surface antigens and in vitro-differentiation into the adipogenic, osteogenic and chondrogenic lineages. In addition, transcriptome analyses were performed. Results: Bone marrow prepared in the absence of heparin showed a comparable processability as bone marrow in the presence of heparin. Notably, no coagulation was observed. The number of mononuclear cells retrieved from the density gradient was independent of heparin addition. Moreover, none of the phenotypic or functional parameters assessed correlated with the addition of heparin. Transcriptome and quantitative realtime PCR analyses demonstrated that some selected genes were up- or downregulated in the cells isolated with the addition of heparin. However, this statement held true for only some but not all of the donors investigated in the present study. Conclusions: No correlation with heparin addition was observed with respect to the parameters assessed in the present study. This implies the absence of consequences both for the results of in vitro-investigations and also during in vivo-application, thereby ruling out heparin as a potential source of disparate results that are described in the literature.

Project description:Systemic infusion of bone marrow-derived mesenchymal stem cells (BMSCs) has become a promising strategy for disease treatment and tissue regeneration. Strategies to enhance the efficiency of BMSC cell therapy are crucial to promote its clinical application. Here, we aimed to improve BMSC cell therapy by inhibiting the BMSC-induced coagulation reaction. Intravenous injection of gradient BMSCs into mice showed that BMSCs were not fully compatible with blood. Large doses of BMSCs induced a series of symptoms of respiratory failure and heart failure. Histological and homeostasis analysis confirmed that large doses of BMSCs induced disseminated intravascular thrombosis, exhaustion of platelets and coagulation factors, and prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT). Similar to mouse BMSCs, goat and human BMSCs also induced coagulation reactions in vitro and in vivo. The coagulation was induced mostly by tissue factor, the overexpression of which enhanced the procoagulant activity of BMSCs during in vitro culture. Notably, clinical doses of BMSCs in cell therapy also induced mild and reversible coagulation, which increased BMSC lung embolism and clearance. Anticoagulation treatment by heparin (400 U/kg) prevented BMSC-induced coagulation and the acute adverse effects of large-dose BMSCs infusion efficiently. Importantly, heparin treatment led to decreased BMSC lung embolism and enhanced migration and maintenance of BMSCs to target organs in cell therapy. Based on an experimental colitis model, we confirmed that heparin treatment enhanced the effect of BMSC therapy efficiently to reduce mortality, prevent weight loss, suppress inflammation reaction and alleviate tissue injury. In conclusion, BMSCs possess procoagulant activity that could induce disseminated coagulation and thrombosis in recipients. Anticoagulation treatment by heparin is a practical strategy to improve both the safety and therapeutic effect of BMSC therapy.

Project description:Ruxolitinib is a JAK2/JAK1 inhibitor that blocks the inflammatory JAK-STAT signaling pathway. Ruxolitinib has been demonstrated to be effective in the treatment of steroid-resistant acute graft-versus-host disease (GVHD). Ruxolitinib's effect on inflammatory cells of hematopoietic origin is known. However, its effect on nonhematopoietic cell types with immune-modulating and antigen-presenting cell competency plausibly involved in pathogenesis of GVHD has not been explored. Mesenchymal stromal cells (MSCs) are CD45- nonhematopoietic cells of the bone marrow with immune modulatory functions in vivo. MSCs' immunobiology largely depends on their responsiveness to IFNγ. We aimed to define the effect of ruxolitinib on the immunobiology of MSCs that are modulated by IFNγ. Human bone marrow derived MSCs, peripheral blood mononuclear cells (PBMCs), and primary bone marrow aspirates were analyzed for their sensitivity to ruxolitinib-mediated blocking of IFNγ-induced STAT-1 phosphorylation and downstream effector molecules, utilizing Western blot, flow cytometry, secretome analysis, and phosflow techniques. IFNγ-induced cytostatic effects on MSCs are reversed by ruxolitinib. Ruxolitinib inhibits IFNγ and secretome of activated peripheral PBMC-induced STAT-1 phosphorylation on human bone marrow derived MSCs. In addition, ruxolitinib inhibits IFNγ-induced pro-GVHD pathways on MSCs, which includes HLAABC(MHCI), HLADR(MHCII), CX3CL1, and CCL2. IFNγ-induced immunosuppressive molecules IDO and PDL-1 were also inhibited by ruxolitinib on MSCs. Comparative analysis with PBMCs has demonstrated that MSCs are as equal as to HLADR+ PBMC populations in responding to ruxolitinib-mediated inhibition of IFNγ-induced STAT-1 phosphorylation. Ex vivo analysis of human marrow aspirates has demonstrated that ruxolitinib blocks IFNγ-induced STAT-1 phosphorylation in CD45+/-HLADR+/- populations at different levels, which is depending on their sensitivity to IFNγ responsiveness. These results inform the hypothesis that ruxolitinib's immune-modulatory effects in vivo may pharmacologically involve marrow and tissue-resident MSCs. Ruxolitinib affects the immunobiology of MSCs equivalent to professional HLADR+ antigen presenting cells, which collectively mitigate GVHD.

Project description:Mesenchymal stromal cells (MSCs) are non-hematopoietic cells that have a broad therapeutic potential. To obtain sufficient cells for clinical application, they must be expanded ex vivo. In the initial expansion protocols described, fetal calf serum (FCS) was used as the reference growth supplement, but more recently different groups started to replace FCS with platelet lysate (PL). We investigated in this study the impact of the culture supplement on gene expression of MSCs. Human bone marrow derived MSCs were expanded in vitro in FCS and PL supplemented medium. We found that MSCs expanded in PL-containing medium (PL-MSCs) express typical MSC immunomorphological features and can migrate, as their counterparts expanded in FCS-containing medium, through a layer of endothelial cells in vitro. Additionally, they show an increased proliferation rate compared to MSCs expanded in FCS medium (FCS-MSCs). RNA sequencing performed for MSCs cultured in both types of expansion medium revealed a large impact of the choice of growth supplement on gene expression: 1974 genes were at least twofold up- or downregulated. We focused on impact of genes involved in apoptosis and senescence. Our data showed that PL-MSCs express more anti-apoptotic genes and FCS-MSCs more pro-apoptotic genes. FCS-MSCs showed upregulation of senescence-related genes after four passages whereas this was rarer in PL-MSCs at the same timepoint. Since PL-MSCs show higher proliferation rates and anti-apoptotic gene expression, they might acquire features that predispose them to malignant transformation. We screened 10 MSC samples expanded in PL-based medium for the presence of tumor-associated genetic variants using a 165 gene panel and detected only 21 different genetic variants. According to our analysis, none of these were established pathogenic mutations. Our data show that differences in culture conditions such as growth supplement have a significant impact on the gene expression profile of MSCs and favor the use of PL over FCS for expansion of MSCs.

Dataset Information

Heparin Anticoagulant for Human Bone Marrow Does Not Influence In Vitro Performance of Human Mesenchymal Stromal Cells.

Publications

Heparin Anticoagulant for Human Bone Marrow Does Not Influence In Vitro Performance of Human Mesenchymal Stromal Cells.

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