Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regene... more Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process. This report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production. The strategy for quality control testing depends on the product's cell composition, the manufacturing process and the indication and ta...
The possible different therapeutic efficacy of human mesenchymal stem cells (hMSC) derived from u... more The possible different therapeutic efficacy of human mesenchymal stem cells (hMSC) derived from umbilical cord blood (CB), adipose tissue (AT) or bone marrow (BM) for the treatment of myocardial infarction (MI) remains unexplored. This study was to assess the regenerative potential of hMSC from different origins and to evaluate the role of CD105 in cardiac regeneration. Male SCID mice underwent LAD-ligation and received the respective cell type (400.000/per animal) intramyocardially. Six weeks post infarction, cardiac catheterization showed significant preservation of left ventricular functions in BM and CD105(+)-CB treated groups compared to CB and nontreated MI group (MI-C). Cell survival analyzed by quantitative real time PCR for human GAPDH and capillary density measured by immunostaining showed consistent results. Furthermore, cardiac remodeling can be significantly attenuated by BM-hMSC compared to MI-C. Under hypoxic conditions in vitro, remarkably increased extracellular aci...
Among the mechanisms by which somatic stem cells may improve left ventricular function in ischaem... more Among the mechanisms by which somatic stem cells may improve left ventricular function in ischaemic heart disease are pro-survival stimuli mediated by secreted factors. This phenomenon is frequently referred to, but remains poorly understood. We therefore investigated the non-regenerative cardioprotective effects of cord blood mesenchymal stromal cells (CBMSCs) in vitro and sought to identify relevant intracellular signalling pathways. Conditioned medium from CBMSCs and fibroblasts was prepared, and secreted factors were analysed by Luminex(®) immunobead assay. Murine cardiomyocyte-derived HL-1 cells were subjected to simulated ischaemia by glucose and serum deprivation and hypoxia in CBMSC-conditioned or cell-free control medium or in medium conditioned by foreskin fibroblasts. The proportions of vital, apoptotic and necrotic cells (poly-caspase activity, annexin V and ethidium homodimer-III staining) were quantified using a high-content imaging system. Metabolic activity and proliferation rate were determined via 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and 5-bromo-2-deoxyuridine assays. Phosphorylation of Akt, extracellular-signal-regulated kinase (ERK)1/2, signal transducer and activator of transcription 3 (STAT3) and glycogen synthase kinase 3β was determined by western blot, and experiments were repeated in the presence of specific small-molecule inhibitors (Wortmannin, UO126 and Stattic). CBMSC medium reduced the proportion of dead HL-1 cardiomyocytes from 39 ± 3 to 28 ± 1% (P < 0.05) and the rate of late apoptotic cells to 68 ± 2% of that in control medium (P < 0.001). Metabolic activity was increased by 12 ± 1% compared with control (P < 0.05), while in fibroblast medium it was not (5 ± 2%, P = 1). This was associated with increased phosphorylation of Akt (2-fold, P < 0.05), ERK1/2 (3-fold, P < 0.01) and STAT3 (12-fold, P < 0.001). Combined blocking of the phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt and mitogen-activated protein kinase/ERK signalling abolished the protective CBMSC effect, while blocking the pathways individually had no effect. Inhibition of STAT3 phosphorylation drastically lowered HL-1 cell viability in control medium, but not in medium conditioned by CBMSCs. The factors released by CBMSCs protect cardiomyocyte-like HL-1 cells from simulated ischaemia more than those released from fibroblasts. While CBMSC-triggered Akt and ERK1/2 activation provides protection in a compensatory manner, STAT3 is crucial for cardiomyocyte survival in ischaemia, but is not a key mediator of cytoprotective stem cell actions.
From 4 to 5 April 2008, international experts met for the second time in Tubingen, Germany, to pr... more From 4 to 5 April 2008, international experts met for the second time in Tubingen, Germany, to present and discuss the latest proceedings in research on non-hematopoietic stem cells (NHSC). This report presents issues of basic research including characterization, isolation, good manufacturing practice (GMP)-like production and imaging as well as clinical applications focusing on the regenerative and immunomodulatory capacities of NHSC.
Journal of tissue engineering and regenerative medicine, Jan 15, 2015
The use of fetal bovine serum (FBS) as a culture medium supplement in cell therapy and clinical t... more The use of fetal bovine serum (FBS) as a culture medium supplement in cell therapy and clinical tissue engineering is challenged by immunological concerns and the risk of disease transmission. Here we tested whether human, thrombin-activated, pooled, platelet-rich plasma (tPRP) can be substituted for FBS in the engineering of osteogenic and vasculogenic grafts, using cells from the stromal vascular fraction (SVF) of human adipose tissue. SVF cells were cultured under perfusion flow into porous hydroxyapatite scaffolds for 5 days, with the medium supplemented with either 10% tPRP or 10% FBS and implanted in an ectopic mouse model. Following in vitro culture, as compared to FBS, the use of tPRP did not modify the fraction of clonogenic cells or the different cell phenotypes, but increased by 1.9-fold the total number of cells. After 8 weeks in vivo, bone tissue was formed more reproducibly and in higher amounts (3.7-fold increase) in constructs cultured with tPRP. Staining for human-s...
Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regene... more Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process. This report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production. The strategy for quality control testing depends on the product's cell composition, the manufacturing process and the indication and ta...
The possible different therapeutic efficacy of human mesenchymal stem cells (hMSC) derived from u... more The possible different therapeutic efficacy of human mesenchymal stem cells (hMSC) derived from umbilical cord blood (CB), adipose tissue (AT) or bone marrow (BM) for the treatment of myocardial infarction (MI) remains unexplored. This study was to assess the regenerative potential of hMSC from different origins and to evaluate the role of CD105 in cardiac regeneration. Male SCID mice underwent LAD-ligation and received the respective cell type (400.000/per animal) intramyocardially. Six weeks post infarction, cardiac catheterization showed significant preservation of left ventricular functions in BM and CD105(+)-CB treated groups compared to CB and nontreated MI group (MI-C). Cell survival analyzed by quantitative real time PCR for human GAPDH and capillary density measured by immunostaining showed consistent results. Furthermore, cardiac remodeling can be significantly attenuated by BM-hMSC compared to MI-C. Under hypoxic conditions in vitro, remarkably increased extracellular aci...
Among the mechanisms by which somatic stem cells may improve left ventricular function in ischaem... more Among the mechanisms by which somatic stem cells may improve left ventricular function in ischaemic heart disease are pro-survival stimuli mediated by secreted factors. This phenomenon is frequently referred to, but remains poorly understood. We therefore investigated the non-regenerative cardioprotective effects of cord blood mesenchymal stromal cells (CBMSCs) in vitro and sought to identify relevant intracellular signalling pathways. Conditioned medium from CBMSCs and fibroblasts was prepared, and secreted factors were analysed by Luminex(®) immunobead assay. Murine cardiomyocyte-derived HL-1 cells were subjected to simulated ischaemia by glucose and serum deprivation and hypoxia in CBMSC-conditioned or cell-free control medium or in medium conditioned by foreskin fibroblasts. The proportions of vital, apoptotic and necrotic cells (poly-caspase activity, annexin V and ethidium homodimer-III staining) were quantified using a high-content imaging system. Metabolic activity and proliferation rate were determined via 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and 5-bromo-2-deoxyuridine assays. Phosphorylation of Akt, extracellular-signal-regulated kinase (ERK)1/2, signal transducer and activator of transcription 3 (STAT3) and glycogen synthase kinase 3β was determined by western blot, and experiments were repeated in the presence of specific small-molecule inhibitors (Wortmannin, UO126 and Stattic). CBMSC medium reduced the proportion of dead HL-1 cardiomyocytes from 39 ± 3 to 28 ± 1% (P < 0.05) and the rate of late apoptotic cells to 68 ± 2% of that in control medium (P < 0.001). Metabolic activity was increased by 12 ± 1% compared with control (P < 0.05), while in fibroblast medium it was not (5 ± 2%, P = 1). This was associated with increased phosphorylation of Akt (2-fold, P < 0.05), ERK1/2 (3-fold, P < 0.01) and STAT3 (12-fold, P < 0.001). Combined blocking of the phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt and mitogen-activated protein kinase/ERK signalling abolished the protective CBMSC effect, while blocking the pathways individually had no effect. Inhibition of STAT3 phosphorylation drastically lowered HL-1 cell viability in control medium, but not in medium conditioned by CBMSCs. The factors released by CBMSCs protect cardiomyocyte-like HL-1 cells from simulated ischaemia more than those released from fibroblasts. While CBMSC-triggered Akt and ERK1/2 activation provides protection in a compensatory manner, STAT3 is crucial for cardiomyocyte survival in ischaemia, but is not a key mediator of cytoprotective stem cell actions.
From 4 to 5 April 2008, international experts met for the second time in Tubingen, Germany, to pr... more From 4 to 5 April 2008, international experts met for the second time in Tubingen, Germany, to present and discuss the latest proceedings in research on non-hematopoietic stem cells (NHSC). This report presents issues of basic research including characterization, isolation, good manufacturing practice (GMP)-like production and imaging as well as clinical applications focusing on the regenerative and immunomodulatory capacities of NHSC.
Journal of tissue engineering and regenerative medicine, Jan 15, 2015
The use of fetal bovine serum (FBS) as a culture medium supplement in cell therapy and clinical t... more The use of fetal bovine serum (FBS) as a culture medium supplement in cell therapy and clinical tissue engineering is challenged by immunological concerns and the risk of disease transmission. Here we tested whether human, thrombin-activated, pooled, platelet-rich plasma (tPRP) can be substituted for FBS in the engineering of osteogenic and vasculogenic grafts, using cells from the stromal vascular fraction (SVF) of human adipose tissue. SVF cells were cultured under perfusion flow into porous hydroxyapatite scaffolds for 5 days, with the medium supplemented with either 10% tPRP or 10% FBS and implanted in an ectopic mouse model. Following in vitro culture, as compared to FBS, the use of tPRP did not modify the fraction of clonogenic cells or the different cell phenotypes, but increased by 1.9-fold the total number of cells. After 8 weeks in vivo, bone tissue was formed more reproducibly and in higher amounts (3.7-fold increase) in constructs cultured with tPRP. Staining for human-s...
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Papers by Karen Bieback