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Differential properties of human bone marrow and cardiac derived stromal cells supporting proliferation of hematopoietic cells
S32
Poster abstracts
proliferation capacity/functionality still remains elusive. Many possible effects of exercise on the release of progenitors from the bone marrow are currently discussed, including increased shear stress, a hypoxic environment, blood parameters such as norepinephrine (NE) or blood lactate concentration (La). The same factors might also impact the proliferative capacity of CPCs. The aim of this study was to evaluate the influence of ergometry-induced La and/or NE concentrations on the functionality of CPCs in vitro. Methods: Venous blood from 7 subjects (26.1 4.8 yrs) at rest was used for secondary CFU-GM assays incubated with La and/or NE. A subgroup of 6 subjects was randomly chosen to cover a possible drop-out rate. Acquired plasma baseline values of NE in vivo were about 5*10-9 mol/l before ergometry (baseline) and 5*10-8 mol/l after ergometry and were used for cell culture incubation. Results: CPC incubated with a NE concentration of 5*10-8 mol/l showed a significant decrease in CPC functionality in vitro (p < 0.05) compared to baseline, whereas a La concentration simulating baseline conditions did not show any significant effect. Simultaneously adding both substances at an ergometry-induced concentration to the cell culture, however, a trend was visible (p ¼ 0.08). Conclusion: NE levels equivalent to exhausting exercise significantly reduces CPC functionality, besides a known influence on CPC mobilization. The combination of La and NE does not significantly affect CPC functionality, although a decreasing trend could be recognized. These results suggest an influence of exercise-induced stress parameters on the body’s regeneration.
Mesenchymal stem cells (MSCs) are known to display not only stem cell multipotency, but also robust anti-inflammatory and regenerative properties. Autologous and allogeneic MSCs have been applied in a range of animal models of disease and human conditions including acute myocardial infarction and heart failure. However, a critical limitation in the field is the availability of cell sources that are both accessible and effective for cell therapy. This study compares the secretion of growth factors, immunosuppressive effects and differentiation capability of human MSCs derived from umbilical cord (UC), amniotic (AM), chorionic (Ch) or Decidual (Dc) membranes. We assessed the characteristic MSC phenotype with the expression of the specific surface markers. Trilineage differentiation and 5azacytidine (5-aza) induced cardiac differentiation were assessed by the corresponding stainings and real time PCRs. Growth factor secretion was also quantified by ELISA. All our cell sources rendered a typical MSC immunophenotype and a comparable immunosuppressive effect, though the yield of AM cultures was lower at early passages. Ch-MSCs were the only source to express all three factors (VEGF, FGF-1 and HGF). FGF-1 and HGF were also detected in UCMSCs conditioned media. D-MSCs were poor secretors and only moderate level of VEGF was detected. While cardiac myosin relative expression after 5aza treatment was undetectable in AM-MSCs, Ch-MSCs showed (80-100) and 1000X higher expression than UC and Dc-MSCs respectively. These results suggest that Ch and UC derived MSCs are potential candidate for cardiovascular regenerative cell therapy approach. Their evaluation in a related animal model is under evaluation.
104 DIFFERENTIAL PROPERTIES OF HUMAN BONE MARROW AND CARDIAC DERIVED STROMAL CELLS SUPPORTING PROLIFERATION OF HEMATOPOIETIC CELLS I McNiece, S Sivajothi The University of Texas MD Anderson Cancer Center, Houston, TX
106 PRIMING WITH SECRETED LIGANDS FROM HUMAN STROMAL PROGENITOR CELLS (P75MSCS) PROMOTES GRAFTS OF ADULT CARDIAC STEM/PROGENITOR CELLS AFTER MYOCARDIAL INFARCTION JL Spees University of Vermont, Colchester, VT
Bone marrow (BM) stromal cells (also termed mesenchymal stem cells; MSC) have been extensively studied and shown to control differentiation of hematopoietic stem cells (HSCs) in part through secreted growth factors. In addition, MSCs support the growth of tumor cells both in vitro and in vivo. As MSCs are being evaluated in a number of clinical trials for regenerative medicine approaches, it is critical to understand the role of stromal cells in different tissue environments. Stromal cells can be isolated from cardiac tissue and have an identical appearance in culture to MSCs. We have compared human cardiac stromal cells (CStrCs) to human BM-MSCs and demonstrate that CStrCs have a similar morphology and surface marker expression as BM-MSCs. To test the functional properties of CStrCs we cocultured cord blood (CB) cells on CStrCs and these cultures failed to expand the CB cells. As we have previously reported, co-culture of CB on BMMSCs resulted in expansion of hematopoietic cells. The effect of the CStrCs was through a cytostatic mechanism as removal of the CB cells from the CStrCs after 7 days of co-culture resulted in hematopoietic cell expansion and detection of colony forming cells by methycellulose assay. These data suggest that the function of cardiac derived stromal cells are distinct to BM derived stromal cells and provides insight into the basic biology of the different tissues. We have also demonstrated that CStrCs inhibit tumor cell proliferation. Tumor development in cardiac tissue is a rare event and these data suggest that CStrCs may play a key role in inhibiting proliferation of hematopoietic cells and tumor cells. Clinical trials are presently evaluating the use of BM-MSCs to repair ischemic tissue and the replacement of CStrCs with BM-MSCs may lead to a tumor supportive environment in cardiac tissue.
105 EVALUATION OF DIFFERENT SOURCES OF BIRTH-TISSUEDERIVED MESENCHYMAL STEM CELLS FOR THEIR CARDIOVASCULAR REGENERATIVE POTENTIAL L Salazar1, P González2, R Armijo3, P Maffud1, D Seguel4, J Acevedo1, F Figueroa1, J Bartolucci1,5, M Khoury1 1 Universidad de Los Andes, miRNA and stem cell laboratory, Santiago, Chile, 2Pontificia Universidad Catolica de Valparaiso, Valparaiso, Chile, 3Universidad Santo Tomas, Santiago, Chile, 4Universidad de Santiago de Chile, Santiago, Chile, 5Clinica santa Maria, Santiago, Chile
Transplantation of culture-expanded adult stem/progenitor cells often results in poor cellular engraftment, survival, and migration into sites of tissue injury. Mesenchymal cells such as fibroblasts and other stromal cells secrete factors that protect injured tissues, promote tissue repair, and support many types of stem/ progenitor cells in culture. We hypothesized secreted factors in conditioned medium (CdM) from adult human bone marrow-derived multipotent stromal cells (MSCs) could be used to prime adult cardiac stem/progenitor cells (CSCs/ CPCs) and improve graft success after myocardial infarction (MI). CdM from MSCs isolated by plastic adherence or by magnetic sorting against the p75 lowaffinity nerve growth factor receptor (p75MSCs) phosphorylated STAT3 and Akt in adult rat CPCs, supporting their proliferation under normoxic conditions and survival under hypoxic conditions (1% oxygen). Priming CSCs with 30 p75MSC CdM for 30 min prior to transplantation into sub-epicardial tissue 1 day after MI markedly increased engraftment 1 week after MI compared with priming in vehicle. Screening CdM with neutralizing/blocking antibodies identified 2 peptide ligands in p75MSC CdM that protected CPCs. The peptides acted synergistically, promoting CPC survival during hypoxia in culture. Similar to priming with CdM, priming of CSCs with both peptides for 30 min prior to transplantation promoted robust sub-epicardial engraftment, survival and migration of CSC derivatives 1 week after MI. Our results indicate that short-term priming of human CSCs with secreted ligands from p75MSCs may improve graft success and cardiac regeneration in patients with MI. 107 BASES FOR A COMBINED CELLULAR THERAPY TO IMPROVE CHRONIC SPINAL CORD INJURY OUTCOME GA Moviglia1, M Moviglia-Brandolino1, G Albanese1, N Blasetti1, V Mellisi2, S Piccone1, C Gaeta1 1 CIITT, Buenos Aires, Argentina, 2National University of La Plata, Buenos Aires, Argentina Introduction: Different authors agree that, in acute condition, Th1 cells which infiltrate the damaged tissue, favors spinal cord recovery after injury. Two and a half weeks after injury Th1 cells are replaced by Th2 cells. In turn Th2 cells inhibit SCI recovery. During the first and second week post injury there is a blood concentration peak of MSC. Looking for if these facts may that Th1 cells influence the MSC differentiation into Neuroblasts (NSC) we have tested this hypothesis in vitro.
Poster abstracts
proliferation capacity/functionality still remains elusive. Many possible effects of exercise on the release of progenitors from the bone marrow are currently discussed, including increased shear stress, a hypoxic environment, blood parameters such as norepinephrine (NE) or blood lactate concentration (La). The same factors might also impact the proliferative capacity of CPCs. The aim of this study was to evaluate the influence of ergometry-induced La and/or NE concentrations on the functionality of CPCs in vitro. Methods: Venous blood from 7 subjects (26.1 4.8 yrs) at rest was used for secondary CFU-GM assays incubated with La and/or NE. A subgroup of 6 subjects was randomly chosen to cover a possible drop-out rate. Acquired plasma baseline values of NE in vivo were about 5*10-9 mol/l before ergometry (baseline) and 5*10-8 mol/l after ergometry and were used for cell culture incubation. Results: CPC incubated with a NE concentration of 5*10-8 mol/l showed a significant decrease in CPC functionality in vitro (p < 0.05) compared to baseline, whereas a La concentration simulating baseline conditions did not show any significant effect. Simultaneously adding both substances at an ergometry-induced concentration to the cell culture, however, a trend was visible (p ¼ 0.08). Conclusion: NE levels equivalent to exhausting exercise significantly reduces CPC functionality, besides a known influence on CPC mobilization. The combination of La and NE does not significantly affect CPC functionality, although a decreasing trend could be recognized. These results suggest an influence of exercise-induced stress parameters on the body’s regeneration.
Mesenchymal stem cells (MSCs) are known to display not only stem cell multipotency, but also robust anti-inflammatory and regenerative properties. Autologous and allogeneic MSCs have been applied in a range of animal models of disease and human conditions including acute myocardial infarction and heart failure. However, a critical limitation in the field is the availability of cell sources that are both accessible and effective for cell therapy. This study compares the secretion of growth factors, immunosuppressive effects and differentiation capability of human MSCs derived from umbilical cord (UC), amniotic (AM), chorionic (Ch) or Decidual (Dc) membranes. We assessed the characteristic MSC phenotype with the expression of the specific surface markers. Trilineage differentiation and 5azacytidine (5-aza) induced cardiac differentiation were assessed by the corresponding stainings and real time PCRs. Growth factor secretion was also quantified by ELISA. All our cell sources rendered a typical MSC immunophenotype and a comparable immunosuppressive effect, though the yield of AM cultures was lower at early passages. Ch-MSCs were the only source to express all three factors (VEGF, FGF-1 and HGF). FGF-1 and HGF were also detected in UCMSCs conditioned media. D-MSCs were poor secretors and only moderate level of VEGF was detected. While cardiac myosin relative expression after 5aza treatment was undetectable in AM-MSCs, Ch-MSCs showed (80-100) and 1000X higher expression than UC and Dc-MSCs respectively. These results suggest that Ch and UC derived MSCs are potential candidate for cardiovascular regenerative cell therapy approach. Their evaluation in a related animal model is under evaluation.
104 DIFFERENTIAL PROPERTIES OF HUMAN BONE MARROW AND CARDIAC DERIVED STROMAL CELLS SUPPORTING PROLIFERATION OF HEMATOPOIETIC CELLS I McNiece, S Sivajothi The University of Texas MD Anderson Cancer Center, Houston, TX
106 PRIMING WITH SECRETED LIGANDS FROM HUMAN STROMAL PROGENITOR CELLS (P75MSCS) PROMOTES GRAFTS OF ADULT CARDIAC STEM/PROGENITOR CELLS AFTER MYOCARDIAL INFARCTION JL Spees University of Vermont, Colchester, VT
Bone marrow (BM) stromal cells (also termed mesenchymal stem cells; MSC) have been extensively studied and shown to control differentiation of hematopoietic stem cells (HSCs) in part through secreted growth factors. In addition, MSCs support the growth of tumor cells both in vitro and in vivo. As MSCs are being evaluated in a number of clinical trials for regenerative medicine approaches, it is critical to understand the role of stromal cells in different tissue environments. Stromal cells can be isolated from cardiac tissue and have an identical appearance in culture to MSCs. We have compared human cardiac stromal cells (CStrCs) to human BM-MSCs and demonstrate that CStrCs have a similar morphology and surface marker expression as BM-MSCs. To test the functional properties of CStrCs we cocultured cord blood (CB) cells on CStrCs and these cultures failed to expand the CB cells. As we have previously reported, co-culture of CB on BMMSCs resulted in expansion of hematopoietic cells. The effect of the CStrCs was through a cytostatic mechanism as removal of the CB cells from the CStrCs after 7 days of co-culture resulted in hematopoietic cell expansion and detection of colony forming cells by methycellulose assay. These data suggest that the function of cardiac derived stromal cells are distinct to BM derived stromal cells and provides insight into the basic biology of the different tissues. We have also demonstrated that CStrCs inhibit tumor cell proliferation. Tumor development in cardiac tissue is a rare event and these data suggest that CStrCs may play a key role in inhibiting proliferation of hematopoietic cells and tumor cells. Clinical trials are presently evaluating the use of BM-MSCs to repair ischemic tissue and the replacement of CStrCs with BM-MSCs may lead to a tumor supportive environment in cardiac tissue.
105 EVALUATION OF DIFFERENT SOURCES OF BIRTH-TISSUEDERIVED MESENCHYMAL STEM CELLS FOR THEIR CARDIOVASCULAR REGENERATIVE POTENTIAL L Salazar1, P González2, R Armijo3, P Maffud1, D Seguel4, J Acevedo1, F Figueroa1, J Bartolucci1,5, M Khoury1 1 Universidad de Los Andes, miRNA and stem cell laboratory, Santiago, Chile, 2Pontificia Universidad Catolica de Valparaiso, Valparaiso, Chile, 3Universidad Santo Tomas, Santiago, Chile, 4Universidad de Santiago de Chile, Santiago, Chile, 5Clinica santa Maria, Santiago, Chile
Transplantation of culture-expanded adult stem/progenitor cells often results in poor cellular engraftment, survival, and migration into sites of tissue injury. Mesenchymal cells such as fibroblasts and other stromal cells secrete factors that protect injured tissues, promote tissue repair, and support many types of stem/ progenitor cells in culture. We hypothesized secreted factors in conditioned medium (CdM) from adult human bone marrow-derived multipotent stromal cells (MSCs) could be used to prime adult cardiac stem/progenitor cells (CSCs/ CPCs) and improve graft success after myocardial infarction (MI). CdM from MSCs isolated by plastic adherence or by magnetic sorting against the p75 lowaffinity nerve growth factor receptor (p75MSCs) phosphorylated STAT3 and Akt in adult rat CPCs, supporting their proliferation under normoxic conditions and survival under hypoxic conditions (1% oxygen). Priming CSCs with 30 p75MSC CdM for 30 min prior to transplantation into sub-epicardial tissue 1 day after MI markedly increased engraftment 1 week after MI compared with priming in vehicle. Screening CdM with neutralizing/blocking antibodies identified 2 peptide ligands in p75MSC CdM that protected CPCs. The peptides acted synergistically, promoting CPC survival during hypoxia in culture. Similar to priming with CdM, priming of CSCs with both peptides for 30 min prior to transplantation promoted robust sub-epicardial engraftment, survival and migration of CSC derivatives 1 week after MI. Our results indicate that short-term priming of human CSCs with secreted ligands from p75MSCs may improve graft success and cardiac regeneration in patients with MI. 107 BASES FOR A COMBINED CELLULAR THERAPY TO IMPROVE CHRONIC SPINAL CORD INJURY OUTCOME GA Moviglia1, M Moviglia-Brandolino1, G Albanese1, N Blasetti1, V Mellisi2, S Piccone1, C Gaeta1 1 CIITT, Buenos Aires, Argentina, 2National University of La Plata, Buenos Aires, Argentina Introduction: Different authors agree that, in acute condition, Th1 cells which infiltrate the damaged tissue, favors spinal cord recovery after injury. Two and a half weeks after injury Th1 cells are replaced by Th2 cells. In turn Th2 cells inhibit SCI recovery. During the first and second week post injury there is a blood concentration peak of MSC. Looking for if these facts may that Th1 cells influence the MSC differentiation into Neuroblasts (NSC) we have tested this hypothesis in vitro.