Supplemental Material, Supplementary_Table_1 for Combination of Cardiac Progenitor Cells From the... more Supplemental Material, Supplementary_Table_1 for Combination of Cardiac Progenitor Cells From the Right Atrium and Left Ventricle Exhibits Synergistic Paracrine Effects In Vitro by Ryan McQuaig, Parul Dixit, Atsushi Yamauchi, Isabelle Van Hout, Jayanthi Bellae Papannarao, Richard Bunton, Dominic Parry, Philip Davis and Rajesh Katare in Cell Transplantation
Preliminary efficacy data indicates that stem cell transplantation has the potential to enhance m... more Preliminary efficacy data indicates that stem cell transplantation has the potential to enhance myocardial perfusion and/or contractile performance in patients with ischemic heart disease. A significant challenge in cardiovascular regenerative medicine is the identification and selection of the best suited stem/progenitor cell type. Cardiac progenitor cells (CPCs) and cardiac stem cells (CSCs) have been reviewed as the most promising cell types for autologous transplantation. The other extracardiac cell types that have been considered include bone marrow derived stem cells (BMCs), adipose tissue derived stem cells (ADSCs), peripheral blood derived endothelial progenitor cells (EPCs) and skeletal myoblasts. CPCs have not been directly compared with extra-cardiac cell types isolated from same patients for properties associated with cardiac repair. Interestingly, even within the heart the best location to isolate CPCs remains undecided. Moreover, atrial and ventricular CPCs may have phenotypic/functional differences owing to the differences in the atrio-ventricular microenvironment. While comparing different progenitor cell types, it is essential to isolate these cells from the same patients because the donor’s physiological and pathological characteristics may have a profound effect on the quality and quantity of progenitor cells isolated. This project, therefore, is aimed at comparing the functional characteristics of resident CPCs from the right atrial appendage (RAA), the left ventricle (LV) and an extra-cardiac source: EPCs from the peripheral blood – all isolated from the same patients, for the properties associated with cardiac repair. CPCs were isolated and characterised from the RAA and LV tissues from nineteen patients undergoing coronary artery bypass surgery (CABG). Early EPCs were isolated from the same patients from the peripheral blood and characterised for the expression of progenitor cell associated markers. To examine whether RAA and LV CPCs from the same hearts show different functional characteristics, the stem cell surface marker expression, cardiomyocyte differentiation capacity, expansion potential, senescence in culture and migration properties of these cells were examined. The RAA CPCs showed a higher expression of CD90 (a surface protein involved in cell migration and adhesion). In consonance with this, a scratch assay revealed a higher migration potential of RAA CPCs compared to LV CPCs isolated from the same hearts. The ex vivo expansion potential of RAA CPCs was greater than LV CPCs with higher cumulative population doublings after six weeks in culture. In contrast, RAA CPCs underwent greater senescence in culture at the same time point with a higher expression of cyclin dependent kinase inhibitor 2A (CDKN2A), a gene involved in the transcription of the senescence protein P16inka. To determine the differences in response to ischemia, the three progenitor cell types were exposed to hypoxia and serum starvation in vitro and their apoptotic cell death and gene expression was quantified. RAA and LV CPCs exhibited a similar pattern of resistance to apoptotic cell death under ischemia. Interestingly, EPCs exhibited the highest resistance to apoptotic cell death, however, they also showed the lowest proliferation under ‘hypoxia only’ treatment. The RT2 profiler gene array showed comparable gene expression pattern in RAA and LV CPCs, however, EPCs differentially expressed certain genes associated with paracrine properties and cell survival. To compare the paracrine properties of the progenitor cells exposed to ischemia, the secretion of cytokines was quantified in the conditioned medium (CM) from all three cell types. EPCs showed a significantly lower secretion of cytokines compared to CPCs. When human umbilical vein endothelial cells (HUVECs) were treated with the CM from the three cell types, LV CPC CM induced the highest angiogenesis in a tube formation assay. Conversely, ischemic mouse cardiomyocytes (HL-1 cells) treated with CM from the RAA CPCs showed the greatest survival. Further, it was identified that RAA CPC CM mediated its effect via both the phosphatidylinositol 3 kinase-Akt (PI3K-Akt) and protein kinase C epsilon (PKCε) pathways, whereas LV CPC CM and EPC CM mediated their effect by PKCε and PI3K-Akt pathways respectively. In conclusion, this study provides the first evidence that progenitor cells from the atria, ventricle and peripheral blood of the same patients show differences in functional properties associated with cardiac repair. The selection of the best cell type could, therefore, be influenced by the nature of the injury and the type of effect desired
BACKGROUND The heart has an intrinsic ability to regenerate, orchestrated by progenitor or stem c... more BACKGROUND The heart has an intrinsic ability to regenerate, orchestrated by progenitor or stem cells. However, the relative complexity of non-resident cardiac progenitor cell (CPC) therapy makes modulation of resident CPCs a more attractive treatment target. Thiamine analogues improve resident CPC function in pre-clinical models. In this double blinded randomised controlled trial (identifier: ACTRN12614000755639), we examined whether thiamine would improve CPC function in humans. METHODS AND RESULTS High dose oral thiamine (one gram twice daily) or matching placebo was administered 3-5 days prior to coronary artery bypass surgery (CABG). Right atrial appendages were collected at the time of CABG, and CPCs isolated. There was no difference in the primary outcome (proliferation ability of CPCs) between treatment groups. Older age was not associated with decreased proliferation ability. In exploratory analyses, isolated CPCs in the thiamine group showed an increase in the proportion of CD34-/CD105+ (endoglin) cells, but no difference in CD34-/CD90+ or CD34+ cells. Thiamine increased maximum force developed by isolated trabeculae, with no difference in relaxation time or beta-adrenergic responsiveness. CONCLUSION Thiamine does not improve proliferation ability of CPC in patients undergoing CABG, but increases the proportion of CD34-/CD105+ cells. Having not met its primary endpoint, this study provides the impetus to re-examine CPC biology prior to any clinical outcome-based trial examining potential beneficial cardiovascular effects of thiamine.
Cardiovascular diseases, such as ischemic heart disease, remain the most common cause of death wo... more Cardiovascular diseases, such as ischemic heart disease, remain the most common cause of death worldwide. Regenerative medicine with stem cell therapy is a promising tool for cardiac repair. Combination of different cell types has been shown to improve the therapeutic potential, which is thought to be due to synergistic or complimentary reparative effects. We investigated if the combination of cardiac progenitor cells (CPCs) of right atrial appendage (RAA) and left ventricle (LV) that are isolated from the same patient exert synergistic or complimentary paracrine effects for apoptotic cell death and angiogenesis in an in vitro model. Flow cytometry analysis showed that both RAA and LV CPCs expressed the mesenchymal cell markers CD90 and CD105, and were predominantly negative for the hematopoietic cell marker, CD34. Analysis of conditioned media (CM) collected from the CPCs cultured either alone or in combination in serum-deprived hypoxic conditions to simulate ischemia showed marked...
Deciding the best cell type for cardiac regeneration remains a big challenge. No studies have dir... more Deciding the best cell type for cardiac regeneration remains a big challenge. No studies have directly compared the functional efficacy of cardiac progenitor cells (CPCs) with extra-cardiac stem cells isolated from the same patient. We compared the functional characteristics of endothelial progenitor cells (EPCs), right atrial (RAA) CPCs and left ventricular (LV) CPCs isolated from the same patients (n=14). Within the same heart, RAA and LV CPCs exhibited marked differences in surface marker expression, with RAA CPCs exhibiting better expansion potential and migration properties. When subjected to hypoxia and serum starvation to simulate in vivo ischemic environment, RAA and LV CPCs exhibited similar pattern of resistance to apoptotic cell death under ischemia. Interestingly, EPCs exhibited highest resistance to apoptotic cell death, however, they also showed the lowest proliferation under hypoxia. RT-profiler array showed comparable gene expression pattern in RAA and LV CPCs, while they were differentially expressed in EPCs. Further, treating human umbilical vein endothelial cells with conditioned medium (CM) from LV showed maximum angiogenic potential, while cardiomyocytes treated with CM from RAA showed greatest survival under hypoxic conditions. Results from this study provide the first evidence that progenitor cells from different regions exhibit functional differences within the same patient.
Current therapeutic strategies for the treatment of critical limb ischemia (CLI) have only limite... more Current therapeutic strategies for the treatment of critical limb ischemia (CLI) have only limited success. Recent in vitro evidence in the literature, using cell lines, proposes that the peptide hormone ghrelin may have angiogenic properties. In this study, we aim to investigate if ghrelin could promote postischemic angiogenesis in a mouse model of CLI and, further, identify the mechanistic pathway(s) that underpin ghrelin's proangiogenic properties. CLI was induced in male CD1 mice by femoral artery ligation. Animals were then randomized to receive either vehicle or acylated ghrelin (150 μg/kg sc) for 14 consecutive days. Subsequently, synchrotron radiation microangiography was used to assess hindlimb perfusion. Subsequent tissue samples were collected for molecular and histological analysis. Ghrelin treatment markedly improved limb perfusion by promoting the generation of new capillaries and arterioles (internal diameter less than 50 μm) within the ischemic hindlimb that were...
Diabetes promotes progressive loss of cardiac cells, which are replaced by a fibrotic matrix, res... more Diabetes promotes progressive loss of cardiac cells, which are replaced by a fibrotic matrix, resulting in the loss of cardiac function. In the current study we sought to identify if excessive autophagy plays a major role in inducing this progressive loss. Immunofluorescence and western blotting analysis of the right atrial appendages collected from diabetic and non-diabetic patients undergoing coronary artery bypass graft surgery showed a marked increase in the level of autophagy in the diabetic heart, as evidenced by increased expression of autophagy marker LC3B-II and its mediator Beclin-1 and decreased expression of p62, which incorporates into autophagosomes to be efficiently degraded. Moreover, a marked activation of pro-apoptotic caspase-3 was observed. Electron microscopy showed increased autophagosomes in the diabetic heart. In vivo measurement of autophagic flux by choloroquine injection resulted in further enhancement of LC3B-II in the diabetic myocardium, confirming incr...
The overall clinical cardiac regeneration experience suggests that stem cell therapy can be safel... more The overall clinical cardiac regeneration experience suggests that stem cell therapy can be safely performed, but it also underlines the need for reproducible results for their effective use in a real-world scenario. One of the significant challenges is the identification and selection of the best suited stem cell type for regeneration therapy. Bone marrow mononuclear cells, bone marrow-derived mesenchymal stem cells, resident or endogenous cardiac stem cells, endothelial progenitor cells and induced pluripotent stem cells are some of the stem cell types which have been extensively tested for their ability to regenerate the lost myocardium. While most of these cell types are being evaluated in clinical trials for their safety and efficacy, results show significant heterogeneity in terms of efficacy. The enthusiasm surrounding regenerative medicine in the heart has been dampened by the reports of poor survival, proliferation, engraftment, and differentiation of the transplanted cells...
Introduction: Deciding the best cell type for cardiac regeneration remains a challenge, however, ... more Introduction: Deciding the best cell type for cardiac regeneration remains a challenge, however, no studies have directly compared the functional efficacy of cardiac stem cells (CSCs) with extra- cardiac SCs isolated from the same patient. Objective: To compare the functional characteristics of endothelial progenitor cells (EPCs), right atrial (RAA) CSCs and left ventricular (LV) CSCs isolated from the same patients. Methods and Results: Flow-cytometry analysis of CSCs (n=12 patients) revealed higher expression of CD90 (involved in cell adhesion and migration) in RAA CSCs (CD90 & CD105 ++ve cells: 71±15% in RAA vs. 47±27% in LV, P<0.05). However, scratch assay showed superior wound closure in LV CSCs (80±10% in LV vs. 60±5% in RAA, P<0.05) suggesting that LV CSCs have a better migration potential. Moreover, the expansion properties of RAA CSCs were better than LV CSCs in terms of higher cumulative population doublings in culture. To simulate in vivo ischemic environment, cells...
Supplemental Material, Supplementary_Table_1 for Combination of Cardiac Progenitor Cells From the... more Supplemental Material, Supplementary_Table_1 for Combination of Cardiac Progenitor Cells From the Right Atrium and Left Ventricle Exhibits Synergistic Paracrine Effects In Vitro by Ryan McQuaig, Parul Dixit, Atsushi Yamauchi, Isabelle Van Hout, Jayanthi Bellae Papannarao, Richard Bunton, Dominic Parry, Philip Davis and Rajesh Katare in Cell Transplantation
Preliminary efficacy data indicates that stem cell transplantation has the potential to enhance m... more Preliminary efficacy data indicates that stem cell transplantation has the potential to enhance myocardial perfusion and/or contractile performance in patients with ischemic heart disease. A significant challenge in cardiovascular regenerative medicine is the identification and selection of the best suited stem/progenitor cell type. Cardiac progenitor cells (CPCs) and cardiac stem cells (CSCs) have been reviewed as the most promising cell types for autologous transplantation. The other extracardiac cell types that have been considered include bone marrow derived stem cells (BMCs), adipose tissue derived stem cells (ADSCs), peripheral blood derived endothelial progenitor cells (EPCs) and skeletal myoblasts. CPCs have not been directly compared with extra-cardiac cell types isolated from same patients for properties associated with cardiac repair. Interestingly, even within the heart the best location to isolate CPCs remains undecided. Moreover, atrial and ventricular CPCs may have phenotypic/functional differences owing to the differences in the atrio-ventricular microenvironment. While comparing different progenitor cell types, it is essential to isolate these cells from the same patients because the donor’s physiological and pathological characteristics may have a profound effect on the quality and quantity of progenitor cells isolated. This project, therefore, is aimed at comparing the functional characteristics of resident CPCs from the right atrial appendage (RAA), the left ventricle (LV) and an extra-cardiac source: EPCs from the peripheral blood – all isolated from the same patients, for the properties associated with cardiac repair. CPCs were isolated and characterised from the RAA and LV tissues from nineteen patients undergoing coronary artery bypass surgery (CABG). Early EPCs were isolated from the same patients from the peripheral blood and characterised for the expression of progenitor cell associated markers. To examine whether RAA and LV CPCs from the same hearts show different functional characteristics, the stem cell surface marker expression, cardiomyocyte differentiation capacity, expansion potential, senescence in culture and migration properties of these cells were examined. The RAA CPCs showed a higher expression of CD90 (a surface protein involved in cell migration and adhesion). In consonance with this, a scratch assay revealed a higher migration potential of RAA CPCs compared to LV CPCs isolated from the same hearts. The ex vivo expansion potential of RAA CPCs was greater than LV CPCs with higher cumulative population doublings after six weeks in culture. In contrast, RAA CPCs underwent greater senescence in culture at the same time point with a higher expression of cyclin dependent kinase inhibitor 2A (CDKN2A), a gene involved in the transcription of the senescence protein P16inka. To determine the differences in response to ischemia, the three progenitor cell types were exposed to hypoxia and serum starvation in vitro and their apoptotic cell death and gene expression was quantified. RAA and LV CPCs exhibited a similar pattern of resistance to apoptotic cell death under ischemia. Interestingly, EPCs exhibited the highest resistance to apoptotic cell death, however, they also showed the lowest proliferation under ‘hypoxia only’ treatment. The RT2 profiler gene array showed comparable gene expression pattern in RAA and LV CPCs, however, EPCs differentially expressed certain genes associated with paracrine properties and cell survival. To compare the paracrine properties of the progenitor cells exposed to ischemia, the secretion of cytokines was quantified in the conditioned medium (CM) from all three cell types. EPCs showed a significantly lower secretion of cytokines compared to CPCs. When human umbilical vein endothelial cells (HUVECs) were treated with the CM from the three cell types, LV CPC CM induced the highest angiogenesis in a tube formation assay. Conversely, ischemic mouse cardiomyocytes (HL-1 cells) treated with CM from the RAA CPCs showed the greatest survival. Further, it was identified that RAA CPC CM mediated its effect via both the phosphatidylinositol 3 kinase-Akt (PI3K-Akt) and protein kinase C epsilon (PKCε) pathways, whereas LV CPC CM and EPC CM mediated their effect by PKCε and PI3K-Akt pathways respectively. In conclusion, this study provides the first evidence that progenitor cells from the atria, ventricle and peripheral blood of the same patients show differences in functional properties associated with cardiac repair. The selection of the best cell type could, therefore, be influenced by the nature of the injury and the type of effect desired
BACKGROUND The heart has an intrinsic ability to regenerate, orchestrated by progenitor or stem c... more BACKGROUND The heart has an intrinsic ability to regenerate, orchestrated by progenitor or stem cells. However, the relative complexity of non-resident cardiac progenitor cell (CPC) therapy makes modulation of resident CPCs a more attractive treatment target. Thiamine analogues improve resident CPC function in pre-clinical models. In this double blinded randomised controlled trial (identifier: ACTRN12614000755639), we examined whether thiamine would improve CPC function in humans. METHODS AND RESULTS High dose oral thiamine (one gram twice daily) or matching placebo was administered 3-5 days prior to coronary artery bypass surgery (CABG). Right atrial appendages were collected at the time of CABG, and CPCs isolated. There was no difference in the primary outcome (proliferation ability of CPCs) between treatment groups. Older age was not associated with decreased proliferation ability. In exploratory analyses, isolated CPCs in the thiamine group showed an increase in the proportion of CD34-/CD105+ (endoglin) cells, but no difference in CD34-/CD90+ or CD34+ cells. Thiamine increased maximum force developed by isolated trabeculae, with no difference in relaxation time or beta-adrenergic responsiveness. CONCLUSION Thiamine does not improve proliferation ability of CPC in patients undergoing CABG, but increases the proportion of CD34-/CD105+ cells. Having not met its primary endpoint, this study provides the impetus to re-examine CPC biology prior to any clinical outcome-based trial examining potential beneficial cardiovascular effects of thiamine.
Cardiovascular diseases, such as ischemic heart disease, remain the most common cause of death wo... more Cardiovascular diseases, such as ischemic heart disease, remain the most common cause of death worldwide. Regenerative medicine with stem cell therapy is a promising tool for cardiac repair. Combination of different cell types has been shown to improve the therapeutic potential, which is thought to be due to synergistic or complimentary reparative effects. We investigated if the combination of cardiac progenitor cells (CPCs) of right atrial appendage (RAA) and left ventricle (LV) that are isolated from the same patient exert synergistic or complimentary paracrine effects for apoptotic cell death and angiogenesis in an in vitro model. Flow cytometry analysis showed that both RAA and LV CPCs expressed the mesenchymal cell markers CD90 and CD105, and were predominantly negative for the hematopoietic cell marker, CD34. Analysis of conditioned media (CM) collected from the CPCs cultured either alone or in combination in serum-deprived hypoxic conditions to simulate ischemia showed marked...
Deciding the best cell type for cardiac regeneration remains a big challenge. No studies have dir... more Deciding the best cell type for cardiac regeneration remains a big challenge. No studies have directly compared the functional efficacy of cardiac progenitor cells (CPCs) with extra-cardiac stem cells isolated from the same patient. We compared the functional characteristics of endothelial progenitor cells (EPCs), right atrial (RAA) CPCs and left ventricular (LV) CPCs isolated from the same patients (n=14). Within the same heart, RAA and LV CPCs exhibited marked differences in surface marker expression, with RAA CPCs exhibiting better expansion potential and migration properties. When subjected to hypoxia and serum starvation to simulate in vivo ischemic environment, RAA and LV CPCs exhibited similar pattern of resistance to apoptotic cell death under ischemia. Interestingly, EPCs exhibited highest resistance to apoptotic cell death, however, they also showed the lowest proliferation under hypoxia. RT-profiler array showed comparable gene expression pattern in RAA and LV CPCs, while they were differentially expressed in EPCs. Further, treating human umbilical vein endothelial cells with conditioned medium (CM) from LV showed maximum angiogenic potential, while cardiomyocytes treated with CM from RAA showed greatest survival under hypoxic conditions. Results from this study provide the first evidence that progenitor cells from different regions exhibit functional differences within the same patient.
Current therapeutic strategies for the treatment of critical limb ischemia (CLI) have only limite... more Current therapeutic strategies for the treatment of critical limb ischemia (CLI) have only limited success. Recent in vitro evidence in the literature, using cell lines, proposes that the peptide hormone ghrelin may have angiogenic properties. In this study, we aim to investigate if ghrelin could promote postischemic angiogenesis in a mouse model of CLI and, further, identify the mechanistic pathway(s) that underpin ghrelin's proangiogenic properties. CLI was induced in male CD1 mice by femoral artery ligation. Animals were then randomized to receive either vehicle or acylated ghrelin (150 μg/kg sc) for 14 consecutive days. Subsequently, synchrotron radiation microangiography was used to assess hindlimb perfusion. Subsequent tissue samples were collected for molecular and histological analysis. Ghrelin treatment markedly improved limb perfusion by promoting the generation of new capillaries and arterioles (internal diameter less than 50 μm) within the ischemic hindlimb that were...
Diabetes promotes progressive loss of cardiac cells, which are replaced by a fibrotic matrix, res... more Diabetes promotes progressive loss of cardiac cells, which are replaced by a fibrotic matrix, resulting in the loss of cardiac function. In the current study we sought to identify if excessive autophagy plays a major role in inducing this progressive loss. Immunofluorescence and western blotting analysis of the right atrial appendages collected from diabetic and non-diabetic patients undergoing coronary artery bypass graft surgery showed a marked increase in the level of autophagy in the diabetic heart, as evidenced by increased expression of autophagy marker LC3B-II and its mediator Beclin-1 and decreased expression of p62, which incorporates into autophagosomes to be efficiently degraded. Moreover, a marked activation of pro-apoptotic caspase-3 was observed. Electron microscopy showed increased autophagosomes in the diabetic heart. In vivo measurement of autophagic flux by choloroquine injection resulted in further enhancement of LC3B-II in the diabetic myocardium, confirming incr...
The overall clinical cardiac regeneration experience suggests that stem cell therapy can be safel... more The overall clinical cardiac regeneration experience suggests that stem cell therapy can be safely performed, but it also underlines the need for reproducible results for their effective use in a real-world scenario. One of the significant challenges is the identification and selection of the best suited stem cell type for regeneration therapy. Bone marrow mononuclear cells, bone marrow-derived mesenchymal stem cells, resident or endogenous cardiac stem cells, endothelial progenitor cells and induced pluripotent stem cells are some of the stem cell types which have been extensively tested for their ability to regenerate the lost myocardium. While most of these cell types are being evaluated in clinical trials for their safety and efficacy, results show significant heterogeneity in terms of efficacy. The enthusiasm surrounding regenerative medicine in the heart has been dampened by the reports of poor survival, proliferation, engraftment, and differentiation of the transplanted cells...
Introduction: Deciding the best cell type for cardiac regeneration remains a challenge, however, ... more Introduction: Deciding the best cell type for cardiac regeneration remains a challenge, however, no studies have directly compared the functional efficacy of cardiac stem cells (CSCs) with extra- cardiac SCs isolated from the same patient. Objective: To compare the functional characteristics of endothelial progenitor cells (EPCs), right atrial (RAA) CSCs and left ventricular (LV) CSCs isolated from the same patients. Methods and Results: Flow-cytometry analysis of CSCs (n=12 patients) revealed higher expression of CD90 (involved in cell adhesion and migration) in RAA CSCs (CD90 & CD105 ++ve cells: 71±15% in RAA vs. 47±27% in LV, P<0.05). However, scratch assay showed superior wound closure in LV CSCs (80±10% in LV vs. 60±5% in RAA, P<0.05) suggesting that LV CSCs have a better migration potential. Moreover, the expansion properties of RAA CSCs were better than LV CSCs in terms of higher cumulative population doublings in culture. To simulate in vivo ischemic environment, cells...
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