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SERUM-FREE CULTURE CONDITIONS SUPPORT THE THERAPEUTIC EFFICACY OF EX VIVO PROLIFERATED HUMAN CARDIAC STEM CELLS
Abstracts
SMEM media, and then cells were isolated and co-incubated for 2 hrs at 37 C with Dynabeads coated with c-kit antibody. These magnetic beads were washed to remove c-kit- cells, and then remaining c-kit+ cells were plated. At 70% confluency, cells were transfected with miR-301a or scrambled negative control, and then protein and RNA was collected 48hrs later. We performed qRT-PCR and Western blot analysis to investigate Dicer expression and expression of pro-fibrotic markers. + RESULTS: miR-301a transfection of c-kit CSCs significantly reduced expression of both Dicer1a and Dicer 1b. In addition, miR-301a over-expression decreased the mRNA level of myosin heavy chain 9, and in the protein levels of non-muscle myosin IIA and alpha-smooth muscle actin. CONCLUSION: These results provide insight into a potential cellular mechanism that influences the differentiation of AFs, BM-MPCs, and c-kit+ CSCs which could be caused by changes in Dicer, the key protein in activating micro-RNAs. 275 GENETIC DELETION OF SOLUBLE EPOXIDE HYDROLASE PROTECTS CARDIAC MITOCHONDRIA FROM LPS-INDUCED TOXICITY KL Jamieson, M Akhonkh, V Samokhvalov, JM Seubert Edmonton, Alberta BACKGROUND:
Lipopolysaccharide (LPS) is a bacterial wall endotoxin producing many pathophysiological conditions including myocardial inflammation leading to cardiotoxicity. Arachidonic acid is a polyunsaturated fatty acid that can be metabolized to cardioprotective epoxyeicosatrienoic acids (EETs) by cytochrome P450 epoxygenases. These metabolites are subsequently hydrolyzed to less bioactive dihydroxyeicosatrienoic acids (DHET) by soluble epoxide hydrolase (sEH). EETs are known to trigger a wide range of pathways protecting cellular structures, reducing cell death and promoting anti-inflammatory reactions in various cell types. We have recently demonstrated that EETs protect rat neonatal cardiomyocytes against LPS-induced cytotoxicity. Increased exposure to LPS as an environmental toxin can have a negative impact on the heart resulting in cardiovascular complications. The goal of this study is to investigate whether inhibition of sEH, genetically or pharmacologically, will influence mitochondrial function following LPS exposure. METHODS: Age matched 2 month old sEH null (KO) and littermate wild-type (WT) mice were injected with LPS (10mg/kg) then sacrificed after 6 or 24 hrs. Hearts and blood were collected to assess inflammatory response and mitochondrial function. Mitochondrial function was evaluated by measuring the levels of ATP in the myocardium and respiratory activity of isolated cardiac mitochondria using a Clarktype electrode. The levels of glucose and inflammatory cytokines in blood were also assessed. RESULTS: Our data demonstrated that LPS-triggered a massive inflammatory response beginning at 6h and until 24h in WT mice. This coincided with pronounced hypoglycemic
S145
response and compromised mitochondrial function in the WT mice. In contrast, sEH-KO mice were protected against LPS-induced cardiotoxicity. These animals did not develop hypoglycemia and no loss in body weight was detected. The levels of inflammatory markers (TNFa, MCP-1) were only modestly elevated and significantly lower than WT mice treated with LPS. Mitochondrial function was preserved in hearts from sEH KO mice based on higher respiratory control ratios compared to WT. CONCLUSION: Deletion soluble epoxide hydrolase provides protective effects against LPS-induced cardiotoxicity maintaining mitochondrial function. Our data suggest that inhibition of sEH to elevate endogenous levels of EETs is a potential therapeutic approach to limiting LPS-induced cardiotoxicity. CIHR
276 SERUM-FREE CULTURE CONDITIONS SUPPORT THE THERAPEUTIC EFFICACY OF EX VIVO PROLIFERATED HUMAN CARDIAC STEM CELLS ST Mount, DR Davis Ottawa, Ontario BACKGROUND:
Autologous cardiac stem cell (CSC) therapies represent an emerging treatment option for patients with congestive heart failure. While previous work demonstrated that serum-free, xeno-free culture conditions support the initial outgrowth of ex vivo proliferated CSCs from plated cardiac biopsies with negligible effect on in vitro angiogenesis and stem cell recruitment, the effect of expanding this CSC product to meaningful clinical doses on in vitro and in vivo measures of regeneration are unknown. METHODS/RESULTS: Human CSCs were cultured from atrial appendages or myocardial biopsies donated by patients undergoing clinically indicated cardiac procedures. Although culture of ventricular myocardial biopsies within serum free xeno free conditions provided robust numbers of cells (1.70.9 million cells per 3214 mg tissue, p¼0.40 compared to standard serum culture), direct clinical application (requiring 20-30 million cells) was limited by a constant initial output return proportional to the amount of tissue plated. Straightforward culture of plated CSCs within adherent culture conditions provided a 5.51.1 fold increase in cell numbers over 1 week with equivalent cardiac (4.10.3%, p¼0.77) and mesenchymal (525%, p¼0.11) progenitor cell content as compared to standard serum culture conditions. Following surgical ligation of the left anterior descending coronary artery in NOD-SCID mice, animals were randomized 1 week later to receive echocardiographic guided intra-myocardial injection of the initial outgrowth from plated cardiac samples (standard or serum free CSC product). As indicated by the ejection fraction measured 4 weeks after experimental infarction, mice receiving the serum-free CSC outgrowth from plated cardiac
S146
samples showed functional improvements equivalent to those receiving standard CSCs (483 vs. 402%, p¼0.07). Further, post-mortem analysis of treated hearts by qPCR for retained human alu sequences revealed no effect for cell product culture delivered on long-term CSC engraftment (4.92.4 vs. 5.44.4 cells per mg left ventricle tissue, p¼0.92). CONCLUSION: These data suggest that serum-free culture methods can produce a cell product with equivalent regenerative potential. Transitioning CSCs to clinically acceptable protocols provides a more uniform and functionally equivalent cell product ready for clinical delivery. These findings represent the first published evidence that altered serum-free xeno-free culture conditions can provide a superior CSC cell product with the potential for ready translation to clinical use.
Canadian Journal of Cardiology Volume 31 2015
hASC-SBs exhibited greater pro-angiogenic potential as VEGF levels were increased also compared to hASC-MLs. CONCLUSION: hASC represent a promising cell source for CCT and cardiac repair. Their paracrine, therapeutic potential can be optimized. Our findings clearly showed that hASCs cultured as 3D structures (i.e. as SBs) exhibit an improvement in both anti-inflammatory and angiogenic properties. Spheroid body formation thus represents an effective alternative to enhance the therapeutic potential of hASCs.
277 ENHANCEMENT OF THE PARACRINE POTENTIAL OF HUMAN ADIPOSE DERIVED STEM CELLS WHEN CULTURED AS SPHEROID BODIES C Sid-Otmane, H Ly Montréal, Québec BACKGROUND:
Ischemic heart disease remains a leading cause of mortality and morbidity worldwide. Cardiac cell therapy (CCT) is a promising therapeutic strategy to help in cardiac repair. Adipose tissue constitutes an important reservoir for the stem cells. Both preclinical and clinical data have shown that adipose derived stem cells (ASCs) could improve cardiac function and volumes, mostly through a paracrine mechanism. STUDY AIM: The objective of our in vitro study is to characterize and compare the secretion profile of ASCs, when cultured under standard conditions (i.e. as a monolayer (ML)) versus in a three-dimension (3-D) structure (i.e. as a spheroid body (SB)). METHODS: Human ASCs (hASCs) were expanded in standard culture conditions in a monolayer form. ASCs were characterized according to both surface markers expression (assessed by immunofluorescence) and their ability to maintain multilineage differentiation. Alternatively, ASCs were also cultured as 3-D structure as spheroid bodies (SBs), by using the hanging drop technique. Luminex and ELISA assays were conducted to quantify key anti-inflammatory and angiogenic mediators to compare the two study groups. RESULTS: hASCs expressed similar surface markers as those documented for bone marrow MSCs including CD44, CD105 and CD90. Their ability to differentiate into adipogenic, chondrogenic and osteogenic lineage were unaltered. Paracrine activity of hASCs was enhanced when cultured as hASC-SBs. SBs secreted higher levels of anti-inflammatory cytokines such as MCP-1, IL-6 and IL-10, in a time dependent manner (Figure). Similarly,
278 REPETITIVE TARGETED DELIVERY OF GDF11 BY ULTRASOUNDMEDIATED CATIONIC MICROBUBBLE DESTRUCTION REJUVENATES AND PROTECTS THE AGED MOUSE HEART GK Du, J Wu, Z Shao, S Li, J Wu, NC Ni, RD Weisel, J Tian, R Li Toronto, Ontario BACKGROUND:
Preclinical studies demonstrated dramatic improvements in ventricular function with the implantation of stem cells into the damaged myocardium. However, stem cells in aged recipients have reduced regenerative potential, and aged tissues have diminished renewal capacity even with potent stem cells. Growth differentiation factor 11 (GDF11), a member of the TGF-b superfamily, has been shown to reverse age-related cardiac hypertrophy and skeletal muscular dysfunction, remodel the cerebral vasculature of aged mice and enhance olfactory neurogenesis. However, systemic delivery of recombinant GDF11 protein may not be the ideal method to rejuvenate specific organs. To evaluate a noninvasive method to protect aged hearts from ischemic injury, we tested whether a GDF11 expression plasmid construct could be delivered via ultrasound-targeted microbubble destruction (UTMD) using our effective cationic microbubbles (CMBs). METHODS & RESULTS: Young (aged 3 months) and old (aged 20-22 months) C57BL/6 mice were used to evaluate the expression of GDF11 mRNA and protein at baseline and after ischemia/reperfusion (I/R) injury. GDF11 mRNA and protein expression at baseline in old mice was significantly
SMEM media, and then cells were isolated and co-incubated for 2 hrs at 37 C with Dynabeads coated with c-kit antibody. These magnetic beads were washed to remove c-kit- cells, and then remaining c-kit+ cells were plated. At 70% confluency, cells were transfected with miR-301a or scrambled negative control, and then protein and RNA was collected 48hrs later. We performed qRT-PCR and Western blot analysis to investigate Dicer expression and expression of pro-fibrotic markers. + RESULTS: miR-301a transfection of c-kit CSCs significantly reduced expression of both Dicer1a and Dicer 1b. In addition, miR-301a over-expression decreased the mRNA level of myosin heavy chain 9, and in the protein levels of non-muscle myosin IIA and alpha-smooth muscle actin. CONCLUSION: These results provide insight into a potential cellular mechanism that influences the differentiation of AFs, BM-MPCs, and c-kit+ CSCs which could be caused by changes in Dicer, the key protein in activating micro-RNAs. 275 GENETIC DELETION OF SOLUBLE EPOXIDE HYDROLASE PROTECTS CARDIAC MITOCHONDRIA FROM LPS-INDUCED TOXICITY KL Jamieson, M Akhonkh, V Samokhvalov, JM Seubert Edmonton, Alberta BACKGROUND:
Lipopolysaccharide (LPS) is a bacterial wall endotoxin producing many pathophysiological conditions including myocardial inflammation leading to cardiotoxicity. Arachidonic acid is a polyunsaturated fatty acid that can be metabolized to cardioprotective epoxyeicosatrienoic acids (EETs) by cytochrome P450 epoxygenases. These metabolites are subsequently hydrolyzed to less bioactive dihydroxyeicosatrienoic acids (DHET) by soluble epoxide hydrolase (sEH). EETs are known to trigger a wide range of pathways protecting cellular structures, reducing cell death and promoting anti-inflammatory reactions in various cell types. We have recently demonstrated that EETs protect rat neonatal cardiomyocytes against LPS-induced cytotoxicity. Increased exposure to LPS as an environmental toxin can have a negative impact on the heart resulting in cardiovascular complications. The goal of this study is to investigate whether inhibition of sEH, genetically or pharmacologically, will influence mitochondrial function following LPS exposure. METHODS: Age matched 2 month old sEH null (KO) and littermate wild-type (WT) mice were injected with LPS (10mg/kg) then sacrificed after 6 or 24 hrs. Hearts and blood were collected to assess inflammatory response and mitochondrial function. Mitochondrial function was evaluated by measuring the levels of ATP in the myocardium and respiratory activity of isolated cardiac mitochondria using a Clarktype electrode. The levels of glucose and inflammatory cytokines in blood were also assessed. RESULTS: Our data demonstrated that LPS-triggered a massive inflammatory response beginning at 6h and until 24h in WT mice. This coincided with pronounced hypoglycemic
S145
response and compromised mitochondrial function in the WT mice. In contrast, sEH-KO mice were protected against LPS-induced cardiotoxicity. These animals did not develop hypoglycemia and no loss in body weight was detected. The levels of inflammatory markers (TNFa, MCP-1) were only modestly elevated and significantly lower than WT mice treated with LPS. Mitochondrial function was preserved in hearts from sEH KO mice based on higher respiratory control ratios compared to WT. CONCLUSION: Deletion soluble epoxide hydrolase provides protective effects against LPS-induced cardiotoxicity maintaining mitochondrial function. Our data suggest that inhibition of sEH to elevate endogenous levels of EETs is a potential therapeutic approach to limiting LPS-induced cardiotoxicity. CIHR
276 SERUM-FREE CULTURE CONDITIONS SUPPORT THE THERAPEUTIC EFFICACY OF EX VIVO PROLIFERATED HUMAN CARDIAC STEM CELLS ST Mount, DR Davis Ottawa, Ontario BACKGROUND:
Autologous cardiac stem cell (CSC) therapies represent an emerging treatment option for patients with congestive heart failure. While previous work demonstrated that serum-free, xeno-free culture conditions support the initial outgrowth of ex vivo proliferated CSCs from plated cardiac biopsies with negligible effect on in vitro angiogenesis and stem cell recruitment, the effect of expanding this CSC product to meaningful clinical doses on in vitro and in vivo measures of regeneration are unknown. METHODS/RESULTS: Human CSCs were cultured from atrial appendages or myocardial biopsies donated by patients undergoing clinically indicated cardiac procedures. Although culture of ventricular myocardial biopsies within serum free xeno free conditions provided robust numbers of cells (1.70.9 million cells per 3214 mg tissue, p¼0.40 compared to standard serum culture), direct clinical application (requiring 20-30 million cells) was limited by a constant initial output return proportional to the amount of tissue plated. Straightforward culture of plated CSCs within adherent culture conditions provided a 5.51.1 fold increase in cell numbers over 1 week with equivalent cardiac (4.10.3%, p¼0.77) and mesenchymal (525%, p¼0.11) progenitor cell content as compared to standard serum culture conditions. Following surgical ligation of the left anterior descending coronary artery in NOD-SCID mice, animals were randomized 1 week later to receive echocardiographic guided intra-myocardial injection of the initial outgrowth from plated cardiac samples (standard or serum free CSC product). As indicated by the ejection fraction measured 4 weeks after experimental infarction, mice receiving the serum-free CSC outgrowth from plated cardiac
S146
samples showed functional improvements equivalent to those receiving standard CSCs (483 vs. 402%, p¼0.07). Further, post-mortem analysis of treated hearts by qPCR for retained human alu sequences revealed no effect for cell product culture delivered on long-term CSC engraftment (4.92.4 vs. 5.44.4 cells per mg left ventricle tissue, p¼0.92). CONCLUSION: These data suggest that serum-free culture methods can produce a cell product with equivalent regenerative potential. Transitioning CSCs to clinically acceptable protocols provides a more uniform and functionally equivalent cell product ready for clinical delivery. These findings represent the first published evidence that altered serum-free xeno-free culture conditions can provide a superior CSC cell product with the potential for ready translation to clinical use.
Canadian Journal of Cardiology Volume 31 2015
hASC-SBs exhibited greater pro-angiogenic potential as VEGF levels were increased also compared to hASC-MLs. CONCLUSION: hASC represent a promising cell source for CCT and cardiac repair. Their paracrine, therapeutic potential can be optimized. Our findings clearly showed that hASCs cultured as 3D structures (i.e. as SBs) exhibit an improvement in both anti-inflammatory and angiogenic properties. Spheroid body formation thus represents an effective alternative to enhance the therapeutic potential of hASCs.
277 ENHANCEMENT OF THE PARACRINE POTENTIAL OF HUMAN ADIPOSE DERIVED STEM CELLS WHEN CULTURED AS SPHEROID BODIES C Sid-Otmane, H Ly Montréal, Québec BACKGROUND:
Ischemic heart disease remains a leading cause of mortality and morbidity worldwide. Cardiac cell therapy (CCT) is a promising therapeutic strategy to help in cardiac repair. Adipose tissue constitutes an important reservoir for the stem cells. Both preclinical and clinical data have shown that adipose derived stem cells (ASCs) could improve cardiac function and volumes, mostly through a paracrine mechanism. STUDY AIM: The objective of our in vitro study is to characterize and compare the secretion profile of ASCs, when cultured under standard conditions (i.e. as a monolayer (ML)) versus in a three-dimension (3-D) structure (i.e. as a spheroid body (SB)). METHODS: Human ASCs (hASCs) were expanded in standard culture conditions in a monolayer form. ASCs were characterized according to both surface markers expression (assessed by immunofluorescence) and their ability to maintain multilineage differentiation. Alternatively, ASCs were also cultured as 3-D structure as spheroid bodies (SBs), by using the hanging drop technique. Luminex and ELISA assays were conducted to quantify key anti-inflammatory and angiogenic mediators to compare the two study groups. RESULTS: hASCs expressed similar surface markers as those documented for bone marrow MSCs including CD44, CD105 and CD90. Their ability to differentiate into adipogenic, chondrogenic and osteogenic lineage were unaltered. Paracrine activity of hASCs was enhanced when cultured as hASC-SBs. SBs secreted higher levels of anti-inflammatory cytokines such as MCP-1, IL-6 and IL-10, in a time dependent manner (Figure). Similarly,
278 REPETITIVE TARGETED DELIVERY OF GDF11 BY ULTRASOUNDMEDIATED CATIONIC MICROBUBBLE DESTRUCTION REJUVENATES AND PROTECTS THE AGED MOUSE HEART GK Du, J Wu, Z Shao, S Li, J Wu, NC Ni, RD Weisel, J Tian, R Li Toronto, Ontario BACKGROUND:
Preclinical studies demonstrated dramatic improvements in ventricular function with the implantation of stem cells into the damaged myocardium. However, stem cells in aged recipients have reduced regenerative potential, and aged tissues have diminished renewal capacity even with potent stem cells. Growth differentiation factor 11 (GDF11), a member of the TGF-b superfamily, has been shown to reverse age-related cardiac hypertrophy and skeletal muscular dysfunction, remodel the cerebral vasculature of aged mice and enhance olfactory neurogenesis. However, systemic delivery of recombinant GDF11 protein may not be the ideal method to rejuvenate specific organs. To evaluate a noninvasive method to protect aged hearts from ischemic injury, we tested whether a GDF11 expression plasmid construct could be delivered via ultrasound-targeted microbubble destruction (UTMD) using our effective cationic microbubbles (CMBs). METHODS & RESULTS: Young (aged 3 months) and old (aged 20-22 months) C57BL/6 mice were used to evaluate the expression of GDF11 mRNA and protein at baseline and after ischemia/reperfusion (I/R) injury. GDF11 mRNA and protein expression at baseline in old mice was significantly