Effect of the stop-flow technique on cardiac retention of c-kit positive human cardiac stem cells after intracoronary infusion in a porcine model of chronic ischemic cardiomyopathy.

It is commonly thought that the optimal method for intracoronary administration of cells is to stop coronary flow during cell infusion, in order to prolong cell/vascular wall contact, enhance adhesion, and promote extravasation of cells into the interstitial space. However, occlusion of a coronary artery with a balloon involves serious risks of vascular damage and/or dissection, particularly in non-stented segments such as those commonly found in patients with heart failure. It remains unknown whether the use of the stop-flow technique results in improved donor cell retention. Acute myocardial infarction was produced in 14 pigs. One to two months later, pigs received 10 million indium-111 oxyquinoline (oxine)-labeled c-kit(pos) human cardiac stem cells (hCSCs) via intracoronary infusion with (n = 7) or without (n = 7) balloon inflation. Pigs received cyclosporine to prevent acute graft rejection. Animals were euthanized 24 h later and hearts harvested for radioactivity measurements. With the stop-flow technique, the retention of hCSCs at 24 h was 5.41 ± 0.80 % of the injected dose (n = 7), compared with 4.87 ± 0.62 % without coronary occlusion (n = 7), (P = 0.60). When cells are delivered intracoronarily in a clinically relevant porcine model of chronic ischemic cardiomyopathy, the use of the stop-flow technique does not result in greater myocardial cell retention at 24 h compared with non-occlusive infusion. These results have practical implications for the design of cell therapy trials. Our observations suggest that the increased risk of complications secondary to coronary manipulation and occlusion is not warranted.

Differential Expression of Long Noncoding RNAs in Murine Myoblasts After Short Hairpin RNA-Mediated Dysferlin Silencing In Vitro: Microarray Profiling.

BACKGROUND: Long noncoding RNAs (lncRNAs) are noncoding RNA transcripts greater than 200 nucleotides in length and are known to play a role in regulating the transcription of genes involved in vital cellular functions. We hypothesized the disease process in dysferlinopathy is linked to an aberrant expression of lncRNAs and messenger RNAs (mRNAs). OBJECTIVE: In this study, we compared the lncRNA and mRNA expression profiles between wild-type and dysferlin-deficient murine myoblasts (C2C12 cells). METHODS: LncRNA and mRNA expression profiling were performed using a microarray. Several lncRNAs with differential expression were validated using quantitative real-time polymerase chain reaction. Gene Ontology (GO) analysis was performed to understand the functional role of the differentially expressed mRNAs. Further bioinformatics analysis was used to explore the potential function, lncRNA-mRNA correlation, and potential targets of the differentially expressed lncRNAs. RESULTS: We found 3195 lncRNAs and 1966 mRNAs that were differentially expressed. The chromosomal distribution of the differentially expressed lncRNAs and mRNAs was unequal, with chromosome 2 having the highest number of lncRNAs and chromosome 7 having the highest number of mRNAs that were differentially expressed. Pathway analysis of the differentially expressed genes indicated the involvement of several signaling pathways including PI3K-Akt, Hippo, and pathways regulating the pluripotency of stem cells. The differentially expressed genes were also enriched for the GO terms, developmental process and muscle system process. Network analysis identified 8 statistically significant (P<.05) network objects from the upregulated lncRNAs and 3 statistically significant network objects from the downregulated lncRNAs. CONCLUSIONS: Our results thus far imply that dysferlinopathy is associated with an aberrant expression of multiple lncRNAs, many of which may have a specific function in the disease process. GO terms and network analysis suggest a muscle-specific role for these lncRNAs. To elucidate the specific roles of these abnormally expressed noncoding RNAs, further studies engineering their expression are required.

Flipped Classrooms in Physician Assistant Education.

PURPOSE: This study assessed the impact of flipped classrooms on physician assistant (PA) students' performance and opinions. METHODS: Students completed quizzes and an opinion survey in Genetics, Human Pathophysiology (HPP), Clinical Medicine (CM) (n = 105) and Physical Exam (PE) (n = 98) courses. RESULTS: In Genetics and PE, the quiz scores were significantly higher for flipped classrooms (Genetics 95.00 ± 6.56; PE 83.09 ± 11.47) compared to the traditional lectures (Genetics 90.00 ± 10.53; PE 55.43 ± 16.66). In HPP, students performed better with traditional lectures (86.54 ± 8.82) compared to the flipped classrooms (75.12 ± 8.54). In CM, students' gain score was significantly higher for flipped classrooms (37.85 ± 16.73) than for traditional lectures (20.97 ± 15.55). The opinion surveys showed that the students surveyed preferred traditional lectures over flipped classrooms in Genetics (4.58 ± 0.46 vs. 2.29 ± 0.71) and HPP (4.14 ± 0.35 vs. 2.09 ± 0.53). CONCLUSION: Flipped classrooms improved the quality of learning in courses that deliver a hands-on skill or use case-based scenarios. They may not be an ideal choice for courses that require explanation of intricate scientific concepts.

Transcription factor-induced activation of cardiac gene expression in human c-kit+ cardiac progenitor cells.

Although transplantation of c-kit+ cardiac progenitor cells (CPCs) significantly alleviates post-myocardial infarction left ventricular dysfunction, generation of cardiomyocytes by exogenous CPCs in the recipient heart has often been limited. Inducing robust differentiation would be necessary for improving the efficacy of the regenerative cardiac cell therapy. We assessed the hypothesis that differentiation of human c-kit+ CPCs can be enhanced by priming them with cardiac transcription factors (TFs). We introduced five different TFs (Gata4, MEF2C, NKX2.5, TBX5, and BAF60C) into CPCs, either alone or in combination, and then examined the expression of marker genes associated with the major cardiac cell types using quantitative RT-PCR. When introduced individually, Gata4 and TBX5 induced a subset of myocyte markers. Moreover, Gata4 alone significantly induced smooth muscle cell and fibroblast markers. Interestingly, these gene expression changes brought by Gata4 were also accompanied by morphological changes. In contrast, MEF2C and NKX2.5 were largely ineffective in initiating cardiac gene expression in CPCs. Surprisingly, introduction of multiple TFs in different combinations mostly failed to act synergistically. Likewise, addition of BAF60C to Gata4 and/or TBX5 did not further potentiate their effects on cardiac gene expression. Based on our results, it appears that GATA4 is able to potentiate gene expression programs associated with multiple cardiovascular lineages in CPCs, suggesting that GATA4 may be effective in priming CPCs for enhanced differentiation in the setting of stem cell therapy.

C-Kit Promotes Growth and Migration of Human Cardiac Progenitor Cells via the PI3K-AKT and MEK-ERK Pathways.

A recent phase I clinical trial (SCIPIO) has shown that autologous c-kit+ cardiac progenitor cells (CPCs) improve cardiac function and quality of life when transplanted into patients with ischemic heart disease. Although c-kit is widely used as a marker of resident CPCs, its role in the regulation of the cellular characteristics of CPCs remains unknown. We hypothesized that c-kit plays a role in the survival, growth, and migration of CPCs. To test this hypothesis, human CPCs were grown under stress conditions in the presence or absence of SCF, and the effects of SCF-mediated activation of c-kit on CPC survival/growth and migration were measured. SCF treatment led to a significant increase in cell survival and a reduction in cell death under serum depletion conditions. In addition, SCF significantly promoted CPC migration in vitro. Furthermore, the pro-survival and pro-migratory effects of SCF were augmented by c-kit overexpression and abrogated by c-kit inhibition with imatinib. Mechanistically, c-kit activation in CPCs led to activation of the PI3K and the MAPK pathways. With the use of specific inhibitors, we confirmed that the SCF/c-kit-dependent survival and chemotaxis of CPCs are dependent on both pathways. Taken together, our findings suggest that c-kit promotes the survival/growth and migration of human CPCs cultured ex vivo via the activation of PI3K and MAPK pathways. These results imply that the efficiency of CPC homing to the injury site as well as their survival after transplantation may be improved by modulating the activity of c-kit.

Safety of intracoronary infusion of 20 million C-kit positive human cardiac stem cells in pigs.

BACKGROUND: There is mounting interest in using c-kit positive human cardiac stem cells (c-kit(pos) hCSCs) to repair infarcted myocardium in patients with ischemic cardiomyopathy. A recent phase I clinical trial (SCIPIO) has shown that intracoronary infusion of 1 million hCSCs is safe. Higher doses of CSCs may provide superior reparative ability; however, it is unknown if doses >1 million cells are safe. To address this issue, we examined the effects of 20 million hCSCs in pigs. METHODS: Right atrial appendage samples were obtained from patients undergoing cardiac surgery. The tissue was processed by an established protocol with eventual immunomagnetic sorting to obtain in vitro expanded hCSCs. A cumulative dose of 20 million cells was given intracoronarily to pigs without stop flow. Safety was assessed by measurement of serial biomarkers (cardiac: troponin I and CK-MB, renal: creatinine and BUN, and hepatic: AST, ALT, and alkaline phosphatase) and echocardiography pre- and post-infusion. hCSC retention 30 days after infusion was quantified by PCR for human genomic DNA. All personnel were blinded as to group assignment. RESULTS: Compared with vehicle-treated controls (n=5), pigs that received 20 million hCSCs (n=9) showed no significant change in cardiac function or end organ damage (assessed by organ specific biomarkers) that could be attributed to hCSCs (P>0.05 in all cases). No hCSCs could be detected in left ventricular samples 30 days after infusion. CONCLUSIONS: Intracoronary infusion of 20 million c-kit positive hCSCs in pigs (equivalent to ~40 million hCSCs in humans) does not cause acute cardiac injury, impairment of cardiac function, or liver and renal injury. These results have immediate translational value and lay the groundwork for using doses of CSCs >1 million in future clinical trials. Further studies are needed to ascertain whether administration of >1 million hCSCs is associated with greater efficacy in patients with ischemic cardiomyopathy.

c-kit+ Cardiac stem cells alleviate post-myocardial infarction left ventricular dysfunction despite poor engraftment and negligible retention in the recipient heart.

Although transplantation of c-kit+ cardiac stem cells (CSCs) has been shown to alleviate left ventricular (LV) dysfunction induced by myocardial infarction (MI), the number of exogenous CSCs remaining in the recipient heart following transplantation and their mechanism of action remain unclear. We have previously developed a highly sensitive and accurate method to quantify the absolute number of male murine CSCs in female recipient organs after transplantation. In the present study, we used this method to monitor the number of donor CSCs in the recipient heart after intracoronary infusion. Female mice underwent a 60-min coronary occlusion followed by reperfusion; 2 days later, 100,000 c-kit+/lin- syngeneic male mouse CSCs were infused intracoronarily. Only 12.7% of the male CSCs present in the heart immediately (5 min) after infusion were still present in the heart at 24 h, and their number declined rapidly thereafter. By 35 days after infusion, only ∼ 1,000 male CSCs were found in the heart. Significant numbers of male CSCs were found in the lungs and kidneys, but only in the first 24 h. The number of CSCs in the lungs increased between 5 min and 24 h after infusion, indicating recirculation of CSCs initially retained in other organs. Despite the low retention and rapid disappearance of CSCs from the recipient heart, intracoronary delivery of CSCs significantly improved LV function at 35 days (Millar catheter). These results suggest that direct differentiation of CSCs alone cannot account for the beneficial effects of CSCs on LV function; therefore, paracrine effects must be the major mechanism. The demonstration that functional improvement is dissociated from survival of transplanted cells has major implications for our understanding of cell therapy. In addition, this new quantitative method of stem cell measurement will be useful in testing approaches of enhancing CSC engraftment and survival after transplantation.