Science-based assessment of source materials for cell-based medicines: report of a stakeholders workshop.

Human pluripotent stem cells (hPSCs) have the potential to transform medicine. However, hurdles remain to ensure safety for such cellular products. Science-based understanding of the requirements for source materials is required as are appropriate materials. Leaders in hPSC biology, clinical translation, biomanufacturing and regulatory issues were brought together to define requirements for source materials for the production of hPSC-derived therapies and to identify other key issues for the safety of cell therapy products. While the focus of this meeting was on hPSC-derived cell therapies, many of the issues are generic to all cell-based medicines. The intent of this report is to summarize the key issues discussed and record the consensus reached on each of these by the expert delegates.

Stable expression of miR-200c alone is sufficient to regulate TCF8 (ZEB1) and restore E-cadherin expression.

Regulation of the transcription factor TCF8 (ZEB1) by the microRNA miR-200c and was first identified using a bioinformatic and relative quantitative PCR based approach.(1) Using stable ectopic expression of miR-200c we then demonstrated loss of TCF8 (ZEB1) mRNA and restoration of its primary regulatory target, E-cadherin.(2) Recently, other members of the miR-200 'family' and an additional unrelated microRNA, miR-205, have been reported to be essential for the regulation of TCF8 (ZEB1) and restoration of E-cadherin.(3-5) To investigate, we repeated our initial method(s) and generated individual stable cell-lines, expressing the miR-200 'family' members; miR-200c, miR-200b, miR-141 and the related miR-205. Of these lines, miR-200b produced no mature transcript and miR-205 and miR-141 cells were either morphologically similar to controls or showed some slight changes respectively. However no reduction in TCF8/ZEB1 mRNA or restoration of E-cadherin could be detected in either line. In contrast, cells expressing miR-200c had a significantly altered morphology from mesenchymal to epithelial and restored expression of E-cadherin. These contrasting findings demonstrate that, as transient transfection is in essence an RNAi experiment, results should be treated with caution when investigating microRNAs and that an examination of microRNAs with similar seed regions requires stable ectopic expression to accurately reflect the endogenous mechanism(s).

Autologous skeletal myoblast transplantation improved hemodynamics and left ventricular function in chronic heart failure dogs.

BACKGROUND: Previous studies have suggested that autologous skeletal myoblast transplantation (ASMT) improves left ventricular (LV) function in small animals after myocardial infarction. We tested the effects of ASMT on hemodynamics, LV function and remodeling in coronary microembolization-induced chronic heart failure (CHF) in conscious dogs. METHODS: Nineteen dogs were continuously instrumented with LV pressure sensors and mid-myocardial sonomicrometry crystals for dP/dt(max) and LV volume determination. Each dog underwent baseline assessment in a conscious state. CHF (20% to 30% reduction in dP/dt(max) and LV end-diastolic pressure >16 mm Hg) was created by daily coronary microembolizations via a continuously implanted coronary catheter. Skeletal muscle biopsy was performed and myoblasts were isolated and expanded. Then 2.7 x 10(8) to 8.3 x 10(8) myoblasts were injected into the infarcted region of 11 dogs after establishment of CHF. Saline injection (sham) was performed in 8 control dogs. Animals were evaluated every 2 weeks for up to 10 weeks. Global ejection fraction was determined by echocardiography. The end-systolic pressure-end-systolic volume relationship (ESPVR) was analyzed by the Sonomicrometic system. RESULTS: Compared with saline injection, ASMT significantly increased dP/dt(max) (105 +/- 9% vs 97 +/- 7%, values were expressed as percentage change from baseline CHF, p = 0.013) and ejection fraction (46 +/- 3% vs 40 +/- 2%, p = 0.034) at 10 weeks after myoblast transplantation. There was a significant leftward and upward shift of the ESPVR back toward normal at 10 weeks after myoblast transplantation (p = 0.034). Three animals labeled with BrdU myoblasts showed no histologic evidence of viable engraftment. CONCLUSIONS: ASMT provided mild improvements in hemodynamics and LV function and reduced LV remodeling in conscious dogs with CHF.

Influence of peroxynitrite on energy metabolism and cardiac function in a rat ischemia-reperfusion model.

Ischemia-reperfusion generates peroxynitrite (ONOO-), which interacts with many of the systems altered by ischemia-reperfusion. This study examines the influence of endogenously produced ONOO- on cardiac metabolism and function. Nitro-L-arginine (an inhibitor of ONOO- biosynthesis) and urate (a scavenger of ONOO-) were utilized to investigate potential pathophysiological roles for ONOO- in a rat Langendorff heart model perfused with glucose-containing saline at constant pressure and exposed to 30 min of ischemia followed by 60 min of reperfusion. In this model, ischemia-reperfusion decreased contractile function (e.g., left ventricular developed pressure), cardiac work (rate-pressure product), efficiency of O2 utilization, membrane-bound creatine kinase activity, and NMR-detectable ATP and creatine phosphate without significantly altering the recovery of coronary flow, heart rate, lactate release, and muscle pH. Treatment with urate and nitro-L-arginine produced a substantial recovery of left ventricular developed pressure, rate-pressure product, efficiency of O2 utilization, creatine kinase activity, and NMR-detectable creatine phosphate and a partial recovery of ATP. The pattern of effects observed in this study and in previously published work with similar models suggests that ONOO- may alter key steps in the efficiency of mitochondrial high-energy phosphate generation.