Safety and efficacy of intracoronary artery administration of human bone marrow-derived mesenchymal stem cells in STEMI of Lee-Sung pigs—A preclinical study for supporting the feasibility of the OmniMSC-AMI phase I clinical trial

BackgroundThis study tested whether early left intracoronary arterial (LAD) administration of human bone marrow-derived mesenchymal stem cells (hBMMSCs, called OmniMSCs) in acute ST-segment elevation myocardial infarction (STEMI) of Lee-Sung pigs induced by 90 min balloon-occluded LAD was safe and effective.Methods and resultsYoung male Lee-Sung pigs were categorized into SC (sham-operated control, n = 3), AMI-B (STEMI + buffer/21 cc/administered at 90 min after STEMI, n = 6), and AMI-M [acute myocardial infarction (AMI) + hBMMSCs/1.5 × 107/administered at 90 min after STEMI, n = 6] groups. By 2 and 5 months after STEMI, the cardiac magnetic resonance imaging demonstrated that the muscle scar score (MSS) and abnormal cardiac muscle exercise score in the infarct region were significantly increased in the AMI-B than in the SC group that were significantly reversed in the AMI-M group, whereas the left ventricular ejection function by each month (from 1 to 5) displayed an opposite pattern of MSS among the groups (all p < 0.001). By 5 months, histopathological findings of infarct and fibrosis areas and isolectin-B4 exhibited an identical pattern, whereas the cellular expressions of troponin-I/troponin-T/von Willebrand factor exhibited an opposite pattern of MSS among the groups (all p < 0.001). The ST-segment resolution (>80%) was significantly earlier (estimated after 6-h AMI) in the AMI-M group than in the AMI-B group (p < 0.001). The protein expressions of inflammation (IL-1β/TNF-α/NF-κB)/oxidative stress (NOX-1/NOX-2/oxidized protein)/apoptosis (cleaved caspase-3/cleaved PARP)/DNA damage (γ-H2AX) displayed an identical pattern to MSS among the groups, whereas the protein expressions of angiogenesis factors (SDF-1α/VEGF) were significantly and progressively increased from SC, AMI-B, to AMI-M groups (all p < 0.001).ConclusionEarly intra-LAD transfusion of OmniMSC treatment effectively reduced the infarct size and preserved LV function in porcine STEMI

CBARA1 plays a role in stemness and proliferation of human embryonic stem cells.

Human embryonic stem cells (hESCs) are capable of unlimited self-renewal and can generate almost all of the cells in the body. Although some pluripotency factors have been identified, much remains unclear regarding the molecules and mechanisms that regulate hESC self-renewal and pluripotency. In this study, we identified a mitochondrial gene, CBARA1, that is expressed in undifferentiated hESCs and that is down-regulated rapidly after cellular differentiation. To study its role in hESCs, endogenous CBARA1 expression was knocked down using shRNA. CBARA1 knockdown in hESCs resulted in down-regulation of Oct4 and Nanog expression, attenuated cell growth, and G0/G1 phase cell cycle arrest; however, knockdown did not noticeably affect apoptosis. Taken together, these results suggest that CBARA1 is a marker for undifferentiated hESCs that plays a role in maintaining stemness, cell cycle progression, and proliferation

Expression of CBARA1 in hESCs following spontaneous differentiation.

<p>(A–C) Flow cytometry shows the percentage and mean fluorescent intensity (in brackets) of CBARA1- or Oct4-positive cells in undifferentiated, 3-day, or 7-day differentiated cells. (D–E) Representative images showing CBARA1 and Oct4 immunostaining in undifferentiated and in 3- or 7-day spontaneously differentiated cells. One graph or image that was representative of three independent experiments is shown for the TW1 (A, D), HES3 (B, E), and TW5 (C, F) hESC lines. Scale bar = 100 µm.</p

Effect of CBARA1 on hESC proliferation and apoptosis.

<p>(A) Graphs show a reduced cell proliferation in CBARA1 shRNA vector-transduced hES3 cells for up to 5 days after puromycin selection compared to the non-target shRNA control. Statistical analysis was performed using the Student’s t-test. *Difference compared to non-target shRNA control (<i>P</i><0.01). (B) Cell cycle analysis shows the cell cycle distribution of non-target shRNA vector-transduced, CBARA1 shRNA vector-transduced, and non-transduced hESCs at the G0/G1, S, and G2/M phases. One graph is shown representative of two independent experiments. The percentage of cells in each phase of the cell cycle was calculated using MODFIT-LT software. (C) Annexin V/PI flow cytometry analysis showing the percentage of early apoptotic cells (Annexin V+/PI- in the lower right quadrant) in CBARA1 shRNA-treated, non-target shRNA-treated, non-transduced, and unstained control hESCs. One graph is shown that is representative of two independent experiments. Abbreviations: PI, propidium iodide.</p

CBARA1 is a marker for undifferentiated hESCs.

<p>(A) Quantitative PCR was used to determine CBARA1 and Oct4 mRNA levels in undifferentiated TW1 hESCs and noggin-induced differentiated cells. Gene expression was normalized to GAPDH expression and is presented as the fold-change compared to expression in undifferentiated hESCs. The bars show the means ± standard deviations of three independent experiments. *Significant difference compared to undifferentiated hESCs (<i>P</i><0.01). (B) Western blot showing CBARA1 protein levels in undifferentiated and 7-day differentiated hESCs. α-tubulin was used as a loading control. The relative reduction of CBARA1 in 7-day differentiated cells was quantified by densitometry as shown under the blots. (C) Representative images show CBARA1 and Oct4 immunostaining in undifferentiated or 7-day differentiated hESCs. Abbreviations: Undiff, undifferentiated hESC; Diff, differentiated hESC. Scale bar = 50 µm.</p

Effect of CBARA1 on hESC stemness.

<p>(A) Representative phase-contrast images show CBARA1 shRNA vector-transduced, non-target shRNA vector-transduced, and non-transduced hES3 cells. (B–C) Quantitative PCR and flow cytometry show reduced CBARA1 and Oct4 mRNA and protein expression in CBARA1 shRNA vector-transduced hESCs compared to non-target shRNA vector-transduced and non-transduced controls. Gene expression was normalized to that of GAPDH and is presented as fold-change compared to the non-target shRNA control. Bars represent the means ± standard deviations of three independent experiments. *Difference compared to non-target shRNA control (<i>P</i><0.05). **Difference compared to non-target shRNA control (<i>P</i><0.01) (D) Representative images show CBARA1 and Oct4 immunostaining in non-target shRNA vector-transduced, CBARA1 shRNA vector-transduced, and non-transduced hESCs. Regions of cell differentiation are indicated with arrows. (E) RT-PCR shows the expression of specific markers for the three germ layers in non-transduced hESCs and in hESCs transduced with CBARA1 shRNA or non-target shRNA vectors. Lane 1, HES3 non-target shRNA; lane2, HES3 CBARA1 shRNA; lane 3, H9 non-target shRNA; lane4, H9 CBARA1 shRNA; lane5, negative control. Scale bar = 100 µm.</p