Abstract
Our team has previously shown the isolation and culture of a mesenchymal cell population from the right atrial appendage (RAA) of living human donor tissues. The cells are CD44/105/166+, CD45/19/11b- and secrete growth factors (including VEGF and IGF-1) comparable to BM-MSCs. Their secretome preserved EF% and dP/dTmax in a mouse MI model and stimulated angiogenesis in a mouse limb ischemia model.
In the current study, we specifically investigated using these cell-derived extracellular vesicles (EV) as a therapeutic. First, we validated our isolation technique and profiled EV miRNA cargo using PCR arrays. We then assessed the ability of EVs to protect human cardiomyocytes in a hypoxic (1% O2) hiPSC-CM model. Lastly, we used RNA-seq to compare the transcriptome of EV-treated hiPSC-CMs against hypoxic and normoxic controls.
Results confirmed isolation of high purity EVs, showing (by cryoEM) ~120 nm bilayer membrane-bound vesicles, positive for CD81, CD63, ALIX, TSG101 and other EV markers (by antibody array). In the EV cargo we identified 363 known miRNAs present in 3 donor cell isolates, including hsa-miR-21-5p, -202-5p, -1260a and -125b. Target prediction of the most abundant miRNAs ranked GO:0010667 “negative regulation of cardiac muscle cell apoptotic process” as highly significant (26.3%, P = 1.6 x10-8), as well as other predicted pathways related to angiogenesis, cardiac development and regulation of inflammation.
A single, transient, treatment of donor EVs (66 ng EV protein/μl) reduced LDH leakage from hypoxic hiPSC-CMs, preserved morphology, and increased viability at 48 hrs and 7 days after injury. RNA-seq of the EV-treated hiPSC-CMs revealed strong upregulation of pathways related to PI3k-akt, calcium signaling and ECM binding compared to hypoxic hiPSC-CMs with vehicle control. We are currently validating key RNA-seq results.
In conclusion, we analyzed and confirmed cardioprotective properties of EVs derived from human RAA samples using a human model system. Combined with our published animal studies, these data indicate that this cell population is a clinically-relevant source of donor cells for cardiac cell therapy. The EVs are also an attractive cell-free potential therapeutic option.
In the current study, we specifically investigated using these cell-derived extracellular vesicles (EV) as a therapeutic. First, we validated our isolation technique and profiled EV miRNA cargo using PCR arrays. We then assessed the ability of EVs to protect human cardiomyocytes in a hypoxic (1% O2) hiPSC-CM model. Lastly, we used RNA-seq to compare the transcriptome of EV-treated hiPSC-CMs against hypoxic and normoxic controls.
Results confirmed isolation of high purity EVs, showing (by cryoEM) ~120 nm bilayer membrane-bound vesicles, positive for CD81, CD63, ALIX, TSG101 and other EV markers (by antibody array). In the EV cargo we identified 363 known miRNAs present in 3 donor cell isolates, including hsa-miR-21-5p, -202-5p, -1260a and -125b. Target prediction of the most abundant miRNAs ranked GO:0010667 “negative regulation of cardiac muscle cell apoptotic process” as highly significant (26.3%, P = 1.6 x10-8), as well as other predicted pathways related to angiogenesis, cardiac development and regulation of inflammation.
A single, transient, treatment of donor EVs (66 ng EV protein/μl) reduced LDH leakage from hypoxic hiPSC-CMs, preserved morphology, and increased viability at 48 hrs and 7 days after injury. RNA-seq of the EV-treated hiPSC-CMs revealed strong upregulation of pathways related to PI3k-akt, calcium signaling and ECM binding compared to hypoxic hiPSC-CMs with vehicle control. We are currently validating key RNA-seq results.
In conclusion, we analyzed and confirmed cardioprotective properties of EVs derived from human RAA samples using a human model system. Combined with our published animal studies, these data indicate that this cell population is a clinically-relevant source of donor cells for cardiac cell therapy. The EVs are also an attractive cell-free potential therapeutic option.
| Original language | English |
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| Publication status | Published - Oct 2023 |