Abstract Body (Do not enter title and authors here): ABSTRACT
Background: It is well-known that cardiomyocytes (CMs) in adult mammalian hearts lose capacity to proliferate. Although cardiogenic potential of human induced-pluripotent stem-cells (hiPSCs) is well-recognized, hiPSCs cannot be administered directly due to their tumorigenicity. Acellular products that replicate the regenerative activity of hiPSCs may be more readily translated to the clinic. Here, we assessed the potency of hiPSC-derived nanovesicles (hiPSC-NVs) for cardiac regeneration.
Methods and Results: A hypo-immunogenic human induced pluripotent stem cell (hiPSC) line was created using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 gene editing to knockout β2-microglobulin in hiPSCs (^B2MKOhiPSCs) and used for manufacturing nanovesicles (^B2MKOhiPSC-NVs). Approximately 9,500 ^B2MKOhiPSC-NVs were produced from a single ^B2MKOhiPSC. Proteomic analyses indicated that, compared to ^B2MKOhiPSCs, the cargos of ^B2MKOhiPSC-NVs were enriched in spindle and chromosomal proteins, as well as proteins that regulate the cell cycle. When administrated to hiPSCs derived CMs (hiPSC-CMs), ^B2MKOhiPSC-NVs increased hiPSC-CM mitosis and cytokinesis via the YAP pathway, and were hypoimmunogenic when co-cultured with human CD8⁺T cells or delivered to C57BL/6 mice. Furthermore, when 0.9% NaCl or 0.9% NaCl containing ^B2MKOhiPSC-NVs was intramyocardially injected into C57BL/6 mice hearts after cardiac ischemia/reperfusion injury, cardiac function and infarct size, assessed 4 weeks later, were significantly improved in the ^B2MKOhiPSC-NV group, with increased mouse CM cell cycle activity. Thus, the proteins in the ^B2MKOhiPSC-NV cargos convergently upregulated YAP signaling to induce CM cell cycle activity.
Conclusions: The use of hiPSCs as parental cells for manufacturing NVs can easily scale up production and has great potential for cardiac regeneration.