R31 - Hypoimmunogenic hPSC-derived cardiac organoids evade host immune rejection and promote cardiac functional recovery

Introduction: : Cardiovascular disease (CVD) is the leading cause of death worldwide and bears an immense economic burden. CVD such as myocardial infarction (MI) and ischemia reperfusion (I/R) are associated with extensive cardiac cell death and restricted heart function. As adult heart tissue has limited regenerative capacity, human pluripotent stem cells (hPSCs) have emerged as a promising cell source for cardiac regenerative strategies due to their proven scalability and ability to differentiate into functional cardiac cells. hPSC-CMs have been shown to restore the contractile function of infarcted hearts in numerous models, including non-human primates. Furthermore, preliminary data released from ongoing clinical trials have indicated therapeutic potency of hPSC-CMs to treat heart failure patients. However, hPSC-derived cell-based therapies are limited in translational use due to the threat of host immune rejection of transplanted cells. Transplantations of allogenic cells require careful HLA-matching and the use of immunosuppressant treatments that put the patient at risk for adverse side effects. In recent years, the development of a single, universal hPSC line that can evade allogeneic rejection has emerged as a promising cell source for hPSC-derived cardiac regenerative therapies.

Materials and

Methods: : 19-9-11 hiPSCs (wild type (WT) hPSCs) were edited by our collaborator Dr. Xiaojun Lance Lian via genetic deletion of the B2M gene as well as knock-in with HLA-E fusion molecule (termed “B2M-/- HLA-E+ ‘UDET hPSCs’”). Four cell types from both WT and UDET hPSCs were differentiated and characterized based off previously established protocols: hPSC-derived cardiomyocytes (hPSC-CMs), cardiac fibroblasts (hPSC-cFbs), endothelial cells (hPSC-ECs), and pericytes (hPSC-PCs). NK and T cell cytotoxicity assays were performed following the co-culture of isolated allogeneic human NK or activated T cells with target WT and UDET hPSC-derived cells via LDH assays. WT and UDET cardiac organoids were fabricated by seeding hPSC-derived cells at a ratio of 70% hPSC-CMs: 10% hPSC-cFbs: 15% hPSC-ECs: 5% hPSC-PCs in low-attachment agarose molds to facilitate self-assembly. Bulk RNA sequencing and subsequent analysis was performed to compare WT and UDET hPSC-derived cardiac cells as well as WT and UDET engineered cardiac organoids. We next examined the in vivo immune response to cardiac organoids following injection of organoids under the kidney capsule of humanized NSG mice via kidney histology and flow cytometry of circulating T cells 14 days after organoid transplantation. Finally, WT and UDET cardiac organoids were injected at 1.5 x 106 total cells into an athymic rat model of acute cardiac I/R injury. Cardiac function of rats was characterized via echocardiography and explanted rat hearts were evaluated with histological and immunofluorescent staining.

Results, Conclusions, and Discussions:: Following prolonged culture (>30 passages), gene-edited B2M-/- HLA-E+ “UDET hPSCs” maintained pluripotency, a normal karyotype, and gene edits as characterized by flow cytometry. We verified that both WT and UDET hPSCs could be differentiated into hPSC-CMs, hPSC-cFbs, hPSC-ECs, and hPSC-PCs via their expression of characteristic markers of cTnT, vimentin, CD31, and NG2, respectively (Figure 1A-D). In vitro cytotoxicity assays revealed that UDET hPSC-derived cell types evaded both NK- and T cell-mediated cell lysis compared to their WT counterparts (Figure 1E-F). WT and UDET cardiac organoids similarly and robustly aggregated into spontaneously contracting spherical microtissues approximately 250µm in diameter (Figure 2A-D). Bulk RNA sequencing analyses revealed that WT and UDET hPSC-derived cardiac cell types and cardiac organoids display only subtle differences and are nearly transcriptomically identical, excluding variation in the expression of HLA-E, suggesting minimal off-target effects due to hPSC gene editing (Figure 2E). Furthermore, transcriptomic comparison of cardiac organoids to 2D hPSC-CMs as well as aggregate 2D cell transcriptomic data from all hPSC-derived cardiac cell types demonstrated that the microenvironment provided in 3D cardiac organoids enhances key biological processes involved in promoting graft survival and integration within the ischemic heart (Figure 2F-K). Following engraftment of WT and UDET cardiac organoids in a rat model of myocardial ischemia/reperfusion (I/R) injury, functional recovery as well as attenuation of maladaptive left ventricular remodeling was observed at similar extents in WT and UDET cardiac organoid-treated rats (Figure 3A-G). Notably, when transplanted under the kidney capsule in humanized NSG mice, hypoimmunogenic cardiac organoids demonstrated superior graft retention and immune evasion compared to WT organoids (Figure 3H-K).
In summary, we validated the ability of UDET-derived cells to evade immune cell-mediated lysis, showing that UDET-derived cells were significantly less vulnerable to immune cell killing. Furthermore, we demonstrated the similar capacity of wild type and UDET-derived cardiac organoids to improve I/R-injured rat heart function and structure compared to controls. Altogether, our findings underscore the potential of hypoimmunogenic hPSC-derived cardiac organoids as a clinically relevant approach for allogenic cardiac regenerative therapy, eliminating the need for immunosuppression while promoting effective cardiac recovery.

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