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Hypoimmunogenic hPSC-derived cardiac organoids for immune evasion and heart repair [bulkRNA-seq]

ABSTRACT: Human pluripotent stem cell (hPSC)-derived cardiac therapies hold great promise for heart 41 regeneration but face major translational barriers due to allogeneic immune rejection. Here, we 42 engineered hypoimmunogenic hPSCs using a two-step CRISPR-Cas9 strategy: (1) B2M 43 knockout, eliminating HLA class I surface expression, and (2) knock-in of HLA-E or HLA-G trimer 44 constructs in the AAVS1 safe harbor locus to confer robust immune evasion. Hypoimmunogenic 45 hPSCs maintained pluripotency, efficiently differentiated into cardiac cell types that resisted both 46 T and NK cell-mediated cytotoxicity in vitro, and self-assembled into engineered cardiac 47 organoids. Comprehensive analyses of the hypoimmunogenic cells and organoids revealed 48 preservation of transcriptomic, structural, and functional properties with minimal off-target effects 49 from gene editing. In vivo, hypoimmunogenic cardiac organoids restored contractile function in 50 infarcted rat hearts and demonstrated superior graft retention and immune evasion in humanized 51 mice compared to wild-type counterparts. These findings establish the therapeutic potential of 52 hypoimmunogenic hPSC-CMs in the cardiac organoid platform, laying the foundation for off-the shelf cardiac cell therapies to treat cardiovascular disease, the leading cause of death worldwide.

ORGANISM(S): Homo sapiens

PROVIDER: GSE299171 | GEO | 2025/06/08

REPOSITORIES: GEO

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Project description:Genetically engineered human pluripotent stem cells (hPSCs) have been proposed as a source for transplantation therapies and are rapidly becoming valuable tools for human disease modeling. However, many of the potential applications are still limited by the lack of robust differentiation paradigms that allow for the isolation of defined functional tissues. These challenges could be overcome by the use of adult tissue stem cells derived from hPSCs, as their restricted potential could limit the differentiation towards other undesired linages, and allow in vitro expansion and long- term propagation of fully differentiated tissue. To isolate adult stem cells from hPSCs, we applied genome-editing to generate an LGR5-GFP reporter system and subsequently developed a differentiation protocol for human intestinal tissue comprising an adult stem cell niche and all major cell types of the adult intestine. This novel derivation protocol is highly robust and even permits the isolation of intestinal organoids without the LGR5 reporter. Transcriptional profiling, electron microscopy and functional analysis revealed that such human organoid cultures could be derived with high purity, and a composition and morphology similar to that of cultures obtained from human biopsies. Importantly, hPSC-derived organoids responded to the canonical signaling pathways that control self-renewal and differentiation in the adult human intestinal stem cell compartment. With our ability to genetically engineer hPSCs using site-specific nucleases, this adult stem cell system provides a novel platform by which to study human intestinal disease in vitro. RNA from primary organoid samples was isolated from organoid lines that were both cultured for 1-6 months and derived from duodenum, ileum, or rectum biopsies of human subjects as described previously (Sato et al., Gastroenterology 2011) grown in media called WENR+inhibitors. RNA was also isolated from various steps in the culturing and differentiation protocol.

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Hypoimmunogenic hPSC-derived cardiac organoids for immune evasion and heart repair [bulkRNA-seq]

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