It is demonstrated that porcine expanded potential stem cells, derived from preimplantation embryos, provide a robust and versatile platform for generating donor cells for xenotransplantation and for functionally evaluating genetic modifications, thereby advancing the prospects of xenotransplantation.
Abstract
Xenotransplantation utilizing pig donors offers a promising solution to organ shortages, but immune incompatibilities across species remain a major challenge. Current reliance on porcine primary fibroblasts for genome editing is limited by low inefficiency in complex gene editing and difficulties in immunophenotyping edited cells. In this study, we demonstrate that porcine expanded potential stem cells (pEPSCs), derived from preimplantation embryos, provide a robust and versatile platform for generating donor cells for xenotransplantation. These pluripotent cells can differentiate into both embryonic and extraembryonic lineages, maintain genetic stability through multiple edits, and enable precise genome modifications. We performed multiple gene knockouts in pEPSCs targeting key immune rejection genes and precisely inserted a genetic cassette to facilitate streamlined introduction of human cDNAs via cassette exchange. The engineered pEPSCs retained their pluripotency and stability even after multiple rounds of genome editing. When differentiated into endothelial cells, they exhibited high immunogenicity, serving as a rapid and quantitative platform for immune response assessment. Importantly, the edited endothelial cells exhibited substantially reduced immunogenicity, confirming the functional impact of the genetic modifications. Although we have not yet generated live pigs from these gene‐edited pEPSCs via somatic cell nuclear transfer (SCNT), our findings establish pEPSCs as a novel and improved platform for generating genetically engineered pig donors and for functionally evaluating genetic modifications, thereby advancing the prospects of xenotransplantation.
The persistent shortage of human organs for transplantation has intensified efforts to develop alternative sources, specifically xenotransplantation and exogenesis. Xenotransplantation uses genetically engineered pigs to provide organs, tissues, and cells for clinical use. Significant progress has occurred in developing multigene-modified pigs that lack glycan xenoantigens while expressing human complement and coagulation regulators. These modifications have successfully mitigated hyperacute, antibody-mediated, and cellular rejection in preclinical nonhuman primate models. Recent compassionate-use cases in humans have demonstrated the feasibility of heart, kidney, and liver xenotransplantation, although achieving long-term survival remains a challenge. Complementing this approach, exogenesis aims to generate human-compatible organs within animal hosts through interspecies chimerism. Although advances in establishing organ niches and overcoming xeno-barriers have yielded preliminary success in heart, pancreas, and muscle development, formidable immune and developmental hurdles remain. Together, these approaches offer promising strategies to expand the donor organ pool and address the growing global demand for transplantation. Further advances in genetic engineering, immune modulation, and developmental biology, supported by rigorous preclinical and clinical evaluation, will be critical for widespread translation. This review outlines the current progress, major challenges, and future directions in xenogeneic and exogenic organ generation.
Asghar Ali, M. Kurome, Daniel Reichart et al.· Transplantation· 0 citations
Hematopoietic stem and progenitor cells (HSPCs) gene therapy may transform the therapeutic landscape for inherited hematological disorders and already offers potentially curative options for some diseases, including inborn errors of immunity and β-hemoglobinopathies. Its continued success relies on further refinement of gene transfer technologies, gene editing tools such as CRISPR-Cas, and optimized ex vivo HSPC manipulation protocols that ensure robust, long-term engraftment and clonal diversity with reduced-toxicity, non-genotoxic conditioning strategies. Here, we review recent developments and refinements in gene transfer and editing technologies for HSPCs, while also discussing the critical limitations and hurdles to clinical translation, as recently presented at the New Investigator Committee Gene Therapy webinar. Future directions must prioritize integrating technological innovation with the development of equitable and simplified models to reduce costs and ensure that these life-saving cellular therapies reach patients worldwide. Teaser abstract Hematopoietic stem and progenitor cells (HSPCs) gene therapy is advancing rapidly, with lentiviral gene transfer, genome editing, and emerging in vivo delivery approaches expanding the therapeutic landscape for inherited hematologic disorders. Recent clinical successes have demonstrated the potential for durable correction, while ongoing refinements continue to improve safety, efficacy, and feasibility. Key challenges remain in genotoxicity, conditioning toxicity, manufacturing scalability, and equitable global access.
Kohei Shiroshita, A. Stolz, C. Malouf et al.· Experimental Hematology· 0 citations
Evidence that cell-type-specific promoters lose fidelity when paired with neurogenic transgene payloads and that published in vivo reprogramming efficiencies may be substantially confounded by promoter leakage in the absence of formal lineage tracing are synthesized.
Mariam Abdelnaby, A. Galiakberova, E. Dashinimaev· International Journal of Mol...· 0 citations
This review discusses how organoids and OoC-based platforms are being leveraged to study and enhance cell fate reprogramming, repair, and regeneration across multiple tissues and outlines current limitations, including scalability, standardization, and biomaterial constraints.
Hrithiha Sriramulu, Hyunsung Woo, Anavi Kaul et al.· Current Opinion in Genetics...· 1 citation
A new method for surgically removing training examples from a model reveals that as datasets grow, the link between what a model learns and what it produces dissolves.
