Introduction: Exogenesis is a promising strategy for generating humanized organs by replacing specific cell lineages with human cells in genetically engineered pig embryos that harbor organogenesis-disabled niches. Embryo complementation with patient-specific stem cells has the potential to produce transplantable xeno-organs while further reducing immunological and physiological barriers. However, achieving high integration of human cells into developing organs in evolutionarily distant species remains a major challenge. Insufficient chimerism is often due to a developmental mismatch, causing stem cells to be competitively eliminated from the blastocyst or tissue niche. In order to improve interspecies chimerism, we employed human expanded pluripotent stem cells (EPSCs) alongside refined injection and in vitro culture protocols for porcine embryos.
Methods: Here, we applied four modified expanded potential culture conditions (EPSCM, EPSCM-1, EPSCM-2 and EPSCM-8C) that enable conversion of human primed induced pluripotent stem cells (hiPSCs) to earlier developmental states, resembling the morula and 8-cell embryo stages. We assessed the expression of pluripotency- and totipotency-associated genes in these lines and compared their chimeric competency in parthenogenetic blastocysts. The development of interspecies chimeric embryos was monitored over two days using the Primo Vision system. Stem cell survival, proliferation, and integration in porcine embryos were confirmed through immunofluorescence imaging.
Results: Human primed iPSCs converted into stable EPSC lines exhibited a consistent dome-shaped morphology over more than 20 passages and showed upregulation of pluripotency markers (SOX2, OCT4, NANOG, KLF17, DPPA3 and DPPA5) and totipotency-associated genes (TPRX1, MAEL, DUXA and DUXB). Normal porcine blastocyst formation was observed after the injection of ten GFP-labeled human PSCs derived from four modified EPSC conditions, resulting in a 94% recovery rate after two days. Quantification of GFP-positive human cells per blastocyst revealed that cells cultured in EPSCM-2 conditions displayed the highest chimeric competency, contributing 15-40 cells per embryo after two days. Expression of the cell-adhesion molecule E-cadherin confirmed that the injected hPSCs formed functional adherens junctions with cells of the host blastocyst. The viability test of the injected cells within chimeric blastocysts, after PARP and CASPASE staining revealed that only a few human PSCs underwent apoptosis.
Conclusion: We demonstrated that hEPSCs, owing to their enhanced pluripotent properties and differentiation potential, represent promising donor cells for the generation of interspecies chimeras. Our long-term goal is to produce functional, rejection-free humanized pig hearts using genetically modified Auckland xeno-embryos for preclinical studies and, ultimately, clinical transplantation trials.