Human-Mouse Chimerism for Organogenesis Without Neural or Germline Contributions

Human pluripotent cells, including embryonic stem cells (ESCs), have been used for interspecies blastocyst complementation, with the potential to generate human organs in animals for clinical applications. However, ethical concerns exist as human cells may contribute to the germline and central nervous system (CNS) in chimeric animals. To prevent such concerns, here we generated BLIMP1 and PAX6 knockout human ESCs (hESCs) that are capable of contributing to all cell and tissue types, except the germline and CNS in human-mouse chimeras. To overcome the low chimerism efficiency of hESCs, we also engineered BLIMP1^-/- /PAX6^-/- hESCs with a doxycycline-inducible system to express the anti-apoptotic gene BCL2 (iBCL2) and injected them into Igf1r^-/- mouse blastocysts, which remarkably enhanced the chimerism efficiency. To test organogenesis potential, mesenchymal stem cells were generated from BLIMP1^-/- /PAX6^-/- /iBCL2⁺ hESCs injected into Sox9^+/- mouse blastocysts, which rescued Sox9 haploinsufficiency skeletal defects and contributed to mesenchymal tissues of the chimeric fetuses and neonates. Thus, we provide a simple and effective approach to generating human-animal chimeras that are free of ethical concerns.