ERK-independent African Green monkey pluripotent stem cells in a putative chimera-competent state

Generating human organs inside interspecies chimeras might one day produce patient-specific organs for clinical applications, but further advances in identifying human chimera-competent pluripotent stem (PS) cells are needed. Moreover, the potential for human PS cells to contribute to the brains in human-animal chimeras raises ethical questions. The use of non-human primate (NHP) chimera-competent PS cells would allow one to test interspecies organogenesis strategies while also bypassing such ethical concerns. Here, we provide the first evidence for a putative chimera-competent pluripotent state in NHPs. Using histone deacetylase (HDAC) and selective kinase inhibition, we converted the PS cells of an Old World monkey, the African Green monkey (aGM), to an ERK-independent cellular state that can be propagated in culture conditions similar to those that sustain chimera-competency in rodent cells. The obtained stem cell lines indefinitely self-renew in MEK inhibitor-containing culture media lacking serum replacement and FGF. Compared to conventional PS cells, the novel stem cells express elevated levels of KLF4, exhibit more intense nuclear staining for TFE3, and manifest increased mitochondrial membrane depolarization. These data are preliminary but indicate that the key to deriving primate chimera-competent PS cells is to shield cells from the activation of ERK, PKC, and WNT signaling. Because of the similarity of aGMs to humans, the more ethically palatable use of NHP cells, and the more similar gestation length between aGMs and large animals such as sheep, the aGM cell lines described herein will serve as a useful tool for evaluating the efficacy and safety of interspecies organogenesis strategies. Future studies will examine chimera-competency and generalizability to human cells.