Controlling the switch from neurogenesis to pluripotency during marmoset monkey somatic cell reprogramming with self-replicating mRNAs and small molecules

Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of cell-based therapies for many currently incurable diseases. However, the safety and efficacy of potential iPSC-based treatments need to be verified in relevant animal disease models before their application in the clinic. Moreover, in order to reduce possible risks for the patients, it is necessary to use reprogramming approaches that ensure to the greatest extent possible the genomic integrity of the cells. Here, we report the derivation of iPSCs from common marmoset monkeys (Callithrix jacchus) using self-replicating mRNA vectors based on the Venezuelan equine encephalitis virus (VEE-mRNAs). By transfection of marmoset fetal fibroblasts with Tomato-modified VEE-mRNAs carrying the human OCT4, KLF4, SOX2, and c-MYC (VEE-OKS-iM-iTomato) and culture in medium supplemented with two small molecule inhibitors, we first established intermediate primary colonies with neural progenitor-like properties. In the second reprogramming step, we converted these colonies into transgene-free pluripotent stem cells by further culturing them with customized marmoset iPSC medium in feeder-free conditions. The resulting cell lines possess pluripotency characteristics, such as expression of various pluripotency markers, long-term self-renewal, stable karyotype, and ability to differentiate into derivatives of the three primary germ layers in vitro and in vivo. Our experiments reveal a novel paradigm for flexible reprogramming of somatic cells, where primary colonies obtained by a single VEE-mRNA transfection can be directed either towards the neural lineage or further reprogrammed to pluripotency. These results (i) will further enhance the role of the common marmoset as animal disease model for preclinical testing of iPSC-based therapies and (ii) establish an in vitro system to experimentally address developmental signal transduction pathways in primates.