Controlling the Switch from Neurogenesis to Pluripotency during Marmoset Monkey Somatic Cell Reprogramming with Self-Replicating mRNAs and Small Molecules

Abstract

Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of cell-based therapies; 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. 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 fibroblasts with VEE-mRNAs carrying the human OCT4, KLF4, SOX2, and c-MYC and culture in the presence of small molecule inhibitors CHIR99021 and SB431542, 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. Our experiments revealed a novel paradigm for flexible reprogramming of somatic cells, where primary colonies obtained by a single VEE-mRNA transfection can be directed either toward the neural lineage or further reprogrammed to pluripotency. These results (1) will further enhance the role of the common marmoset as animal disease model for preclinical testing of iPSC-based therapies and (2) establish an in vitro system to experimentally address developmental signal transduction pathways in primates.

Generation of marmoset primordial germ cell–like cells under chemically defined conditions

“…WNT inhibition is required to maintain cyESCs and cjiPSCs on MEFs ( Sakai et al, 2020 ; Seita et al, 2023 ). In our hands, the combination of CHIR and IWR-1 contributes to the stabilisation of NHP cell lines, which are per se more unstable in feeder-free culture than their human counterparts ( Petkov et al, 2020 ; Stauske et al, 2020 ). Consistently, CHIR/IWR-1 can act synergistically to promote self-renewal of human ESCs and mouse epiblast stem cells by first activating (CHIR) and then stabilising (IWR-1) β-catenin in the cytoplasm ( Kim et al, 2013 ).…”

“…To generate cjPGCLCs from cjiPSCs, we tested whether cjiPSCs (DPZ_cjiPSC#2, DPZ_cjiPSC#3, and DPZ_cjiPSC#5) are competent to be directly induced into cjPGCLCs. CjiPSCs were adapted to a modified UPPS medium containing forskolin, LIF, and a low concentration of ActA, which improves the stability of cjiPSCs ( Petkov et al, 2020 ). Dissociated cjiPSCs were reaggregated in ultra-low attachment wells to allow the formation of EBs in the presence of BMP4, EGF, SCF, and LIF.…”

“…Marmoset foetal fibroblasts were extracted from foetuses at day 70–74 of gestation used in an unrelated project ( Wolff et al, 2019 ) licensed by the Lower Saxony’s State Office of Consumer Protection and Food Safety (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit; LAVES; license number 42502-04-16/2129). These fibroblasts were previously also used for the generation of iPSCs in another study ( Petkov et al, 2020 ). Neonatal skin fibroblasts were extracted from one male and one female animal.…”

“…For the generation of PGCLCs, cjiPSCs were transferred to modified UPPS medium ( Petkov et al, 2020 ) as previously described. Briefly, cjiPSCs were cultured in StemMACS iPS-Brew XF medium (130-104-368; Miltenyi Biotec) supplemented with 1 μM IWR-1 (I0161-5MG; Sigma-Aldrich), 0.5 μM CHIR (Stem Cell Institute (SCI)), 10 μM forskolin (S2449; Selleckchem), and 1 ng/ml ActA (120-14P; PeproTech).…”

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

“…NOTE: Due to its robustness, the method presented here can be applied to any primate iPSC line. In this article, we describe cerebral organoid production from human (iLonza2.2) 29 , chimpanzee (Sandra A) 30 , rhesus macaque (iRh33.1) 29 , and common marmoset (cj_160419_5) 31 iPSC lines. The culture conditions are summarized in Table 1 .…”