Project description: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.
Project description:Induced pluripotent stem cells (iPSCs) give rise to neural stem/progenitor cells, serving as a good source for neural regeneration. Here, we established transgene-free (TF) iPSCs from dental stem cells (DSCs) and determined their capacity to differentiate into functional neurons in vitro. Generated TF iPSCs from stem cells of apical papilla and dental pulp stem cells underwent two methods-embryoid body-mediated and direct induction, to guide TF-DSC iPSCs along with H9 or H9 Syn-GFP (human embryonic stem cells) into functional neurons in vitro. Using the embryoid body-mediated method, early stage neural markers PAX6, SOX1, and nestin were detected by immunocytofluorescence or reverse transcription-real time polymerase chain reaction (RT-qPCR). At late stage of neural induction measured at Weeks 7 and 9, the expression levels of neuron-specific markers Nav1.6, Kv1.4, Kv4.2, synapsin, SNAP25, PSD95, GAD67, GAP43, and NSE varied between stem cells of apical papilla iPSCs and H9. For direct induction method, iPSCs were directly induced into neural stem/progenitor cells and guided to become neuron-like cells. The direct method, while simpler, showed cell detachment and death during the differentiation process. At early stage, PAX6, SOX1 and nestin were detected. At late stage of differentiation, all five genes tested, nestin, ?III-tubulin, neurofilament medium chain, GFAP, and Nav, were positive in many cells in cultures. Both differentiation methods led to neuron-like cells in cultures exhibiting sodium and potassium currents, action potential, or spontaneous excitatory postsynaptic potential. Thus, TF-DSC iPSCs are capable of undergoing guided neurogenic differentiation into functional neurons in vitro, thereby may serve as a cell source for neural regeneration.
Project description:After the first report of induced pluripotent stem cells (iPSCs), considerable efforts have been made to develop more efficient methods for generating iPSCs without foreign gene insertions. Here we show that Sendai virus vector, an RNA virus vector that carries no risk of integrating into the host genome, is a practical solution for the efficient generation of safer iPSCs. We improved the Sendai virus vectors by introducing temperature-sensitive mutations so that the vectors could be easily removed at nonpermissive temperatures. Using these vectors enabled the efficient production of viral/factor-free iPSCs from both human fibroblasts and CD34(+) cord blood cells. Temperature-shift treatment was more effective in eliminating remaining viral vector-related genes. The resulting iPSCs expressed human embryonic stem cell markers and exhibited pluripotency. We suggest that generation of transgene-free iPSCs from cord blood cells should be an important step in providing allogeneic iPSC-derived therapy in the future.
Project description:The common marmoset (marmoset; Callithrix jacchus) shows anatomical and physiological features that are in common with humans. Establishing induced pluripotent stem cells (iPSCs) from marmosets holds promise for enhancing the utility of the animal model for biomedical and preclinical studies. However, in spite of the presence of some previous reports on marmoset iPSCs, the reprogramming technology in marmosets is still under development. In particular, the efficacy of RNA-based reprogramming has not been thoroughly investigated. In this study, we attempted RNA-based reprogramming for deriving iPSCs from marmoset fibroblasts. Although we failed to derive iPSC colonies from marmoset fibroblasts by using a conventional RNA-based reprogramming method previously validated in human fibroblasts, we succeeded in deriving colony-forming cells with a modified induction medium supplemented with a novel set of small molecules. Importantly, following one-week culture of the colony-forming cells in conventional embryonic stem cell (ESC) medium, we obtained iPSCs which express endogenous pluripotent markers and show a differentiation potential into all three germ layers. Taken together, our results indicate that RNA-based reprogramming, which is valuable for deriving transgene-free iPSCs, is applicable to marmosets. Overall design: RNA seq of marmoset fibroblasts, ESCs and iPSCs
Project description:The common marmoset (marmoset; Callithrix jacchus) harbors various desired features as a non-human primate (NHP) model for neuroscience research. Recently, efforts have been made to induce neural cells in vitro from marmoset pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), which are characterized by their capacity to differentiate into all cell types from the three germ layers. Successful generation of marmoset neural cells is not only invaluable for understanding neural development and for modeling neurodegenerative and psychiatric disorders, but is also necessary for the phenotypic screening of genetically-modified marmosets. However, differences in the differentiation propensity among PSC lines hamper the applicability and the reproducibility of differentiation methods. To overcome this limitation, we evaluated the efficacy of small molecules for neural differentiation of marmoset ESCs (cjESCs) and iPSCs using multiple differentiation methods. By immunochemical and transcriptomic analyses, we confirmed that our methods using the small molecules are efficient for various differentiation protocols by either enhancing the yield of a mixture of neural cells including both neurons and glial cells, or a pure population of neurons. Collectively, our findings optimized in vitro neural differentiation methods for marmoset PSCs, which would ultimately help enhance the utility of the animal model in neuroscience. Overall design: RNA seq of marmoset ESCs, ESC-derived neural differentiated cells and cortex