| Induced pluripotent stem cells ( iPSCs ) are adult cells that have been genetically reprogrammed to an embryonic stem cell–like state by being forced to express genes and factors for maintaining the properties of embryonic stem cells. iPSCs have the ability for self-renewal and differentiation into different kinds of cell types. Although additional research is needed, iPSCs are already an useful tool for drug development and modeling of diseases, hoping to use them in transplantation medicine. iPSCs hold great promise for regenerative medicine. For the application of iPSCs to forms of autologous cell therapy, suitable animal models are required. Potentially, any mammalian species could serve as a translational model for this kind of autologous cell therapy, but nonhuman primates are particularly suited for such studies because of the aspects of their anatomy and physiology that they share with humans. Among nonhuman primates, the marmoset is especially relevant for biomedical research because of well developed models in this species for neurodegenerative disorders (Parkinson's, Alzheimer's and Huntington's disease). Forms of experimental cell therapy have been developed for models of neural injury in the marmoset and the marmoset has been proposed as an ideal model for elucidation of safety and efficiency of new technologies in regenerative medicine.iPSCs have been derived from somatic cells of mouse, human, rhesus monkey, rat, pig, dog and rabbit. It's another milestone in the history of stem cell development. But there is no report about marmoset iPSCs. iPSCs is still in their infancy, there are some problems to be solved, for example, low efficiency of reprogramming. So far, people focused on reprogramming or inducing methods, but there has been little work on optimizing conditions for coinfection with retroviral vectors.In this study, we opimized the methods for cell infection with higher concentration polybrene (Flocculation) combine with spinoculation by infecting human (HPF) and mouse (3T3) fibroblast. In addition, we derived marmoset iPSCs using the optimized retroviral transduction with human Oct4, Sox2, Klf4 and c-Myc. Meanwhile, we established the feeder free condition of marmoset iPSCs, also Pho-kinase Y27632 promote the survival and proliferation of dissociated marmoset iPSCs.1. In this study, we used amphotropic retroviral vectors produced by the Plat-A cell line to investigate coinfection efficiency (defined as cells that are both red and green as a percentage of all cells infected) using green and red fluorescent proteins (pLEGFP-N1 and pMXs-dsRed2). Primary human fibroblasts and 3T3 cells were used as target cells. We tested the effect of spinoculation, concentration by centrifugation at 10,000g or by flocculation using Polybrene, concentration by Polybrene flocculation and optimized spinoculation on the coinfection rates. The results showed that unconcentrated vector preparations produced a coinfection rate of 4.0±0.1% (PHF) and 3.4±0.2% (3T3). Combining the two processes, concentration by Polybrene flocculation and optimized spinoculation increased the coinfection rate to 53.0±0.6% (PHF) and 35.0±0.4% (3T3) separately.2. In this investigation, we produced marmoset iPSCs using optimized retroviral transduction with human Oct4, Sox2, Klf4 and c-Myc. After transduction, we treated cells with 1mM Valproic acid (VPA) for 12 days. Starting with a population of 4×105 MF, we obtained around 100 colonies with iPSCs-like morphology. Of those, 30 were expanded sufficiently to be cryopreserved. From those, 8 were characterized in more detail. All the 8 clones fulfil the critical criteria for successful reprogramming: they exhibit typical iPSCs morphology; they are alkaline phosphatase positive; they express high levels of Nanog, Oct4 and Sox2 mRNAs, while the corresponding vector genes are silenced; they are immunoreactive for Oct4, TRA-1-81 and SSEA-4; and when implanted into immunodeficient mice (RAG2-/-, c-/-) these clones produce teratomas which have derivatives of all three germ layers (endoderm,α-fetoprotein; ectoderm,βIII-tubulin; mesoderm, smooth muscle actin). These experiments provide the evident that iPSCs technology can be adapted for use in the marmoset, as a future model of autologous cell therapy.3. In this study, we investigated the effect of condition medium produced from MEF and STO on marmoset iPSCs, feeder free condition can support marmoset iPSCs. meanwhile, we compared effect of different concentration of FBS (10%, 15%, 20%), condition medium (35% and 17.5%) and different concentration of bFGF (20 ng/ml,100 ng/ml and 150 ng/ml) on iPSCs. The results showed that mamoset iPSCs under different feeder free condition expressed the Oct4, Sox2, Nanog at the similar levels that were comparable to that under MEF feeder condition. They are alkaline phosphatase positive, they are immunoreactive for Oct4, TRA-1-81 and SSEA-4; and when implanted into immunodeficient mice (RAG2-/-, c-/-) they produce teratomas that have derivatives of all three germ layers; Also, they differentiated to neurons in vitro.4. In this study, we investigated the effect of Y27632 on marmoset iPSCs. Y27632 significantly improved the efficiency of colony formation from single cells without changing pluripotent state and karyotype in feeder free culture for miPSCs. Dramatically increased cloning efficiency from 5.0±0.4 % to 33.0±6.7 %. In vitro, miPSCs gave rise to neurons. Measurement of proliferation by means of BrdU (bromodeoxyuridine) incoporation revealed that Y27632 promote miPSCs proliferation. |
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