Project description:Epstein-Barr virus (EBV)-based episomal vector system enables persistent transgene expression, which is advantageous for efficient derivation of transgene-free induced pluripotent stem cells (iPSCs) without viral transduction. Here, we report establishment of an iPSC line from somatic fibroblasts of a neonatal common marmoset monkey (marmoset; Callithrix jacchus) using an all-in-one episomal vector that we newly developed. The established iPSC line, named NM-iPS, showed standard characteristics of pluripotency such as pluripotency-related marker expression, three germ layer differentiation, and normal karyotype (2n = 46). The novel iPSC line would be a useful resource for stem cell research using non-human primates.
Project description:The analysis compares primary fibroblasts initially used for reprogramming, established marmoset ES cells and a marmoset iPS cell line which was generated witha non-viral approach using a six-factor-in-one-vector approach
Project description:The analysis compares primary fibroblasts initially used for reprogramming, established marmoset ES cells and a marmoset iPS cell line which was generated witha non-viral approach using a six-factor-in-one-vector approach Cells were grown under standard conditions for marmoset pluripotent stem cells and primary fibroblasts without additional treatment. For each cell type at least four independent cell preparations were used.
Project description:We report the liver progenitor cell related gene expression of established marmoset liver progenitor cell line and provide the whole gene expression transcriptomes of marmoset liver.
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.
Project description:Here we describe an episomal, one-vector system which allows the generation of cell populations displaying homogenous, inducible gene inactivation by RNA interference in a one step procedure. A dual tet-repressor/activator system tightly controls a bi-directional promoter, which simultaneously drives expression of microRNAs and a fluorescent marker protein. We demonstrate the effectiveness of this vector by knockdown of p53 expression in a human cell line which resulted in the expected loss of G1-arrest after DNA damage. The generation of a cell pool homogenously expressing the ectopic microRNAs was achieved in 1 week without the need for viral infections. Induction of microRNA expression did not elicit an interferon response. Furthermore, the vector was adapted for convenient ligation-free transfer of microRNA cassettes from public libraries. This conditional knockdown-system should prove useful for many research and gene therapeutic applications.
Project description:Extended pluripotent stem cells (EPSCs) derived from mice and humans showed an enhanced potential for chimeric formation. By exploiting transcriptomic techniques, we assessed the differences in gene expression profile between extended EPSCs derived from mice and humans, and those newly derived from the common marmoset (marmoset; Callithrix jacchus). Although the marmoset EPSC-like cells displayed a unique colony morphology distinct from murine and human EPSCs, they displayed a pluripotent state akin to embryonic stem cells (ESCs), as confirmed by gene expression and immunocytochemical analyses of pluripotency markers and three-germ-layer differentiation assay. Importantly, the marmoset EPSC-like cells showed interspecies chimeric contribution to mouse embryos, such as E6.5 blastocysts in vitro and E8.5 epiblasts in vivo in mouse development. Also, we discovered that the perturbation of gene expression of the marmoset EPSC-like cells from the original ESCs resembled that of human EPSCs. Thus, we established the efficacy of the method for the derivation of marmoset EPSCs
Project description:Analysis of the episomal backbone's influence on gene expression. The first hypothesis tested in the present study is that the episomal EBNA vectors, which rely on the EBNA-1 oncoprotein for episomal maintenance, have a greater influence on the cells' expression profiles than S/MAR vectors. The second hypothesis tested was that when bacterial sequences are removed from the episomal vector backbone, the gene disturbance is minimal.