MicroRNA profile analysis of sorted cells during different reprogramming stages
ABSTRACT: Mouse embryonic fibroblasts were reprogrammed using Oct4, Sox2, Klf4 and cMyc genes. At day 5, cells were sorted as Thy1 positive and Thy1 negative populations. microRNA expression profile from Thy1+ and Thy1- cells was compared with original MEFs (D0) to identify significantly changed microRNAs during initial stage of reprogramming
Project description:Mouse embryonic fibroblasts were reprogrammed using Oct4, Sox2, Klf4 and cMyc genes. At day 5, cells were sorted as Thy1 positive and Thy1 negative populations. microRNA expression profile from Thy1+ and Thy1- cells was compared with original MEFs (D0) to identify significantly changed microRNAs during initial stage of reprogramming
Project description:To date different cell types of various mammalian species have been reprogrammed to induced pluripotent stem cells (iPSCs) using Yamanaka's cocktail of transcription factors (Oct4, Klf4, Sox2, and cMyc). It has been shown that several primary human cancer cell lines could be reprogrammed to iPSCs. We sought if immortalized mouse fibroblast cell lines could also be reprogrammed to iPSCs. The approach of generating iPSCs from such cells should be valuable in different experimental settings as it allows clonally derive cell lines carrying mutations whose impact on reprogramming could be next evaluated. Therefore, we investigated reprogramming of widely used immortalized cell lines (NIH3T and STO), as well as of de novo immortalized fibroblast line (tKM) with the use of highly effective lentiviral polycistronic OKSM expression system. Our reprogramming experiments have shown that in contrast to mouse embryonic fibroblasts (MEFs), none of the immortalized cell lines can be reprogrammed to pluripotent state. Contrary to colonies derived from MEFs, those derived from the immortalized cells lines (1) developed much later, (2) contained large round cells, not typical for iPSCs, and (3) were negative for trusted markers of matured iPSCs, Nanog and SSEA1. Immortalized cell lines NIH3T and STO are known to be mostly aneuploid, whereas tKM population includes cells with normal karyotype, however, neither cell type can be reprogrammed. Thus our data argue that aneuploidy per se is not a reason for the observed refractoriness of mouse immortalized cells to reprogramming to pluripotent state.
Project description:To reprogram mouse embryonic fibroblasts (MEFs) to induced Pluripotent Stem Cells (iPSCs), we constructed the PiggyBac (PB) transposon carrying the four Yamanaka factor cDNAs controlled by a CAG promoter (PB-CAG-OCKS, Oct4, cMyc, Klf4 and Sox2). As the baseline reprogramming control, we transfected the PB-CAG-OCKS transposon into Oct4-reporter MEFs and plated the cells on STO feeder cells (4F-iPS). To examine the effects of miR-25 on reprogramming, in addition to the Yamanaka factors, we co-transfected the PB-CAG-OCKS plasmid with the PB-CAG-miR-25 plasmid and selected for puromycin resistance (2.0 mg/ml) (25-iPS). We then performed genome-wide gene expression microarray analysis on the iPS cells generated and compared the expression profiles to those of Oct4-reporter MEFs and wildtype ES cells.
Project description:It has been suggested that the transcription factor Nanog is essential for the establishment of pluripotency during the derivation of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. However, successful reprogramming to pluripotency with a growing list of divergent transcription factors, at ever increasing efficiencies, suggests that there may be many distinct routes to a pluripotent state. Here, we have investigated whether Nanog is necessary for reprogramming murine fibroblasts under highly efficient conditions using the canonical reprogramming factors Oct4, Sox2, Klf4 and cMyc. In agreement with prior results, the efficiency of reprogramming Nanog-/- fibroblasts was significantly lower than that of control fibroblasts. However, in contrast to previous findings, we were able to reproducibly generate iPS cells from Nanog-/- fibroblasts that effectively contributed to chimeric mice. Thus while Nanog may be an important mediator of reprogramming it is not required for establishing pluripotency in the mouse, even under standard conditions. In order to further evaluate the equivalency of Nanog null iPSC to nanog null ESCs, we have performed RNAseq on two independent nanog null iPSC lines, as well as Nanog Null ESC, WT ESC and iPSCs as well as MEFs. As a negativve control for reprogramming we have analyzed a partially reprogrammed iPSC line. 2-4 biological replicates each of 7 conditions (WT MEFs, WT ESC, WT iPSC, WT partially reprogrammed iPSC (piPS), Nanog null ESC, Nanog null iPSC clone G2 and Nanog null iPSC clone G5)
Project description:Ectopic expression of defined sets of transcription factors in somatic cells enables them to adopt the qualities of pluripotency. Mouse embryonic fibroblasts (MEFs) are the classic target cell used to elucidate the core principles of nuclear reprogramming. However, their phenotypic and functional heterogeneity represents a major hurdle for mechanistic studies aimed at defining the molecular nature of cellular plasticity. We show that reducing the complexity of MEFs by flow cytometry allows the isolation of discrete cell subpopulations that can be efficiently reprogrammed to pluripotency with fewer genes. Using these FACS-sorted cells, we performed a systematic side-by-side analysis of the reprogramming efficiency with different two- and three-factor combinations of Oct4, Sox2 and Klf4. We show that introduction of exogenous Oct4 with either Sox2 or Klf4 does not directly convert MEFs to a pluripotent state. Instead, each combination of factors disrupts the normal cellular homeostasis and establishes transient states characterized by the concurrent expression of mixed lineage markers. These cells convert into induced pluripotent stem cells in a stochastic fashion. Our data suggest that there is a partial functional redundancy between Sox2 and Klf4 in the disruption of cellular homeostasis and activation of regulatory networks that define pluripotency.
Project description:The detailed mechanism of reprogramming somatic cells into induced pluripotent stem cells (iPSCs) remains largely unknown. Partially reprogrammed iPSCs are informative and useful for understanding the mechanism of reprogramming but remain technically difficult to generate in a predictable and reproducible manner. Using replication-defective and persistent Sendai virus (SeVdp) vectors, we analyzed the effect of decreasing the expression levels of OCT4, SOX2, KLF4, and c-MYC and found that low KLF4 expression reproducibly gives rise to a homogeneous population of partially reprogrammed iPSCs. Upregulation of KLF4 allows these cells to resume reprogramming, indicating that they are paused iPSCs that remain on the path toward pluripotency. Paused iPSCs with different KLF4 expression levels remain at distinct intermediate stages of reprogramming. This SeVdp-based stage-specific reprogramming system (3S reprogramming system) is applicable for both mouse and human somatic cells and will facilitate the mechanistic analysis of reprogramming.
Project description:MEFs with or without four factor transduction (Oct4, Sox2, Klf4 and cMyc) were transfected with siControl or siWisp1 at 50nM. Total RNAs were extracted at day2 after transfection and mRNA expression profile was then analyzed.
Project description:Transcription factor-based reprogramming can lead to the successful switching of cell fates. We have recently reported that mouse embryonic fibroblasts (MEFs) can be directly reprogrammed into induced neural stem cells (iNSCs) after the forced expression of Brn4, Sox2, Klf4, and Myc. Here, we tested whether iNSCs could be further reprogrammed into induced pluripotent stem cells (iPSCs). The two factors Oct4 and Klf4 were sufficient to induce pluripotency in iNSCs. Immunocytochemistry and gene expression analysis showed that iNSC-derived iPSCs (iNdiPSCs) are similar to embryonic stem cells at the molecular level. In addition, iNdiPSCs could differentiate into cells of all three germ layers, both in vitro and in vivo, proving that iNdiPSCs are bona fide pluripotent cells. Furthermore, analysis of the global gene expression profile showed that iNdiPSCs, in contrast to iNSCs, do not retain any MEF transcriptional memory even at early passages after reprogramming. Overall, our results demonstrate that iNSCs can be reprogrammed to pluripotency and suggest that cell fate can be redirected numerous times. Importantly, our findings indicate that the induced pluripotent cell state may erase the donor-cell type epigenetic memory more efficiently than other induced somatic cell fates.
Project description:How metastases develop is not well understood and no genetic mutations have been reported as specific metastatic drivers. Here we have addressed the idea that epigenetic reprogramming by GLI-regulated pluripotent stemness factors promotes metastases. Using primary human colon cancer cells engrafted in mice, we find that transient expression of OCT4, SOX2, KLF4 +/- cMYC establishes an enhanced pro-metastatic state in the primary tumor that is stable through sequential engraftments and is transmitted through clonogenic cancer stem cells. Metastatic reprogramming alters NANOG methylation and stably boosts NANOG and NANOGP8 expression. Metastases and reprogrammed EMT-like phenotypes require endogenous NANOG, but enhanced NANOG is not sufficient to induce these phenotypes. Finally, reprogrammed tumors enhance GLI2, and we show that GLI2(high) and AXIN2(low), which are markers of the metastatic transition of colon cancers, are prognostic of poor disease outcome in patients. We propose that metastases arise through epigenetic reprogramming of cancer stem cells within primary tumors.
Project description:Somatic cells are reprogrammed to induced pluripotent stem cells (iPSCs) by overexpression of a combination of defined transcription factors. We generated iPSCs from mouse embryonic fibroblasts (with Oct4-GFP reporter) by transfection of pCX-OSK-2A (Oct4, Sox2, and Klf4) and pCX-cMyc vectors. We could generate partially reprogrammed cells (XiPS-7), which maintained more than 20 passages in a partially reprogrammed state; the cells expressed Nanog but were Oct4-GFP negative. When the cells were transferred to serum-free medium (with serum replacement and basic fibroblast growth factor), the XiPS-7 cells converted to Oct4-GFP-positive iPSCs (XiPS-7c, fully reprogrammed cells) with ESC-like properties. During the conversion of XiPS-7 to XiPS-7c, we found several clusters of slowly reprogrammed genes, which were activated at later stages of reprogramming. Our results suggest that partial reprogrammed cells can be induced to full reprogramming status by serum-free medium, in which stem cell maintenance- and gamete generation-related genes were upregulated. These long-term expandable partially reprogrammed cells can be used to verify the mechanism of reprogramming.