Nanog Independent Reprogramming to iPSCs with Canonical Factors
ABSTRACT: 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: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:Induced pluripotent stem (iPS) cells can be obtained through the introduction of defined factors into somatic cells. The combination of Oct4, Sox2 and Klf4 (OSK) constitutes the minimal requirement for generating iPS cells from mouse embryonic fibroblasts (MEFs). Through the genomic analyses of ESC genes that have roles in pluripotency and fusion-mediated somatic cell reprogramming, we identified Tbx3 as a transcription factor that significantly improves the quality of iPS cells. Induced-PS cells generated with OSK + Tbx3 (OSKT) are superior in both germ cell contribution to the gonads and germ-line transmission frequency. However, global gene expression profiling could not distinguish between OSK and OSKT iPS cells. Genome-wide ChIP-sequencing analysis of Tbx3 binding sites in ESCs suggests that Tbx3 regulates pluripotency-associated and reprogramming factors, in addition to sharing many common downstream regulatory targets with Oct4, Sox2, Nanog and Smad1. ChIP-seq of Tbx3 binding in mouse ESCs
Project description:In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of nonproductive and staggered productive intermediates arise at different reprogramming time points1-11. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells12,13 prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that during reprogramming the cells progressively lose donor cell identity and gradually acquire iPS cell properties1,2,7,8,10. Here, we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200 that are absent in fibroblasts and iPS cells. Single cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic reprogramming phase2. Expression profiling revealed that the transcriptional regulators Nr0b1 and Etv5 are specifically expressed in this early reprogramming state, preceding activation of key pluripotency regulators such as Rex1, Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus mark some of the earliest known regulators of iPS cell induction. Our study shows an ordered sequence of transitions during the earliest steps of iPS cell reprogramming that deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition. Samples for poised (CD73+ or CD49d+) and non-poised (CD73-) reprogramming samples were FACS sorted 6 and 9 days after induction of Klf4, Oct4, Sox2 and cMyc in Rosa-rtTA +/- mouse embryonic fibroblasts (MEFs). 'Total' populations are expression analyses for unsorted populations analyzed at the same time points. Control populations were also sampled: mouse embryonic fibroblasts (MEFs), partially reprogrammed cells (SC4) and mouse embryonic stem cell (ESC).
Project description:We show here that singular loss of the Bright/Arid3A transcription factor leads to reprograming of mouse embryonic fibroblasts (MEFs) and enhancement of standard four-factor (4F) reprogramming. Bright-deficient MEFs bypass senescence and, under standard embryonic stem cell (ESC) culture conditions, spontaneously form clones that in vitro express pluripotency markers, differentiate to all germ lineages, and in vivo form teratomas and chimeric mice. We demonstrate that BRIGHT binds directly to the promoter/enhancer regions of Oct4, Sox2, and Nanog to contribute to their repression in both MEFs and ESCs. Thus, elimination of the BRIGHT barrier may provide an approach for somatic cell reprogramming.
Project description:Induction of pluripotency in differentiated cells through the exogenous expression of the transcription factors Oct4, Sox2, Klf4 and cellular Myc involves reprogramming at the epigenetic level. Histones and their metabolism governed by histone chaperones constitute an important regulator of epigenetic control. We hypothesized that histone chaperones facilitate or inhibit the course of reprogramming. For the first time, we report here that the downregulation of histone chaperone Aprataxin PNK-like factor (APLF) promotes reprogramming by augmenting the expression of E-cadherin (Cdh1), which is implicated in the mesenchymal-to-epithelial transition (MET) involved in the generation of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts (MEFs). Downregulation of APLF in MEFs expedites the loss of the repressive MacroH2A.1 (encoded by H2afy) histone variant from the Cdh1 promoter and enhances the incorporation of active histone H3me2K4 marks at the promoters of the pluripotency genes Nanog and Klf4, thereby accelerating the process of cellular reprogramming and increasing the efficiency of iPSC generation. We demonstrate a new histone chaperone (APLF)-MET-histone modification cohort that functions in the induction of pluripotency in fibroblasts. This regulatory axis might provide new mechanistic insights into perspectives of epigenetic regulation involved in cancer metastasis.
Project description:Pluripotency can be induced in differentiated murine and human cells by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors. Using these inducible constructs, we derived induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs) and found that transgene silencing is a prerequisite for normal cell differentiation. We have analyzed the timing of known pluripotency marker activation during mouse iPS cell derivation and observed that alkaline phosphatase (AP) was activated first, followed by stage-specific embryonic antigen 1 (SSEA1). Expression of Nanog and the endogenous Oct4 gene, marking fully reprogrammed cells, was only observed late in the process. Importantly, the virally transduced cDNAs needed to be expressed for at least 12 days in order to generate iPS cells. Our results are a step toward understanding some of the molecular events governing epigenetic reprogramming.
Project description:Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4 and Myc (OSKM) into somatic cells. Though iPSCs are pluripotent, they frequently exhibit high variation in their quality as measured by chimera contribution and tetraploid (4n) complementation. Thus, improving the quality of iPSCs is an indispensable prerequisite for future iPSC-based therapy. Here we show that one major determinant for iPSCs quality is the selection of the reprogramming factors combination. Ectopic expression of Sall4, Nanog, Esrrb and Lin28 (SNEL) in MEFs efficiently generated high quality iPSCs as compared to other combinations of factors. SNEL-iPSCs produced approximately 5 times more efficiently “all-iPSC” mice compared to OSKM-iPSCs. While differentially methylated regions, transcript number of master regulators, establishment of ESC-specific super enhancers, and global aneuploidy were comparable between the lines, aberrant expression of 1,765 genes, trisomy of chromosome 8 and abnormal H2A.X deposition were frequently observed in poor quality OSKM-iPSCs. For high-quality iPSCs, H2A.X pattern of SNEL is most similar to that of ESC, OSK and OSKM have more devoid regions than SNEL iPSCs. Compare H2A.X deposition pattern of the OSKM 4-factor iPS cell lines (4N-), SNEL 4-factor iPS cell lines (4N+) with ChIP-Seq. The same background ES cell line as the control line.
Project description:The reprogramming of differentiated cells to an embryonic stem cell-like state provides a powerful system to explore fundamental mechanisms of development, including how mammalian cells establish and maintain pluripotency and long-term self-renewal capability. Based on the similarities between embryonic stem cells and cancer cells, we investigated the potential role of the retinoblastoma tumor suppressor and cell cycle regulator RB in the reprogramming of fibroblasts into induced pluripotent stem cells (iPS cells). Herein we demonstrate that loss of RB function leads to both an acceleration of the reprogramming process and the generation of more iPS clones from fibroblasts. This effect is largely due to a restrictive role for RB at the early stages of reprogramming. Surprisingly, however, RB inactivation does not enhance the formation of iPS clones by accelerating the proliferation of cells undergoing reprogramming. Rather, a genome-wide investigation of RB targets indicates that RB binds to regulatory regions of pluripotency genes such as Sox2 and Oct4 and contributes to their full repression in differentiated cells. This effect correlates with the maintenance of a repressive chromatin structure at these loci. Accordingly, Rb-deficient fibroblasts can be reprogrammed into iPS cells even in the absence of exogenous Sox2, which is normally required to initiate reprogramming from fibroblasts. These experiments identify a novel barrier in the reprogramming process, mainly the repression of certain pluripotency genes such as Sox2 by RB, which provides a new link between tumor suppressor mechanisms and cellular reprogramming. RNAseq from MEFs with 2 biological replicates (save CP), Rb ChIPseq from MEFs with 2 biological replicates, Histone H3 modification ChIPseq from MEFs with 1 biological replicate
Project description:Somatic cell reprogramming can be achieved by cell fusion with embryonic stem cells (ESCs), nuclear transfer into oocytes, or forced expression of transcription factors essential for ESC identity. Reprogramming by transcription factors is a less efficient and slower process than that by other methods. Identification of a gene set capable of driving rapid and proper reprogramming to induced pluripotent stem cells (iPSCs) is an important issue. Here, we show that the efficiency and kinetics of iPSC reprogramming are dramatically improved by combined transduction of Jarid2, a gene highly expressed in both ESCs and oocytes and genes encoding its associated proteins. We demonstrate that forced expression of Jarid2 promotes iPSC reprogramming by suppressing the expression of Arf, a known reprogramming barrier, and that the N-terminal half of JARID2 is sufficient for such promotion. Moreover, Jarid2 accelerated retroviral transgene silencing and Nanog promoter demethylation, confirming its promoting activity. We further reveal that JARID2 physically interacts with ESRRB, SALL4A and PRDM14, and show that these JARID2-associated proteins synergistically and robustly facilitate iPSC reprogramming in a Jarid2-dependent manner. Our findings provide an insight into the important roles of Jarid2 during reprogramming, and suggest that the JARID2-associated protein network contributes to overcoming reprogramming barriers. We used microarrays to detect the up- and down-regulated genes in MEFs transduced with Jarid2 compared to empty vector control. Total RNA was extracted from mouse embrionc fibroblasts (MEFs) transduced with empty vector (control) or the indicated genes at 3 days post-transduction, and two iPSC clones from MEFs transduced with OSKM plus Jarid2. Gene expression profile was analyzed by the Affymetrix GeneChip microarray system.
Project description:One of the hurdles for practical application of induced pluripotent stem cells (iPSC) is the low efficiency and slow process of reprogramming. Octamer-binding transcription factor 4 (Oct4) has been shown to be an essential regulator of embryonic stem cell (ESC) pluripotency and key to the reprogramming process. To identify small molecules that enhance reprogramming efficiency, we performed a cell-based high-throughput screening of chemical libraries. One of the compounds, termed Oct4-activating compound 1 (OAC1), was found to activate both Oct4 and Nanog promoter-driven luciferase reporter genes. Furthermore, when added to the reprogramming mixture along with the quartet reprogramming factors (Oct4, Sox2, c-Myc, and Klf4), OAC1 enhanced the iPSC reprogramming efficiency and accelerated the reprogramming process. Two structural analogs of OAC1 also activated Oct4 and Nanog promoters and enhanced iPSC formation. The iPSC colonies derived using the Oct4-activating compounds along with the quartet factors exhibited typical ESC morphology, gene-expression pattern, and developmental potential. OAC1 seems to enhance reprogramming efficiency in a unique manner, independent of either inhibition of the p53-p21 pathway or activation of the Wnt-?-catenin signaling. OAC1 increases transcription of the Oct4-Nanog-Sox2 triad and Tet1, a gene known to be involved in DNA demethylation.