Project description:Transcriptional profiling of Oct4, Sox2, Lin28 and Nanog (OSLN) reprogramming cell of empty vector compared to HIF2M-NM-1 over-expression at the early stage (day 12) and the late stage(day 30) using fibroblasts MRC5 and IMR90 Eight-condition experiment, empty vector vs. HIF2M-NM-1 over-expression cells, at early stage vs. late stage, and fibroblasts MRC5 vs. IMR90
Project description:We recently found that DNA repair-related gene expression could be altered by reprogramming as well as the increased expression of genes that accurately convey genomic information, such as homologous recombination (HR) and mismatch repair (MMR), and the decreased expression of error-prone translesion synthesis (TLS) polymerase. Here, we confirmed this change in expression in another cell-line and found that such alteration was maintained by overlapping passages as well as OCT3/4 and NANOG. Our findings suggest that changes in the expression of DNA repair-related genes associated with reprogramming and their maintenance can be novel indicators of the quality control of the cells exhibiting pluripotency.
Project description:The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is lengthy and inefficient. The development of a reprogramming system that allows rapid and synchronous reprogramming to pluripotency is imperative for understanding the mechanism of iPSC formation and for future therapeutic applications. We have recently reported that a short expression in mouse primary B cells of the transcription factor C/EBPα before the induction of pluripotency factors increases the iPSC reprogramming efficiency >100-fold, involving 95% of the cells within a week. Here we present a dataset containing the time course of gene expression during this process as determined by microarray and RNA-seq techniques.
Project description:The SWI/SNF (SWItch/Sucrose NonFermentable or BAF, Brg/Brahma-associated factors) complexes are epigenetic modifiers of chromatin structure and undergo progressive changes in subunit composition during cellular differentiation. For example, in embryonic stem cells, esBAF contains Brg1 and Baf155, while their homologs, Brm and Baf170, are present in BAF of somatic cells. In this study, we sought to determine whether Brm and Baf170 play any roles in induced pluripotent stem cell (iPSC) reprogramming by using shRNA-mediated knockdown studies in the mouse model. We found that knocking down Brm during early, mid, and late stages (days 3, 6, and 9 after initial iPSC induction) and knocking down Baf170 during late-stage (day 9) reprogramming improve the numbers of iPSC colonies formed. We further showed that inhibition of these somatic BAF components also promotes complete reprogramming of partially reprogrammed somatic cells (pre-iPSCs). Finally, we found that the expression of Brm and Baf170 during reprogramming was regulated by Jak/Stat3 activity. Taken together, these data suggest that inhibiting somatic BAF improves complete reprogramming by facilitating the activation of the pluripotency circuitry.
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:Introduction:Many studies have reported that human-induced pluripotent stem (hiPS)/embryonic stem (hES) cells have an exceptional ability to repair damaged DNA. Moreover, unlike differentiated cells, hES cells have features and mechanisms such as apoptosis-prone mitochondria, which prevent any changes in genetic information caused by DNA damage to be transmitted to their descendants. Type-A (dark) spermatogonia and cancer stem cells are thought to be dormant. However, hiPS/hES cells, the so-called stem cells used in regenerative medicine, generally have a high proliferative capacity. This suggests that in these cells, oxidative DNA damage associated with vigorous proliferation and DNA scission associated with replication occur frequently. Although pluripotency according to change of genomic structure is well studied, the change of DNA repair through reprogramming has not been well studied. Methods:We analyzed the expression of DNA repair-related genes in hiPS cells using microarray and western blotting analyses and assessed changes in PARP activity through reprogramming. Results:Through reprogramming, hiPS cells were found to upregulate poly (ADP-ribose) polymerase (PARP) activity and genes regulating homologous recombination (HR). Simultaneously, the expression level of genes involved in non-homologous end joining (NHEJ) was not high, suggesting that at least at the gene expression level, frequently occurring DNA scission is preferentially dealt with via HR instead of NHEJ. Also, reflecting the high proliferative activity, genes related to mismatch repair (MMR) were upregulated through reprogramming. Conversely, error-prone polymerase was downregulated through reprogramming. These are also likely to be the mechanisms preventing changes in genetic information. Conclusions:High PARP activity and HR-related gene expression in hiPS cells were achieved through reprogramming and likely facilitate precise genome editing in these cells in exchange for a high possibility of cell death.
Project description:The protein level of OCT4, a core pluripotency transcriptional factor, is vital for embryonic stem cell (ESC) maintenance, differentiation and somatic cell reprogramming. Although OCT4 protein is regulated at multiple scales, the role and regulatory mechanisms of OCT4 ubiquitination in reprogramming remains elusive. We identified the five lysine residues as ubiquitination sites on OCT4, and found that destruction of the ubiquitination can enhance OCT4 activity in reprogramming. We used microarrays to detail the global gene expression underlying cellular reprogramming induced by two sets of reprogramming factor combinations, including O(WT)SK and O(5R)SK, and identified distinct pattern of gene expression change during this process. Overall design: Reprogramming cells, induced by transcription factor combination of O(WT)SK or O(5R)SK, were collected at successive stages at day 3, 5, and 7 for RNA extraction and hybridization on Affymetrix microarrays.
Project description:BACKGROUND: Somatic cells can be reprogrammed to induced-pluripotent stem cells (iPSCs) by introducing few reprogramming factors, which challenges the long held view that cell differentiation is irreversible. However, the mechanism of induced pluripotency is still unknown. METHODS: Inspired by the phenomenological reprogramming model of Artyomov et al (2010), we proposed a novel Markov model, stepwise reprogramming Markov (SRM) model, with simpler gene regulation rules and explored various properties of the model with Monte Carlo simulation. We calculated the reprogramming rate and showed that it would increase in the condition of knockdown of somatic transcription factors or inhibition of DNA methylation globally, consistent with the real reprogramming experiments. Furthermore, we demonstrated the utility of our model by testing it with the real dynamic gene expression data spanning across different intermediate stages in the iPS reprogramming process. RESULTS: The gene expression data at several stages in reprogramming and the reprogramming rate under several typically experiment conditions coincided with our simulation results. The function of reprogramming factors and gene expression change during reprogramming could be partly explained by our model reasonably well. CONCLUSIONS: This lands further support on our general rules of gene regulation network in iPSC reprogramming. This model may help uncover the basic mechanism of reprogramming and improve the efficiency of converting somatic cells to iPSCs.
Project description:Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka, who first generated iPSCs by retroviral transduction of four reprogramming factors, several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However, the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study, three different strategies, based on retroviral vectors, episomal vectors, and Sendai virus vectors, were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells, including the expression of alkaline phosphatase and stemness maker genes, and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion, the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs.
Project description:Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we examined defined intermediate cell populations poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcriptional wave and can be rescued by elevated expression of all four factors. The establishment of bivalent domains occurs gradually after the first wave, whereas changes in DNA methylation take place after the second wave when cells acquire stable pluripotency. This integrative analysis allowed us to identify genes that act as roadblocks during reprogramming and surface markers that further enrich for cells prone to forming iPSCs. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming.