Human transcription factors responsive to initial reprogramming are predominantly legitimate during iPSC reprogramming
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ABSTRACT:
PROVIDER: PRJNA668803 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12815316.fastq.gz | Fastqsanger.gz | |||
| SRR12815317.fastq.gz | Fastqsanger.gz | |||
| SRR12815318.fastq.gz | Fastqsanger.gz | |||
| SRR12815319.fastq.gz | Fastqsanger.gz | |||
| SRR12815320.fastq.gz | Fastqsanger.gz |
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Project description:The four transcription factors OCT4, SOX2, KLF4 and MYC (OSKM) together can convert human fibroblasts to induced pluripotent stem cells (iPSCs). It is, however, perplexing that they can do so only for a rare population of the starting cells with a long latency. Transcription factors (TFs) define identities of both the starting fibroblasts and the end product, iPSCs, and are of paramount importance for the reprogramming process as well. It is critical to upregulate or activate iPSC-enriched TFs while downregulate or silence the fibroblast-enriched TFs. This report explores the initial TF responses to OSKM as the molecular underpinnings for both the potency aspect and limitation sides of the OSKM reprogramming. The author first defined the TF reprogramome, i.e., the full complement of TFs to be reprogrammed. Most TFs were resistant to OSKM reprogramming at the initial stages, which agrees with the inefficiency and long latency of iPSC reprogramming. Surprisingly, the current analyses revealed that the majority of the TFs (83 genes) that did respond to OSKM induction underwent legitimate reprogramming. The initial legitimate transcriptional responses of TFs to OSKM reprogramming were observed from fibroblasts from a different individual. Such early biased legitimate reprogramming of the responsive TFs aligns well with the robustness aspect of the otherwise inefficient and stochastic OSKM reprogramming
2020-12-08 | GSE159410 | GEO
Project description:Yamanaka reprogramming is revolutionary but inefficient, slow and stochastic. The underlying molecular events for these mixed outcomes of iPSC reprogramming is still unclear. Previous studies about transcriptional responses to reprogramming overlooked human reprogramming, and are compromised by the fact that only a rare population proceeds towards pluripotency and a significant amount of the collected transcriptional data may not represent the positive reprogramming. We recently developed a concept of reprogramome, which allows one to study the early transcriptional responses to the Yamanaka factors in the perspective of reprogramming legitimacy of a gene response to reprogramming. Using RNA-seq, this study scored 579 genes successfully reprogrammed within 48 hours, indicating the potency of the reprogramming factors. This report also tallied 438 genes reprogrammed significantly but insufficiently up to 72 hours, indicating a positive drive with some inadequacy of the Yamanaka factors. In addition, 953 member genes within the reprogramome were transcriptionally irresponsive to reprogramming, showing the inability of the reprogramming factors to directly act on these genes. Furthermore, there were 305 genes undergoing six types of aberrant reprogramming: over, wrong, and unwanted up- or down- reprogramming, revealing significant negative impacts of the Yamanaka factors. The mixed findings about the initial transcriptional responses to the reprogramming factors shed new insights into the robustness as well as limitations of the Yamanaka factors.
2020-06-07 | GSE148158 | GEO
Project description:The four transcription factors OCT4, SOX2, KLF4, and MYC (OSKM) together can convert human fibroblasts to induced pluripotent stem cells (iPSCs). It is, however, perplexing that they can do so only for a rare population of the starting cells with a long latency. Transcription factors (TFs) define identities of both the starting fibroblasts and the end product, iPSCs, and are also of paramount importance for the reprogramming process. It is critical to upregulate or activate the iPSC-enriched TFs while downregulate or silence the fibroblast-enriched TFs. This report explores the initial TF responses to OSKM as the molecular underpinnings for both the potency aspects and the limitation sides of the OSKM reprogramming. The authors first defined the TF reprogramome, i.e., the full complement of TFs to be reprogrammed. Most TFs were resistant to OSKM reprogramming at the initial stages, an observation consistent with the inefficiency and long latency of iPSC reprogramming. Surprisingly, the current analyses also revealed that most of the TFs (at least 83 genes) that did respond to OSKM induction underwent legitimate reprogramming. The initial legitimate transcriptional responses of TFs to OSKM reprogramming were also observed in the reprogramming fibroblasts from a different individual. Such early biased legitimate reprogramming of the responsive TFs aligns well with the robustness aspect of the otherwise inefficient and stochastic OSKM reprogramming.
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Project description:Different stage of iPSC reprogramming
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Project description:A PIANO (Proper, Insufficient, Aberrant, and NO reprogramming) response to the Yamanaka factors in the initial stages of human iPSC reprogramming
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Project description:RNA seq data during different point of ipsc reprogramming from Dox TetOn OSKM system
2024-07-29 | GSE227745 | GEO
Project description:Despite rapid progress in characterizing transcription factor-driven reprogramming of somatic cells to an induced pluripotent stem (iPS) cell state, many mechanistic questions still remain. To gain insight into the earliest events in the reprogramming process, we systematically analyzed the transcriptional and epigenetic changes that occur during early factor induction after discrete numbers of divisions. We observed rapid, genome-wide changes in the euchromatic histone modification, H3K4me2, at more than a thousand loci including large subsets of pluripotency or developmentally related gene promoters and enhancers. In contrast, patterns of the repressive H3K27me3 modification remained largely unchanged except for focused depletion specifically at positions where H3K4 methylation is gained. These chromatin regulatory events precede transcriptional changes within the corresponding loci. Our data provide evidence for an early, organized, and population-wide epigenetic response to ectopic reprogramming factors that clarify the temporal order through which somatic identity is reset during reprogramming. Gene expression was measured by Affymetric microarrays during the initial phase of the reprogramming of mouse embryonic fibroblasts.
2011-01-03 | E-GEOD-26096 | biostudies-arrayexpress
Project description:The differentiated state of somatic cells provides barriers for the efficient derivation of induced pluripotent stem cells (iPSCs). To address why some cell types reprogram more readily than others, we studied the effect of combined modulation of cellular signaling pathways. This revealed that inhibition of TGFβ together with activation of Wnt signaling in presence of ascorbic acid allows >80% of murine fibroblasts to acquire pluripotency after one week of reprogramming factor expression. In contrast, hepatic progenitors and blood progenitors predominantly required only TGFβ inhibition or canonical Wnt activation, respectively, to reprogram at efficiencies approaching 100%. Strikingly, blood progenitors reactivated endogenous pluripotency loci in a highly synchronous manner. We further demonstrate that expression of specific chromatin-modifying enzymes and reduced TGFβ/MAP kinase activity are intrinsic properties associated with the unique reprogramming response of these cells. Together, our observations define novel cell type-specific requirements for the rapid and synchronous reprogramming of somatic cells. For the study of reprogramming intermediates, cultures of reprogrammable MEFs at day 4 in presence of dox and compounds were harvested and stained with biotinylated anti-SSEA1 antibody (MC-480, eBioscience), followed by APC-conjugated Streptavidin and then anti-APC microbeads (Milteny Biotec) and enrichment using MACS separation columns (Milteny Biotec) according to manufacturer’s instructions. Total RNA extracted from SSEA1+ cells (>90% purity) with the RNeasy mini kit (QIAGEN) with a RIN value > 8 was subjected to transcriptional analyses with Affymetrix mouse genome 430 2.0 mRNA expression microarrays followed by bioinformatic analyses.
2014-09-11 | E-GEOD-59865 | biostudies-arrayexpress
Project description:Factor induced reprogramming is a slow and inefficient process with only rare cells progressing towards induced pluripotent stem cells (iPSCs). Owing to these restraints, mechanistic studies have been limited to analyses of heterogeneous bulk populations undergoing reprogramming and partially reprogrammed cell lines. Here, by combining surface markers (Thy1, SSEA1) and an Oct4-GFP fluorescent reporter allele, we analyzed defined intermediate cell populations poised to becoming iPSCs at the transcriptional and epigenetic levels using genome-wide and single cell technologies. We found that factor-induced reprogramming elicits two discernible transcriptional waves that are characterized by the initial extinction of the somatic gene expression program and the concomitant acquisition of an ESC-like proliferative and metabolic state, followed by the activation of an embryonic pluripotent state primed for differentiation. The first wave is mostly driven by gene activation through c-Myc and gene repression by Klf4, whereas the second wave is a result of gradually activated Oct4/Sox2 targets in cooperation with Klf4 targets and other downstream regulators. While microRNA expression and enrichment for individual histone modifications (H3K4me3 or H3K27me3 enriched promoters) mirrored the observed biphasic transcriptional pattern, the establishment of bivalent domains (H3K4me3/H3K27me3 enriched promoters) occurred more gradually. In contrast, changes in DNA methylation took place predominantly at the end of reprogramming when cells assumed a stable pluripotent state. Cells that became refractory to reprogramming activated the first but failed to initiate the second transcriptional wave. However, introduction of additional copies of the reprogramming transgenes into these cells rescued their ability to form iPSCs, indicating that suboptimal transcription factor levels are a limiting factor for efficient iPSC formation. This integrative analysis allowed us to identify novel genes and microRNAs that enhance reprogramming and surface markers that further subdivide intermediate cell populations. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming and provide a valuable resource of molecules that may act as roadblocks during iPSC formation. Time series design with samples sorted into subpopulations according to surface markers.
2012-12-20 | E-GEOD-42477 | biostudies-arrayexpress
Project description:Factor induced reprogramming is a slow and inefficient process with only rare cells progressing towards induced pluripotent stem cells (iPSCs). Owing to these restraints, mechanistic studies have been limited to analyses of heterogeneous bulk populations undergoing reprogramming and partially reprogrammed cell lines. Here, by combining surface markers (Thy1, SSEA1) and an Oct4-GFP fluorescent reporter allele, we analyzed defined intermediate cell populations poised to becoming iPSCs at the transcriptional and epigenetic levels using genome-wide and single cell technologies. We found that factor-induced reprogramming elicits two discernible transcriptional waves that are characterized by the initial extinction of the somatic gene expression program and the concomitant acquisition of an ESC-like proliferative and metabolic state, followed by the activation of an embryonic pluripotent state primed for differentiation. The first wave is mostly driven by gene activation through c-Myc and gene repression by Klf4, whereas the second wave is a result of gradually activated Oct4/Sox2 targets in cooperation with Klf4 targets and other downstream regulators. While microRNA expression and enrichment for individual histone modifications (H3K4me3 or H3K27me3 enriched promoters) mirrored the observed biphasic transcriptional pattern, the establishment of bivalent domains (H3K4me3/H3K27me3 enriched promoters) occurred more gradually. In contrast, changes in DNA methylation took place predominantly at the end of reprogramming when cells assumed a stable pluripotent state. Cells that became refractory to reprogramming activated the first but failed to initiate the second transcriptional wave. However, introduction of additional copies of the reprogramming transgenes into these cells rescued their ability to form iPSCs, indicating that suboptimal transcription factor levels are a limiting factor for efficient iPSC formation. This integrative analysis allowed us to identify novel genes and microRNAs that enhance reprogramming and surface markers that further subdivide intermediate cell populations. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming and provide a valuable resource of molecules that may act as roadblocks during iPSC formation. Time series design with samples sorted into subpopulations according to surface markers. 18 Samples total
2012-12-20 | E-GEOD-42475 | biostudies-arrayexpress