FAM13B knockdown in iCMs
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA913783 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR22806300_1.fastq.gz | Fastqsanger.gz | |||
| SRR22806300_2.fastq.gz | Fastqsanger.gz | |||
| SRR22806301_1.fastq.gz | Fastqsanger.gz | |||
| SRR22806301_2.fastq.gz | Fastqsanger.gz | |||
| SRR22806302_1.fastq.gz | Fastqsanger.gz |
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Project description:Common human genetic variation near the FAM13B gene are associated with atrial fibrillation as well as with the expression level of FAM13B mRNA in human left atrial appendage tissues. To determine the effects of FAM13B gene expression in human cardiomyocytes derived from induced pluripotent stem cells (iCMs from iCell Cardiomyocytes), we used siRNA targeted against FAM13B to knockdown its gene expression (KD) or an siRNA with a scrambled sequence (SCR). Total RNA was prepared from biological triplicate plates of cells and subjected to bulk RNAseq. 100-bp paired-end sequencing was performed on the Illumina HiSeq 2000. The sequence reads were mapped to the human genome using the STAR aligner (version 2.3.0) to derive a digital count of the expression of genes, which were defined using the Ensembl gene catalog (version 69). Genes were filtered to ensure a CPM (counts per million) > 5 in at least 2 samples, resulting in 11,017 genes. Differential expression analyses of FAM13B KD versus control SCR siRNA in iCMs was performed using the limma-voom approach with false discovery rate control.
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Project description:This study aimed to compare the transcriptome profiling (RNA-seq) and chromatin accessibility landscape (ATACseq) of induced cardiomyocytes (iCMs) with Beclin1 (Becn1) knockdown and control iCMs (shNT). we generated ATAC-Seq data from day 3 shNT- and shBecn1-treated MGT or control LacZ transduced fibroblasts. We interrogated the enrichment of ATAC-seq reads at +1 and -1 Kb around TSS (Transcription starting site). Comparing MGT-iCMs with LacZ transduced cells, we found that MGT-iCMs gained numerous open chromatin regions compared to LacZ transduced cells. In total, 22,262 high-confidence open chromatin regions were identified when comparing MGT-shNT iCMs with LacZ-shNT cells, and 11,401 open regions were found in MGT-shBecn1 iCMs when comparing to LacZ-shBecn1 cells. However, MGT-shBecn1 iCMs exhibited minimal differences that did not reach statistical significance when compared with MGT-shNT cells. MGT-shBecn1 iCMs had same number of open chromatin regions as the MGT-shNT cells. In parallel, we performed RNA-seq to profile transcriptome changes among day 3 and day 5 shNT- or shBecn1-treated reprogramming and control cells. Next, we profiled the differentially expressed genes (DEGs) at each time point. Gene set enrichment analysis (GSEA) revealed that shBecn1 significantly up-regulated gene sets related to myogenesis, such as those involved in the formation of contractile fiber and myofibril assembly and down-regulated gene sets related to cell proliferation and inflammation. Interestingly, GSEA analysis demonstrated additional enrichment plots of Wnt/β-Catenin signaling in shBecn1 iCMs.
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Project description:Direct conversion of fibroblasts to induced cardiomyocytes (iCMs) has great potential for regenerative medicine. Recent publications have reported significant progress, but the evaluation of reprogramming has relied upon non-functional measures such as flow cytometry for cardiomyocyte markers or GFP expression driven by a cardiomyocyte-specific promoter. The issue is one of practicality: the most stringent measures - electrophysiology to detect cell excitation and the presence of spontaneously contracting myocytes - are not readily quantifiable in the large numbers of cells screened in reprogramming experiments. However, excitation and contraction are linked by a third functional characteristic of cardiomyocytes: the rhythmic oscillation of intracellular calcium levels. We set out to optimize direct conversion of fibroblasts to iCMs with a quantifiable calcium reporter to rapidly assess functional transdifferentiation. We constructed a reporter system in which the calcium indicator GCaMP is driven by the cardiomyocyte-specific Troponin T promoter. Using calcium activity as our primary outcome measure, we compared several published combinations of transcription factors along with novel combinations in mouse embryonic fibroblasts. The most effective combination consisted of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT). This combination is >50-fold more efficient than GMT alone and produces iCMs with cardiomyocyte marker expression, robust calcium oscillation, and spontaneous beating that persists for weeks following inactivation of reprogramming factors. HNGMT is also significantly more effective than previously published factor combinations for the transdifferentiation of adult mouse cardiac fibroblasts to iCMs. Quantification of calcium function is a convenient and effective means for the identification and evaluation of cardiomyocytes generated by direct reprogramming. Using this stringent outcome measure, we conclude that HNGMT produces iCMs more efficiently than previously published methods. Mouse embryonic fibroblasts were treated with different combinations of transcription factors to drive transdifferentiation to induced cardiomyocytes (iCMs). Putative iCMs were enriched by zeocin selection. Zeocin resistance was conferred to iCMs via the TroponinT-GCaMP5-Zeo lentiviral reporter.
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