Modeling Down syndrome AML by introducing disese-specific mutations in iPSCs
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PROVIDER: PRJNA779425 | ENA |
REPOSITORIES: ENA
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Project description:Down syndrome AML is characterized by the presence of the pathgnonomic mutation in GATA1, which cooperates with other somatic mutations. In this study, we utilzed iPSCs derived from Down syndrome individuals bearing trisomy 21 and introduced disease-specific mutations using CRISPR-Cas9. Hematopoeitic stem and progenitor cells (HSPCs) obtained by hematopoeitic differentiation of these iPSCs, and megakaryocytes generated from the HSPCs (by culturing in megakaryocyte-specific media) were used for RNA sequencing analysis.
2022-03-02 | GSE188568 | GEO
Project description:Severe congenital neutropenia (CN) is a pre-leukemia syndrome that, in the majority of patients, is caused by heterogeneous ELANE mutations encoding neutrophil elastase (NE). To study leukemogenesis associated with CN we generated CN and CN/AML patient-specific induced pluripotent stem cells (iPSCs). Additional mutations in leukemia-relevant genes, CSF3R and RUNX1, were introduced using CRISPR/Cas9 gene-editing. Consequently, we performed in vitro embryoid body (EB)-based hematopoietic and myeloid differentiation of generated iPSC lines. On day 14-17 of EB-based differentiation, iPSC-derived CD45+CD34+ cells were harvested and mRNA was isolated using RNeasy Mini- or Micro Kit (Qiagen). Sequencing libraries were prepared using the TruSeq RNA Sample Prep Kit (Illumina). Poly (A) selected single-read and pair-read sequencing libraries were sequenced on the Illumina platform in order to compare the transcriptomes of CN and CN/AML iPSCs-derived HSPCs from 2 CN/AML patients. Next, we identified that BAALC knockout resulted in a dramatic induction of granulocytic differentiation and a significant reduction in proliferation of CN/AML iPSC-derived HSPCs. To identify BAALC-dependent leukemia-associated gene expression, we compared the transcriptomes of CN/AML iPSCs before and after BAALC KO using a similar approach described above for CN and CN/AML iPSCs-derived HSPCs.
2021-03-08 | E-MTAB-10162 | biostudies-arrayexpress
Project description:RNA-seq of CN, CN/AML, and CN/AML BAALC KO iPSCs-derived HSPCs
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Project description:Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) resets the epigenetic landscapes that mark the aging clock, and consequently cells differentiated from iPSCs resemble fetal cells rather than adult or aged cells. The lack of proper cellular aging in cells differentiated from iPSCs presents a significant problem in iPSC-based disease models to investigate the pathological progression of age-associated diseases such as neurodegeneration. To address this caveat, we introduced cellular senescence into iPSC-based cell models in this study, as senescence has been shown to play a critical role not only in aging but also in neurodegeneration. We created an inducible CRISPR interference (CRISPRi) targeting TERF2, a major component of the telomere protecting Shelterin complex. We demonstrated that suppression of TERF2 robustly activated DNA damage response, the p53/p21 signaling, cellular senescence and inflammatory response in iPSCs. The inducible approach allows the temporal control of senescence activation over the course of differentiation of iPSCs to desired cell types. We applied the CRISPRi-TERF2 system to differentiation of iPSCs into neural progenitor cells (NPCs) and showed that suppression of TERF2 efficiently activated DNA damage response, the p53/p21 signaling and senescence in differentiated NPCs. The inducible cell model of cellular senescence generated in this study has broad application in investigation of cellular senescence in the progression of age-related diseases and improvement of disease modeling with a proper cellular aging context to facilitate drug discovery.
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Project description:Induced pluripotent stem cells (iPSCs) are similar to embryonic stem cells and can be generated from somatic cells. We have generated episomal plasmid-based and integration-free iPSCs (E-iPSCs) from human fetal foreskin fibroblast cells (HFF1). E-iPSCs were fully characterized and their transcriptomes are more similar to that of hESCs (R2 = 0.9363) in comparison to viral-derived HFF1-iPSCs (R2 = 0.8176). We used an E-iPSC-line to model hepatogenesis in vitro. The differentiation of iPSCs into hepatocyte-like cells (HLCs) is a three-step process, from the undifferentiated E-iPSC to definitive endoderm (DE), hepatic endoderm (HE) and ultimately HLCs. The HLCs were characterized biochemically, i.e. glycogen storage, ICG uptake and release, UREA and bile acid production, as well as CYP3A4 activity. Ultra-structure analysis by electron microscopy revealed the presence of lipid and glycogen storage, tight junctions and bile canaliculi- all typical features of hepatocytes. Furthermore, the transcriptome of undifferentiated E-iPSC, DE, HE and HLCs were compared to that of fetal liver and primary human hepatocytes (PHH). K-means clustering identified 100 clusters which include developmental stage-specific groups of genes, e.g. OCT4 expression at the undifferentiated stage, SOX17 marking the DE stage, DLK and HNF6 the HE stage, HNF4a and Albumin is specific to HLCs, fetal liver and adult liver (PHH) stage. The lack of viral DNA integrations in these E-iPSCs endow them superior to viral-derived iPSCs for (i) modeling gene regulatory networks associated with hepatogenesis and gastrulation in general, (ii) toxicology studies and (iii) drug screening platforms.
2017-09-06 | GSE66282 | GEO
Project description:DNA methyltransferases DNMT3A- and DNMT3B-mediated de novo DNA methylation critically regulates epigenomic and transcriptomic patterning during development. The hotspot DNMT3A mutations at the site of Arg822 (R882) promote macro-oligomer formation, leading to aberrant DNA methylation that in turn contributes to pathogenesis of acute myeloid leukemia (AML). However, the molecular basis underlying the hotspot mutation-induced functional mis-regulation of DNMT3A remains unclear. Here, we report the crystal structure of DNMT3A methyltransferase (MTase) domain, revealing a molecular basis for its DNMT3B-distinct oligomerization behavior. Introducing DNMT3B-converting mutations to DNMT3A R882 mutants also led to structure determination of R882H- and R882C-mutated DNMT3A, which show enhanced intermolecular contacts than wild-type DNMT3A. Consistently, our in vitro and genomic DNA methylation analyses reveal that the DNMT3B-converting mutations eliminate the gain-of-function effect of the DNMT3A R882 mutations in cells. Together, this study provides mechanistic insights into DNMT3A R882 mutation-triggered aberrant oligomerization and DNA hypomethylation in AML, with important implications in cancer therapy.
2023-12-31 | GSE225828 | GEO
Project description:To ameliorate the cumbersome processes of reprogramming and subsequent gene-editing in vulnerable iPSCs, we have developed a greatly simplified one-step procedure, simultaneously introducing reprogramming and gene-editing components into human fibroblast cells. This not only serves to save time, labor, and costs, but opens up a new arena of research that is beyond the current application of gene-editing methodologies due to restrictive reprogramming concerns, inhibitory pluripotency maintenance requirements, and vulnerability of single-cell dissociated iPSCs. We generated iPSCs (mALK2-iPSCs) derived from Fibrodysplasia ossificans progressiva (FOP)-fibroblast cells carrying the ACVR1 p.R206H mutation and gene-corrected ALK2-iPSCs (cALK2-iPSCs). To identify gene-correction effects on global gene expression, gene expression profiling was measured in cALK2-iPSCs and mALK2-iPSCs, calibrated to WT-iPSCs with duplication.
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