Cell type determination for cardiac differentiation occurs after seeding of human induced pluripotent stem cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE198729 | GEO | 2022/03/19
REPOSITORIES: GEO
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Project description:Human induced pluripotent stem (hiPS) cells and human embryonic stem (hES) cells differentiate into cells of the endothelial lineage, but derivation of cells with human umbilical cord blood endothelial colony forming cell (ECFC)-like properties has not been reported. Here we describe a novel serum- and stromal cell-free ECFC differentiation protocol for the derivation of clinically relevant numbers of ECFCs (> 108) from hiPS and hES cells. We identified NRP-1+CD31+ selected cells that displayed a stable endothelial phenotype exhibiting high clonal proliferative potential, extensive replicative capacity, formation of human vessels that inosculated with host vasculature upon transplantation, but lacking in teratoma formation in vivo. We also identified NRP-1-VEGF165-KDR-mediated activation of KDR as a critical mechanism for the emergence and derivation of ECFCs from hiPS and hES cells. This protocol advances the field by generating highly replicative but stable endothelial cells for use as a potential cell therapy for human clinical disorders. Transcriptome sequencing of undifferentiated day 0 hiPS cells, Day 3 differentiated hiPS-derived mesoderm proginator cells, Day 12 hiPS-derived NRP-1+CD31+ cells, Day 12 H9-hES-derived NRP-1+CD31+ cells and cord blood-derived Endothelial colony forming cells.
2014-07-18 | E-GEOD-49074 | biostudies-arrayexpress
Project description:Direct reprogramming of human fibroblasts to a pluripotent state has been achieved through ectopic expression of the transcription factors OCT4, SOX2, and either cMYC and KLF4 or NANOG and LIN28. Little is known, however, about the mechanisms by which reprogramming occurs, which is in part limited by the low efficiency of conversion. To this end, we sought to create a doxycycline-inducible lentiviral system to convert primary human fibroblasts and keratinocytes into human induced pluripotent stem (hiPS) cells. hiPS cells generated with this system were molecularly and functionally similar to human embryonic stem (hES) cells, demonstrated by gene expression profiles, DNA methylation status, and differentiation potential. While expression of the viral transgenes was required for several weeks in fibroblasts, we found that 10 days was sufficient for the reprogramming of keratinocytes, suggesting that the kinetics of reprogramming are cell-type dependent. Using our inducible system, we developed a strategy to induce hiPS cell formation at high frequency by generating differentiated cells that contain the viral transgenes in a pattern that enables successful induction of pluripotency. Upon addition of doxycycline to differentiated hiPS-derived cells, we obtained “secondary” hiPS cells at a frequency at least 100-fold greater than the initial conversion. The ability to reprogram cells with high efficiency provides a unique platform to dissect the underlying molecular and biochemical processes that accompany nuclear reprogramming.
2008-09-12 | GSE12390 | GEO
Project description:Direct reprogramming of human fibroblasts to a pluripotent state has been achieved through ectopic expression of the transcription factors OCT4, SOX2, and either cMYC and KLF4 or NANOG and LIN28. Little is known, however, about the mechanisms by which reprogramming occurs, which is in part limited by the low efficiency of conversion. To this end, we sought to create a doxycycline-inducible lentiviral system to convert primary human fibroblasts and keratinocytes into human induced pluripotent stem (hiPS) cells. hiPS cells generated with this system were molecularly and functionally similar to human embryonic stem (hES) cells, demonstrated by gene expression profiles, DNA methylation status, and differentiation potential. While expression of the viral transgenes was required for several weeks in fibroblasts, we found that 10 days was sufficient for the reprogramming of keratinocytes, suggesting that the kinetics of reprogramming are cell-type dependent. Using our inducible system, we developed a strategy to induce hiPS cell formation at high frequency by generating differentiated cells that contain the viral transgenes in a pattern that enables successful induction of pluripotency. Upon addition of doxycycline to differentiated hiPS-derived cells, we obtained âsecondaryâ hiPS cells at a frequency at least 100-fold greater than the initial conversion. The ability to reprogram cells with high efficiency provides a unique platform to dissect the underlying molecular and biochemical processes that accompany nuclear reprogramming. Experiment Overall Design: Total RNA was isolated from control HUES lines, hBJ (donor line) 4 primary HIPS lines and 1 secondary HIPS line using an RNeasy kit (Qiagen). Samples were processed as independent triplicates. RNA probes for microarray hybridization were prepared and hybridized to Affymetrix HGU133 plus 2 oligonucleotide microarrays. Data was extracted and analyzed using Rosetta Resolver system. During importation, the data was subjected to background correction, intrachip normalization, and the Rosetta Resolver Affymetrix GeneChip error model (Weng et al, 2006). For the generation of intensity plots, genes that showed greater than a two-fold difference in expression value (p<0.01) in HUES8 hES cells and BJ fibroblasts were noted (19,663 probes) and their expression analyzed. A hierarchical clustering was performed.
2008-10-25 | E-GEOD-12390 | biostudies-arrayexpress
Project description:Purpose: To induce hiPS-CMs maturation by using engineering technics, including culturing on the anisotropic pattern, has been widely explored. However, the underlying mechanisms of the benefits driven by the aligned topographic stimuli are still pending. To obtain insights into the underlying molecular pathways/signaling involved in the facilitation of hiPS-CMs maturation driven by specific topographic stimuli, we performed RNA-seq for the hiPS-CMs samples after culturing on the different patterns Methods: hiPS-CMs cultured on the flat bottom 24-well plate (MS-80240; Sumilon)/random nanofiber substrate (NanoECM, 2401; Funakoshi)/aligned nanofiber substrate (NanoAligned, 2402; Funakoshi) for one week. Then the cells were harvested as Flat, Random, Align group samples. Total RNA was extracted using the RNeasy Plus mini kit (740990.250; Takara), the concentration of RNA was measured by using NanoDrop (2000/2000c Spectrophotometers, Thermo Fisher). The Illumina package bcl2fastq software was used for base-calling. The raw reads were mapped to the human reference genome sequences (GRCh38) using TopHat ver. 2.1.1 in combination with Bowtie2 ver. 2.3.4.1 Results: The principal component analysis(PCA) analysis revealed that the hiPS-CMs from flat and random patterns showed the most variance. And the differentially expressed genes (DEGs) were detected with theDESeq2 package, showed that the flat group samples occupied most of the enrichment gene expression. The enriched DEGs termed by Gene Ontology(GO) Biological Process revealed that the upregulated genes in flat group are mainly related to the regulation of cell movement, including extracellular matrix organization, cell adhesion, and cell migration. The well-known cardiac maturation markers such as MYH7 and TNNI3 showed significantly upregulated in align group, as well as cardiac structural(MLC2, TNNT2, GJA1), and calcium handling relevant genes( CASQ2, CAMK2B, CAV3) also showed a higher expression than that of in flat group. The most up-regulated gene sets related pathways in align group include cardiac development and heart morphogenesis, negative regulation of binding, and microtubule-based process. In contrast, the relative down-regulated gene enriched pathways in align group are mostly involved in KRT gene family, which also plays a role in cell movement Conclusions: hiPS-CMs by culturing on the aligned pattern for a short-term have facilitated maturation of hiPS-CMs. The up-regulated gene sets in align group related to regulating cell cycle pathways. The genome-wide expression analysis provided insights into the underlying molecular pathways/signaling involved in the specific topographic stimuli induced maturation of hiPS-CMs
2021-02-10 | GSE162707 | GEO
Project description:Directed differentiation of human induced pluripotent stem cells creates billions of patient-specific cells. However, the differentiated derivatives of hiPS cells are immature relative to adult counterparts. Proliferative progenitor cells in these protocols are also therapeutically promising, but progenitors with enhanced potential for maturation have not been discovered. We hypothesized that brief epigenetic and innate immune modulation with polyinosinc cytidilic acid (pIC) could create cardiac progenitors with enhanced later maturation. Progenitor transcriptomic analysis revealed increases in cardiac crescent-like proliferative notch signaling by Jagged1, and activated progenitors spontaneously gave rise to cardiomyocytes with enhanced maturity and conductive microtissue with arrhythmogenic resistance. Furthermore, activated cardiac progenitors improved survival in animals after myocardial infarction compared to untreated progenitors. Our data suggest that future organization and maturation of cardiomyocytes are impacted by earlier pre-cardiomyocyte developmental signaling, and that progenitors modifying these pathways have translational promise. The forward epigenetic and immune modulation approach here utilized may be applicable to other cell lineages as well.
2018-08-01 | GSE98941 | GEO
Project description:The developmental potential of human pluripotent stem cells suggests that they can produce disease-relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Towards this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines. We used microarrays to compare the gene expression profiles between human ES cell lines, iPS cell lines, fibroblasts and embryoid bodies, and to identify cell-line specific outlier genes. 20 human ES cell lines (HUES1, HUES3, HUES6, HUES8, HUES9, HUES13, HUES28, HUES44, HUES45, HUES48, HUES49, HUES53, HUES62, HUES63, HUES64, HUES65, HUES66, H1, H7, H9), 12 human iPS cell lines (hiPS 11a, hiPS 11b, hiPS 11c, hiPS 15b, hiPS 17a, hiPS 17b, hiPS 18a, hiPS 18b, hiPS 18c, hiPS 20b, hiPS 27b, hiPS 27e), 6 fibroblast cell lines (hFib 11, hFib 15, hFib 17, hFib 18, hFib 20, hFib 27), 5 embryoid bodies (hEB16d HUES1, hEB16d HUES3, hEB16d HUES6, hEB16d HUES45, hEB16d H1)
2011-02-03 | E-GEOD-25970 | biostudies-arrayexpress
Project description:dCas9KRAB and gRNA targeting HERVK TEs were used to repress the TEs in stem cells (hiPS) and in differentiated induced neurons (iN). As control, dCas9KRAB alone without gRNA was used.
2020-01-24 | GSE144151 | GEO
Project description:Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human embryonic stem (hES) cells differ significantly, which affects hiPS cell function. These differences include epigenetic memory and aberrations that emerge during reprogramming, for which the mechanisms remain unknown. Here we characterized the persistence and emergence of these epigenetic differences by performing genome-wide DNA methylation profiling throughout primed and naive reprogramming of human somatic cells to hiPS cells. We found that reprogramming-induced epigenetic aberrations emerge midway through primed reprogramming, whereas DNA demethylation begins early in naive reprogramming. Using this knowledge, we developed a transient-naive-treatment (TNT) reprogramming strategy that emulates the embryonic epigenetic reset. We show that the epigenetic memory in hiPS cells is concentrated in cell of origin-dependent repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. TNT reprogramming reconfigures these domains to a hES cell-like state and does not disrupt genomic imprinting. Using an isogenic system, we demonstrate that TNT reprogramming can correct the transposable element overexpression and differential gene expression seen in conventional hiPS cells, and that TNT-reprogrammed hiPS and hES cells show similar differentiation efficiencies. Moreover, TNT reprogramming enhances the differentiation of hiPS cells derived from multiple cell types. Thus, TNT reprogramming corrects epigenetic memory and aberrations, producing hiPS cells that are molecularly and functionally more similar to hES cells than conventional hiPS cells. We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory.
2023-10-25 | GSE246103 | GEO
Project description:Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human embryonic stem (hES) cells differ significantly, which affects hiPS cell function. These differences include epigenetic memory and aberrations that emerge during reprogramming, for which the mechanisms remain unknown. Here we characterized the persistence and emergence of these epigenetic differences by performing genome-wide DNA methylation profiling throughout primed and naive reprogramming of human somatic cells to hiPS cells. We found that reprogramming-induced epigenetic aberrations emerge midway through primed reprogramming, whereas DNA demethylation begins early in naive reprogramming. Using this knowledge, we developed a transient-naive-treatment (TNT) reprogramming strategy that emulates the embryonic epigenetic reset. We show that the epigenetic memory in hiPS cells is concentrated in cell of origin-dependent repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. TNT reprogramming reconfigures these domains to a hES cell-like state and does not disrupt genomic imprinting. Using an isogenic system, we demonstrate that TNT reprogramming can correct the transposable element overexpression and differential gene expression seen in conventional hiPS cells, and that TNT-reprogrammed hiPS and hES cells show similar differentiation efficiencies. Moreover, TNT reprogramming enhances the differentiation of hiPS cells derived from multiple cell types. Thus, TNT reprogramming corrects epigenetic memory and aberrations, producing hiPS cells that are molecularly and functionally more similar to hES cells than conventional hiPS cells. We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory.
2023-08-11 | GSE239579 | GEO
Project description:Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human embryonic stem (hES) cells differ significantly, which affects hiPS cell function. These differences include epigenetic memory and aberrations that emerge during reprogramming, for which the mechanisms remain unknown. Here we characterized the persistence and emergence of these epigenetic differences by performing genome-wide DNA methylation profiling throughout primed and naive reprogramming of human somatic cells to hiPS cells. We found that reprogramming-induced epigenetic aberrations emerge midway through primed reprogramming, whereas DNA demethylation begins early in naive reprogramming. Using this knowledge, we developed a transient-naive-treatment (TNT) reprogramming strategy that emulates the embryonic epigenetic reset. We show that the epigenetic memory in hiPS cells is concentrated in cell of origin-dependent repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. TNT reprogramming reconfigures these domains to a hES cell-like state and does not disrupt genomic imprinting. Using an isogenic system, we demonstrate that TNT reprogramming can correct the transposable element overexpression and differential gene expression seen in conventional hiPS cells, and that TNT-reprogrammed hiPS and hES cells show similar differentiation efficiencies. Moreover, TNT reprogramming enhances the differentiation of hiPS cells derived from multiple cell types. Thus, TNT reprogramming corrects epigenetic memory and aberrations, producing hiPS cells that are molecularly and functionally more similar to hES cells than conventional hiPS cells. We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory.
2023-08-11 | GSE178779 | GEO