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Comparison of gene-specific DNA methylation patterns in equine induced pluripotent stem cell lines with cells derived from equine adult and fetal tissues.

ABSTRACT: Cellular pluripotency is associated with expression of the homeobox transcription factor genes NANOG, SOX2, and POU5F1 (OCT3/4 protein). Some reports suggest that mesenchymal progenitor cells (MPCs) may express increased quantities of these genes, creating the possibility that MPCs are more "pluripotent" than other adult cell types. The objective of this study was to determine whether equine bone marrow-derived MPCs had gene expression or DNA methylation patterns that differed from either early fetal-derived or terminally differentiated adult cells. Specifically, this study compared DNA methylation of the NANOG and SOX2 promoter regions and concurrent gene expression of NANOG, SOX2, and POU5F1 in equine induced pluripotent stem (iPS) cells, fetal fibroblasts, fetal brain cells, adult chondrocytes, and MPCs. Results indicate that NANOG and POU5F1 were not detectable in appreciable quantities in tissues other than the equine iPS cell lines. Equine iPS cells expressed large quantities of all three genes examined. Significantly increased quantities of SOX2 were noted in iPS cells and both fetal-derived cell types compared with adult cells. MPCs and adult chondrocytes expressed equivalent, low quantities of SOX2. Further, NANOG and SOX2 expression inversely correlated with the DNA methylation pattern in the promoter region, such that as gene expression increased, DNA methylation decreased. The equine iPS cell lines examined demonstrated DNA methylation and gene expression patterns that were consistent with pluripotency features described in other species. Results do not support previous reports that NANOG, SOX2, and POU5F1 are poised for increased activity in MPCs compared with other adult cells.

SUBMITTER: Hackett CH

PROVIDER: S-EPMC3376462 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Comparison of gene-specific DNA methylation patterns in equine induced pluripotent stem cell lines with cells derived from equine adult and fetal tissues.

Hackett Catherine H CH Greve Line L Novakofski Kira D KD Fortier Lisa A LA

Stem cells and development 20111121 10

Cellular pluripotency is associated with expression of the homeobox transcription factor genes NANOG, SOX2, and POU5F1 (OCT3/4 protein). Some reports suggest that mesenchymal progenitor cells (MPCs) may express increased quantities of these genes, creating the possibility that MPCs are more "pluripotent" than other adult cell types. The objective of this study was to determine whether equine bone marrow-derived MPCs had gene expression or DNA methylation patterns that differed from either early ...[more]

PMID: 21988203

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Project description:IntroductionPluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patient-specific induced pluripotent stem cells (iPSCs) has become possible in multiple species.ObjectivesWe hypothesize that different cell types respond differently to the reprogramming process; thus, the goals of this study were to isolate and characterize equine adult and fetal cells and induce these cells to pluripotency for future regenerative and translational purposes.MethodsAdult equine fibroblasts (eFibros) and mesenchymal cells derived from the bone marrow (eBMmsc), adipose tissue (eADmsc), and umbilical cord tissue (eUCmsc) were isolated, their multipotency was characterized, and the cells were induced in vitro into pluripotency (eiPSCs). eiPSCs were generated through a lentiviral system using the factors OCT4, SOX2, c-MYC, and KLF4. The morphology and in vitro pluripotency maintenance potential (alkaline phosphatase detection, embryoid body formation, in vitro spontaneous differentiation, and expression of pluripotency markers) of the eiPSCs were characterized. Additionally, a miRNA profile analysis of the mesenchymal and eiPSCs was performed.ResultsMultipotent cells were successfully isolated, but the eBMmsc failed to generate eiPSCs. The eADmsc-, eUCmsc-, and eFibros-derived iPSCs were positive for alkaline phosphatase, OCT4 and NANOG, were exclusively dependent on bFGF, and formed embryoid bodies. The miRNA profile revealed a segregated pattern between the eiPSCs and multipotent controls: the levels of miR-302/367 and the miR-92 family were increased in the eiPSCs, while the levels of miR-23, miR-27, and miR-30, as well as the let-7 family were increased in the nonpluripotent cells.ConclusionsWe were able to generate bFGF-dependent iPSCs from eADmsc, eUCmsc, and eFibros with human OSKM, and the miRNA profile revealed that clonal lines may respond differently to the reprogramming process.

Project description:BackgroundDNA methylation is an important epigenetic control mechanism that has been shown to be associated with gene silencing through the course of development, maturation and aging. However, only limited data are available regarding the relationship between methylation and gene expression in human development.ResultsWe analyzed the methylome and transcriptome of three human fetal liver samples (gestational age 20-22 weeks) and three adult human liver samples. Genes whose expression differed between fetal and adult numbered 7,673. Adult overexpression was associated with metabolic pathways and, in particular, cytochrome P450 enzymes while fetal overexpression reflected enrichment for DNA replication and repair. Analysis for DNA methylation using the Illumina Infinium 450 K HumanMethylation BeadChip showed that 42% of the quality filtered 426,154 methylation sites differed significantly between adult and fetal tissue (q ≤ 0.05). Differences were small; 69% of the significant sites differed in their mean methylation beta value by ≤0.2. There was a trend among all sites toward higher methylation in the adult samples with the most frequent difference in beta being 0.1. Characterization of the relationship between methylation and expression revealed a clear difference between fetus and adult. Methylation of genes overexpressed in fetal liver showed the same pattern as seen for genes that were similarly expressed in fetal and adult liver. In contrast, adult overexpressed genes showed fetal hypermethylation that differed from the similarly expressed genes. An examination of gene region-specific methylation showed that sites proximal to the transcription start site or within the first exon with a significant fetal-adult difference in beta (>0.2) showed an inverse relationship with gene expression.ConclusionsNearly half of the CpGs in human liver show a significant difference in methylation comparing fetal and adult samples. Sites proximal to the transcription start site or within the first exon that show a transition from hypermethylation in the fetus to hypomethylation or intermediate methylation in the adult are associated with inverse changes in gene expression. In contrast, increases in methylation going from fetal to adult are not associated with fetal-to-adult decreased expression. These findings indicate fundamentally different roles for and/or regulation of DNA methylation in human fetal and adult liver.

Project description:BackgroundTendon injuries occur frequently in human and equine athletes. Treatment options are limited, and the prognosis is often poor with functionally deficient scar tissue resulting. Fetal tendon injuries in contrast are capable of healing without forming scar tissue. Embryonic stem cells (ESCs) may provide a potential cellular therapeutic to improve adult tendon regeneration; however, whether they can mimic the properties of fetal tenocytes is unknown. To this end, understanding the unique expression profile of normal adult and fetal tenocytes is crucial to allow validation of ESC-derived tenocytes as a cellular therapeutic.MethodsEquine adult, fetal and ESC-derived tenocytes were cultured in a three-dimensional environment, with histological, morphological and transcriptomic differences compared. Additionally, the effects on gene expression of culturing adult and fetal tenocytes in either conventional two-dimensional monolayer culture or three-dimensional culture were compared using RNA sequencing.ResultsNo qualitative differences in three-dimensional tendon constructs generated from adult, fetal and ESCs were found using histological and morphological analysis. However, genome-wide transcriptomic analysis using RNA sequencing revealed that ESC-derived tenocytes' transcriptomic profile more closely resembled fetal tenocytes as opposed to adult tenocytes. Furthermore, this study adds to the growing evidence that monolayer cultured cells' gene expression profiles converge, with adult and fetal tenocytes having only 10 significantly different genes when cultured in this manner. In contrast, when adult and fetal tenocytes were cultured in 3D, large distinctions in gene expression between these two developmental stages were found, with 542 genes being differentially expressed.ConclusionThe information provided in this study makes a significant contribution to the investigation into the differences between adult reparative and fetal regenerative cells and supports the concept of using ESC-derived tenocytes as a cellular therapy. Comparing two- and three-dimensional culture also indicates three-dimensional culture as being a more physiologically relevant culture system for determining transcriptomic difference between the same cell types from different developmental stages.

Project description:BackgroundDNA methylation is an epigenetic modification that plays an important role during mammalian development. Around birth in humans, the main site of red blood cell production moves from the fetal liver to the bone marrow. DNA methylation changes at the β-globin locus and a switch from fetal to adult hemoglobin production characterize this transition. Understanding this globin switch may improve the treatment of patients with sickle cell disease and β-thalassemia, two of the most common Mendelian diseases in the world. The goal of our study was to describe and compare the genome-wide patterns of DNA methylation in fetal and adult human erythroblasts.MethodsWe used the Illumina HumanMethylation 450 k BeadChip to measure DNA methylation at 402,819 CpGs in ex vivo-differentiated erythroblasts from 12 fetal liver and 12 bone marrow CD34+ donors.ResultsWe identified 5,937 differentially methylated CpGs that overlap with erythroid enhancers and binding sites for erythropoiesis-related transcription factors. Combining this information with genome-wide association study results, we show that erythroid enhancers define particularly promising genomic regions to identify new genetic variants associated with fetal hemoglobin (HbF) levels in humans. Many differentially methylated CpGs are located near genes with unanticipated roles in red blood cell differentiation and proliferation. For some of these new candidate genes, we confirm the correlation between DNA methylation and gene expression levels in red blood cell progenitors. We also provide evidence that DNA methylation and genetic variation at the β-globin locus independently control globin gene expression in adult erythroblasts.ConclusionsOur DNA methylome maps confirm the widespread dynamic changes in DNA methylation that occur during human erythropoiesis. These changes tend to happen near erythroid enhancers, further highlighting their importance in erythroid regulation and HbF production. Finally, DNA methylation may act independently of the transcription factor BCL11A to repress fetal hemoglobin production. This provides cues on strategies to more efficiently re-activate HbF production in sickle cell disease and β-thalassemia patients.

Project description:Progressive vision loss, caused by retinal degenerative (RD) diseases such as age-related macular degeneration, retinitis pigmentosa, and Leber congenital amaurosis, severely impacts quality of life and affects millions of people. Finding efficient treatment for blinding diseases is among the greatest unmet clinical needs. The evagination of optic vesicles from developing pluripotent stem cell-derived neuroepithelium and self-organization, lamination, and differentiation of retinal tissue in a dish generated considerable optimism for developing innovative approaches for treating RD diseases, which previously were not feasible. Retinal organoids may be a limitless source of multipotential retinal progenitors, photoreceptors (PRs), and the whole retinal tissue, which are productive approaches for developing RD disease therapies. In this study we compared the distribution and expression level of molecular markers (genetic and epigenetic) in human fetal retina (age 8-16 weeks) and human embryonic stem cell (hESC)-derived retinal tissue (organoids) by immunohistochemistry, RNA-seq, flow cytometry, and mass-spectrometry (to measure methylated and hydroxymethylated cytosine level), with a focus on PRs to evaluate the clinical application of hESC-retinal tissue for vision restoration. Our results revealed high correlation in gene expression profiles and histological profiles between human fetal retina (age 8-13 weeks) and hESC-derived retinal tissue (10-12 weeks). The transcriptome signature of hESC-derived retinal tissue from retinal organoids maintained for 24 weeks in culture resembled the transcriptome of human fetal retina of more advanced developmental stages. The histological profiles of 24 week-old hESC-derived retinal tissue displayed mature PR immunophenotypes and presence of developing inner and outer segments. Collectively, our work highlights the similarity of hESC-derived retinal tissue at early stages of development (10 weeks), and human fetal retina (age 8-13 weeks) and it supports the development of regenerative medicine therapies aimed at using tissue from hESC-derived retinal organoids (hESC-retinal implants) for mitigating vision loss.

Project description:The development of 3D cerebral organoid technology using human-induced pluripotent stem cells (iPSCs) provides a promising platform to study how brain diseases are appropriately modeled and treated. So far, understanding of the characteristics of organoids is still in its infancy. The current study profiled, for the first time, the electrophysiological properties of organoids at molecular and cellular levels and dissected the potential age equivalency of 2-month-old organoids to human ones by a comparison of gene expression profiles among cerebral organoids, human fetal and adult brains. Cerebral organoids exhibit heterogeneous gene and protein markers of various brain cells, such as neurons, astrocytes, and vascular cells (endothelial cells and smooth muscle cells) at 2 months, and increases in neural, glial, vascular, and channel-related gene expression over a 2-month differentiation course. Two-month organoids exhibited action potentials, multiple channel activities, and functional electrophysiological responses to the anesthetic agent propofol. A bioinformatics analysis of 20,723 gene expression profiles showed the similar distance of gene profiles in cerebral organoids to fetal and adult brain tissues. The subsequent Ingenuity Pathway Analysis (IPA) of select canonical pathways related to neural development, network formation, and electrophysiological signaling, revealed that only calcium signaling, cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) signaling in neurons, glutamate receptor signaling, and synaptogenesis signaling were predicted to be downregulated in cerebral organoids relative to fetal samples. Nearly all cerebral organoid and fetal pathway phenotypes were predicted to be downregulated compared with adult tissue. This novel study highlights dynamic development, cellular heterogeneity and electrophysiological activity. In particular, for the first time, electrophysiological drug response recapitulates what occurs in vivo, and neural characteristics are predicted to be highly similar to the human brain, further supporting the promising application of the cerebral organoid system for the modeling of the human brain in health and disease. Additionally, the studies from these characterizations of cerebral organoids in multiple levels and the findings from gene comparisons between cerebral organoids and humans (fetuses and adults) help us better understand this cerebral organoid-based cutting-edge platform and its wide uses in modeling human brain in terms of health and disease, development, and testing drug efficacy and toxicity.

Dataset Information

Comparison of gene-specific DNA methylation patterns in equine induced pluripotent stem cell lines with cells derived from equine adult and fetal tissues.

Publications

Comparison of gene-specific DNA methylation patterns in equine induced pluripotent stem cell lines with cells derived from equine adult and fetal tissues.

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