scholarly journals Dynamic Methylation of an L1 Transduction Family during Reprogramming and Neurodifferentiation

2019 ◽  
Vol 39 (7) ◽  
Author(s):  
Carmen Salvador-Palomeque ◽  
Francisco J. Sanchez-Luque ◽  
Patrick R. J. Fortuna ◽  
Adam D. Ewing ◽  
Ernst J. Wolvetang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
De Novo ◽  
Germ Line ◽  
Somatic Mosaicism ◽  
Induced Pluripotent Stem Cell ◽  
Early Embryo ◽  
Individual Genome ◽  
Induced Pluripotent

ABSTRACT The retrotransposon LINE-1 (L1) is a significant source of endogenous mutagenesis in humans. In each individual genome, a few retrotransposition-competent L1s (RC-L1s) can generate new heritable L1 insertions in the early embryo, primordial germ line, and germ cells. L1 retrotransposition can also occur in the neuronal lineage and cause somatic mosaicism. Although DNA methylation mediates L1 promoter repression, the temporal pattern of methylation applied to individual RC-L1s during neurogenesis is unclear. Here, we identified a de novo L1 insertion in a human induced pluripotent stem cell (hiPSC) line via retrotransposon capture sequencing (RC-seq). The L1 insertion was full-length and carried 5ʹ and 3ʹ transductions. The corresponding donor RC-L1 was part of a large and recently active L1 transduction family and was highly mobile in a cultured-cell L1 retrotransposition reporter assay. Notably, we observed distinct and dynamic DNA methylation profiles for the de novo L1 and members of its extended transduction family during neuronal differentiation. These experiments reveal how a de novo L1 insertion in a pluripotent stem cell is rapidly recognized and repressed, albeit incompletely, by the host genome during neurodifferentiation, while retaining potential for further retrotransposition.

scholarly journals Investigation of de novo mutations in a schizophrenia case-parent trio by induced pluripotent stem cell-based in vitro disease modeling: convergence of schizophrenia- and autism-related cellular phenotypes

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Edit Hathy ◽  
Eszter Szabó ◽  
Nóra Varga ◽  
Zsuzsa Erdei ◽  
Csongor Tordai ◽  
...  
Keyword(s):  
Stem Cell ◽  
Dentate Gyrus ◽  
Pluripotent Stem Cell ◽  
Disease Modeling ◽  
De Novo ◽  
Disease Onset ◽  
Induced Pluripotent Stem Cell ◽  
Synaptic Function ◽  
De Novo Mutations ◽  
Induced Pluripotent

Abstract Background De novo mutations (DNMs) have been implicated in the etiology of schizophrenia (SZ), a chronic debilitating psychiatric disorder characterized by hallucinations, delusions, cognitive dysfunction, and decreased community functioning. Several DNMs have been identified by examining SZ cases and their unaffected parents; however, in most cases, the biological significance of these mutations remains elusive. To overcome this limitation, we have developed an approach of using induced pluripotent stem cell (iPSC) lines from each member of a SZ case-parent trio, in order to investigate the effects of DNMs in cellular progenies of interest, particularly in dentate gyrus neuronal progenitors. Methods We identified a male SZ patient characterized by early disease onset and negative symptoms, who is a carrier of 3 non-synonymous DNMs in genes LRRC7, KHSRP, and KIR2DL1. iPSC lines were generated from his and his parents’ peripheral blood mononuclear cells using Sendai virus-based reprogramming and differentiated into neuronal progenitor cells (NPCs) and hippocampal dentate gyrus granule cells. We used RNASeq to explore transcriptomic differences and calcium (Ca2+) imaging, cell proliferation, migration, oxidative stress, and mitochondrial assays to characterize the investigated NPC lines. Results NPCs derived from the SZ patient exhibited transcriptomic differences related to Wnt signaling, neuronal differentiation, axonal guidance and synaptic function, and decreased Ca2+ reactivity to glutamate. Moreover, we could observe increased cellular proliferation and alterations in mitochondrial quantity and morphology. Conclusions The approach of reprograming case-parent trios represents an opportunity for investigating the molecular effects of disease-causing mutations and comparing these in cell lines with reduced variation in genetic background. Our results are indicative of a partial overlap between schizophrenia and autism-related phenotypes in the investigated family. Limitations Our study investigated only one family; therefore, the generalizability of findings is limited. We could not derive iPSCs from two other siblings to test for possible genetic effects in the family that are not driven by DNMs. The transcriptomic and functional assays were limited to the NPC stage, although these variables should also be investigated at the mature neuronal stage.

scholarly journals Characterization of DNA Methylomic Signatures in Induced Pluripotent Stem Cells During Neuronal Differentiation

2021 ◽  
Vol 9 ◽  
Author(s):  
Jennifer Imm ◽  
Ehsan Pishva ◽  
Muhammadd Ali ◽  
Talitha L. Kerrigan ◽  
Aaron Jeffries ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cell ◽  
Neuronal Differentiation ◽  
Pluripotent Stem Cell ◽  
Interaction Network ◽  
Induced Pluripotent Stem Cell ◽  
Gene Interaction ◽  
Gene Interaction Network ◽  
Induced Pluripotent

In development, differentiation from a pluripotent state results in global epigenetic changes, although the extent to which this occurs in induced pluripotent stem cell-based neuronal models has not been extensively characterized. In the present study, induced pluripotent stem cell colonies (33Qn1 line) were differentiated and collected at four time-points, with DNA methylation assessed using the Illumina Infinium Human Methylation EPIC BeadChip array. Dynamic changes in DNA methylation occurring during differentiation were investigated using a data-driven trajectory inference method. We identified a large number of Bonferroni-significant loci that showed progressive alterations in DNA methylation during neuronal differentiation. A gene–gene interaction network analysis identified 60 densely connected genes that were influential in the differentiation of neurons, with STAT3 being the gene with the highest connectivity.

scholarly journals Investigation of de novo mutations in a schizophrenia case-parent trio by induced pluripotent stem cell based in vitro disease-modeling: Convergence of schizophrenia and autism-related cellular phenotypes

2020 ◽  
Author(s):  
Edit Hathy ◽  
Eszter Szabó ◽  
Nóra Varga ◽  
Zsuzsa Erdei ◽  
Csongor Tordai ◽  
...  
Keyword(s):  
Stem Cell ◽  
Dentate Gyrus ◽  
Pluripotent Stem Cell ◽  
Disease Modeling ◽  
De Novo ◽  
Disease Onset ◽  
Induced Pluripotent Stem Cell ◽  
Synaptic Function ◽  
De Novo Mutations ◽  
Induced Pluripotent

Abstract Background: De novo mutations (DNMs) have been implicated in the etiology of schizophrenia (SZ), a chronic debilitating psychiatric disorder characterized by hallucinations, delusions, cognitive dysfunction and decreased community functioning. Several DNMs have been identified by examining SZ cases and their unaffected parents, however in most cases the biological significance of these mutations remains elusive. To overcome this limitation, we have developed an approach of using induced pluripotent stem cell (iPSC) lines from each member of a SZ case-parent trio, in order to investigate the effects of DNMs in cellular progenies of interest, particularly in dentate gyrus neuronal progenitors.Methods: We identified a male SZ patient characterized by early disease onset and negative symptoms, who is a carrier of 3 non-synonymous DNMs in genes LRRC7, KHSRP, and KIR2DL1. iPSC lines were generated from his and his parents’ peripheral blood mononuclear cells using Sendai virus-based reprogramming and differentiated into neuronal progenitor cells (NPCs) and hippocampal dentate gyrus granule cells. We used RNASeq to explore transcriptomic differences, and calcium (Ca2+) imaging, cell proliferation, migration, oxidative stress, and mitochondrial assays to characterize the investigated NPC lines. Results: NPCs derived from the SZ patient exhibited transcriptomic differences related to Wnt-signaling, neuronal differentiation, axonal guidance and synaptic function, and decreased Ca2+ reactivity to glutamate. Moreover, we could observe increased cellular proliferation, and alterations in mitochondrial quantity and morphology.Conclusions: The approach of reprograming case-parent trios represents an opportunity for investigating the molecular effects of disease-causing mutations, and comparing these in cell lines with reduced variation in genetic background. Our results are indicative of a partial overlap between schizophrenia and autism-related phenotypes in the investigated family.Limitations: Our study investigated only one family, therefore the generalizability of findings is limited. We could not derive iPSCs from two other siblings to test for possible genetic effects in the family that are not driven by DNMs. The transcriptomic and functional assays were limited to the NPC stage, although these variables should also be investigated at the mature neuronal stage.

scholarly journals An Important Role for DNMT3A-Mediated DNA Methylation in Cardiomyocyte Metabolism and Contractility

Circulation ◽  
2020 ◽  
Vol 142 (16) ◽  
pp. 1562-1578
Author(s):  
Alexandra Madsen ◽  
Grit Höppner ◽  
Julia Krause ◽  
Marc N. Hirt ◽  
Sandra D. Laufer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Stem Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Heart Tissue ◽  
Engineered Heart Tissue ◽  
Induced Pluripotent

Background: DNA methylation acts as a mechanism of gene transcription regulation. It has recently gained attention as a possible therapeutic target in cardiac hypertrophy and heart failure. However, its exact role in cardiomyocytes remains controversial. Thus, we knocked out the main de novo DNA methyltransferase in cardiomyocytes, DNMT3A, in human induced pluripotent stem cells. Functional consequences of DNA methylation-deficiency under control and stress conditions were then assessed in human engineered heart tissue from knockout human induced pluripotent stem cell–derived cardiomyocytes. Methods: DNMT3A was knocked out in human induced pluripotent stem cells by CRISPR/Cas9gene editing. Fibrin-based engineered heart tissue was generated from knockout and control human induced pluripotent stem cell–derived cardiomyocytes. Development and baseline contractility were analyzed by video-optical recording. Engineered heart tissue was subjected to different stress protocols, including serum starvation, serum variation, and restrictive feeding. Molecular, histological, and ultrastructural analyses were performed afterward. Results: Knockout of DNMT3A in human cardiomyocytes had three main consequences for cardiomyocyte morphology and function: (1) Gene expression changes of contractile proteins such as higher atrial gene expression and lower MYH7/MYH6 ratio correlated with different contraction kinetics in knockout versus wild-type; (2) Aberrant activation of the glucose/lipid metabolism regulator peroxisome proliferator-activated receptor gamma was associated with accumulation of lipid vacuoles within knockout cardiomyocytes; (3) Hypoxia-inducible factor 1α protein instability was associated with impaired glucose metabolism and lower glycolytic enzyme expression, rendering knockout-engineered heart tissue sensitive to metabolic stress such as serum withdrawal and restrictive feeding. Conclusion: The results suggest an important role of DNA methylation in the normal homeostasis of cardiomyocytes and during cardiac stress, which could make it an interesting target for cardiac therapy.

scholarly journals Investigation of de novo mutations in a schizophrenia case-parent trio by induced pluripotent stem cell based in vitro disease-modeling: Convergence of schizophrenia and autism-related cellular phenotypes

2020 ◽  
Author(s):  
Edit Hathy ◽  
Eszter Szabó ◽  
Nóra Varga ◽  
Zsuzsa Erdei ◽  
Csongor Tordai ◽  
...  
Keyword(s):  
Stem Cell ◽  
Dentate Gyrus ◽  
Pluripotent Stem Cell ◽  
Disease Modeling ◽  
De Novo ◽  
Disease Onset ◽  
Induced Pluripotent Stem Cell ◽  
Synaptic Function ◽  
De Novo Mutations ◽  
Induced Pluripotent

Abstract Background: De novo mutations (DNMs) have been implicated in the etiology of schizophrenia (SZ), a chronic debilitating psychiatric disorder characterized by hallucinations, delusions, cognitive dysfunction and decreased community functioning. Several DNMs have been identified by examining SZ cases and their unaffected parents, however in most cases the biological significance of these mutations remains elusive. To overcome this limitation, we have developed an approach of using induced pluripotent stem cell (iPSC) lines from each member of a SZ case-parent trio, in order to investigate the effects of DNMs in cellular progenies of interest, particularly in dentate gyrus neuronal progenitors. Methods: We identified a male SZ patient characterized by early disease onset and negative symptoms, who is a carrier of 3 non-synonymous DNMs in genes LRRC7, KHSRP, and KIR2DL1. iPSC lines were generated from his and his parents’ peripheral blood mononuclear cells using Sendai virus-based reprogramming and differentiated into neuronal progenitor cells (NPCs) and hippocampal dentate gyrus granule cells. We used RNASeq to explore transcriptomic differences, and calcium (Ca 2+ ) imaging, cell proliferation, migration, oxidative stress, and mitochondrial assays to characterize the investigated NPC lines. Results: NPCs derived from the SZ patient exhibited transcriptomic differences related to Wnt-signaling, neuronal differentiation, axonal guidance and synaptic function, and decreased Ca 2+ reactivity to glutamate. Moreover, we could observe increased cellular proliferation, and alterations in mitochondrial quantity and morphology. Conclusions: The approach of reprograming case-parent trios represents an opportunity for investigating the molecular effects of disease-causing mutations, and comparing these in cell lines with reduced variation in genetic background. Our results are indicative of a partial overlap between schizophrenia and autism-related phenotypes in the investigated family. Limitations: Our study investigated only one family, therefore the generalizability of findings is limited. We could not derive iPSCs from two other siblings to test for possible genetic effects in the family that are not driven by DNMs. The transcriptomic and functional assays were limited to the NPC stage, although these variables should also be investigated at the mature neuronal stage.

Sign in / Sign up

Export Citation Format

Share Document