Pressure-Driven Mitochondrial Transfer Pipeline Generates Mammalian Cells of Desired Genetic Combinations and Fates
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE115871 | GEO | 2020/12/29
REPOSITORIES: GEO
Similar Datasets
Project description:It has been reported that human mesenchymal stem cells (MSCs) can transfer mitochondria to the cells with severely compromised mitochondrial function. We tested whether MSCs transfer mitochondria to the cells under several different conditions of mitochondrial dysfunction, including human pathogenic mitochondrial DNA (mtDNA) mutations. Using biochemical selection methods, we found that exponentially growing cells in restrictive media (uridine and bromodeoxyuridine [BrdU]+) after coculture of MSCs (uridine-independent and BrdU-sensitive) and 143B-derived cells with severe mitochondrial dysfunction induced by either long-term ethidium bromide treatment or short-term rhodamine 6G (R6G) treatment (uridine-dependent but BrdU-resistant). The exponentially growing cells had nuclear DNA fingerprint patterns identical to 143B, and a sequence of mtDNA identical to the MSCs. Since R6G causes rapid and irreversible damage to mitochondria without the removal of mtDNA, the mitochondrial function appears to be restored through a direct transfer of mitochondria rather than mtDNA alone. Conditioned media, which were prepared by treating mtDNA-less 143B 0 cells under uridine-free condition, induced increased chemotaxis in MSC, which was also supported by transcriptome analysis. A chemotaxis inhibitory agent blocked mitochondrial transfer phenomenon in the above condition. However, we could not find any evidence of mitochondrial transfer to the cells harboring human pathogenic mtDNA mutations (A3243G mutation or 4,977 bp deletion). Thus, the mitochondrial transfer is limited to the condition of a near total absence of mitochondrial function. Elucidation of the mechanism of mitochondrial transfer will help us create a potential “cell therapy-based mitochondrial restoration or mitochondrial gene therapy” for human diseases caused by mitochondrial dysfunction. time series
2011-11-22 | E-GEOD-33831 | biostudies-arrayexpress
Project description:Mitochondrial biogenesis is under the control of two different genetic systems: the nuclear genome (nDNA) and the mitochondrial genome (mtDNA). mtDNA is a circular genome of 16.6 kb encoding 13 of the approximately 90 subunits that form the respiratory chain, the remaining ones being encoded by the nuclear genome (nDNA). Eukaryotic cells are able to monitor and respond to changes in mitochondrial function through alterations in nuclear gene expression, a phenomenon first defined in yeast and known as retrograde regulation. With this experiment we aimed to identify the set of nuclear genes that significantly change their expression level in response to depletion of mtDNA. Experiment Overall Design: We used Affymetrix HG-U133A GeneChips to study the transcriptome of two human cell lines, 143BTK- and A549, which had been entirely depleted of mtDNA (rho0 cells), and compared it with the corresponding undepleted parental cells (rho+ cells). Three independent biological replicates were analyzed for each cell line and treatment group.
2009-05-11 | E-GEOD-14900 | biostudies-arrayexpress
Project description:There is evidence for the on-going recurrent transfer of mitochondrial DNA (mtDNA) into the nucleus in both germ line and somatic cells. However, the outcomes associated with the transfer of mtDNA into somatic cell nuclei are poorly understood. High-resolution Chromosome Conformation Capture (HiC) techniques, which are used to identify global patterns of chromatin interactions, regularly capture physical interactions between mitochondrial and nuclear DNA (i.e. mito-nDNA interactions) in mammalian cells. These mito-nDNA interactions are routinely considered a consequence of nonspecific ligation events during chromatin library preparation. Here, we have evaluated mito-nDNA interactions captured by HiC in six human cell lines, and by Circular Chromosome Conformation Capture (4C) in mouse cortical astrocytes. We show that mito-nDNA interactions are statistically significant and shared between biological and technical replicates in the HiC and 4C experiments. The most frequent interactions between mtDNA and nuclear loci in the HiC and 4C data occur with repetitive DNA sequences including the centromeric regions in the six human cell lines and 18S rDNA in mouse cortical astrocytes. Such findings confirm previous observations of mtDNA forming interactions with rDNA genes in budding yeast and centomeres in rat bone marrow cells. Finally the mitochondrial D-loop tends to be enriched in the captured mito-nDNA interactions. Collectively our results imply a degree of selective regulation in the identity of the interacting mitochondrial partners confirming that mito-nDNA interactions in mammalian cells are not random.
2017-11-21 | GSE77610 | GEO
Project description:HEK293 cells with CRISPR KO were analyzed using RNA-Seq. Abstract: Mitochondrial DNA copy number (mtDNA-CN) has been associated with a variety of aging-related diseases, including all-cause mortality. However, the mechanism by which mtDNA-CN influences disease is not currently understood. One such mechanism may be through regulation of nuclear gene expression via the modification of nuclear DNA (nDNA) methylation. To investigate this hypothesis, we assessed the relationship between mtDNA-CN and nDNA methylation in 2,507 African American (AA) and European Americans (EA) participants from the Atherosclerosis Risk in Communities (ARIC) study using the Infinium Human Methylation 450K Beadchip (485,764 CpGs). Thirty-four independent CpGs were associated with mtDNA-CN at genome-wide significance (P<5x10-8). To validate our findings we assayed an additional 2,528 participants from the Cardiovascular Health Study (CHS) (N=533) and Framingham Heart Study (FHS) (N=1995). Meta-analysis across all cohorts identified 6 mtDNA-CN associated CpGs to be validated across cohorts at genome-wide significance (P<5x10-8). Additionally, over half of these CpGs were associated with phenotypes known to be associated with mtDNA-CN, including CHD, CVD, and mortality. Experimental modification of mtDNA-CN through knockout via CRISPR-Cas9 of TFAM, a regulator of mtDNA replication, demonstrated that modulation of mtDNA-CN directly drives changes in nDNA methylation and gene expression of specific CpGs and nearby transcripts. Strikingly, the ‘neuroactive ligand receptor interaction’ KEGG pathway was found to be highly overrepresented in the ARIC cohort (P= 5.24x10-12), as well as the TFAM knockout methylation (P=4.41x10-4) and expression (P=4.30x10-4) studies. These results demonstrate that changes in mtDNA-CN influence nDNA methylation at specific loci and result in differential gene expression of specific genes, including those acting in the ‘neuroactive ligand receptor interaction’ pathway that may impact human health and disease via altered cell signaling.
2019-07-10 | GSE134048 | GEO
Project description:Mitochondrial biogenesis is under the control of two different genetic systems: the nuclear genome (nDNA) and the mitochondrial genome (mtDNA). mtDNA is a circular genome of 16.6 kb encoding 13 of the approximately 90 subunits that form the respiratory chain, the remaining ones being encoded by the nuclear genome (nDNA). Eukaryotic cells are able to monitor and respond to changes in mitochondrial function through alterations in nuclear gene expression, a phenomenon first defined in yeast and known as retrograde regulation. With this experiment we aimed to identify the set of nuclear genes that significantly change their expression level in response to depletion of mtDNA. Keywords: case-control
2009-05-01 | GSE14900 | GEO
Project description:It has been reported that human mesenchymal stem cells (MSCs) can transfer mitochondria to the cells with severely compromised mitochondrial function. We tested whether MSCs transfer mitochondria to the cells under several different conditions of mitochondrial dysfunction, including human pathogenic mitochondrial DNA (mtDNA) mutations. Using biochemical selection methods, we found that exponentially growing cells in restrictive media (uridine- and bromodeoxyuridine [BrdU]+) after coculture of MSCs (uridine-independent and BrdU-sensitive) and 143B-derived cells with severe mitochondrial dysfunction induced by either long-term ethidium bromide treatment or short-term rhodamine 6G (R6G) treatment (uridine-dependent but BrdU-resistant). The exponentially growing cells had nuclear DNA fingerprint patterns identical to 143B, and a sequence of mtDNA identical to the MSCs. Since R6G causes rapid and irreversible damage to mitochondria without the removal of mtDNA, the mitochondrial function appears to be restored through a direct transfer of mitochondria rather than mtDNA alone. Conditioned media, which were prepared by treating mtDNA-less 143B rho0 cells under uridine-free condition, induced increased chemotaxis in MSC, which was also supported by transcriptome analysis. A chemotaxis inhibitory agent blocked mitochondrial transfer phenomenon in the above condition. However, we could not find any evidence of mitochondrial transfer to the cells harboring human pathogenic mtDNA mutations (A3243G mutation or 4,977 bp deletion). Thus, the mitochondrial transfer is limited to the condition of a near total absence of mitochondrial function. Elucidation of the mechanism of mitochondrial transfer will help us create a potential “cell therapy-based mitochondrial restoration or mitochondrial gene therapy” for human diseases caused by mitochondrial dysfunction.
2011-11-22 | GSE33831 | GEO
Project description:Mitochondrial DNA copy number (mtDNA-CN) has been associated with a variety of aging-related diseases, including all-cause mortality. However, the mechanism by which mtDNA-CN influences disease is not currently understood. One such mechanism may be through regulation of nuclear gene expression via the modification of nuclear DNA (nDNA) methylation. To investigate this hypothesis, we assessed the relationship between mtDNA-CN and nDNA methylation in 2,507 African American (AA) and European Americans (EA) participants from the Atherosclerosis Risk in Communities (ARIC) study using the Infinium Human Methylation 450K Beadchip (485,764 CpGs). Thirty-four independent CpGs were associated with mtDNA-CN at genome-wide significance (P<5x10-8). To validate our findings we assayed an additional 2,528 participants from the Cardiovascular Health Study (CHS) (N=533) and Framingham Heart Study (FHS) (N=1995). Meta-analysis across all cohorts identified 6 mtDNA-CN associated CpGs to be validated ac ross cohorts at genome-wide significance (P<5x10-8). Additionally, over half of these CpGs were associated with phenotypes known to be associated with mtDNA-CN, including CHD, CVD, and mortality. Experimental modification of mtDNA-CN through knockout via CRISPR-Cas9 of TFAM, a regulator of mtDNA replication, demonstrated that modulation of mtDNA-CN directly drives changes in nDNA methylation and gene expression of specific CpGs and nearby transcripts. Strikingly, the ‘neuroactive ligand receptor interaction’ KEGG pathway was found to be highly overrepresented in the ARIC cohort (P= 5.24x10-12), as well as the TFAM knockout methylation (P=4.41x10-4) and expression (P=4.30x10-4) studies. These results demonstrate that changes in mtDNA-CN influence nDNA methylation at specific loci and result in differential gene expression of specific genes, including those acting in the ‘neuroactive ligand receptor interaction’ pathway that may impact human health and disease via altered cell signaling.
2019-07-09 | GSE133994 | GEO
Project description:Mitochondria are vital due to their principal role in energy production via oxidative phosphorylation (OXPHOS)1. Mitochondria carry their own genome (mtDNA) encoding critical genes involved in OXPHOS, therefore, mtDNA mutations cause fatal or severely debilitating disorders with limited treatment options. 2. Clinical manifestations of mtDNA disease vary based on mutation type and heteroplasmy levels i.e. presence of mutant and normal mtDNA within each cell. 3,4. We evaluated therapeutic concepts of generating genetically corrected pluripotent stem cells for patients with mtDNA mutations. We initially generated multiple iPS cell lines from a patient with mitochondrial encephalomyopathy and stroke-like episodes (MELAS) caused by a heteroplasmic 3243A>G mutation and a patient with Leigh disease carrying a homoplasmic 8993T>G mutation (Leigh-iPS). Due to spontaneous mtDNA segregation in proliferating fibroblasts, isogenic MELAS iPS cell lines were recovered containing exclusively wild type (wt) mtDNA with normal metabolic function. As expected, all iPS cells from the patient with Leigh disease were affected. Using somatic cell nuclear transfer (SCNT; Leigh-NT1), we then simultaneously replaced mutated mtDNA and generated pluripotent stem cells from the Leigh patient fibroblasts. In addition to reversing to a normal 8993G>T, oocyte derived donor mtDNA (human haplotype D4a) in Leigh-NT1 differed from the original haplotype (F1a) at a additional 47 nucleotide sites. Leigh-NT1 cells displayed normal metabolic function compared to impaired oxygen consumption and ATP production in Leigh-iPS cells or parental fibroblasts (Leigh-fib). We conclude that natural segregation of heteroplasmic mtDNA allows the generation of iPS cells with exclusively wild type mtDNA. Moreover, SCNT offers mitochondrial gene replacement strategy for patients with homoplasmic mtDNA disease.
2015-07-16 | GSE61390 | GEO
Project description:Somatic mitochondrial DNA (mtDNA) mutations contribute to the pathogenesis of age-related disorders, including myelodysplastic syndromes (MDS). The accumulation of mitochondria harboring mtDNA mutations in patients with these disorders suggests a failure of normal mitochondrial quality-control systems. The mtDNA-mutator mice acquire somatic mtDNA mutations via a targeted defect in the proofreading function of the mtDNA polymerase, PolgA, and develop macrocyticanemia similar to that of patients with MDS. We observed an unexpected defect in clearance of dysfunctional mitochondria at specific stages during erythroid maturation in hematopoietic cells from aged mtDNA-mutator mice. Mechanistically, aberrant activation of mechanistic target of rapamycin signaling and phosphorylation of uncoordinated 51-like kinase (ULK) 1 in mtDNA-mutator mice resulted in proteasome mediated degradation of ULK1 and inhibition of autophagy in erythroid cells. To directly evaluate the consequence of inhibiting autophagy on mitochondrial function in erythroid cells harboring mtDNA mutations in vivo, we deleted Atg7 from erythroid progenitors of wildtype and mtDNA-mutator mice. Genetic disruption of autophagy did not cause anemia in wild-type mice but accelerated the decline in mitochondrial respiration and development of macrocytic anemia in mtDNA-mutator mice. These findings highlight a pathological feedback loop that explains how dysfunctional mitochondria can escape autophagy-mediated degradation and propagate in cells predisposed to somatic mtDNA mutations, leading to disease. We used microarrays to identify expression profiles and pathways that are differentially activated or suppressed in Ter119+ bone marrow cells isolated from phlebotomized wildtype or Polg mutant mice
2015-04-21 | E-GEOD-57761 | biostudies-arrayexpress
Project description:Somatic mitochondrial DNA (mtDNA) mutations contribute to the pathogenesis of age-related disorders, including myelodysplastic syndromes (MDS). The accumulation of mitochondria harboring mtDNA mutations in patients with these disorders suggests a failure of normal mitochondrial quality-control systems. The mtDNA-mutator mice acquire somatic mtDNA mutations via a targeted defect in the proofreading function of the mtDNA polymerase, PolgA, and develop macrocytic anemia similar to that of patients with MDS. We observed an unexpected defect in clearance of dysfunctional mitochondria at specific stages during erythroid maturation in hematopoietic cells from aged mtDNA-mutator mice. Mechanistically, aberrant activation of mechanistic target of rapamycin signaling and phosphorylation of uncoordinated 51-like kinase (ULK) 1 in mtDNA-mutator mice resulted in proteasome mediated degradation of ULK1 and inhibition of autophagy in erythroid cells. To directly evaluate the consequence of inhibiting autophagy on mitochondrial function in erythroid cells harboring mtDNA mutations in vivo, we deleted Atg7 from erythroid progenitors of wildtype and mtDNA-mutator mice. Genetic disruption of autophagy did not cause anemia in wild-type mice but accelerated the decline in mitochondrial respiration and development of macrocytic anemia in mtDNA-mutator mice. These findings highlight a pathological feedback loop that explains how dysfunctional mitochondria can escape autophagy-mediated degradation and propagate in cells predisposed to somatic mtDNA mutations, leading to disease.
2015-04-21 | GSE57761 | GEO