Ananya Ghosal, MAKAUT(WB)
The two main reasons for an increase in the application of Induced Pluripotent Stem Cells (iPSCs) are the development of organoids and differentiated cells. Few concerns like the pre-existing or reprogramming-related occurrence of tumorigenic mutations, the age-related burden of somatic mutation hamper the therapeutic use of iPSCs. Mitochondrial DNA (mtDNA) is an area of genetic variability which affects the quality of iPSCs. Mitochondria DNA (mtDNA) has a higher mutagenesis rate than nuclear DNA and circular multicopy, small, double-stranded molecule of DNA within mitochondria.
The host of multiple pathogenic mutations causes a huge variety of clinical phenotypes, distinguished by removal by autophagy, the propensity to apoptosis, increased oxidative stress, oxidative phosphorylation, organelle dynamic, and Calcium mishandling. Moreover, Mitochondrial DNA (mtDNA) follows some rules which include threshold effects miotic segregation, individual cell clonal expansion of mutant molecules, germline bottleneck, homo-hetero polyplasmy, and maternal inheritance. In the global function of individual cells and iPSCs, the characteristics of mtDNA are reflected in higher-order complexities.
Some studies show that iPSC quality effected by mtDNA mutagenesis. Clonal expansion in a single cell of private heteroplasmic mtDNA variants is a critical issue and called universal heteroplasmy. The mtDNA metagenesis is related to the bottleneck effects, cell reprogramming, and genetic drift due to mtDNA copy number reduction which characterizes iPSCs. The deep next-generation sequencing has decreased the detection limit of heteroplasmy low-level heteroplasmy can be maintained and transmitted within families.
The procedure of the study:
- Genetic characterization of fibroblast– Fibroblast is extracted from five healthy age-matched subjects and 13 patients who are affected by the mitochondrial disorder. The patients were listed into the mitochondrial group (M1 to M6) consisting of either heteroplasmic or homoplasmic mt-DNA mutations, or a nuclear group where mutations occur in nuclear genes that are coding for mitochondrial proteins, generation, and characterization. The newly produced or already generated iPSCs are used. The generation of iPSCs and NARP from fibroblast extracted from a three-generation family carrying the homoplasmic mutation. mtDNA Sanger sequence analysis and characterization were performed. The determination of mutation load in fibroblast and extracted iPSCs by mini-sequencing.
- CGH array– The verification of the integrity of nuclear DNA was done by array comparative genomic hybridization (CGH).
- mtDNA sequencing– Next Generation Sequencing (NGS) approach the sequence analysis of the entire mtDNA molecule. Mitochondrial DNA (mtDNA) molecules are primed in two segments.
- Statistical analysis– By Mann-Whitney unpaired test reprogramming method and distinguishing between mean values of variants concerning to age of cell line donors were assessed. The effect of age on the number of variants in the cell lines was assessed by univariate linear regression analysis.
- Respiratory activity– NPCs were controlled with an XF96 extracellular flux analyzer and Oxygen Consumption Rate (OCR) was measured in DOA.
Findings of the study:
The inducible pluripotent stem cells (iPSCs) is the technique that allows the production of pluripotent patient-specific cell lines used for drug screening, disease modeling, and cell therapy. To allow iPSCs utilization integrity of nuclear DNA is mandatory. Quality control of mtDNA is minimal in the iPSCs validation process. mtDNA deep sequencing was performed during the change from fibroblast to reprogrammed iPSC and NPCS extracted from patients and controls affected by mitochondrial disorders. The potentially pathogenic mtDNA variants fluctuate between disappearing, emerging, and having some functional inference.
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Reference: Palombo, F., Peron, C., Caporali, L., Iannielli, A., Maresca, A., Di Meo, I., Fiorini, C., Segnali, A., Sciacca, F. L., Rizzo, A., Levi, S., Suomalainen, A., Prigione, A., Broccoli, V., Carelli, V., & Tiranti, V. (2021). The relevance of mitochondrial DNA variants fluctuation during reprogramming and neuronal differentiation of human iPSCs. Stem Cell Reports, S2213671121003246. https://doi.org/10.1016/j.stemcr.2021.06.016
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