iPSC Mitochondrial DNA Analysis
iPSC mtDNA monitoring for research and clinical applications
- Induced Pluripotent Stem Cell (iPSC) technology is becoming an effective tool in therapeutic applications (1). Consequently, the genetic integrity of iPSCs after propagation and culture becomes critically important. Recent studies clearly show that mitochondrial genomes in stem cell clones derived from the same patient differ (2, 3). Specifically, one study investigated the accumulation of somatic mitochondrial genome (mtDNA) mutations in skin fibroblasts, blood, and iPSCs derived from young and elderly subjects (24–72 years old). It was demonstrated that the accumulation of mtDNA mutations of somatic origin in iPSCs cells increased with age and led to respiratory defects (2,3). This important observation warrants the need to monitor mtDNA mutations by sequence analysis of the mitochondrial genome (4) of each iPSC clone whether it is to be used for research or for clinical applications.
- Authentication and contamination of cell lines by mtDNA sequencing
Human cultured cell lines are used in a number of biomedical research and clinical applications, including cancer research, drug discovery, genetics and biobanking. However, misidentified human and animal cell lines continue to be used today despite multiple and repeated warnings, articles and letters by prominent scientists in the field calling for authentication (5). Cell line authentication can be achieved by examining polymorphic mtDNA markers. These highly discriminative markers, identified by sequencing the cells’ mitochondrial genomes, can then be used for the detection and identification of contaminating human cells.
- Genes (37): MT-ATP6, MT-ATP8, MT-CO1, MT-CO2, MT-CO3, MT-CYB, MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, MT-TA, MT-TC, MT-TD, MT-TE, MT-TF, MT-TG, MT-TH, MT-TI, MT-TK, MT-TL1, MT-TL2, MT-TM, MT-TN, MT-TP, MT-TQ, MT-TR, MT-TS1, MT-TS2, MT-TT, MT-TV, MT-TW, MT-TY, MT-RNR1, and MT-RNR2
• 8103 or 8104 (for research applications)
• 8100 (for clinical applications)
• iPSC clones derived from original cell pools or sources where mitochondrial heteroplasmy is known or suspected (e.g. from blood or pooled skin fibroblasts obtained from an older adult)
• Authentication of cell lines, and verification of the absence of contaminating cells
Test Info Sheet: iPSC Mitochondrial DNA Analysis
Requisition: Mitochondrial Test Requisition Form and/or Contact ApolloGen Diagnostic Laboratory
- Turn-Around Time: 2 Weeks
• Cell Pellet: 100,000 cells provided as a frozen cell pellet shipped on dry ice
• DNA: 500 ng of total DNA (at 25 ng/µL or higher concentration) in water or TE shipped on frozen ice packs
• Blood: 3-5 mL blood (1 mL minimum in a Lavender Top / EDTA Tube) shipped at room temperature
Other Specimens: Contact ApolloGen Diagnostic Laboratory
- Test Results: Results typically are provided as a table that lists the variants and heteroplasmies found
Pricing: Please contact us at (949) 916-8886 or firstname.lastname@example.org for current pricing
- Methodology: Long-range PCR followed by Next-Generation Sequencing (NGS)
Mitochondrial DNA Analysis for Cell Authentication (RUO)
Comprehensive Mitochondrial Genome Analysis
1. Takahashi, K. & Yamanaka, S. A decade of transcription factor-mediated reprogramming to pluripotency. Nat Rev Mol Cell Biol 17, 183–193 (2016).
2. Kang, E. et al. Age-related accumulation of somatic mitochondrial DNA mutations in adult-derived human iPSCs. Cell Stem Cell 18, 625-636 (2016).
3. Perales-Clemente, E. et al. Natural underlying mtDNA heteroplasmy as a potential source of intra-person hiPSC variability. EMBO J 35, 1979-1990 (2016)
5. Marx, V. Cell-line authentication demystified. Nat Methods 11, 483-488 (2014).
6. Tang, S. & Huang, T. Characterization of mitochondrial DNA heteroplasmy using a parallel sequencing system. Biotechniques 48, 287–296 (2010).
7. Huang, T. Next generation sequencing to characterize mitochondrial genomic DNA heteroplasmy. Curr Protoc Hum Genet Chapter 19, Unit19.8 (2011).