Background <p>Mitochondrial DNA (mtDNA) mutations contribute to respiratory dysfunction and cause mitochondrial diseases. The pathologies of these multisystemic inherited diseases are poorly understood. Mutations in the mitochondrial tRNA gene are one of the most frequent mtDNA mutations and are associated with various clinical symptoms such as diabetes mellitus, hearing loss, cardiomyopathy, exercise intolerance, and found in patients with mitochondrial disorders. Human induced pluripotent stem cells (hiPSCs), generated by reprogramming patient-specific somatic cells are recognized as useful tools for disease modeling and serve to better understand the multisystemic pathologies associated with mitochondrial tRNA mutations.</p> Methods <p>hiPSCs were generated from control BJ and two fibroblast lines with mitochondrial tRNA mutations using non-modified reprogramming and immune evasion mRNAs and microRNAs. Expression of hiPSC-associated intracellular and cell surface markers were identified by immunofluorescence and flow cytometry. Sanger sequencing was used to detect the mutation in all hiPSCs. Mitochondrial network morphology analysis was conducted to detect and quantify mitochondrial structures. The mitochondrial respiration ability, glycolytic function, and composite bioenergetics health index (BHI) were measured by the Seahorse Bioscience XFe96 extracellular flux analyzer.</p> Results <p>Reprogrammed hiPSCs expressed pluripotent stem cell markers including transcription factors POU5F1, NANOG, and SOX2, and cell surface markers SSEA4, TRA-1-60, and TRA-1-81 at the protein level. Sanger sequencing analysis confirmed the presence of mutations in both hiPSCs. Cytogenetic analyses confirmed the presence of normal karyotypes in both hiPSCs. Mitochondrial morphological analysis indicates the presence of hyperfused mitochondria in diseased hiPSC lines. The composite BHI values, a measure of mitochondrial dysfunction that was based on comprehensive bioenergetics analysis of OXPHOS and glycolysis, demonstrated that the mitochondrial functional defects were severe in both the hiPSC lines exhibiting tRNA mutations.</p> Conclusion <p>Overall, the hiPSCs exhibited variable mitochondrial morphology and respiratory dysfunction that have the potential to alter hiPSC differentiation potential, cell fate, and tissue development. These results indicate the potential significance of using hiPSCs and their derivatives to assess mitochondrial morphology and key bioenergetics parameters as a means to better understand early developmental defects in mitochondrial disorders in children.</p>