INTRODUCTION: Mitochondrial remodeling supports the metabolic and transcriptional shifts required for stem cell differentiation. A key component of this process is mitochondrial biogenesis, which governs mtDNA replication and mitochondrial expansion. However, how biogenesis is regulated during early embryoid body (EB) formation remains poorly characterized.

AIM(S): This study aimed to evaluate mitochondrial DNA (mtDNA) content and the expression of mitochondrial biogenesis regulators in early human EBs.

METHOD(S): Unguided 14-day embryoid bodies were generated from a human induced pluripotent stem cell (iPSC) line using only Essential 6 media. mtDNA copy number was measured by quantitative PCR using ND1/HBB and ND5/SERPINA1 ratios. Expression of PPARGC1A, NRF1, and TFAM was assessed by reverse transcription qPCR (RT-qPCR) and normalized to GAPDH. Three biological replicates were analyzed. Data were tested for normality and compared using unpaired, two-tailed t-tests in GraphPad Prism.

RESULTS: Compared to the iPSC line, embryoid bodies showed a significant decrease in mtDNA copy number. This reduction was accompanied by increased expression of PPARGC1A, stable levels of NRF1, and significantly reduced TFAM expression, suggesting a potential disconnect between mitochondrial biogenesis initiation and mtDNA maintenance.

CONCLUSIONS: These findings suggest a transcriptional uncoupling within the mitochondrial biogenesis pathway, with upstream activation occurring in the absence of downstream mtDNA maintenance. This may reflect a regulatory pause in mitochondrial transcription during early lineage transition.

FINANCIAL SUPPORT: Supported by the National Science Centre Grant No. 2019/35/B/NZ3/04383