Granulosa cells are key somatic components of ovarian follicles, playing essential roles in steroidogenesis, follicular maturation, and oocyte development. The team had previously shown that granulosa cells from young mice (2–3 months old) could be efficiently reprogrammed by chemical means into chemically induced pluripotent stem cells (GC‑CiPSCs), which could then be differentiated into PGCLCs and eventually into functional oocytes capable of producing healthy offspring.
However, granulosa cells themselves undergo senescence with reproductive ageing – featuring mitochondrial dysfunction, telomere shortening, elevated oxidative stress, and other hallmarks. The key question was whether these age‑related features would be "remembered" after reprogramming and impair subsequent germ‑cell differentiation. To address this, the researchers established GC‑CiPSC lines from granulosa cells of reproductively aged mice (11–12 months old).
First, they confirmed that aged granulosa cells indeed showed typical senescence markers: increased PCNA and p16 expression, reduced DRP1 (a key mitochondrial fission protein), decreased mtDNA copy number, lower ATP levels, elevated reactive oxygen species (ROS), and reduced mitochondrial membrane potential. Unexpectedly, however, these senescent granulosa cells could still be successfully reprogrammed into iPSCs, with pluripotency gene expression profiles comparable to those of embryonic stem cells (ESCs) and young GC‑CiPSCs.
But when differentiated into PGCLCs, aged GC‑CiPSCs showed a marked efficiency defect – their PGCLC induction efficiency was far lower than that of young GC‑CiPSCs and ESCs. Attempts to rescue this defect by supplementing with mitochondrial enhancers such as NMN and α‑ketoglutarate (α‑KG) proved unsuccessful. Even activating DRP1 with sodium palmitate (NaPA) to promote mitochondrial fission did not improve PGCLC induction. This suggests that the impaired differentiation capacity of aged GC‑CiPSCs is not due to mitochondrial dysfunction alone, but involves multiple factors including nuclear ageing.
Transcriptomic analysis provided a key clue: genes upregulated in aged GC‑CiPSCs were enriched in the ERK/MAPK signalling pathway. Given the well‑known role of ERK/MAPK in regulating somatic versus germline fate decisions, the team applied the MEK inhibitor PD0325901 during PGCLC induction. The results were encouraging: ERK/MAPK inhibition nearly doubled the PGCLC induction efficiency from aged GC‑CiPSCs. Immunofluorescence staining showed increased numbers of cells positive for the germ‑cell markers STELLA and VASA, and a decrease in cells positive for the somatic marker GATA4, in PD0325901‑treated PGCLCs.
Further transcriptomic analysis revealed that PD0325901 treatment reversed changes in gene expression related to MAPK cascade regulation and cellular senescence in aged PGCLCs, while upregulating genes associated with mitochondrial metabolism. These findings indicate that aberrant activation of the ERK/MAPK pathway is a key mechanism underlying the reduced germ‑cell differentiation potential of iPSCs derived from aged granulosa cells.
Significance and outlook
This study is the first to systematically evaluate the germ‑cell differentiation potential of iPSCs derived from senescent granulosa cells, and to reveal the multifactorial nature of the observed defects. Although chemical reprogramming can reset epigenetic states and pluripotency gene expression, age‑related mitochondrial damage and nuclear ageing are apparently "remembered" to some extent, limiting the cells' ability to differentiate into germ cells.
Importantly, the study shows that ERK/MAPK inhibition can partially overcome this barrier, offering a feasible strategy to improve germ‑cell differentiation efficiency from aged donor‑derived iPSCs. This has significant implications for future efforts to use autologous cells – especially from older women – for germ‑cell regeneration.
The authors note that while mitochondrial function is essential for differentiation, simply restoring it is insufficient to rescue the age‑related decline in reprogramming potential. Future strategies will need to target both mitochondrial and nuclear ageing simultaneously to more effectively restore germ‑cell differentiation capacity.
The study was co‑authored by PhD students Dai Heng and Kairang Jin, among others, and was supported by the National Key Research and Development Program of China and the National Natural Science Foundation of China.
About Science China Life Sciences
Science China Life Sciences is a monthly journal co‑sponsored by the Chinese Academy of Sciences and the National Natural Science Foundation of China, and co‑published with the Chinese Society of Biochemistry and Molecular Biology. It is dedicated to publishing innovative and cutting‑edge research across all areas of the life sciences.
