International Ph.D. Program in Cell Therapy and Regenerative Medicine, College of Medicine, Taipei Medical University, Taipei, 11031, Taiwan.
Laboratory, Vinmec International Hospital, Minh Khai Street, Hai Ba Trung, Hanoi, Vietnam.
J Biomed Sci. 2024 Oct 11;31(1):95. doi: 10.1186/s12929-024-01085-8.
Primary ovarian insufficiency (POI) is an early decline in ovarian function that leads to ovarian failure. Conventional treatments for POI are inadequate, and treatments based on mesenchymal stem cells (MSCs) have emerged as an option. However, the lack of consideration of the estrogen niche in ovarian tissue significantly reduces the therapeutic efficacy, with an unclear mechanism in the MSCs in POI treatment. Furthermore, the disruption of circadian rhythm associated with POI has not been previously addressed.
Conditioned medium (CM) and estradiol-conditioned medium (E2-CM) were generated from estrogen receptor positive MSCs (ERpcMSCs). Chemotherapy-induced POI models were established using C57BL/6 mice (in vivo) and KGN cells (in vitro) treated with cyclophosphamide (CTX) or 4-hydroperoxycyclophosphamide (4-OOH-CP). Gene/protein expressions were detected using RT-qPCR, Western blotting, and immunohistochemistry assays. Locomotor activity was monitored for behavioral circadian rhythmicity. Cytokine arrays and miRNA analysis were conducted to analyze potential factors within CM/E2-CM.
The secretome of ERpcMSCs (CM and E2-CM) significantly reduced the CTX-induced defects in ovarian folliculogenesis and circadian rhythm. CM/E2-CM also reduced granulosa cell apoptosis and rescued angiogenesis in POI ovarian tissues. E2-CM had a more favorable effect than the CM. Notably, ERpcMSC secretome restored CTX-induced circadian rhythm defects, including the gene expressions associated with the ovarian circadian clock (e.g., Rora, E4bp4, Rev-erbα, Per2 and Dbp) and locomotor activity. Additionally, the cytokine array analysis revealed a significant increase in cytokines and growth factors associated with immunomodulation and angiogenesis, including angiogenin. Neutralizing the angiogenin in CM/E2-CM significantly reduced its ability to promote HUVEC tube formation in vitro. Exosomal miRNA analysis revealed the miRNAs involved in targeting the genes associated with POI rescue (PTEN and PDCD4), apoptosis (caspase-3, BIM), estrogen synthesis (CYP19A1), ovarian clock regulation (E4BP4, REV-ERBα) and fibrosis (COL1A1).
This study is the first to demonstrate that, in considering the estrogen niche in ovarian tissue, an estrogen-priming ERpcMSC secretome achieved ovarian regeneration and restored the circadian rhythm in a CTX-induced POI mouse model. The potential factors involved include angiogenin and exosomal miRNAs in the ERpcMSC secretome. These findings offer insights into potential stem cell therapies for chemotherapy-induced POI and circadian rhythm disruption.
原发性卵巢功能不全（POI）是卵巢功能的早期衰退，导致卵巢衰竭。POI 的常规治疗效果不佳，基于间充质干细胞（MSCs）的治疗方法应运而生。然而，由于卵巢组织中雌激素生态位未被充分考虑，治疗效果显著降低，其在 POI 治疗中 MSC 的作用机制尚不清楚。此外，POI 相关的昼夜节律紊乱也尚未得到解决。
从雌激素受体阳性 MSCs（ERpcMSCs）中生成条件培养基（CM）和雌二醇条件培养基（E2-CM）。使用环磷酰胺（CTX）或 4-羟环磷酰胺（4-OOH-CP）处理 C57BL/6 小鼠（体内）和 KGN 细胞（体外），建立化疗诱导的 POI 模型。使用 RT-qPCR、Western blot 和免疫组织化学检测基因/蛋白表达。监测运动活动以检测行为昼夜节律性。进行细胞因子阵列和 miRNA 分析以分析 CM/E2-CM 中的潜在因素。
ERpcMSC 的分泌组（CM 和 E2-CM）显著减轻了 CTX 诱导的卵巢卵泡发生和昼夜节律缺陷。CM/E2-CM 还减少了 POI 卵巢组织中的颗粒细胞凋亡并挽救了血管生成。E2-CM 比 CM 更有效。值得注意的是，ERpcMSC 分泌组恢复了 CTX 诱导的昼夜节律缺陷，包括与卵巢昼夜节律相关的基因表达（如 Rora、E4bp4、Rev-erbα、Per2 和 Dbp）和运动活动。此外，细胞因子阵列分析显示，与免疫调节和血管生成相关的细胞因子和生长因子显著增加，包括血管生成素。在 CM/E2-CM 中中和血管生成素显著降低了其在体外促进 HUVEC 管形成的能力。外泌体 miRNA 分析显示，miRNA 参与靶向与 POI 挽救相关的基因（PTEN 和 PDCD4）、凋亡（caspase-3、BIM）、雌激素合成（CYP19A1）、卵巢时钟调节（E4BP4、REV-ERBα）和纤维化（COL1A1）。
这项研究首次表明，在考虑卵巢组织中的雌激素生态位时，经雌激素预处理的 ERpcMSC 分泌组可实现卵巢再生，并恢复 CTX 诱导的 POI 小鼠模型中的昼夜节律。潜在的相关因素包括 ERpcMSC 分泌组中的血管生成素和外泌体 miRNA。这些发现为化疗诱导的 POI 和昼夜节律紊乱的潜在干细胞治疗提供了思路。
