Gizer Merve, Önen Selin, Erol Özgür Doğuş, Aerts-Kaya Fatima, Reçber Tuba, Nemutlu Emirhan, Korkusuz Petek
Department of Stem Cell Sciences, Graduate School of Health Sciences, Hacettepe University, Ankara, 06100, Turkey.
METU MEMS Center, Ankara, 06530, Turkey.
Biol Res. 2025 Mar 12;58(1):13. doi: 10.1186/s40659-025-00596-4.
Male factor infertility (MFI) is responsible for 50% of infertility cases and in 15% of the cases sperm is absent due to germ cell aplasia. Human induced pluripotent stem cell (hiPSC)-derived spermatogonial stem cells (hSSCs) could serve as an autologous germ cell source for MFI in patients with an insufficient sperm yield for assisted reproductive technology (ART). The endocannabinoid system (ECS) has been implicated to play a role in mouse embryonic stem cells (mESCs) and the human testicular environment. However, the contribution of the ECS in hiPSCs and hiPSC-derived hSSCs is currently unknown. Here, we aimed to assess whether hiPSCs and hiPSC-derived hSSCs are regulated by components of the ECS and whether manipulation of the ECS could increase the yield of hiPSC-derived SSCs and serve as an autologous cell-based source for treatment of MFI.
We reprogrammed human dermal fibroblasts (hDFs) to hiPSCs, induced differentiation of hSSC from hiPSCs and evaluated the presence of ECS ligands (AEA, 2-AG) by LC/MS, receptors (CB1R, CB2R, TRPV1, GPR55) by qPCR, flow cytometry and immunofluorescent labeling. We then examined the efficacy of endogenous and synthetic selective ligands (ACPA, CB65, CSP, ML184) on proliferation of hiPSCs using real-time cell analysis (RTCA) and assessed the effects of on CB2R agonism on hiPSC pluripotency and differentiation to hSSCs.
hiPSCs from hDFs expressed the pluripotency markers OCT4, SOX2, NANOG, SSEA4 and TRA-1-60; and could be differentiated into ID4+, PLZF + hSSCs. hiPSCs and hiPSC-derived hSSCs secreted AEA and 2-AG at 10 - 10 M levels. Broad expression of all ECS receptors was observed in both hiPSCs and hiPSC-derived hSSCs, with a higher CB2R expression in hSSCs in comparison to hiPSCs. CB2R agonist CB65 promoted proliferation and differentiation of hiPSCs to hiPSC-hSSCs in comparison to AEA, 2-AG, ACPA, CSP and ML184. The EC of CB65 was determined to be 2.092 × 10 M for support of pluripotency and preservation of stemness on hiPSCs from 78 h. CB65 stimulation at EC also increased the yield of ID4 + hSSCs, PLZF + SSPCs and SCP3 + spermatocytes from day 10 to 12.
We demonstrated here for the first time that stimulation of CB2R results in an increased yield of hiPSCs and hiPSC-derived hSSCs. CB65 is a potent CB2R agonist that can be used to increase the yield of hiPSC-derived hSSCs offering an alternative source of autologous male germ cells for patients with MFI. Increasing the male germ/stem cell pool by CB65 supplementation could be part of the ART-associated protocols in MFI patients with complete germ cell aplasia.
男性因素导致的不育（MFI）占不育病例的50%，在15%的病例中，由于生殖细胞发育不全而无精子。对于辅助生殖技术（ART）中精子产量不足的MFI患者，人诱导多能干细胞（hiPSC）来源的精原干细胞（hSSC）可作为自体生殖细胞来源。内源性大麻素系统（ECS）已被证明在小鼠胚胎干细胞（mESC）和人类睾丸环境中发挥作用。然而，ECS在hiPSC和hiPSC来源的hSSC中的作用目前尚不清楚。在此，我们旨在评估hiPSC和hiPSC来源的hSSC是否受ECS成分的调节，以及操纵ECS是否可以提高hiPSC来源的SSC产量，并作为治疗MFI的自体细胞来源。
我们将人皮肤成纤维细胞（hDF）重编程为hiPSC，诱导hiPSC分化为hSSC，并通过液相色谱/质谱（LC/MS）评估ECS配体（AEA、2-AG）的存在，通过定量聚合酶链反应（qPCR）、流式细胞术和免疫荧光标记评估受体（CB1R、CB2R、TRPV1、GPR55）的存在。然后，我们使用实时细胞分析（RTCA）检测内源性和合成选择性配体（ACPA、CB65、CSP、ML184）对hiPSC增殖的功效，并评估CB2R激动剂对hiPSC多能性和向hSSC分化的影响。
来自hDFs的hiPSC表达多能性标志物OCT4、SOX2,、NANOG、SSEA4和TRA-1-60；并且可以分化为ID4+、PLZF+ hSSC。hiPSC和hiPSC来源的hSSC以10-10 M的水平分泌AEA和2-AG。在hiPSC和hiPSC来源的hSSC中均观察到所有ECS受体的广泛表达，与hiPSC相比，hSSC中CB2R表达更高。与AEA、2-AG、ACPA、CSP和ML184相比，CB2R激动剂CB65促进了hiPSC向hiPSC-hSSC的增殖和分化。CB65的半数有效浓度（EC）被确定为2.092×10 M，用于支持78小时后hiPSC中的多能性和干性维持。在EC水平的CB65刺激也增加了第10天到第12天ID4+ hSSC、PLZF+ SSPC和SCP3+精母细胞的产量。
我们首次证明刺激CB2R可提高hiPSC和hiPSC来源 的hSSC产量。CB65是一种有效的CB2R激动剂，可用于提高hiPSC来源的hSSC产量，为MFI患者提供自体雄性生殖细胞的替代来源。通过补充CB65增加雄性生殖/干细胞库可能是完全生殖细胞发育不全的MFI患者ART相关方案的一部分。
