Nikmahzar Aghbibi, Koruji Morteza, Jahanshahi Mehrdad, Khadivi Farnaz, Shabani Maryam, Dehghani Sanaz, Forouzesh Mehdi, Jabari Ayob, Feizollahi Narjes, Salem Maryam, Ghanami Gashti Nasrin, Abbasi Yasaman, Abbasi Mehdi
Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Stem Cell and Regenerative Medicine Center & Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
Artif Organs. 2023 Dec;47(12):1818-1830. doi: 10.1111/aor.14643. Epub 2023 Sep 12.
Development of organoids using human primary testicular cells has remained a challenge due to the complexity of the mammalian testicular cytoarchitecture and culture methods. In this study, we generated testicular organoids derived from human primary testicular cells. Then, we evaluated the effect of stem cell factor (SCF) on cell differentiation and apoptosis in the testicular organoid model.
The testicular cells were harvested from the three brain-dead donors. Human spermatogonial stem cells (SSCs) were characterized using immunocytochemistry (ICC), RT-PCR and flow cytometry. Testicular organoids were generated from primary testicular cells by hanging drop culture method and were cultured in three groups: control group, experimental group 1 (treated FSH and retinoic acid (RA)), and experimental group 2 (treated FSH, RA and SCF), for five weeks. We assessed the expression of SCP3 (Synaptonemal Complex Protein 3) as a meiotic gene, PRM2 (Protamine 2) as a post-meiotic marker and apoptotic genes of Bax (BCL2-Associated X Protein) and Bcl-2 (B-cell lymphoma 2), respectively by using RT-qPCR. In addition, we identified the expression of PRM2 by immunohistochemistry (IHC).
Relative expression of SCP3, PRM2 and Bcl-2 were highest in group 2 after five weeks of culture. In contrast, BAX expression level was lower in experimental group 2 in comparison with other groups. IHC analyses indicated the highest expression of PRM2 as a postmeiotic marker in group 2 in comparison to 2D culture and control groups but not find significant differences between experimental group 1 and experimental group 2 groups. Morphological evaluations revealed that organoids are compact spherical structures and in the peripheral region composed of uncharacterized elongated fibroblast-like cells.
Our findings revealed that the testicular organoid culture system promote the spermatogonial stem cell (SSC) differentiation, especially in presence of SCF. Developed organoids are capable of recapitulating many important properties of a stem cell niche.
由于哺乳动物睾丸细胞结构和培养方法的复杂性，利用人原代睾丸细胞开发类器官仍然是一项挑战。在本研究中，我们从人原代睾丸细胞中生成了睾丸类器官。然后，我们在睾丸类器官模型中评估了干细胞因子（SCF）对细胞分化和凋亡的影响。
从三名脑死亡供体中获取睾丸细胞。使用免疫细胞化学（ICC）、逆转录-聚合酶链反应（RT-PCR）和流式细胞术对人精原干细胞（SSCs）进行鉴定。通过悬滴培养法从原代睾丸细胞中生成睾丸类器官，并将其分为三组培养：对照组、实验组1（用促卵泡激素（FSH）和视黄酸（RA）处理）和实验组2（用FSH、RA和SCF处理），共培养五周。我们分别使用逆转录定量聚合酶链反应（RT-qPCR）评估减数分裂基因联会复合体蛋白3（SCP3）、减数分裂后标志物鱼精蛋白2（PRM2）以及凋亡基因Bax（BCL2相关X蛋白）和Bcl-2（B细胞淋巴瘤2）的表达。此外，我们通过免疫组织化学（IHC）鉴定PRM2的表达。
培养五周后，实验组2中SCP3、PRM2和Bcl-2的相对表达最高。相比之下，实验组2中BAX表达水平低于其他组。免疫组织化学分析表明，与二维培养组和对照组相比，实验组2中作为减数分裂后标志物的PRM2表达最高，但实验组组1和实验组2之间未发现显著差异。形态学评估显示，类器官是紧密的球形结构，周边区域由未鉴定的细长成纤维细胞样细胞组成。
我们的研究结果表明，睾丸类器官培养系统可促进精原干细胞（SSC）分化，尤其是在存在SCF的情况下。所开发的类器官能够重现干细胞微环境的许多重要特性。
