ヒト造精幹細胞とセルトーリ細胞の三次元共培養法を開発した。

Three-dimensional co-culture of human spermatogonial stem cells with Sertoli cells in soft agar culture system supplemented by growth factors and Laminin.

• Ayob Jabari
• Mohammad Ali Sadighi Gilani
• Morteza Koruji
• Keykavos Gholami
• Mojtaba Mohsenzadeh
• Tayebeh Rastegar
• Farnaz Khadivi
• Nasrin Ghanami Gashti
• Aghbibi Nikmahzar
• Sina Mojaverrostami
• Ali Talebi
• Sepideh Ashouri Movassagh
• Mohammad Jafar Rezaie
• Mehdi Abbasi

抄録

ヒト造精幹細胞（SSC）の in vitro 増殖・分化のための三次元（3D）培養系の適用は、精子形成過程の研究や、特に思春期前の癌患者における男性不妊症の管理に有用なツールである。本研究の主な目的は、ラミニンと成長因子を添加した軟寒天培養系（SACS）でセルトリ細胞と共培養したヒトSSCの増殖を調べることであった。脳死患者の精巣から精巣細胞を分離し、2 次元培養系で 3 週間培養した。3 週間後、異種移植により機能的な SSC を評価し、免疫細胞化学、フローサイトメトリー、RT-PCR により細胞の同定を行った。その後、ＳＳＣＳとＳｅｒｔｏｒｉ細胞をＳＡＣＳの上層部に３週間移植した。3 週間後、コロニー数、SSC および Sertoli 細胞マーカー、アポトーシス遺伝子の発現を評価した。その結果、移植されたSSCはレシピエントマウスの半毛細管の基底膜に移行することがわかった。移植されたSSCとSertoli細胞のPLZF, α6-Integrin, Vimentinタンパク質の発現は、2次元培養系と3次元培養系で観察された。ラミニンと成長因子を添加したSACSでは、PLZF, α6-Integrin, Bcl2, コロニー数の発現率が非添加群に比べて有意に高かった(P"Zs_202F"≤"Zs_202F"0.01)が、c-kitとBaxの発現率は非添加群に比べて有意に低かった(P"Zs_202F"≤"Zs_202F"0.05)。この3次元共培養システムは、アポトーシスを減少させ、ヒトSSCの増殖を増加させることができた。したがって、このシステムは、思春期前の男性癌や無精子症の男性におけるヒトSSCの増殖を増加させ、体外移植の成功とin vitro精子形成に十分な数のSSCを得るために利用することができる。

Application of a three-dimensional (3D) culture system for in vitro proliferation and differentiation of human spermatogonial stem cells (SSCs) is a useful tool for the investigation of the spermatogenesis process and the management of male infertility particularly in prepubertal cancer patients. The main purpose of this study was to investigate the proliferation of human SSCs co-cultured with Sertoli cells in soft agar culture system (SACS) supplemented by Laminin and growth factors. Testicular cells were isolated from testes of brain-dead patients and cultured in two-dimensional (2D) culture system for 3 weeks. After 3 weeks, functional SSCs were evaluated by xenotransplantation and also identification of cells was assessed by immunocytochemistry, flow cytometry, and RT-PCR. Then, SSCs and Sertoli cells were transferred to the upper layer of SACS for 3 weeks. After 3 weeks, the number of colonies and the expression of specific SSCs and Sertoli cell markers, as well as apoptotic genes were evaluated. Our results showed that transplanted SSCs, migrated into the basement membrane of seminiferous tubules of recipient mice. The expression of PLZF, α6-Integrin, and Vimentin proteins in SSCs and Sertoli cells were observed in 2D and 3D culture systems. The expression rate of PLZF, α6-Integrin, Bcl2, and colony number in SACS supplemented by Laminin and growth factors group were significantly higher than non-supplemented groups (P ≤ 0.01), but the expression rate of c-kit and Bax in supplemented group were significantly lower than non-supplemented groups (P ≤ 0.05). This 3D co-culture system decreased apoptosis and increased propagation of human SSCs. Therefore, this designed system can be utilized to increase the proliferation of human SSCs in prepubertal male cancer and azoospermic men to obtain an adequate SSCs number to outotransplant success and in vitro spermatogenesis.

Copyright © 2020. Published by Elsevier GmbH.