Xeno-Free Propagation of Spermatogonial Stem Cells from Infant Boys
Xeno-Free Propagation of Spermatogonial Stem Cells from Infant Boys
复制标题
DOI：
10.3390/ijms20215390
复制
发表时间：
2019-11-01
影响因子：
5.6
通讯作者：
Andersen, Claus Yding
中科院分区：
文献类型：
--
作者：
Dong, Lihua;Gul, Murat;Andersen, Claus Yding
精原干细胞（SSC）移植疗法是一种很有前途的策略，可以帮助生育能力受损的青春期前男孩恢复精子发生。然而，由于冷冻保存的睾丸组织中的 SSC 数量有限，因此需要 SSC 的繁殖。这种繁殖必须在无异源条件下进行才能用于临床应用。在无异源条件下，使用人血小板裂解物和营养源，从两个男孩的婴儿睾丸组织（7 毫克和 10 毫克）中繁殖 SSC。我们通过定量实时聚合酶链式反应 (PCR) 和免疫反应测定法，使用 SSC 标记物未分化胚胎细胞转录因子 1 (UTF1)、泛素羧基末端水解酶同工酶 L1 (UCHL1)、GDNF 受体 α-1 (GFR α -1) F α 和早幼粒细胞白血病锌指蛋白 (PLZF) 验证了 SSC 样细胞簇 (SSCLC)。通过使用绿色荧光细胞连接器 PKH67 进行预标记，并将 SSCLC 异种移植到白消安处理的无菌免疫缺陷小鼠中，研究了增殖的 SSC 的功能。初代传代 2 周后出现 SSC 样细胞簇 (SSCLC)。 SSCLC 与 SSC 类似，因为 UTF1、UCHL1、GFR α 1 和 PLZF 均呈阳性。经过2.5个月的培养期，从一份样本中总共收获了1300万个细胞用于异种移植。 3-6 周时，在小鼠生精小管中鉴定并验证了标记的人类繁殖的 SSC，证实移植的细胞含有 SSCLC。目前的无异源临床培养方案允许从男婴中繁殖 SSC。
Spermatogonial stem cell (SSC) transplantation therapy is a promising strategy to renew spermatogenesis for prepubertal boys whose fertility is compromised. However, propagation of SSCs is required due to a limited number of SSCs in cryopreserved testicular tissue. This propagation must be done under xeno-free conditions for clinical application. SSCs were propagated from infant testicular tissue (7 mg and 10 mg) from two boys under xeno-free conditions using human platelet lysate and nutrient source. We verified SSC-like cell clusters (SSCLCs) by quantitative real-time polymerase chain reaction (PCR) and immune-reaction assay using the SSC markers undifferentiated embryonic cell transcription factor 1 (UTF1), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), GDNF receptor alpha-1 (GFR alpha -1) F alpha and promyelocytic leukaemia zinc finger protein (PLZF). The functionality of the propagated SSCs was investigated by pre-labelling using green fluorescent Cell Linker PKH67 and xeno-transplantation of the SSCLCs into busulfan-treated, therefore sterile, immunodeficient mice. SSC-like cell clusters (SSCLCs) appeared after 2 weeks in primary passage. The SSCLCs were SSC-like as the UTF1, UCHL1, GFR alpha 1 and PLZF were all positive. After 2.5 months' culture period, a total of 13 million cells from one sample were harvested for xenotransplantation. Labelled human propagated SSCs were identified and verified in mouse seminiferous tubules at 3-6 weeks, confirming that the transplanted cells contain SSCLCs. The present xeno-free clinical culture protocol allows propagation of SSCs from infant boys.