hTERT在人诱导多能干细胞体外分化为巨核细胞中的作用*

doi:10.3969/j.issn.1671-2587.2025.05.004

临床输血与检验 ›› 2025, Vol. 27 ›› Issue (5): 604-612.DOI: 10.3969/j.issn.1671-2587.2025.05.004

hTERT在人诱导多能干细胞体外分化为巨核细胞中的作用^*

岳伟^1,2, 杨玥², 周梅², 李津杞², 杨怡³, 李彦欣³, 钱宝华², 何啸¹, 顾海慧²

¹盐城市第一人民医院输血科,江苏盐城 224005;
²中国人民解放军海军军医大学第一附属医院输血科,上海 200433;
³上海交通大学医学院附属上海儿童医学中心转化所,国家卫健委儿童血液肿瘤重点实验室,上海 200000

收稿日期:2025-09-01 修回日期:2025-09-11 出版日期:2025-10-20 发布日期:2025-10-11
通讯作者: 顾海慧,主要从事临床输血及细胞治疗与干细胞来源血小板方面研究,（E-mail）haihuigu@126.com。共同通信作者：何啸,主要从事输血检验与临床输血相关研究,（E-mail）ycyyhexiao@sina.com。
作者简介:岳伟,主要从事临床输血与体外分化血小板研究,（E-mail）270195150@qq.com。并列第一作者：杨玥,主要从事输血检验与体外分化血小板研究,（E-mail）978257160@qq.com。
基金资助:
^*本课题受国家自然科学基金面上项目（No.81970165）资助

Role of hTERT in the Differentiation of Human iPSCs into Megakaryocytes in vitro

YUE Wei^1,2, YANG Yue², ZHOU Mei², LI Jinqi², YANG Yi³, LI Yanxin³, QIAN Baohua², HE Xiao¹, GU Haihui²

¹Department of Transfusion Medicine, The First People's Hospital of Yancheng, Jiangsu 224005;
²Department of Transfusion Medicine, The First Affiliated Hospital of Naval Medical University, Shanghai 200433;
³Pediatric Translational Medicine Institute, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Key Laboratory of Pediatric Hematology & Oncology of National Health Commission, Shanghai 200000

Received:2025-09-01 Revised:2025-09-11 Online:2025-10-20 Published:2025-10-11

摘要/Abstract

摘要： 目的建立多西环素（Dox）诱导人端粒酶逆转录酶（hTERT）稳定过表达的人诱导多能干细胞（iPSCs）,探讨hTERT对iPSCs向巨核细胞（MK）分化及巨核细胞增殖能力的影响。方法将Dox诱导型hTERT双载体转染人iPSCs,通过嘌呤霉素筛选和单克隆扩增获得稳定细胞系后,利用荧光显微镜、RT-qPCR及Western blot验证hTERT过表达水平。采用Spin-EB方法体外培养人iPSCs诱导分化生成巨核细胞,利用流式细胞术、细胞计数和显微观察评估hTERT-iPSCs的分化效率及增殖能力,通过透射电镜、瑞氏-吉姆萨染色及免疫荧光等方法检测生成巨核细胞的成熟度、形态与功能。结果成功建立Dox诱导型hTERT-iPSCs稳定株,iPSCs体外培养添加Dox诱导后,hTERT-iPSCs细胞的GFP阳性率、hTERT mRNA（P<0.05）及蛋白表达显著上调。hTERT-iPSCs体外培养分化的细胞可持续培养至35天,但GFP阳性率呈时间依赖性下降。将Dox诱导时间提前至体外培养分化第8天可显著促进细胞增殖（P<0.001）,并维持hTERT表达（P<0.01）。形态学和功能评估显示,hTERT-iPSCs生成的巨核细胞的大小、典型细胞器及血小板样颗粒释放无显著差异。结论 hTERT促进iPSCs体外培养巨核系分化,于分化第8天启动Dox诱导可有效促进iPSCs来源巨核细胞的长期增殖,同时维持其成熟的形态学特征和功能。该策略为体外大规模制备巨核细胞及血小板提供了有效途径。

关键词: 人端粒酶逆转录酶, 诱导多能干细胞, 巨核细胞分化, 四环素调控系统, 多西环素, 慢病毒

Abstract: Objective To establish a human induced pluripotent stem cells (iPSCs) line with doxycycline-inducible, stable over-expression of human telomerase reverse transcriptase (hTERT) and investigate how hTERT affects iPSC differentiation into megakaryocytes (MKs), as well as their proliferative capacity. Methods A doxycycline-inducible dual-vector encoding hTERT was transfected into human iPSCs. Stable clones were obtained via puromycin selection and single-colony expansion, and hTERT induction was verified by fluorescence microscopy, RT-qPCR, and Western blot. IPSCs differentiation into megakaryocytes was triggered by the spin-EB method; Flow cytometry, cell counting and microscopy were employed to assess the differentiation efficiency and proliferative capacity of hTERT-iPSCs, while ultrastructure, morphology and functional maturity of the generated megakaryocytes were evaluated by transmission electron microscopy, Wright-Giemsa staining and immunofluorescence. Results We successfully established a Dox-inducible hTERT-iPSC stable line. After Dox induction in vitro, the GFP positivity rate, hTERT mRNA (P<0.05), and protein expression of hTERT-iPSCs were significantly upregulated. The cells differentiated from hTERT-iPSCs could be continuously cultured for up to 35 days, but the GFP positivity rate decreased in a time-dependent manner. Advancing the timing of Dox induction to day 8 of in vitro differentiation significantly promoted cell proliferation (P<0.001) and maintained hTERT expression (P<0.01). Morphological and functional assessments revealed no significant differences in the size, typical organelles, or platelet-like particle release of megakaryocytes generated from hTERT-iPSCs. Conclusion hTERT promotes the megakaryocytic differentiation of iPSCs in vitro. Initiating Dox induction on day 8 of differentiation effectively enhances the long-term proliferation of iPSC-derived megakaryocytes while maintaining their mature morphological characteristics and functions. This strategy provides a robust and scalable platform for in vitro production of megakaryocytes and platelets.

Key words: Human telomerase reverse transcriptase, Induced pluripotent stem cells, Megakaryocyte differentiation, Tetracycline-inducible system, Doxycycline, Lentivirus

中图分类号:

R329.2

岳伟, 杨玥, 周梅, 李津杞, 杨怡, 李彦欣, 钱宝华, 何啸, 顾海慧. hTERT在人诱导多能干细胞体外分化为巨核细胞中的作用^*[J]. 临床输血与检验, 2025, 27(5): 604-612.

YUE Wei, YANG Yue, ZHOU Mei, LI Jinqi, YANG Yi, LI Yanxin, QIAN Baohua, HE Xiao, GU Haihui. Role of hTERT in the Differentiation of Human iPSCs into Megakaryocytes in vitro[J]. JOURNAL OF CLINICAL TRANSFUSION AND LABORATORY MEDICINE, 2025, 27(5): 604-612.

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hTERT在人诱导多能干细胞体外分化为巨核细胞中的作用^*

Role of hTERT in the Differentiation of Human iPSCs into Megakaryocytes in vitro

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