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

doi:10.3969/j.issn.1671-2587.2025.05.004

JOURNAL OF CLINICAL TRANSFUSION AND LABORATORY MEDICINE ›› 2025, Vol. 27 ›› Issue (5): 604-612.DOI: 10.3969/j.issn.1671-2587.2025.05.004

Previous Articles Next Articles

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

Abstract

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

CLC Number:

R329.2

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.

References

[1] IZADY E,SALTANATPOUR Z,LIU L P,et al.Toward in vitro production of platelet from induced pluripotent stem cells[J]. Stem Cell Rev Rep,2022,18(7):2376-2387.
[2] SUGIMOTO N,ETO K.Generation and manipulation of human iPSC-derived platelets[J]. Cell Mol Life Sci, 2021,78(7):3385-3401.
[3] KIM K M,ALBAIRA K I,KANG J,et al.Cell-based artificial platelet production: historical milestones, emerging trends,and future directions[J]. Blood Res, 2025,60(1):32.
[4] KRISCH L,BRACHTL G,HOCHMANN S,et al.Improving human induced pluripotent stem cell-derived megakaryocyte differentiation and platelet production[J]. Int J Mol Sci,2021,22(15). DOI:10.3390/ijms22158224.
[5] NAKAMURA S,TAKAYAMA N,HIRATA S,et al.Expandable megakaryocyte cell lines enable clinically applicable generation of platelets from human induced pluripotent stem cells[J]. Cell Stem Cell,2014,14(4): 535-548.
[6] MOREAU T,EVANS A L,VASQUEZ L,et al.Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming[J]. Nat Commun,2016,7:11208.
[7] CHEN L,LIU J K,CHEN K Y,et al.SET domain containing 2 promotes megakaryocyte polyploidization and platelet generation through methylation of α-tubulin[J]. J Thromb Haemost,2024,22(6):1727-1741.
[8] AVANZI M P,GOLDBERG F,DAVILA J,et al.Rho kinase inhibition drives megakaryocyte polyploidization and proplatelet formation through MYC and NFE2 downregulation[J]. Br J Haematol,2014,164(6):867-876.
[9] LINDSEY S,PAPOUTSAKIS E T.The aryl hydrocarbon receptor (AHR) transcription factor regulates megakaryocytic polyploidization[J]. Br J Haematol,2011, 152(4):469-484.
[10] STRASSEL C,BROUARD N,MALLO L,et al.Aryl hydrocarbon receptor-dependent enrichment of a megakaryocytic precursor with a high potential to produce proplatelets[J]. Blood,2016,127(18):2231-2240.
[11] HUANG W H,GU H H,ZHAN Z Y,et al.The plant hormone abscisic acid stimulates megakaryocyte differentiation from human iPSCs in vitro[J]. Platelets, 2022,33(3):462-470.
[12] 顾海慧,岳伟,杨玥,等. 脱落酸对诱导多能干细胞巨核系血小板分化的影响[J]. 临床输血与检验,2023,25(3): 420-424.
[13] EICKE D,BAIGGER A,SCHULZE K,et al.Large-scale production of megakaryocytes in microcarrier-supported stirred suspension bioreactors[J]. Sci Rep,2018,8(1): 10146.
[14] ITO Y,NAKAMURA S,SUGIMOTO N,et al. Turbulence activates platelet biogenesis to enable clinical scale ex vivo production[J]. Cell,2018,174(3):636-648.e18.
[15] AUTEXIER C,LUE N F.The structure and function of telomerase reverse transcriptase[J]. Annu Rev Biochem, 2006,75:493-517.
[16] ROAKE C M,ARTANDI S E.Regulation of human telomerase in homeostasis and disease[J]. Nat Rev Mol Cell Biol,2020,21(7):384-397.
[17] ZHOU J Z,DING D Q,WANG M,et al.Telomerase reverse transcriptase in the regulation of gene expression[J]. BMB Rep,2014,47(1):8-14.
[18] KUMAR N,SETHI G.Telomerase and hallmarks of cancer: An intricate interplay governing cancer cell evolution[J]. Cancer Lett,2023,578:216459.
[19] MAIDA Y,MASUTOMI K.Telomerase reverse transcriptase moonlights: Therapeutic targets beyond telomerase[J]. Cancer Sci,2015,106(11):1486-1492.
[20] ZHAO H Q,ZHAO H M,JI S F.A mesenchymal stem cell aging framework,from mechanisms to strategies[J]. Stem Cell Rev Rep,2024,20(6):1420-1440.
[21] LIU M D,ZHANG Y N,JIAN Y P,et al.The regulations of telomerase reverse transcriptase (TERT) in cancer[J]. Cell Death Dis,2024,15(1):90.
[22] TOMÁS-LOBA A,FLORES I,FERNÁNDEZ-MARCOS P J,et al. Telomerase reverse transcriptase delays aging in cancer-resistant mice[J]. Cell,2008,135(4):609-622.
[23] GU H H,HUANG X,XU J,et al.Optimizing the method for generation of integration-free induced pluripotent stem cells from human peripheral blood[J]. Stem Cell Res Ther,2018,9(1):163.
[24] LI S S,SONG L L,ZHANG Y W,et al.Optimizing the method for differentiation of macrophages from human induced pluripotent stem cells[J]. Stem Cells Int,2022, 2022:6593403.
[25] LIU Y F,WANG Y,GAO Y X,et al.Efficient generation of megakaryocytes from human induced pluripotent stem cells using food and drug administration-approved pharmacological reagents[J]. Stem Cells Transl Med, 2015,4(4):309-319.
[26] 黄韦华,顾海慧,臧艳,等. 一种人诱导多能干细胞分化生成巨核细胞的优化方案[J]. 中国输血杂志,2022,35(9): 900-903.
[27] 岳伟,杨玥,钱宝华,等. 构建诱导型永生化基因整合载体与功能验证[J]. 中国输血杂志,2024,37(12):1341-1349.
[28] PARK Y P,CHOI S C,CHO M Y,et al.Modulation of telomerase activity and human telomerase reverse transcriptase expression by caspases and bcl-2 family proteins in Cisplatin-induced cell death[J]. Korean J Lab Med,2006,26(4):287-293.
[29] LIN H H,MENSCH J,HASCHKE M,et al.Establishment and characterization of hTERT immortalized Hutchinson-gilford progeria fibroblast cell lines[J]. Cells,2022, 11(18). DOI:10.3390/cells11182784.
[30] HINDUL N L,JHITA A,OPREA D G,et al.Construction of a human hTERT RPE-1 cell line with inducible Cre for editing of endogenous genes[J]. Biol Open,2022, 11(2). DOI:10.1242/bio.059056.
[31] YANG J,CHANG E,CHERRY A M,et al.Human endothelial cell life extension by telomerase expression[J]. J Biol Chem,1999,274(37):26141-26148.
[32] LEE K M,CHOI K H,OUELLETTE M M.Use of exogenous hTERT to immortalize primary human cells[J]. Cytotechnology,2004,45(1/2):33-38.
[33] NOETZLI L J,FRENCH S L,MACHLUS K R.New insights into the differentiation of megakaryocytes from hematopoietic progenitors[J]. Arterioscler Thromb Vasc Biol,2019,39(7):1288-1300.
[34] WU M Y,LIU S Q,GAO Y X,et al.Conditional gene knockout and reconstitution in human iPSCs with an inducible Cas9 system[J]. Stem Cell Res,2018,29:6-14.
[35] TREICHEL S,FILIPPI M D.Linking cell cycle to hematopoietic stem cell fate decisions[J]. Front Cell Dev Biol,2023,11:1231735.
[36] WANG Y M,PEI Y A,SUN Y,et al.Stem cells immortalized by hTERT perform differently from those immortalized by SV40LT in proliferation,differentiation, and reconstruction of matrix microenvironment[J]. Acta Biomater,2021,136:184-198.
[37] ALHAJI S Y,NORDIN N,NGAI S C,et al.Lack of methylation on transgene leads to high level and persistent transgene expression in induced pluripotent stem cells[J]. Gene,2020,758:144958.
[38] CHENG Y H,STREICHER D A,WANING D L,et al.Signaling pathways involved in megakaryocyte-mediated proliferation of osteoblast lineage cells[J]. J Cell Physiol, 2015,230(3):578-586.

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

RichHTML

PDF

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 2

Recommended Articles

Metrics

Comments

About

Author

Journal

Access Statistics

Contact

[1]	HOU Xiaoyu, XIA Wenjun, CHEN Ruichi, SUN Denglian, WU Huang, WEN Aiqing. METTL3-mediated m⁶A Methylation Modification of ITGB3 Promotes Megakaryocyte Differentiation [J]. JOURNAL OF CLINICAL TRANSFUSION AND LABORATORY MEDICINE, 2025, 27(5): 594-603.
[2]	GU Haihui, YUE Wei, YANG Yue, et al. Effect of Abscisic Acid on the Generation of Megakaryocytes and Platelets from Induced Pluripotent Stem Cells in vitro Differentiation [J]. JOURNAL OF CLINICAL TRANSFUSION AND LABORATORY MEDICINE, 2023, 25(3): 420-424.