Gammaitoni Loretta, Weisel Katja C, Gunetti Monica, Wu Kai-Da, Bruno Stefania, Pinelli Silvana, Bonati Antonio, Aglietta Massimo, Moore Malcolm A S, Piacibello Wanda
Department of Oncological Sciences, IRCC-Institute for Cancer Research and Treatment, Laboratory of Clinical Oncology, University of Turin Medical School, Prov 142, 10060 Candiolo, Turin, Italy.
Blood. 2004 Jun 15;103(12):4440-8. doi: 10.1182/blood-2003-09-3079. Epub 2004 Jan 15.
Telomerase activity, telomere length, stem/progenitor cell production, and function of CD34+ cells from cord blood (CB), bone marrow, and mobilized peripheral blood were evaluated in long-term cultures. CB cells were cultured either on OP-9 stromal cells transduced with an adenovector expressing thrombopoietin (TPO) or stimulated by a cytokine cocktail in the absence of stroma, with, in one method, CD34+ cells reisolated at monthly intervals for passage. Continuous expansion of stem cells as measured by in vitro cobblestone area and secondary colony-forming assays was noted for 18 to 20 weeks and by severe combined immunodeficiency (SCID)-repopulating cells (SRCs), capable of repopulating and serially passage in nonobese diabetic/SCID mice, for 16 weeks. Despite this extensive proliferation, telomere length initially increased and only at late stages of culture was evidence of telomere shortening noted. This telomere stabilization correlated with maintenance of high levels of telomerase activity in the CD34+ cell population for prolonged periods of culture. Cytokine-stimulated cultures of adult CD34+ cells showed CD34+ and SRC expansion (6-fold) for only 3 to 4 weeks with telomere shortening and low levels of telomerase. There is clearly a clinical value for a system that provides extensive stem cell expansion without concomitant telomere erosion.
在长期培养中评估了来自脐带血（CB）、骨髓和动员外周血的CD34+细胞的端粒酶活性、端粒长度、干细胞/祖细胞产生及功能。CB细胞要么培养在经表达血小板生成素（TPO）的腺载体转导的OP-9基质细胞上，要么在无基质的情况下用细胞因子混合物刺激，其中一种方法是每月间隔重新分离CD34+细胞进行传代。通过体外卵石区和二次集落形成试验测定，干细胞持续扩增达18至20周，通过严重联合免疫缺陷（SCID）-重建细胞（SRC）测定，其能够在非肥胖糖尿病/ SCID小鼠中重建并连续传代达16周。尽管有这种广泛的增殖，端粒长度最初增加，仅在培养后期才注意到端粒缩短的迹象。这种端粒稳定与在长时间培养中CD34+细胞群体中端粒酶活性的高水平维持相关。成年CD34+细胞的细胞因子刺激培养仅在3至4周内显示CD34+和SRC扩增（6倍），伴有端粒缩短和低水平的端粒酶。对于一个能提供广泛的干细胞扩增而不伴有端粒侵蚀的系统，显然具有临床价值。
