Mazurier Frederic, Gan Olga I, McKenzie Joby L, Doedens Monica, Dick John E
Division of Cell and Molecular Biology, University Health Network, Ste 7-700, 620 University Ave, Toronto, ON, Canada M5G 2C1.
Blood. 2004 Jan 15;103(2):545-52. doi: 10.1182/blood-2003-05-1558. Epub 2003 Sep 22.
Knowledge of the composition and interrelationship of the various hematopoietic stem cells (HSCs) that comprise the human HSC pool and the consequence of culture on each class is required for effective therapies based on stem cells. Clonal tracking of retrovirally transduced HSCs in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice revealed heterogeneity in the repopulation capacity of SCID-repopulating cells (SRCs). However, it is impossible to establish whether HSC heterogeneity is intrinsic or whether the culture conditions required for retroviral transduction induce qualitative and quantitative alterations to SRCs. Here, we report establishment of a clonal tracking method that uses lentivectors to transduce HSCs with minimal manipulation during overnight culture without cytokine stimulation. By serial bone marrow (BM) sampling of mice receiving transplants, short-term SRCs (ST-SRCs) and long-term SRCs (LT-SRCs) were identified on the basis of repopulation dynamics demonstrating that their existence is not an experimental artifact but reflects the state of the HSC pool. However, 4 days of culture in conditions previously used for SRC retroviral transduction significantly reduced SRC number as assessed by clonal analysis. These studies provide a foundation to understand the molecular and cellular determinants of human HSC development and to develop therapies targeted to specific HSC classes.
基于干细胞的有效治疗需要了解构成人类造血干细胞（HSC）库的各种造血干细胞的组成和相互关系，以及培养对每一类细胞的影响。在非肥胖糖尿病/严重联合免疫缺陷（NOD/SCID）小鼠中对逆转录病毒转导的造血干细胞进行克隆追踪，揭示了严重联合免疫缺陷重建细胞（SRC）的重建能力存在异质性。然而，无法确定造血干细胞的异质性是内在的，还是逆转录病毒转导所需的培养条件会导致SRC发生定性和定量的改变。在此，我们报告了一种克隆追踪方法的建立，该方法使用慢病毒载体在无细胞因子刺激的过夜培养过程中以最少的操作转导造血干细胞。通过对接受移植小鼠的连续骨髓（BM）采样，根据重建动力学确定了短期SRC（ST-SRC）和长期SRC（LT-SRC），这表明它们的存在并非实验假象，而是反映了造血干细胞库的状态。然而，通过克隆分析评估，在先前用于SRC逆转录病毒转导的条件下培养4天会显著减少SRC数量。这些研究为理解人类造血干细胞发育的分子和细胞决定因素以及开发针对特定造血干细胞类别的治疗方法奠定了基础。
