Zjablovskaja Polina, Danek Petr, Kardosova Miroslava, Alberich-Jorda Meritxell
Department of Hemato-Oncology, Institute of Molecular Genetics of the ASCR; Childhood Leukaemia Investigation Prague, Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles University.
Department of Hemato-Oncology, Institute of Molecular Genetics of the ASCR.
J Vis Exp. 2018 Feb 21(132):57033. doi: 10.3791/57033.
Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.
对造血干细胞和祖细胞生物学的理解对再生医学和血液学疾病的治疗具有重要意义。尽管使用体内模型或原代培养可以获得最相关的数据，但造血干细胞和祖细胞的低丰度极大地限制了用于其研究的合适技术的范围。因此，使用细胞系能够充分生产生物材料，以进行需要大量细胞的筛选或检测。在此，我们详细描述、展示并解读了用于研究髓系造血和嗜中性粒细胞分化相关过程的增殖和分化检测方法。这些实验采用了32D/G-CSF-R细胞因子依赖性小鼠髓系细胞系，该细胞系在IL-3存在下具有增殖能力，并在G-CSF作用下能够分化。我们提供了处理32D/G-CSF-R细胞的优化方案，并讨论了可能影响所述检测方法和预期结果的主要陷阱和缺点。此外，本文还包含慢病毒和逆转录病毒的生产、滴定以及32D/G-CSF-R细胞转导的方案。我们证明，对这些细胞进行基因操作可成功用于功能和分子研究，从而补充使用原代造血干细胞和祖细胞或体内模型所获得的结果。
