离体 聚乙烯醇基培养物中小鼠造血干细胞的扩增和遗传操作
Summary
这里介绍的是使用 离体 聚乙烯醇基扩增启动、维持和分析小鼠造血干细胞培养物的方案，以及通过慢病毒转导和电穿孔进行遗传操作的方法。
Abstract
自我更新的多能造血干细胞（HSC）是一种重要的细胞类型，因为它们能够在一生中支持造血并在移植后重建整个血液系统。HSC在临床上用于干细胞移植疗法，代表了一系列血液疾病的治愈性治疗。人们对了解调节HSC活性和造血的机制以及开发新的基于HSC的疗法都非常感兴趣。然而，HSCs离体的稳定培养和扩增一直是在易于处理的离体系统中研究这些干细胞的主要障碍。我们最近开发了一种基于聚乙烯醇的培养系统，该系统可以支持可移植小鼠HSC的长期和大规模扩增以及对其进行基因编辑的方法。该协议描述了通过电穿孔和慢病毒转导培养和遗传操作小鼠HSC的方法。该协议有望对对HSC生物学和造血感兴趣的广泛实验血液学家有用。
Introduction
造血系统通过专门的血液和免疫细胞类型支持哺乳动物的一系列基本过程，从氧气供应到对抗病原体。需要持续的血液生成（造血）来支持血液系统稳态，而血液系统稳态由造血干细胞和祖细胞（HSPC）维持1。最原始的造血细胞是造血干细胞（HSC），它具有独特的自我更新和多谱系分化能力2，3。这是一种罕见的细胞群，主要存在于成人骨髓4中，它们以大约每30，000个细胞中一个的频率发生。HSC被认为支持终生造血，并有助于在血液应激后重建造血。这些能力还允许HSC在移植到受辐照的受体后稳定地重建整个造血系统5。这代表了HSC的功能定义，也构成了HSC移植疗法的科学基础，HSC移植疗法是一系列血液和免疫疾病的治愈性治疗方法6。由于这些原因，HSC是实验血液学的主要焦点。
尽管研究重点很大，但稳定地将HSCs从体外扩增7仍然具有挑战性。我们最近开发了第一个用于小鼠HSC8的长期离体扩增培养系统。该方法可以在 4 周培养中将可移植 HSC 扩增 234-899 倍。与其他方法相比，该方案的主要变化是去除血清白蛋白并用合成聚合物代替。聚乙烯醇（PVA）被确定为小鼠HSC培养物8的最佳聚合物，现在也已用于培养其他造血细胞类型9。然而，最近还发现了另一种名为Soluplus（聚乙烯基己内酰胺-醋酸酯-聚乙二醇接枝共聚物）的聚合物，它似乎可以改善克隆HSC扩增10。在使用聚合物之前，使用胎牛血清、牛血清白蛋白级分V或重组血清白蛋白形式的血清白蛋白，但这些对HSC扩增的支持有限，仅支持短期（~1周）离体培养7。然而，应该注意的是，将HSC保持在静止状态的HSC培养方案可以支持更长的离体培养时间11，12。
与其他培养方法相比，基于PVA的培养物的一个主要优点是可以产生的细胞数量以及该方案可用于体外跟踪HSC的时间长度。这克服了实验血液学领域的几个障碍，例如每只小鼠可分离的HSC数量少（只有几千个）以及难以在体内随时间追踪HSC。然而，重要的是要记住，这些培养物刺激HSC增殖，而体内HSC池主要处于稳定状态13。此外，尽管培养物对HSC具有选择性，但随着时间的推移，其他细胞类型确实会随着培养物而积累，并且可移植HSC在1个月后仅代表约34个细胞中的一个。髓系造血祖细胞似乎是这些HSC培养物中的主要污染细胞类型8。然而，我们可以使用这些培养物来富集来自异质细胞群的HSC（例如，c-Kit+骨髓HSPCs14）。它还支持用于遗传操作的HSC的转导或电穿孔14，15，16。为了帮助从异质培养的HSPC群体中鉴定HSC，CD201（EPCR）最近被确定为有用的离体HSC标志物10，17，18，可移植HSC仅限于CD201 + CD150 + c-Kit+ Sca1 + Lineage–部分。
该协议描述了启动，维持和评估基于PVA的小鼠HSC扩增培养的方法，以及使用电穿孔或慢病毒载体转导在这些培养物中进行遗传操作的方案。这些方法有望对一系列实验血液学家有用。
Protocol
Representative Results
Discussion
我们希望该协议为更普遍地研究HSC生物学，造血和血液学提供有用的方法。自从最初开发用于FACS纯化的HSC8的基于PVA的培养方法以来，该方法已经扩展。例如，该方法已被证明适用于富含骨髓的c-Kit和负表面带电板14。它与转导和电穿孔的相容性也已得到证实14，15。这些HSC和c-Kit+ HSPC培养物的体内验证可…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢用于流式细胞术访问的WIMM流式细胞术核心，以及用于慢病毒载体生成的WIMM病毒筛选核心。这项工作由Kay Kendall白血病基金和英国医学研究委员会资助。
Materials
| Equipment | |||
| Dissection kit | Fisher Scientific | 12764416 | |
| Hemocytometer | Appleton Woods Ltd | HC002 | |
| P3 Primary Cell 4D-Nucleofector X Kit | Lonza | V4XP-3024 | |
| Pestle and mortar | Scientific Laboratory Supplies Limited | X18000 | |
| QuadroMACS separator | Miltenyi Biotec | 130-090-976 | |
| Materials | |||
| 5 mL syringe | VWR International Ltd | 720-2519 | |
| 19 G needle | VWR International Ltd | 613-5394 | |
| 50 μm cell strainer | Sysmex | 04-004-2317 | |
| 70 μm cell strainer | Corning | 431751 | |
| Kimtech wipes | VWR International Ltd | 115-2075 | |
| LS MACS column | Miltenyi Biotec | 130-042-401 | |
| Reagents | |||
| Alt-R S.p. Cas9 Nuclease V3, 100 μg | IDT | 1081058 | |
| Animal free recombinant mouse stem cell factor | Peprotech | AF-250-03 | |
| Animal free recombinant mouse thrombopoietin | Peprotech | AF-315-14 | |
| Anti-mouse CD117 APC (clone: 2B8) | ThermoFisher | 17-1171-83 | |
| Anti-mouse CD117 BV421 (clone: 2B8) | Biolegend | 105828 | |
| Anti-mouse CD127 APC/Cy7 (clone: A7R34) | Biolegend | 135040 | |
| Anti-mouse CD127 biotin (clone: A7R34) | Biolegend | 135006 | |
| Anti-mouse CD150 PE/Cy7 (clone: TC15-12F12.2) | Biolegend | 115914 | |
| Anti-mouse CD201 APC (clone: eBio1560) | ThermoFisher | 17-2012-82 | |
| Anti-mouse CD34 FITC (clone: RAM34) | ThermoFisher | 11-0341-85 | |
| Anti-mouse CD4 APC/Cy7 (clone: RM4-5) | Biolegend | 100526 | |
| Anti-mouse CD4 biotin (clone: RM4-5) | Biolegend | 100508 | |
| Anti-mouse CD45R APC/Cy7 (clone: RA3-6B2) | Biolegend | 103224 | |
| Anti-mouse CD45R biotin (clone: RA3-6B2) | Biolegend | 103204 | |
| Anti-mouse CD48 BV421 (clone: HM48-1) | Biolegend | 103428 | |
| Anti-mouse CD8 biotin (clone: 53-6.7) | Biolegend | 100704 | |
| Anti-mouse CD8a APC/Cy7 (clone: 53-6.7) | Biolegend | 100714 | |
| Anti-mouse Ly6G/Ly6C APC/Cy7 (clone: RB6-8C5) | Biolegend | 108424 | |
| Anti-mouse Ly6G/Ly6C biotin (clone: RB6-8C5) | Biolegend | 108404 | |
| Anti-mouse Sca1 PE (clone: D7) | Biolegend | 108108 | |
| Anti-mouse Ter119 APC/Cy7 (clone: TER-119) | Biolegend | 116223 | |
| Anti-mouse Ter119 biotin (clone: TER-119) | Biolegend | 116204 | |
| CellBIND plates, 24-well | Corning | 3337 | negative surface charged |
| CellBIND plates, 96-well | Corning | 3330 | negative surface charged |
| Custom synthetic sgRNA | Synthego, Sigma Aldrich, IDT | Custom order | |
| Fetal bovine serum | Merck Life Science UK Limited | F7524-50ML | |
| Fibronectin Coated plates, 96-well | BD Biosciences | 354409 | |
| Ham's F-12 Nutrient Mix | Gibco | 11765054 | |
| Insulin-Transferrin-Selenium-X (100x) | Gibco | 51500.056 | |
| Phosphate buffered saline | Alfa Aesar | J61196.AP | |
| Polyvinyl alcohol | Sigma Aldrich | P8136 | |
| Propidium Iodide | Enzo Life Sciences (UK) Ltd | EXB-0018 | |
| Streptavidin APC/Cy7 | Biolegend | 405208 | |
| Türks’ solution | Sigma Aldrich | 109277 | |
| Virkon | Mettler-Toledo Ltd | 95015662 |
References
- Laurenti, E., Göttgens, B. From haematopoietic stem cells to complex differentiation landscapes. Nature. 553 (7689), 418-426 (2018).
- Eaves, C. J. Hematopoietic stem cells: concepts, definitions, and the new reality. Blood. 125 (17), 2605-2613 (2015).
- Wilkinson, A. C., Igarashi, K. J., Nakauchi, H. Haematopoietic stem cell self-renewal in vivo and ex vivo. Nature Reviews Genetics. 21 (9), 541-554 (2020).
- Crane, G. M., Jeffery, E., Morrison, S. J. Adult haematopoietic stem cell niches. Nature Reviews Immunology. 17 (9), 573-590 (2017).
- Osawa, M., Hanada, K., Hamada, H., Nakauchi, H. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science. 273 (5272), 242-245 (1996).
- Granot, N., Storb, R. History of hematopoietic cell transplantation: challenges and progress. Haematologica. 105 (12), 2716-2729 (2020).
- Wilkinson, A. C., Nakauchi, H. Stabilizing hematopoietic stem cells in vitro. Current Opinion in Genetics & Development. 64, 1-5 (2020).
- Wilkinson, A. C., et al. Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation. Nature. 571 (7763), 117-121 (2019).
- Nishimura, T., et al. Use of polyvinyl alcohol for chimeric antigen receptor T-cell expansion. Experimental Hematology. 80, 16-20 (2019).
- Becker, H. J., et al. A single cell cloning platform for gene edited functional murine hematopoietic stem cells. bioRxiv. , (2022).
- Kobayashi, H., et al. Environmental optimization enables maintenance of quiescent hematopoietic stem cells ex vivo. Cell Reports. 28 (1), 145-158 (2019).
- Kobayashi, H., Takubo, K. A culture method to maintain quiescent human hematopoietic stem cells. Journal of Visualized Experiments. (171), e61938 (2021).
- Aal Wilson, ., et al. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell. 135 (6), 1118-1129 (2008).
- Ochi, K., Morita, M., Wilkinson, A. C., Iwama, A., Yamazaki, S. Non-conditioned bone marrow chimeric mouse generation using culture-based enrichment of hematopoietic stem and progenitor cells. Nature Communications. 12 (1), 3568 (2021).
- Wilkinson, A. C., et al. Cas9-AAV6 gene correction of beta-globin in autologous HSCs improves sickle cell disease erythropoiesis in mice. Nature Communications. 12 (1), 686 (2021).
- Haney, M. S., et al. Large-scale in vivo CRISPR screens identify SAGA complex members as a key regulators of HSC lineage commitment and aging. bioRxiv. , (2022).
- Che, J. L. C., et al. Identification and characterization of in vitro expanded hematopoietic stem cells. EMBO Reports. 23 (10), e55502 (2022).
- Zhang, Q., Konturek-Ciesla, A., Yuan, O., Bryder, D. Ex vivo expansion potential of murine hematopoietic stem cells: a rare property only partially predicted by phenotype. bioRxiv. , (2022).
- Schindele, P., Wolter, F., Puchta, H. CRISPR guide RNA design guidelines for efficient genome editing. Methods in Molecular Biology. 2166, 331-342 (2020).
- Hanna, R. E., Doench, J. G. Design and analysis of CRISPR-Cas experiments. Nat Biotechnol. 38 (7), 813-823 (2020).
- Wilkinson, A. C., Ishida, R., Nakauchi, H., Yamazaki, S. Long-term ex vivo expansion of mouse hematopoietic stem cells. Nature Protocols. 15 (2), 628-648 (2020).
- Ieyasu, A., et al. An all-recombinant protein-based culture system specifically identifies hematopoietic stem cell maintenance factors. Stem Cell Reports. 8 (3), 500-508 (2017).
