竞争性移植评价造血干细胞健身
Summary
This protocol provides step-by-step guidelines for setting up competitive mouse bone marrow transplant experiments to study hematopoietic stem/progenitor cell function without prior purification of stem cells by cell sorting.
Abstract
The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture.
Introduction
造血是一个再生过程，确保已经通过损伤，辐射和细胞死亡丢失血细胞的补充。这个过程是由造血干细胞（HSC）在很大程度上驻留在成人骨髓确保。此外，造血干细胞可用于在自体免疫疾病，血液恶性肿瘤和免疫缺陷1治疗目的。因此，有必要更好地理解，调节造血干细胞的功能，包括其增殖扩张和它们到达和移植后嫁接接受者的骨髓能力的机制。虽然最近有研究报道一些细胞表面标志物，包括SLAM家族成员CD150和CD48，前瞻性地充实成人造血干细胞和胎儿造血干细胞约50％的纯度2-4，为造血干细胞功能的黄金标准衡量仍然是一个在体内再植测定法确定他们重新建立血液ç能力ELL生产在辐照主机5。
体内克隆再植法最初是由免耕和麦卡洛克6开发，并一直完善和扩大。按照最初的定义，保证造血干细胞通过自我更新和分化终身血细胞生成。造血干细胞的转移到照射受体从而使我们能够评估：其通过不同的血细胞谱系的分析来区分（T淋巴细胞，B淋巴细胞，粒细胞，单核细胞）和它们的通过串行移植自我更新的能力的能力。该试验通常将涉及的造血干细胞的两个群体的功能性和/或数量的比较， 如 ，细胞来自不同基因型或细胞的两只小鼠已经被处理或未处理的与可能影响造血干细胞的维持或膨胀不同的因素来在文化。供者嵌合，或转供体造血干细胞T中的贡献O型血细胞的生产然后可以通过在外周血流式细胞分析和骨髓使用细胞表面标志物或其他方法，将区分供体细胞来自接受者，或主机来确定。最广泛使用的标记物是肯定为此，我们已选择为下面提供的实施例的基因PTPRC或CD45白细胞抗原7的两个等位基因。
克隆再增殖测定法可以是竞争性或非竞争性。在非竞争性设置，控制和测试造血干细胞被转移到单独的受体小鼠和每个细胞类型的结果将是独立于其它的。在竞争激烈的环境，测试和控制的造血干细胞的功能是对竞争者的造血干细胞群体测定。这里所描述的协议使用的竞争的设置，但也可以适用于非竞争的情况。这两种方法都有其优点和局限性，我们将在详细比较他们讨论。我们还描述了不同的方法，以确保在移植造血干细胞的数目权益，解释如何通过有限稀释测定法（LDA），以适应对造血干细胞的定量测定，并同时提供对结果的解释成功和不成功的移植的例子。
Protocol
Representative Results
Discussion
这里所描述的协议被设计来评估针对已知竞争对手的HSCs给体（试验）造血干细胞的相对健康。竞争的情况增加了测定的相对灵敏度（更可能检测到在干细胞的健身中度减少），并提供了照射和喷射的功效的内部技术控制。然而，它不应该被用作HSC健身的绝对值衡量在竞争性重建的下降并不意味着，在没有竞争的造血干细胞不会表现良好。虽然可以说，有竞争力的设置是更好的起点，作为竞争对手…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Roxann河图洛书，阿尔布尔与手续的人物设计和演示服务。研究实验室是由从科尔基金会转型奖的支持下，发现不授予。 419226-2012从加拿大自然科学和（NSERC）工程研究理事会和加拿大创新基金会（CFI领袖基金批准号：31377）。 KMH是Chercheur-布西尔少年的全宗德RECHERCHE魁北克 – 桑特（FRQS）。
Materials
| Microtainer tubes with K2EDTA | BD Biosciences | 365974 | ||
| 20G needle | BD Syringe | For blood sampling from the mandibular vein | ||
| LabQuake Shaker rotisserie | Thermo Scientific | C415110 | Any other rotating mixer will work as well to prevent coagulation of blood samples | |
| Purified anti-mouse CD16/CD32 (clone 2.4G2, Fc Block) | BD Biosciences | 2.50 | 553142 | Alternatively use clone 93 from eBioscience (cat # 14-0161) or Biolegend (cat# 101310) |
| Pe-Cy7-conjugated anti-mouse CD3e (clone 145-2C11) | eBioscience | 0.25 | 25-0031 | For most flow cytometry antibodies, the clone is important but the colours and companies can vary depending on the available equipment |
| PE-conjugated anti-mouse CD19 (clone 1D3) | eBioscience | 0.25 | 12-0193 | |
| APC-eFluor780 (APC-Cy7 equivalent)-conjugated anti-mouse GR1 (clone RB6-8C5) | eBioscience | 0.25 | 47-5931 | |
| FITC-conjugate anti-mouse CD45.1 (clone A20) | eBioscience | 2.50 | 11-0453 | |
| eFluor450-conjugated anti-mouse CD45.2 (clone 104) | eBioscience | 1.00 | 48-0454 | |
| Biotinylated anti-human/mouse CD45R (B220) (clone RA3-6B2) | eBioscience | 1.25 | 13-0452 | |
| Biotinylated anti-mouse CD3e (clone 145-2C11) | eBioscience | 1.25 | 13-0031 | |
| Biotinylated anti-mouse CD11b (clone M1/70) | eBioscience | 1.25 | 13-0112 | |
| Biotinylated anti-mouse GR1 (clone RB6-8C5) | eBioscience | 1.25 | 13-5931 | |
| Biotinylated anti-mouse TER119 (clone TER119) | eBioscience | 0.63 | 13-5921 | |
| V500 streptavidin | BD Biosciences | 0.50 | 561419 | |
| PE-conjugated anti-mouse CD117 (clone 2B8) | BD Biosciences | 0.25 | 553355 | |
| PE-Cy7-conjugated anti-mouse Ly6A/E (Sca1) (clone D7) | BD Biosciences | 0.25 | 558162 | |
| PerCP-eFluor710-conjugated anti-mouse CD135 (clone A2F10) | eBioscience | 0.50 | 46-1351 | |
| Alexa fluor 647-conjugated anti-mouse CD150 (clone TC15-12F12.2) | Biolegend | 0.63 | 115918 | BD Biosciences and eBioscience do not carry the same clone |
| 1ml tuberculin syringe with 27G needle | BD Syringe | 309623 | ||
| 1ml tuberculin syringe with 25G needle | BD Syringe | 309626 | ||
| 70 um cell strainer | BD Falcon | 352350 |
References
- Li, H. W., Sykes, M. Emerging concepts in haematopoietic cell transplantation. Nat Rev Immunol. 12 (6), 403-416 (2012).
- Kiel, M. J., Yilmaz, O. H., Iwashita, T., Terhorst, C., Morrison, S. J. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell. 121 (7), 1109-1121 (2005).
- Kim, I., He, S., Yilmaz, O. H., Kiel, M. J., Morrison, S. J. Enhanced purification of fetal liver hematopoietic stem cells using SLAM family receptors. Blood. 108 (2), 737-744 (2006).
- Mayle, A., Luo, M., Jeong, M., Goodell, M. A. Flow cytometry analysis of murine hematopoietic stem cells. Cytometry A. 83 (1), 27-37 (2013).
- Rossi, L., et al. Less Is More: Unveiling the Functional Core of Hematopoietic Stem Cells through Knockout Mice. Cell Stem Cell. 11 (3), 302-317 (2012).
- Till, J. E., McCulloch, E. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res. 14, 213-222 (1961).
- Shen, F. W., et al. Cloning of Ly-5 cDNA. Proc Natl Acad Sci U S A. 82 (21), 7360-7363 (1985).
- . Identification of GM mice. Laboratory Animals. 37 (suppl 1), 33-35 (2003).
- Rundberg Nilsson, A., Bryder, D., Pronk, C. J. H. Frequency determination of rare populations by flow cytometry: A hematopoietic stem cell perspective. Cytometry Part A. 83A (8), 721-727 (2013).
- Abidin, B. M., Owusu Kwarteng, E., Heinonen, K. M. Frizzled-6 Regulates Hematopoietic Stem/Progenitor Cell Survival and Self-Renewal. J Immunol. 195 (5), 2168-2176 (2015).
- Heinonen, K. M., Vanegas, J. R., Lew, D., Krosl, J., Perreault, C. Wnt4 enhances murine hematopoietic progenitor cell expansion through a planar cell polarity-like pathway. PLoS One. 6 (4), e19279 (2011).
- Oguro, H., Ding, L., Morrison, S. J. SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors. Cell Stem Cell. 13 (1), 102-116 (2013).
- Golde, W. T., Gollobin, P., Rodriguez, L. L. A rapid, simple, and humane method for submandibular bleeding of mice using a lancet. Lab Anim (NY). 34 (9), 39-43 (2005).
- Santaguida, M., et al. JunB protects against myeloid malignancies by limiting hematopoietic stem cell proliferation and differentiation without affecting self-renewal. Cancer Cell. 15 (4), 341-352 (2009).
- Czechowicz, A., Kraft, D., Weissman, I. L., Bhattacharya, D. Efficient transplantation via antibody-based clearance of hematopoietic stem cell niches. Science. 318 (5854), 1296-1299 (2007).
- Zhang, C. C., Lodish, H. F. Murine hematopoietic stem cells change their surface phenotype during ex vivo expansion. Blood. 105 (11), 4314-4320 (2005).
- Benveniste, P., et al. Intermediate-Term Hematopoietic Stem Cells with Extended but Time-Limited Reconstitution Potential. Cell Stem Cell. 6 (1), 48-58 (2010).
- Fazekasde St Groth, B. The evaluation of limiting dilution assays. J Immunol Methods. 49 (2), R11-R23 (1982).
- Louis, I., Heinonen, K. M., Chagraoui, J., Vainio, S., Sauvageau, G., Perreault, C. The signaling protein Wnt4 enhances thymopoiesis and expands multipotent hematopoietic progenitors through beta-catenin-independent signaling. Immunity. 29 (1), 57-67 (2008).
- Cui, Y. Z., et al. Optimal protocol for total body irradiation for allogeneic bone marrow transplantation in mice. Bone Marrow Transplant. 30 (12), 843-849 (2002).
- Benz, C., et al. Hematopoietic Stem Cell Subtypes Expand Differentially during Development and Display Distinct Lymphopoietic Programs. Cell Stem Cell. 10 (3), 273-283 (2012).
- Eppert, K., et al. Stem cell gene expression programs influence clinical outcome in human leukemia. Nat Med. 17 (9), 1086-1093 (2011).
- McIntosh, B. E., et al. Nonirradiated NOD,B6.SCID Il2rgamma-/- Kit(W41/W41) (NBSGW) mice support multilineage engraftment of human hematopoietic cells. Stem Cell Reports. 4 (2), 171-180 (2015).
