应用造血干细胞移植评估骨髓增生异常综合征的起源
Summary
我们描述了利用造血干细胞移植 (HSCT) 来评估基因工程造血细胞的恶性潜能。HSCT 可用于评估体内各种恶性造血细胞, 并产生大量小鼠骨髓增生异常综合征 (MDS) 或白血病, 以评估新的治疗方法。
Abstract
骨髓增生异常综合征 (MDS) 是由无效造血、外周血血细胞减少、发育不良以及转化为急性白血病的倾向所定义的多种造血干细胞紊乱群。NUP98-HOXD13 (NHD13) 转基因小鼠在外周血血细胞减少、发育不良和急性白血病的转化方面概述了人类 MDS。我们以前证明, mds 可以通过移植 mds 骨髓有核细胞 (BMNC) 从基因工程鼠与 mds 转移到野生型受体。为了更清楚地了解 MDS 原细胞, 我们开发了移植特定的、immunophenotypically 定义的造血亚群的方法。本文描述了造血干细胞和祖细胞特定种群的分离和移植过程。在移植后, 我们描述了评估移植的效率和捐献者 MDS 细胞持续性的方法。
Introduction
骨髓增生异常综合征 (MDS) 代表了一系列不同的无性系血症, 其特点是造血功能无效, 形态学证明发育不良, 并倾向于转化为急性髓系白血病 (AML)1,2 ,3,4。无效造血被认为是在骨髓中的成熟逮捕, 并导致外周血血细胞减少, 尽管 hypercellular 骨髓1,3。mds 的发病率在美国每年有不同程度地估计为每10万人2-12 例, mds 的发病率随着年龄的增长而增加, 这是一个重要的条件来理解, 因为美国人口老龄化3, 5。虽然大多数 mds 病例没有明确的病因, 但有些 mds 的病例被认为是由于接触已知的毒性剂, 包括苯等溶剂和癌症化疗6。
mds 患者通常在 mds 细胞7中获得突变。虽然相对少见, 一些 MDS 患者获得了平衡染色体易位涉及基因, 如 NUP98, EVI1, RUNX1, MLL (http://cgap.nci.nih.gov/Chromosomes/Mitelman)。我们的实验室对染色体易位有长期的兴趣, 其中涉及 NUP98 基因8。通过 Vav1 启动子和增强因子调控的 NUP98-HOXD13 (NHD13) 转基因的转基因小鼠显示了 MDS 的所有关键特征, 包括外周血血细胞减少、发育不良的形态学证据以及转化为 AML9.
虽然 mds 已被确认超过60年10, 并被认为是克隆干细胞紊乱, 但在 immunodeficient 小鼠嫁接人 MDS 细胞的努力基本上不成功, 因为 mds 细胞嫁接差11, 12、13、14 、小鼠不发育临床疾病。为了确定哪些造血细胞能够传输 MDS, 我们转向了 NHD13 模型, 并表明我们可以嫁接 mds 作为一个疾病实体, 它显示了人类 mds 的所有基本特征, 包括外周血血细胞减少、发育不良和转换到 AML15。在本报告中, 我们介绍了这些实验的技术细节, 以及进一步分级造血干细胞和前体细胞 (HSPC) 的方法, 旨在确定 MDS 启动细胞。
Protocol
Representative Results
Discussion
虽然 mds 是一种克隆造血干细胞紊乱, 但 mds “茎” 或启动细胞尚未被描绘出来。我们以前证明, MDS 可以移植到小鼠使用骨髓从 NHD13 小鼠的 HSCT, 其特点是 macrocytic 贫血, 白细胞减少, 中性粒细胞, 和形态学证据的发育不良15。此外, 竞争性重写化验发现 NHD13 骨髓细胞的生长优势。综合起来, 这些发现意味着存在 MDS 干细胞或启动细胞。目前正在进行更多的实验, 目的是通过更明确的茎和…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家癌症研究所、国立卫生研究院 (格兰特号010378和 BC 010983) 的校内研究计划的支持。
Materials
| 14 mL round bottom tube | Falcon | 352057 | |
| Hank's balanced salt solution | Lonza | 10-527F | |
| Anti-CD45.2 antibody | Southern Biotech | 1800-15 | LOT# A077-T044O |
| 3 mL Syringe | Monoject | 8881513934 | |
| 27-G needle | BD | 305109 | |
| 20-G needle | BD | 305176 | |
| Lineage Cocktail | Miltenyi | 130-090-858 | LOT# 5170418221 |
| Anti-Biotin antibodies | Miltenyi | 130-113-288 | LOT# 5171109046 |
| 1 mL Syringe | Excelint | 26027 | Insulin Syringe |
| Heating Lamp | Thermo Fisher Scientific | E70001901 | |
| FACS machine | Cytec | FACScan | 2 lasers, 5 color detectors |
| FACS sorting instrument | Beckman Coulter | MOFLO ASTRIOS | 5 lasers, 23 parameters, 6 population sorting simulteneously |
| Propidium Iodide | Thermo Fisher Scientific | P3566 | |
| Gamma Irradiator | Best Theratronics | Gammacell 40 | |
| Blood collection tube | RAM scientific | 76011 | |
| Recipient mice | Charles River | B6-LY5.1/Cr, CD45.1 | |
| NUP98-HOXD13 mice | n/a | C57Bl/6, CD45.2 | Colony maintained at NIH |
| 5 mL round bottom tube | Falcon | 352058 |
Referências
- Corey, S. J., et al. Myelodysplastic syndromes: the complexity of stem-cell diseases. Nature Reviews Cancer. 7 (2), 118-129 (2007).
- Garcia-Manero, G. Myelodysplastic syndromes: 2015 Update on diagnosis, risk-stratification and management. American Journal of Hematology. 90 (9), 831-841 (2015).
- Heaney, M. L., Golde, D. W. Myelodysplasia. The New England Journal of Medicine. 340 (21), 1649-1660 (1999).
- Nimer, S. D. Myelodysplastic syndromes. Blood. 111 (10), 4841-4851 (2008).
- Aul, C., Giagounidis, A., Germing, U. Epidemiological features of myelodysplastic syndromes: results from regional cancer surveys and hospital-based statistics. International Journal of Hematology. 73 (4), 405-410 (2001).
- Pedersen-Bjergaard, J., Christiansen, D. H., Desta, F., Andersen, M. K. Alternative genetic pathways and cooperating genetic abnormalities in the pathogenesis of therapy-related myelodysplasia and acute myeloid leukemia. Leukemia. 20 (11), 1943-1949 (2006).
- Uy, G. L., et al. Dynamic changes in the clonal structure of MDS and AML in response to epigenetic therapy. Leukemia. 31 (4), 872-881 (2017).
- Gough, S. M., Slape, C. I., Aplan, P. D. NUP98 gene fusions and hematopoietic malignancies: common themes and new biologic insights. Blood. 118 (24), 6247-6257 (2011).
- Lin, Y. W., Slape, C., Zhang, Z., Aplan, P. D. NUP98-HOXD13 transgenic mice develop a highly penetrant, severe myelodysplastic syndrome that progresses to acute leukemia. Blood. 106 (1), 287-295 (2005).
- Block, M., Jacobson, L. O., Bethard, W. F. Preleukemic acute human leukemia. Journal of the American Medical Association. 152 (11), 1018-1028 (1953).
- Thanopoulou, E., et al. Engraftment of NOD/SCID-beta2 microglobulin null mice with multilineage neoplastic cells from patients with myelodysplastic syndrome. Blood. 103 (11), 4285-4293 (2004).
- Kerbauy, D. M., Lesnikov, V., Torok-Storb, B., Bryant, E., Deeg, H. J. Engraftment of distinct clonal MDS-derived hematopoietic precursors in NOD/SCID-beta2-microglobulin-deficient mice after intramedullary transplantation of hematopoietic and stromal cells. Blood. 104 (7), 2202-2203 (2004).
- Benito, A. I., et al. NOD/SCID mice transplanted with marrow from patients with myelodysplastic syndrome (MDS) show long-term propagation of normal but not clonal human precursors. Leukemia Research. 27 (5), 425-436 (2003).
- Medyouf, H., et al. Myelodysplastic cells in patients reprogram mesenchymal stromal cells to establish a transplantable stem cell niche disease unit. Cell Stem Cell. 14 (6), 824-837 (2014).
- Chung, Y. J., Choi, C. W., Slape, C., Fry, T., Aplan, P. D. Transplantation of a myelodysplastic syndrome by a long-term repopulating hematopoietic cell. Proceedings of the National Academy of Sciences of the United States of America. 105 (37), 14088-14093 (2008).
- Pietras, E. M., et al. Functionally Distinct Subsets of Lineage-Biased Multipotent Progenitors Control Blood Production in Normal and Regenerative Conditions. Cell Stem Cell. 17 (1), 35-46 (2015).
- Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M., Hoogstraten-Miller, S. Retro-orbital injections in mice. Lab Animal. 40 (5), 155-160 (2011).
- Chung, Y. J., Fry, T. J., Aplan, P. D. Myeloablative hematopoietic stem cell transplantation improves survival but is not curative in a pre-clinical model of myelodysplastic syndrome. PLoS One. 12 (9), e0185219 (2017).
