人脐血巨分化与血小板形成的 CD34+单元格
Summary
高纯度的巨可以从脐带血衍生的 CD34+细胞获得。本文介绍了一种 CD34+单元格隔离和巨区分的方法。
Abstract
血小板的产生主要发生在骨髓的过程中, 称为形成。在形成, 造血祖细胞分化形成血小板前体称为巨, 后者的末期分化, 释放血小板从长细胞质过程称为 proplatelets。巨是罕见的细胞局限于骨髓, 因此很难获得足够数量的实验室使用。在合适的条件下, 通过培养 CD34 的+细胞, 可以在体外实现高效的人巨生产。这里详述的协议描述了由脐带血标本中的磁性细胞分类分离 CD34 的+细胞。介绍了在无血清条件下生产高纯度、成熟巨的必要步骤。还提供了巨分化的表型分析和 proplatelet 形成和血小板生成的测定的详细资料。影响巨分化和/或 proplatelet 形成的效应, 如血小板抗体或生成拟, 可以添加到培养细胞, 以检查生物功能。
Introduction
由于骨髓中的低频率, 在正常的实验室使用中分离出足够数量的初级人类巨 (MK) 是不可行的, 因为它们在0.01% 的有核细胞中占到了1。一种方便的替代方法是在特定生长因子存在的情况下, 对造血干细胞和祖细胞进行体外扩张和分化. 在培养体系中, 有许多细胞因子, 包括干细胞因子、c 型配体和白细胞介素 (IL)-3 和 IL-11 MKs. 生成 (TPO) 是巨文化最有效的生长和分化因子并且是有效的单独或与其他细胞因子, 例如 IL-32。TPO 可以对干细胞种群产生作用, 导致 MKs2的增殖和成熟。
MK 产生血小板从细胞质突起称为 proplatelets 和,在体内,大约 1 x 1011血小板每天形成, 以维持血小板计数 150-400 x 109/l. 血小板生成体外为1000-折叠低于在体内估计3, 这已经产生了许多文化条件使用 CD34+造血祖细胞改善 MK 和血小板生成体外。用于 MK 分化的 CD34+细胞的初始来源是人外周血4。其他细胞来源包括骨髓5,6, 胚胎干细胞/诱导多能干细胞 (ESC/iPSC)7, 脐血 (联合)8,9,10.人骨髓 CD34+ 11和小鼠血统阴性骨髓细胞5 体外产生 MK 和血小板;然而, 缺乏人类骨髓的可用性限制了它作为 CD34 的+细胞的来源。相反, ESC 和 iPSC 代表了体外血小板产生的无限细胞来源。这些细胞的血小板生成需要饲养细胞, 如小鼠 OP9 细胞和较长的培养期。在无馈线条件下获得的血小板似乎不太正常12。iPSC 源性血小板可能在临床上有应用, 因为它们可以扩展到大规模。这个过程需要慢介导转录因子的转导和长期细胞培养13。
CD34 是一个可访问的+单元的来源, 可以在研究设置中随时使用。TPO 单独可以促进脐血衍生 CD34 细胞的分化, 这就产生了高度纯净、成熟的 MKs, 而不需要补充血清或与饲养细胞共培养.其他细胞因子, 如 CD34, 可降低与脐血细胞的分化, 而 Flt-3 配体和 IL-11 促进未成熟巨的产生, 14。本协议描述了从脐血 CD34+细胞在无血清条件下生产高纯度 MK 培养物的情况。
Protocol
该议定书得到了东南悉尼人类研究伦理委员会的批准, 并得到新南威尔士大学人类研究伦理委员会的批准。从健康捐献者获得的脐带血由悉尼脐血库 (悉尼, 新南威尔士州) 提供。这个过程使用了大约100毫升的容量。 注: 使用无菌技术的第二类生物安全柜工作。用70% 乙醇净化脐带血袋的外部。使用无菌器械 (剪刀、镊子) 进行此程序。 1. 脐血细胞的制备和分?…
Representative Results
此协议允许从脐血衍生的 CD34+细胞制备高度纯净的 MK 培养物。脐血中 CD34+单元格的百分比约为 1.3 (图 1A), 每个单核细胞的总数量 (步骤 1.8) 范围从 90-300 x 106每个单元。隔离后的 CD34+/CD45+ 细胞的纯度从90到 99% (图 1B)。MK (定义为 CD41 的+单元格) 在无血清 CD34+单元格区域…
Discussion
本文所述的协议适用于脐血培养中的 MK 和血小板的持续生产。这些细胞可用于研究各种过程, 如药物或生物活动对 MK 增殖、分化、proplatelet 形成和血小板生成的影响。
文中介绍了多种培养基和细胞因子的组合。细胞因子、Flt-3 配体、IL-3 和 IL-6 细胞因子的增加支持 CD34 的增殖.但是, 此扩展在区域性14中导致 MK 纯度降低。这里提出的方法, 使用无血清?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者承认澳大利亚卫生和医学研究理事会 (与六六六相关的项目赠款 1012409) 的支持。
Materials
| Cell Culture Reagents | |||
| Recombinant Human TPO | Miltenyi Biotec | 130-094-013 | |
| StemSpan SFEM II | Stem Cell Technologies | 9605 | Serum-free media for CD34+ cells |
| Name | Company | Catalog Number | Comments |
| CD34 Isolation Reagents | |||
| CD34 MicroBead kit ultrapure | Miltenyi Biotec | 130-100-453 | This kit includes the FcR human IgG blocking reagent and CD34 microbeads. These beads contain the anti-CD34 antibody clone QBEND/10. Use a different anti-CD34 clone for purity check (e.g. clone 8G12). |
| Lymphoprep | Alere Technologies | 1114545 | Lymphocyte separation media (density 1.077 g/mL) |
| Sterile separation buffer (SB) | Miltenyi Biotec | 130-091-221 | This buffer contains phosphate buffered saline (PBS), pH 7.2 containing 0.5% bovine serum albumin and 2 mM EDTA. It can be prepared using sterile, cell culture grade components. De-gas before use because air bubbles can block the column. |
| Name | Company | Catalog Number | Comments |
| Flow Cytometry and Cell Staining Reagents | |||
| PE Mouse anti-Human CD34 | BD Biosciences | 340669 | Clone 8G12. This can be used for CD34 purity check. Final antibody concentration 1:10 dilution. |
| PerCP mouse anti-human CD45 | BD Biosciences | 347464 | 1:10 dilution |
| PerCP isotype control | BD Biosciences | 349044 | 1:10 dilution |
| FITC Mouse anti-Human CD41a | BD Biosciences | 340929 | Final antibody concentration 1:5 dilution. |
| APC Mouse anti-Human CD42b | BD Biosciences | 551061 | This antibody can also be used to detect mature MK (the percentage of positive cells in usually lower than with anti CD42a). Final antibody concentration 1:10 dilution. |
| Alexa Fluor 647 Mouse anti-Human CD42a | AbD Serotec | MCA1227A647T | Currently distributed by Bio-Rad. Final antibody concentration 1:10 dilution. |
| Alexa Fluor 647 Mouse Negative Control | AbD Serotec | MCA928A647 | Currently distributed by Bio-Rad. Isotype control antibody |
| Anti von Willebrand factor rabbit polyclonal | Abcam | AB6994 | 1:200 dilution |
| V450 mouse anti-humna CD41a | BD Biosciences | 58425 | 1: 20 dilution |
| V450 isotype control | BD Biosciences | 580373 | 1:20 dilution |
| PAC1-FITC antibody | BD Biosciences | 340507 | 1:10 dilution |
| Anti CD62p mouse monoclonal | Abcam | AB6632 | 1:200 dilution |
| Alexa Fluor 488 goat anti rabbit IgG | Invitrogen | A11008 | 1:100 dilution |
| Alexa Fluor 594 goat anti mouse IgG | Invitrogen | A11020 | 1:100 dilution |
| Ig Isotype Control cocktail-C | BD Biosciences | 558659 | Isotype control antibody |
| Propidium iodide | Sigma Aldrich | P4864 | |
| CountBright Absolute Counting Beads | Molecular Probes, Invitrogen | C36950 | Counting beads |
| Name | Company | Catalog Number | Comments |
| Materials | |||
| LS columns | Miltenyi Biotec | 130-042-401 | Smaller and larger columns are also commercially available |
| MidiMACS Separator magnet | Miltenyi Biotec | 130-042-302 | |
| MACS MultiStand | Miltenyi Biotec | 130-042-303 | |
| Falcon 5mL round bottom polypropylene FACS tubes, with Snap Cap, Sterile | In Vitro technologies | 352063 | |
| Glass slides | Menzel-Glaser | J3800AMNZ | |
| Mounting media with DAPI | Vector Laboratories | H-1200 | Antifade mounting medium with DAPI |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| Inverted microscope | Leica | DMIRB inverted microscope | |
| Fluorescent microscope | Zeiss | Vert.A1 | |
| Cell analyser | BD Biosciences | FACS Canto II | |
| Cytospin centrifuge | ThermoScientific | Cytospin 4 | |
| Name | Company | Catalog Number | Comments |
| Software | |||
| Cell analyser software | BD Biosciences | FACS Diva Software | |
| Single cell analysis software | Tree Star | FlowJo | |
| Fluorescent microscope software | Zeiss | Zen 2 blue edition |
Referências
- Nakeff, A., Maat, B. Separation of megakaryocytes from mouse bone marrow by velocity sedimentation. Blood. 43 (4), 591-595 (1974).
- Zeigler, F. C., et al. In vitro megakaryocytopoietic and thrombopoietic activity of c-mpl ligand (TPO) on purified murine hematopoietic stem cells. Blood. 84 (12), 4045-4052 (1994).
- Reems, J. -. A., Pineault, N., Sun, S. In Vitro Megakaryocyte Production and Platelet Biogenesis: State of the Art. Transfus. Med. Rev. 24 (1), 33-43 (2010).
- Choi, E., Nichol, J. L., Hokom, M. M., Hornkohl, A. C., Hunt, P. Platelets generated in vitro from proplatelet-displaying human megakaryocytes are functional. Blood. 85 (2), 402-413 (1995).
- Perdomo, J., et al. A monopartite sequence is essential for p45 NF-E2 nuclear translocation, transcriptional activity and platelet production. J Thromb Haemost. 8 (11), 2542-2553 (2010).
- Shim, M. H., Hoover, A., Blake, N., Drachman, J. G., Reems, J. A. Gene expression profile of primary human CD34+CD38lo cells differentiating along the megakaryocyte lineage. Exp Hematol. 32 (7), 638-648 (2004).
- Feng, Q., et al. Scalable generation of universal platelets from human induced pluripotent stem cells. Stem cell reports. 3 (5), 817-831 (2014).
- Bruno, S., et al. In vitro and in vivo megakaryocyte differentiation of fresh and ex-vivo expanded cord blood cells: rapid and transient megakaryocyte reconstitution. Haematologica. 88 (4), 379-387 (2003).
- Iraqi, M., Perdomo, J., Yan, F., Choi, P. Y. I., Chong, B. H. Immune thrombocytopenia: antiplatelet autoantibodies inhibit proplatelet formation by megakaryocytes and impair platelet production in vitro. Haematologica. 100 (5), 623-632 (2015).
- Lev, P. R., et al. Impaired proplatelet formation in immune thrombocytopenia: a novel mechanism contributing to decreased platelet count. Br. J. Haematol. 165 (6), 854-864 (2014).
- Gandhi, M. J., Drachman, J. G., Reems, J. A., Thorning, D., Lannutti, B. J. A novel strategy for generating platelet-like fragments from megakaryocytic cell lines and human progenitor cells. Blood Cells Mol. Dis. 35 (1), 70-73 (2005).
- Lu, S. -. J., et al. Platelets generated from human embryonic stem cells are functional in vitro and in the microcirculation of living mice. Cell Res. 21 (3), 530-545 (2011).
- Moreau, T., et al. Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming. Nature Commun. 7, 11208 (2016).
- De Bruyn, C., Delforge, A., Martiat, P., Bron, D. Ex vivo expansion of megakaryocyte progenitor cells: cord blood versus mobilized peripheral blood. Stem Cells Dev. 14 (4), 415-424 (2005).
- Nimgaonkar, M. T., et al. A unique population of CD34+ cells in cord blood. Stem cells. 13 (2), 158-166 (1995).
- Italiano, J. E., Patel-Hett, S., Hartwig, J. H. Mechanics of proplatelet elaboration. J Thromb Haemost. 5, 18-23 (2007).
- Matsunaga, T., et al. Ex vivo large-scale generation of human platelets from cord blood CD34+ cells. Stem cells. 24 (12), 2877-2887 (2006).
- Sullenbarger, B., Bahng, J. H., Gruner, R., Kotov, N., Lasky, L. C. Prolonged continuous in vitro human platelet production using three-dimensional scaffolds. Exp Hematol. 37 (1), 101-110 (2009).
- Proulx, C., Boyer, L., Hurnanen, D. R., Lemieux, R. Preferential ex vivo expansion of megakaryocytes from human cord blood CD34+-enriched cells in the presence of thrombopoietin and limiting amounts of stem cell factor and Flt-3 ligand. J. Hematother. Stem Cell Res. 12 (2), 179-188 (2003).
- Bornstein, R., Garcia-Vela, J., Gilsanz, F., Auray, C., Cales, C. Cord blood megakaryocytes do not complete maturation, as indicated by impaired establishment of endomitosis and low expression of G1/S cyclins upon thrombopoietin-induced differentiation. Br. J. Haematol. 114 (2), 458-465 (2001).
- Chong, B. H., Choi, P. Y. I., Khachigian, L., Perdomo, J. Drug-induced Immune Thrombocytopenia. Hematol Oncol Clin North Am. 27 (3), (2013).
- Stasi, R., et al. Idiopathic thrombocytopenic purpura: Current concepts in pathophysiology and management. Thromb Haemost. 99 (1), 4-13 (2008).
Citar este artigo
Perdomo, J., Yan, F., Leung, H. H., Chong, B. H. Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells. J. Vis. Exp. (130), e56420, doi:10.3791/56420 (2017).