人脐带血衍生CD34的泛骨髓分化–造血干细胞和祖细胞
Summary
在这里,我们提出了免疫表征表表征和细胞因子诱导分化脐带血衍生CD34–造血干细胞和祖细胞到四个骨髓系的方案。该协议的应用包括研究骨髓疾病突变或小分子对CD34+细胞骨髓分化的影响。
Abstract
人类造血干细胞的体外分化是研究造血干细胞的一个广泛应用的模型。此处描述的方案用于细胞因子诱导的CD34分化 –造血干细胞和祖细胞到四个骨髓体体细胞。CD34–细胞从人类脐带血中分离出来,在细胞因子存在的情况下与MS-5基质细胞共同培养。描述了干细胞和祖细胞的免疫表征特征,以及分化的骨髓体系细胞。使用该协议,CD34+细胞可以用小分子孵育,或用慢病毒转导,以表达骨髓疾病突变,以研究其对骨髓分化的影响。
Introduction
造血干细胞(HSCs)的正常分化对于维持所有血细胞系的生理水平至关重要。在分化期间,在协调响应细胞外线索(包括生长因子和细胞因子)时,HSCs 首先产生具有淋巴-骨髓电位1、2、3 的多能祖细胞 (MPP) 细胞 ,4 (图 1)。MpP 产生常见的骨髓祖体 (CmPs) 和常见的淋巴原代后代 (CLP) 受血统限制。CLP分化成由B、T和自然杀伤细胞组成的淋巴状谱系。CmPs 通过两个更受限的祖体群体(巨核细胞红细胞祖(MEPs)和粒细胞单细胞原代 (GMPs) 生成骨髓系。MEP产生巨核细胞和红细胞,而GMP产生粒细胞和单核细胞。除了通过CmPs产生,兆核细胞也报告直接产生于HC或早期的MpPs通过非规范途径5,6。
造血干细胞和祖细胞(HSPCs)的特点是表面标记CD34和缺乏系系特异性标记(Lin-)。其他通常用于区分 HSC 和骨髓祖体群体的表面标记包括 CD38、CD45RA 和 CD123 2(图1)。HSC 和 Mp 分别为林–/CD34+/CD38–和林–/CD34+/CD38+。骨髓已提交祖细胞群体由CD45RA和CD123的存在或不存在区分。CmP 是林–/CD34+/CD38+/CD45RA–/CD123 lo,GMP 是林–/CD34+/CD38+/CD45RA+/CD123lo,MEP是林–/CD34|/CD38=/CD45RA–/CD123–.
CD34+干细胞和祖细胞的总数可以从人类脐带血 (UCB)、骨髓和外周血获得。CD34–细胞占人类UCB中单核细胞总数的0.02%至1.46%,而骨髓中细胞的百分比在0.5%至5.3%之间,在外周血7、8、9中,其比例要低得多,为±0.01%.UCB衍生CD34+细胞的增殖能力和分化潜力明显高于骨髓或外周血细胞1、10,为获得足够的材料用于分子分析,结合在分化期间对细胞进行免疫表征和形态表征。
脐带血衍生CD34+HSPCs的体外分化是研究正常造血和造血病机制的一种广泛应用模型。当用适当的细胞因子培养时,UCB CD34+ HSPC 可诱导沿骨髓或淋巴体谱11、12、13、14、15进行分化,16.在这里,我们描述了CD34和HSPCs从人类UCB分离和免疫表征表表征,以及它们分化为骨髓体代细胞的协议。这种培养系统基于在MS-5基质细胞存在的情况下,以细胞因子诱导的HSPCs分化,以模拟骨髓中的微环境。培养条件导致CD34+细胞的初始扩张,随后分化为表达四个骨髓系细胞标记的细胞,即粒细胞(CD66b)、单核细胞(CD14)、巨核细胞(CD41)和红细胞(CD235a)。CD34®细胞分化协议的应用包括控制造血的分子机制研究,以及研究骨髓疾病相关突变和小分子对自我更新和HSPC的差异化。
Protocol
用于实验的人类脐带血是由健康人捐赠后,知情同意马里科帕综合卫生系统(MIHS),凤凰城。被除名的单位是通过MIHS和亚利桑那大学之间的材料转移协议获得的。 1. 试剂和缓冲液 注:在无菌条件下在生物安全柜中制备所有试剂和缓冲液。 通过加入7.2 mL的无菌35%牛血清白蛋白(BSA;使用无菌组织培养等级35%BSA)和5 mL无菌0.5M乙烯二胺四乙酸(EDTA)?…
Representative Results
应用上述协议可产生 5.6 (± 0.5) x 108 MDC 和 1 (± 0.3) x 106 CD34+来自脐带血单位 ±100 mL 的细胞。CD34+细胞总数的百分比在 80-90% 之间 (图 2A,B)。Manz等人描述方案的免疫表位分析表明,CD34+细胞通常由+20%的HSC和+72%的MpPs组成,即林-/CD34+/CD38-和林-/CD34|/CD38=分别 (<strong…
Discussion
此处所述的协议适用于 UCB 衍生 CD34和HSPC 到四个骨髓谱系的体外分化。由SCF、TPO、Flt3L和IL3组成的细胞因子混合物的初始孵育刺激CD34+细胞。随后,通过SCF、IL3、Flt3L、EPO和TPO的混合物实现了差异化。在此组合中,SCF、IL3 和 Flt3L 对 CD34+ HSC 的生存和增殖非常重要。 EPO 和 TPO 分别促进对红细胞和巨核细胞的分化,IL3 促进早期粒细胞单细胞的分化前体和成熟细胞13,17,18。<sup class="xr…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢温迪·巴雷特、雷切尔·卡瓦列罗和马里科帕综合卫生系统的加布里埃拉·鲁伊斯为脱认和捐献脐带血单位,姆利纳利尼·卡拉为流动细胞学提供帮助,盖伊·克鲁克斯和克里斯托弗·西特关于前体骨髓分化的建议。这项工作得到了美国国家卫生研究院(R21CA170786和R01GM127464)和美国癌症协会(机构研究补助金74-001-34-IRG)的资助。内容完全由作者负责,不一定代表国家卫生研究院的官方观点。
Materials
| 0.4% Trypan blue solution | Thermo Fisher Scientific | 15250-061 | Dilute working stock to 0.2% in sterile 1x PBS |
| 0.5 M UltraPure Ethylene diamine tetra acetic acid, pH 8.0 | Gibco | 15575-038 | |
| 10x Hanks Balanced Salt Solution (HBSS) | Invitrogen | 14185052 | Dilute to 1x with sterile distilled water & pH to 7.2 |
| 2.5% Trypsin, no phenol red | Thermo Fisher Scientific | 15090046 | Dilute working stock to 1x with sterile 1x PBS |
| 30 µm Pre-separation filters | Miltenyi biotech | 130-041-407 | |
| 35% sterile Bovine serum albumin | Sigma-Aldrich | A7979 | |
| 7-AAD | Biolegend | 420404 | Used as a live/dead stain to eliminate dead cells from FACS analysis |
| Anti-human CD10-FITC antibody (Clone HI10a) | Biolegend | 312207 | Use 1:20 dilution |
| Anti-human CD11b-FITC (activated) antibody (Clone CBRM1/5) | Biolegend | 301403 | Use 1:5 dilution |
| Anti-human CD123-APC antibody (Clone 6H6) | Biolegend | 306012 | Use 1:20 dilution |
| Anti-human CD14-PE antibody (Clone M5E2) | Biolegend | 301806 | Use 1:20 dilution |
| Anti-human CD19-FITC antibody (Clone 4G7) | BD Biosciences | 347543 | Use 1:5 dilution |
| Anti-human CD235a-APC antibody (Clone GA-R2 (HIR2)) | BD Biosciences | 551336 | Use 1:20 dilution |
| Anti-human CD235a-FITC antibody (Clone HIR2) | Biolegend | 306609 | Use 1:50 dilution |
| Anti-human CD34-APC-Cy7 antibody (Clone 581) | Biolegend | 343514 | Use 1:20 dilution |
| Anti-human CD38-PE antibody (Clone HIT2) | Biolegend | 303506 | Use 1:20 dilution |
| Anti-human CD3-FITC antibody (Clone UCHT1) | Biolegend | 300405 | Use 1:20 dilution |
| Anti-human CD41a-PerCP-Cy5.5 antibody (Clone HIP8) | Biolegend | 303720 | Use 1:20 dilution |
| Anti-human CD45Ra-PE-Cy7 antibody (Clone HI100) | Biolegend | 304126 | Use 1:20 dilution |
| Anti-human CD66b-PE-Cy7 antibody (Clone G10F5) | Biolegend | 305116 | Use 1:20 dilution |
| Anti-human CD7-FITC antibody (Clone CD7-6B7) | Biolegend | 343103 | Use 1:20 dilution |
| Dimethyl sulfoxide (DMSO) | Fisher Scientific | BP231-100 | Filter sterilize before use |
| Dulbecco’s Modified Eagle Medium (DMEM) powder with L-Glutamine | Gibco | 12100046 | Reconstitute 1 packet to make 1 L of DMEM media with sodium bicarbonate, 10% FBS & 1% penicillin & streptomycin |
| Fetal bovine serum, Australian source, heat inactivated | Omega Scientific | FB-22 Lot #609716 | |
| Human CD34 microbead kit | Miltenyi biotech | 130-046-702 | |
| Human Thrombopoietin (TPO), research grade | Miltenyi biotech | 130-094-011 | Make a stock of 100 µg/mL in 1x PBS + 0.1% BSA. Use 50 ng/mL for both myeloid differentiation & stimulation medium |
| L-Glutamine | Omega Scientific | GS-60 | 2 mM concentration in stimulation medium |
| LS Columns | Miltenyi biotech | 130-042-401 | |
| MACS Multi stand | Miltenyi biotech | 130-042-303 | |
| MidiMACS magnetic separator | Miltenyi biotech | 130-042-302 | |
| MNC fractionation media (Ficol-Paque PLUS) | GE Healthcare Biosciences | 17-1440-03 | |
| MS-5 cells | Gift from the laboratory of Gay Crooks, UCLA | ||
| Paraformaldehyde | Sigma-Aldrich | P6148 | Heat 800 mL of 1x PBS in a glass beaker on a stir plate in a chemical hood to ~65 °C. Add 10 g of paraformaldehyde powder. To completely dissolve the paraformaldehyde, raise the pH by adding 1 N NaOH. Cool and filter the solution and make up the volume to 1 L with 1x PBS. Adjust the pH to 7.2. |
| Penicillin & Streptomycin | Sigma-Aldrich | P4458-100ml | |
| Poly-L lysine | Sigma-Aldrich | P2636 | Make a 10 mg/mL stock in 1x PBS |
| Recombinant human erythropoietin-alpha (rHu EPO-α) | BioBasic | RC213-15 | Make a stock of 2000 units/mL in 1x PBS + 0.1% BSA. Use 4 units/mL for myeloid differentiation |
| Recombinant human fibronectin fragment (RetroNectin) | Takara | T100B | Use 20 µg/mL diluted in sterile 1x PBS to coat wells prior to stimulation of CD34+ HSCs. |
| Recombinant human Flt-3 ligand (rHu Flt-3L) | BioBasic | RC214-16 | Make a stock of 100 µg/mL in 1x PBS + 0.1% BSA. Use 5 ng/mL for myeloid differentiation & 50 ng/mL in stimulation medium |
| Recombinant human interleukin-3 (rHu IL-3) | BioBasic | RC212-14 | Make a stock of 100 µg/mL in 1x PBS + 0.1% BSA. Use 5 ng/mL for myeloid differentiation & 20 ng/mL in stimulation medium |
| Recombinant human stem cell factor (rHu SCF) | BioBasic | RC213-12 | Make a stock of 100 µg/mL in 1x PBS + 0.1% BSA. Use 5 ng/mL for myeloid differentiation & 50 ng/mL in stimulation medium |
| Serum free medium (X-Vivo-15) | Lonza | 04-418Q | |
| Sodium bicarbonate | Fisher Scientific | BP328-500 | |
| Wright-Giemsa stain, modified | Sigma-Aldrich | WG16-500 | Use according to manufacturer's instructions |
| Equipment | |||
| BD LSR II flow cytometer | BD Biosciences | ||
| Centrifuge | Sorvall Legend RT | ||
| Light microscope | Olympus |
References
- Hao, Q. L., Shah, A. J., Thiemann, F. T., Smogorzewska, E. M., Crooks, G. M. A functional comparison of CD34 + CD38- cells in cord blood and bone marrow. Blood. 86 (10), 3745-3753 (1995).
- Manz, M. G., Miyamoto, T., Akashi, K., Weissman, I. L. Prospective isolation of human clonogenic common myeloid progenitors. Proceedings of the National Academy of Sciences of the United States of America. 99 (18), 11872-11877 (2002).
- Kondo, M., Weissman, I. L., Akashi, K. Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell. 91 (5), 661-672 (1997).
- Seita, J., Weissman, I. L. Hematopoietic stem cell: self-renewal versus differentiation. Wiley Interdisciplinary Reviews: Systems Biology and Medicine. 2 (6), 640-653 (2010).
- Haas, S., et al. Inflammation-Induced Emergency Megakaryopoiesis Driven by Hematopoietic Stem Cell-like Megakaryocyte Progenitors. Cell Stem Cell. 17 (4), 422-434 (2015).
- Sanjuan-Pla, A., et al. Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy. Nature. 502 (7470), 232-236 (2013).
- Bender, J. G., et al. Phenotypic analysis and characterization of CD34+ cells from normal human bone marrow, cord blood, peripheral blood, and mobilized peripheral blood from patients undergoing autologous stem cell transplantation. Clinical Immunology and Immunopathology. 70 (1), 10-18 (1994).
- Fritsch, G., et al. The composition of CD34 subpopulations differs between bone marrow, blood and cord blood. Bone Marrow Transplantation. 17 (2), 169-178 (1996).
- Nimgaonkar, M. T., et al. A unique population of CD34+ cells in cord blood. Stem Cells. 13 (2), 158-166 (1995).
- Hordyjewska, A., Popiolek, L., Horecka, A. Characteristics of hematopoietic stem cells of umbilical cord blood. Cytotechnology. 67 (3), 387-396 (2015).
- Bapat, A., et al. Myeloid Disease Mutations of Splicing Factor SRSF2 Cause G2-M Arrest and Skewed Differentiation of Human Hematopoietic Stem and Progenitor Cells. Stem Cells. 36, 1-13 (2018).
- Yip, B. H., et al. The U2AF1S34F mutation induces lineage-specific splicing alterations in myelodysplastic syndromes. Journal of Clinical Investigation. 127 (6), 2206-2221 (2017).
- Yoo, E. S., et al. Myeloid differentiation of human cord blood CD34+ cells during ex vivo expansion using thrombopoietin, flt3-ligand and/or granulocyte-colony stimulating factor. British Journal of Haematology. 105 (4), 1034-1040 (1999).
- Hao, Q. L., Smogorzewska, E. M., Barsky, L. W., Crooks, G. M. In vitro identification of single CD34+CD38- cells with both lymphoid and myeloid potential. Blood. 91 (11), 4145-4151 (1998).
- Moretta, F., et al. The generation of human innate lymphoid cells is influenced by the source of hematopoietic stem cells and by the use of G-CSF. European Journal of Immunology. 46 (5), 1271-1278 (2016).
- Sanz, E., et al. Ordering human CD34+CD10-CD19+ pre/pro-B-cell and CD19- common lymphoid progenitor stages in two pro-B-cell development pathways. Proceedings of the National Academy of Sciences of the United States of America. 107 (13), 5925-5930 (2010).
- Egeland, T., et al. Myeloid differentiation of purified CD34+ cells after stimulation with recombinant human granulocyte-monocyte colony-stimulating factor (CSF), granulocyte-CSF, and interleukin-3. Blood. 78 (12), 3192-3199 (1991).
- Ogawa, M. Differentiation and proliferation of hematopoietic stem cells. Blood. 81 (11), 2844-2853 (1993).
- Perdomo, J., Yan, F., Leung, H. H. L., Chong, B. H. Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells. Journal of Visualized Experiments. (130), e56420 (2017).
- Palii, C. G., Pasha, R., Brand, M. Lentiviral-mediated knockdown during ex vivo erythropoiesis of human hematopoietic stem cells. Journal of Visualized Experiments. (53), e2813 (2011).
- Davies, C., et al. Silencing of ASXL1 impairs the granulomonocytic lineage potential of human CD34(+) progenitor cells. British Journal of Haematology. 160 (6), 842-850 (2013).
- Caceres, G., et al. TP53 suppression promotes erythropoiesis in del(5q) MDS, suggesting a targeted therapeutic strategy in lenalidomide-resistant patients. Proceedings of the National Academy of Sciences of the United States of America. 110 (40), 16127-16132 (2013).
- Shi, H., et al. ASXL1 plays an important role in erythropoiesis. Scientific Reports. 6, 28789 (2016).
- Mazumdar, C., et al. Leukemia-Associated Cohesin Mutants Dominantly Enforce Stem Cell Programs and Impair Human Hematopoietic Progenitor Differentiation. Cell Stem Cell. 17 (6), 675-688 (2015).
- Chung, K. Y., et al. Enforced expression of an Flt3 internal tandem duplication in human CD34+ cells confers properties of self-renewal and enhanced erythropoiesis. Blood. 105 (1), 77-84 (2005).
- Ambrosini, P., et al. IL-1beta inhibits ILC3 while favoring NK-cell maturation of umbilical cord blood CD34(+) precursors. European Journal of Immunology. 45 (7), 2061-2071 (2015).
- Batard, P., et al. TGF-(beta)1 maintains hematopoietic immaturity by a reversible negative control of cell cycle and induces CD34 antigen up-modulation. Journal of Cell Science. 113, 383-390 (2000).
- Huang, N., Lou, M., Liu, H., Avila, C., Ma, Y. Identification of a potent small molecule capable of regulating polyploidization, megakaryocyte maturation, and platelet production. Journal of Hematology & Oncology. 9 (1), 136 (2016).
Cite This Article
Bapat, A., Keita, N., Sharma, S. Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells. J. Vis. Exp. (150), e59836, doi:10.3791/59836 (2019).