单细胞指数分型与内皮细胞小生境联合培养对胚胎造血干细胞前体的克隆分析
Summary
本文介绍了小鼠胚胎发育过程中造血干细胞前体的克隆分析方法。我们结合内皮细胞共培养和移植对胚胎主动脉-性腺-中区单细胞的指标进行分类, 以表征单造血前体的表型性质和植入电位。
Abstract
在胚胎发育过程中, 研究造血干细胞 (hsc) 成因的能力受到了早期胚胎中 HSC 前体稀有性的限制, 缺乏检测功能, 可以识别长期的多向植入潜能。个人假定的 HSC 前体。在这里, 我们描述的方法, 使功能验证的 HSC 前体在单细胞水平的分离和鉴定。首先, 我们利用索引排序来编目每个单独排序的细胞的精确表型参数, 使用表型标记的组合, 以丰富的 HSC 前体和附加标记的实验分析。其次, 每个指数排序细胞都是由主动脉-性腺-中 (嫁接) 区的血管位质基质共同培养的, 它支持非 hsc 前体的成熟与多向的功能性 hsc, 长期植入电位在移植化验。这种方法使克隆 hemogenic 前体的表型性质与其功能植入电位或其他特性 (如转录剖面) 的相关性, 为 HSC 前驱物的详细分析提供了手段。在单个单元格级别上进行开发。
Introduction
克隆研究揭示了成人 HSCs 长期植入特性的异质性, 为 hsc 亚型和在老化的1中的 hsc 行为变化提供了新的见解。然而, 对胚胎 HSCs 及其前体的类似研究更具挑战性。在早期胚胎发育过程中, HSCs 产生于一种称为 hemogenic 内皮的前体, 在一个瞬态过程中称为内皮细胞到造血转换2。第一个 HSC, 定义的能力, 提供强健, 长期多向植入后移植到条件成年接受者, 直到胚胎天 10.5 (E10.5) 后, 在小鼠胚胎, 在非常低频率3.在它们的发展过程中, hemogenic 内皮引起的 hsc 前体 (hsc) 必须在获得成年 HSC 的特性之前进行成熟, 从而在移植化验中允许有效的植入4,5,6. 模糊了罕见的 HSC 起源的研究, 在从前 hsc7、8出现之前, 已经检测到大量具有红、髓系和淋巴电位的造血祖细胞。因此, 区分前 hsc 与其他造血祖细胞需要的方法, 以无性分离的干细胞, 并提供足够的信号, 他们成熟的 hsc, 以检测其植入的性质, 在移植化验。
已经描述了许多方法, 允许通过 ” 体外” 或体内成熟度检测 hsc。”前体” 方法依赖于胚胎组织的区域性, 例如, 在开发9中检测到第一个 HSC 的 “年会” 区域。在这些方法的基础上, 结合了周年大会组织的分离、分类和再聚合的协议, 允许在 E9.5 到 E11.5 的准主动脉的发育过程中含有 HSC 前体的排序种群的特征。splanchnopleura (P Sp)/股东大会区域4,5,10;然而, 这些方法不适用于克隆分析所需的单细胞水平的前体高通量分析。同样,在体内通过移植进入新生小鼠, 在这种情况下, 假设微环境更适合于早期的 HSC 前体的支持, 也使从蛋黄囊中分类的种群的研究和周年大会/p sp (p sp 是年度股东大会的前体区域), 具有预 HSC 的特点, 但这些方法也未能为单个单元分析1112提供一个健壮的平台。
从 Rafii et . 的研究表明, Akt 活化内皮细胞 (EC) 基质可以提供一个利基基质支持成人 HSC 自我更新体外13,14,15。我们最近确定, 由年会 (Akt) 衍生的欧共体 (年会) 提供了一个适当的体外利基, 以成熟的 hemogenic 前体, 早在 E9 的发展, 对成年嫁接 HSC, 以及随后自更新生成的 HSC16。鉴于该系统采用了简单的2维共培养, 它很容易适应克隆分析的 hemogenic 前体独立的 HSC 潜力。
我们最近报告了一种方法来检测克隆 hemogenic 前体的 HSC 潜力, 结合 hemogenic 前体的小鼠胚胎的指数排序与年会-欧共体共培养和随后的功能分析移植化验17。索引排序是一种荧光活化细胞分选模式, 它记录 (索引) 所有表型参数 (即、正散射 (FSC a)、侧向散射 (SSC a)、荧光参数), 从而使这些特征可以追溯到后续功能分析后的排序。资产管制系统软件记录每个单元的表单信息, 以及放置在其中的96孔板的位置/井。这种技术以前很优雅地用于鉴定成人 hsc 中的异质性, 确定进一步丰富 hsc 长期嫁接子集的表型参数, 并将 hsc 的表形参数与转录相关联。单个单元格级别的属性18,19。在这里, 我们提供了这种方法的详细方法, 使识别独特的表型参数和谱系贡献的前 HSC 在胚胎发育的早期阶段。
Protocol
这里描述的所有方法都得到了弗雷德哈钦森癌症研究中心机构动物护理和使用委员会 (IACUC) 的批准。 1. 为共同文化准备年会-EC 膜 24小时前的年会解剖和排序, 使年会-欧共体文化媒体和过滤消毒。 在水中加入0.1% 明胶 (100 µL/井), 每井96井板, 并在室温下孵化15分钟. 吸入明胶, 将盘子放在组织培养罩中, 除去盖子, 直到水井干涸为止。 将股东大会-EC ?…
Representative Results
图 1A显示了实验设计的示意图。一旦在胶原酶中解剖、汇集和分离了 p-Sp/年会组织, 就会对 VE 钙黏蛋白和 EPCR 的抗体染色, 以进行指数排序。在 VE 钙黏蛋白+EPCR高(图 1B) 中排序的单元格中, 预 HSC 被丰富。其他 fluorochrome 共轭抗体可包括在回顾性分析其他表型参数, 这是记录的每个细胞在索引排序。在 E11 年会的这…
Discussion
研究胚胎发育过程中的 hsc 成因, 需要检测 hemogenic 前体中的 hsc 电位, 但在移植成人接受者中缺乏提供长期多向造血重建的能力。在本议定书中, 我们提出了一种克隆方法的胚胎 hemogenic 前体之间的基质共培养的血管位, 从大会, 支持成熟的前体对 HSC, 并随后功能分析的移植化验。纳入索引排序允许回顾性分析的表型参数, 以表征前 HSC 的性质, 由表面标记的定义包括在化验。因此, 这种方法比其他依?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们要感谢安德鲁. 伯格, 史黛西 Dozono, 和布莱恩 Raden 在弗雷德. 哈钦森流式细胞术核心协助与外地资产管制。这项工作得到了国家卫生研究院 NHLBI UO1 赠款 #HL100395、辅助合作赠款 #HL099997 和 NIDDK 赠款 #RC2DK114777 的支持。布兰登上 Hadland 是支持的亚历克斯的柠檬水站基础和现代希望在车轮基础上。
Materials
| AGM-EC culture media | |||
| Materials for culture of endothelial cells | |||
| TrypLE Express | Gibco | 12605-028 | Use to dissociate AGM-EC monolayers |
| Gelatin 0.1% in water | StemCell Technologies | 7903 | Use to adhere AGM-EC monolayers to plastic |
| 96-well tissue culture plates | Corning | 3599 | Use to co-culture AGM-EC monolayers and index sorted clones |
| Dulbecco's Phosphate Buffered Saline (PBS) | Gibco | 14190-144 | Use for dissection and FACS staining |
| 500 ml filter bottles | Fisher Scientific | 9741202 | Use to sterile filter Endothelial media |
| AGM-EC culture media (500 mls) | |||
| Iscove's Modified Dulbecco's Medium (IMDM) | Gibco | 12440-053 | 400 mls |
| Hyclone Fetal Bovine Serum | Fisher Scientific | SH30088.03 | 100 mls |
| Penicillin Streptomycin | Gibco | 15140-122 | 5 mls |
| Heparin | Sigma | H3149 | 50 mg dissolved in 10 mls media |
| L-glutamine (200 mM) | StemCell Technologies | 7100 | 5 mls |
| Endothelial Mitogen (ECGS) | Alfa Aesar | J64516 (BT-203) | Add 10 mls media to dissolve and add |
| Materials for AGM index sort | |||
| Collagenase (0.25%) | StemCell Technologies | 7902 | Use for dissociation of embryonic tissues |
| 3ml syringe | BD Biosciences | 309657 | Use to sterile filter antibody solutions for FACS |
| 0.22 µM syringe-driven filter | Millipore | SLGP033RS | Use to sterile filter antibody solutions for FACS |
| 5 ml polystyrene tube with cell-strainer cap | Corning | 352235 | Use to remove cell clumps prior to FACS |
| DAPI (prepared as 1 mg/ml stock in H2O) | Millipore | 268298 | Use to exclude dead cells from sort |
| anti-mouse CD16/CD32 (FcR block) | BD Biosciences | 553141 | Use to block non-specific staining to Fc receptors |
| Antibodies for AGM index sort | |||
| Anti-mouse CD144 PE-Cyanine7 | eBioscience | 25-1441-82 | Staining |
| Rat IgG1 kappa Isotype Control, PE-Cyanine7 | eBioscience | 25-4301-81 | Isotype control for CD144 PE-Cyanine7 |
| Anti-mouse CD201 (EPCR) PerCP-eFluor710 | eBioscience | 46-2012-80 | Staining |
| Rat IgG2b kappa Isotype Control, PerCP-eFlour710 | eBioscience | 46-4031-80 | Isotype control for CD201 PerCP-eFluor710 |
| Anti-mouse CD41 PE | BD Biosciences | 558040 | Staining |
| Rat IgG1 kappa Isotype Control, PE | BD Biosciences | 553925 | Isotype control for CD41 PE |
| Anti-mouse CD45 FITC | eBioscience | 11-0451-85 | Staining |
| Rat IgG2b kappa Isotype Control, FITC | eBioscience | 11-4031-81 | Isotype control for CD45 FITC |
| AGM-serum free media (10mls) | |||
| X-Vivo 20 | Lonza | 04-448Q | 10 mls |
| recombinant murine stem cell factor (SCF) | Peprotech | 250-03 | 10 ml (100 mg/ml stock) Final concentration 100 ng/ml |
| recombinant human FLT3 Ligand (FLT3L) | Peprotech | 300-19 | 10 ml (100 mg/ml stock) Final concentration 100 ng/ml |
| recombinant human thrombopoietin (TPO) | Peprotech | 300-18 | 2 ml (100 mg/ml stock) Final concentration 20 ng/ml |
| recombinant murine interleukin-3 (IL3) | Peprotech | 213-13 | 2 ml (100 mg/ml stock) Final concentration 20 ng/ml |
| Materials for Staining co-cultured cells | |||
| 96 well plate, V-bottom | Corning | 3894 | Use for FACS analysis |
| Antibodies for analysis of Index sorted clones co-cultured with endothelial cells | |||
| Anti-mouse CD45 PerCP-Cyanine5.5 | eBioscience | 45-0451-82 | Staining |
| Rat IgG2b kappa Isotype Control, PE-Cyanine5.5 | eBioscience | 35-4031-80 | Isotype control for CD45 PerCP-Cyanine5.5 |
| Anti-mouse CD201 (EPCR) PE | eBioscience | 12-2012-82 | Staining |
| Rat IgG2b kappa Isotype Control, PE | eBioscience | 12-4031-81 | Isotype control for CD201 PE |
| Anti-mouse Ly-6A/E (Sca-1) APC | eBioscience | 17-5981-83 | Staining |
| Rat IgG2a kappa Isotype Control, APC | eBioscience | 17-4321-81 | Isotype control for Ly-6A/E APC |
| Anti-mouse F4/80 FITC | eBioscience | 11-4801-81 | Staining |
| Rat IgG2a kappa Isotype Control, FITC | eBioscience | 11-4321-41 | Isotype control for F4/80 FITC |
| Anti-mouse Ly-6G/C (Gr1) FITC | BD Biosciences | 553127 | Staining |
| Rat IgG2b kappa Isotype Control, FITC | BD Biosciences | 556923 | Isotype control for Ly-6G/C and CD3 FITC |
| Materials for tail vein injection for transplant | |||
| 1/2 ml insulin syringes with 29G 1/2" needles | BD Biosciences | 309306 | Use for tail vein injection for transplantation |
| Antibodies for Peripheral Blood analysis following translant | |||
| Anti-mouse Ly-6G/C (Gr1) PerCP | Biolegend | 108426 | Staining |
| Rat IgG2b kappa Isotype Control, PerCP | Biolegend | 400629 | Isotype control for Ly-6G/C PerCP |
| Anti-mouse F4/80 PE | eBioscience | 12-4801-82 | Staining |
| Rat IgG2a kappa Isotype Control, PE | eBioscience | 12-4321-80 | Isotype control for F4/80 PE |
| Anti-mouse CD3 FITC | BD Biosciences | 555274 | Staining |
| Anti-mouse CD19 APC | BD Biosciences | 550992 | Staining |
| Rat IgG2a kappa Isotype Control, APC | BD Biosciences | 553932 | Isotype control for CD19 APC |
| Anti-mouse CD45.1 (A20) PE-Cyanine7 | eBioscience | 25-0453-82 | Staining |
| Rat IgG2a kappa Isotype Control, PE-Cyanine7 | eBioscience | 25-4321-81 | Isotype control for CD45.1 PE-Cyanine7 |
| Anti-mouse CD45.2 (104) APC-eFluor780 | eBioscience | 47-0454-82 | Staining |
| Rat IgG2a kappa Isotype Control, APC-eFluor780 | eBioscience | 47-4321-80 | Isotype control for CD45.2 APC-eFluor780 |
| Equipment | |||
| BD FACSAria II with DIVA software | BD Biosciences | ||
| BD FACSCanto II with plate reader | BD Biosciences | ||
| Haemocytometer | Fisher Scientific | S17040 | For counting cells |
| Multi-channel pipette | Fisher Scientific | 14-559-417 | For dispensing cells |
| FACS analysis software | FlowJo/BD Biosciences | https://www.flowjo.com/solutions/flowjo | |
References
- Copley, M. R., Beer, P. A., Eaves, C. J. Hematopoietic stem cell heterogeneity takes center stage. Cell Stem Cell. 10 (6), 690-697 (2012).
- Medvinsky, A., Rybtsov, S., Taoudi, S. Embryonic origin of the adult hematopoietic system: advances and questions. Development. 138 (6), 1017-1031 (2011).
- Kumaravelu, P., et al. Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver. Development. 129 (21), 4891-4899 (2002).
- Taoudi, S., et al. Extensive hematopoietic stem cell generation in the AGM region via maturation of VE-cadherin+CD45+ pre-definitive HSCs. Cell Stem Cell. 3 (1), 99-108 (2008).
- Rybtsov, S., et al. Hierarchical organization and early hematopoietic specification of the developing HSC lineage in the AGM region. J Exp Med. 208 (6), 1305-1315 (2011).
- Rybtsov, S., et al. Tracing the origin of the HSC hierarchy reveals an SCF-dependent, IL-3-independent CD43(-) embryonic precursor. Stem Cell Reports. 3 (3), 489-501 (2014).
- McGrath, K. E., et al. Distinct Sources of Hematopoietic Progenitors Emerge before HSCs and Provide Functional Blood Cells in the Mammalian Embryo. Cell Rep. 11 (12), 1892-1904 (2015).
- Lin, Y., Yoder, M. C., Yoshimoto, M. Lymphoid progenitor emergence in the murine embryo and yolk sac precedes stem cell detection. Stem Cells Dev. 23 (11), 1168-1177 (2014).
- Medvinsky, A., Dzierzak, E. Definitive hematopoiesis is autonomously initiated by the AGM region. Cell. 86 (6), 897-906 (1996).
- Sheridan, J. M., Taoudi, S., Medvinsky, A., Blackburn, C. C. A novel method for the generation of reaggregated organotypic cultures that permits juxtaposition of defined cell populations. Genesis. 47 (5), 346-351 (2009).
- Yoder, M. C., Hiatt, K., Mukherjee, P. In vivo repopulating hematopoietic stem cells are present in the murine yolk sac at day 9.0 postcoitus. Proc Natl Acad Sci U S A. 94 (13), 6776-6780 (1997).
- Arora, N., et al. Effect of developmental stage of HSC and recipient on transplant outcomes. Dev Cell. 29 (5), 621-628 (2014).
- Butler, J. M., Rafii, S. Generation of a vascular niche for studying stem cell homeostasis. Methods Mol Biol. 904, 221-233 (2012).
- Butler, J. M., et al. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell. 6 (3), 251-264 (2010).
- Kobayashi, H., et al. Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells. Nat Cell Biol. 12 (11), 1046-1056 (2010).
- Hadland, B. K., et al. Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros-derived hematopoietic stem cells. J Clin Invest. 125 (5), 2032-2045 (2015).
- Hadland, B. K., et al. A Common Origin for B-1a and B-2 Lymphocytes in Clonal Pre- Hematopoietic Stem Cells. Stem Cell Reports. 8 (6), 1563-1572 (2017).
- Wilson, N. K., et al. Combined Single-Cell Functional and Gene Expression Analysis Resolves Heterogeneity within Stem Cell Populations. Cell Stem Cell. 16 (6), 712-724 (2015).
- Schulte, R., et al. Index sorting resolves heterogeneous murine hematopoietic stem cell populations. Exp Hematol. 43 (9), 803-811 (2015).
- Morgan, K., Kharas, M., Dzierzak, E., Gilliland, D. G. Isolation of early hematopoietic stem cells from murine yolk sac and AGM. J Vis Exp. (16), (2008).
- deBruijn, M. F., et al. Hematopoietic stem cells localize to the endothelial cell layer in the midgestation mouse aorta. Immunity. 16 (5), 673-683 (2002).
- Lorsbach, R. B., et al. Role of RUNX1 in adult hematopoiesis: analysis of RUNX1-IRES-GFP knock-in mice reveals differential lineage expression. Blood. 103 (7), 2522-2529 (2004).
- Holmes, R., Zúñiga-Pflücker, J. C. The OP9-DL1 system: generation of T-lymphocytes from embryonic or hematopoietic stem cells in vitro. Cold Spring Harb Protoc. 2009 (2), pdb.prot5156 (2009).
- Yoshimoto, M. The first wave of B lymphopoiesis develops independently of stem cells in the murine embryo. Ann N Y Acad Sci. 1362, 16-22 (2015).
- Kobayashi, M., et al. Functional B-1 progenitor cells are present in the hematopoietic stem cell-deficient embryo and depend on Cbfβ for their development. Proc Natl Acad Sci U S A. 111 (33), 12151-12156 (2014).
- Yoshimoto, M., et al. Autonomous murine T-cell progenitor production in the extra-embryonic yolk sac before HSC emergence. Blood. 119 (24), 5706-5714 (2012).
- Inlay, M. A., et al. Identification of multipotent progenitors that emerge prior to hematopoietic stem cells in embryonic development. Stem Cell Reports. 2 (4), 457-472 (2014).
- Palis, J. Hematopoietic stem cell-independent hematopoiesis: emergence of erythroid, megakaryocyte, and myeloid potential in the mammalian embryo. FEBS Lett. 590 (22), 3965-3974 (2016).
Citer Cet Article
Hadland, B. K., Varnum-Finney, B., Nourigat-Mckay, C., Flowers, D., Bernstein, I. D. Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture. J. Vis. Exp. (135), e56973, doi:10.3791/56973 (2018).