重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划
Summary
The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.
Abstract
This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis “in a dish,” allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.
Introduction
造血是一个复杂的发育过程，其中的造血干细胞（HSCs）出芽存在于各种胚胎造血网站，如主动脉-性腺-中肾和胎盘1,2 hemogenic内皮。无法培养造血干细胞在体外阻止此过程的深入分析，以及这些研究的临床应用。规避此限制，先前的研究试图获得造血干细胞从头或者通过使用重新编程媒体4,5-多能干细胞（的PSCs）3，或在体细胞诱导的可塑性和定向分化的分化。这些研究，但是，不产生临床安全engraftable细胞或允许最终造血发育研究“的菜。”
从体细胞的成纤维细胞p由山及其同事建立的新颖工作以产生诱导性多能干细胞（iPS细胞）rovides转录因子（TF）在重编程的细胞命运6,7-基于过表达策略的框架。这项工作已经促使调查人员在若干领域通过容易获得体细胞重编程TF产生细胞类型的选择。这里所描述的重编程策略的目标是诱导从使用具有转换这些发现对人体系统重新编程特定病人的成纤维细胞，以研究人类造血体外的目标基于TF重新编程的方法和小鼠体细胞hemogenic过程生成患者特异性血制品用于疾病建模，药物检验，和干细胞移植。
第一步，以确保在该小鼠系统适当重新编程是开发担任的读出为CD34的表达，在内皮祖细胞和造血干细胞的已知标记的报道一致。要做到这一点，huCD34-和tTA的TETO-H2BGFP转基因小鼠系被用于与Generate双转基因小鼠胚胎成纤维细胞（MEF），现在表示34 / H2BGFP，发荧光绿于CD34启动子8的激活。这允许各种已知被要求在不同的点造血说明书和发育过程中的TF的筛选。在PMX retrovial载体18开始的TF（通过文献挖掘和GFP标签保留从之前造血干细胞的分析确定描述34 / H2BGFP只），34 / H2BGFP MEF中与AFT024 HSC-支持基质细胞的所有因素和培养转。检测34 / H2BGFP激活后，转录因子随后从重新编程的鸡尾酒删除，直到转录因子的活化记者一组最佳被确定。此初始屏幕之后，将因素转移到DOX诱导pFUW载体系统，以允许转录因子的控制的表达。由于这两个DOX可控系统是不兼容的（34 / H2BGFP细胞和pFUW诱导矢量），由MEF野生型C57BL / 6小鼠被要求。此外，还必须提供适当的微环境，使hemogenesis继续创造多向祖细胞克隆。
试图体细胞重编程到造血干细胞和祖细胞（骨髓干细胞）目前的研究已达到成功的9-11不同层次。迄今为止，小鼠和具有长期和自我更新的重新填充能力的人类移植骨髓干细胞的产生尚未使用同一组转录因子实现。在这个协议中，我们提供先前建立的策略的详细描述可重复地诱导hemogenesis在MEF中。我们证明，引进了全套转录因子（GATA2，Gfi1b，首席财务官和ETV6）的最小的能够煽动体外复杂的发展计划，规定了可发展的造血和造血重新编程的临床应用，可进一步研究12的平台。
Protocol
Representative Results
Discussion
从容易实现的体细胞产生骨髓干细胞从头提供了一个独特的方法来研究体外造血，并有机会可能这个技术应用到人的制度。这个翻译将产生一个新的工具来研究人类血液病在培养皿中，以及提供药物测试平台和基因定位机会以治疗与新疗法或HSC移植众多障碍。在该领域，最近的研究上的能力扩展到产生骨髓干细胞从头 ，展示的重编程过程的几个特征的重要性。这些措施包括起…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by an NHLBI grant to K.M. and I.R.L. (1RO1HL119404). Carlos-Filipe Pereira was the recipient of a Revson Senior Biomedical Fellowship. We gratefully acknowledge the Mount Sinai hESC/iPSC Shared Resource Facility and S. D’Souza for assistance with materials and protocols. We also thank the Mount Sinai Flow Cytometry, Genomics, and Mouse facilities.
Materials
| DMEM | Invitrogen | 11965-092 | |
| 0.45uM filters | Corning | 430625 | |
| Amicon Ultra centrifugal filters | Millipore | UFC900324 | |
| Penicillin/Streptomycin | Invitrogen | 15140-122 | |
| L-Glutamine | Invitrogen | 25030-081 | |
| FBS | Gemini's Benchmark | 100-106 | |
| PBS | Life Technologies | 14190-144 | |
| 18G needles | BD | 305195 | |
| 20G needles | BD | 305175 | |
| 25G needles | BD | 305125 | |
| Collagenase Type I | Sigma | C0130-100MG | |
| TrypLE Express | Invitrogen | 12605-010 | |
| Myelocult media | Stem Cell Technologies | M5300 | |
| SCF | R & D Systems | 455-MC | |
| Flt3L | R & D Systems | 427-FL | |
| IL-3 | R & D Systems | 403-ML | |
| IL-6 | R & D Systems | 406-ML | |
| TPO | R & D Systems | 488-TO | |
| Doxycycline | Sigma | D9891-1G | |
| Polybrene (hexadimethrine bromide) | Sigma | AL-118 | |
| Durapore 0.65uM membrane filters | Millipore | DVPP14250 | |
| Methylcellulose media | Stem Cell Technologies | Methocult M3434 | |
| Hydrocortisone | Stem Cell Technologies | 07904 | |
| C57BL/6 mice | The Jackson Laboratory | 000664 | |
| Gelatin | Sigma | G-1890 100g | |
| pFUW-tetO | Addgene | Plasmid #20321 | |
| Gata2 | Origene | MR226728 | |
| Gfi1b | Origene | MR204861 | |
| cFos | Addgene | Plasmid #19259 | |
| Etv6 | Origene | MR207053 | |
| psPAX2 | Addgene | Plasmid #12260 | |
| pMD2.G | Addgene | Plasmid #12259 | |
| CaCl2 | Sigma | C5670-100g | |
| FUW-M2rtTA | Addgene | Plasmid #20342 | |
| 35 x 10 mm culture dishes | Thermo Scientific | 171099 |
References
- Zovein, A. C., et al. Fate tracing reveals the endothelial origin of hematopoietic stem cells. Cell Stem Cell. 3 (6), 625-636 (2008).
- Bertrand, J. Y., et al. Haematopoietic stem cells derive directly from aortic endothelium during development. Nature. 464 (7285), 108-111 (2010).
- Papapetrou, E. P., Sadelain, M. Reconstructing blood from induced pluripotent stem cells. F1000 Med Rep. 2, (2010).
- Szabo, E., et al. Direct conversion of human fibroblasts to multilineage blood progenitors. Nature. 468 (7323), 521-526 (2010).
- Mitchell, R., et al. Molecular evidence for OCT4-induced plasticity in adult human fibroblasts required for direct cell fate conversion to lineage specific progenitors. Stem Cells. 32 (8), 2178-2187 (2014).
- Takahashi, K., Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126 (4), 663-676 (2006).
- Takahashi, K., et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 131 (5), 861-872 (2007).
- Qiu, J., Papatsenko, D., Niu, X., Schaniel, C., Moore, K. Divisional history and hematopoietic stem cell function during homeostasis. Stem Cell Reports. 2 (4), 473-490 (2014).
- Riddell, J., et al. Reprogramming committed murine blood cells to induced hematopoietic stem cells with defined factors. Cell. 157 (3), 549-564 (2014).
- Sandler, V. M., et al. Reprogramming human endothelial cells to haematopoietic cells requires vascular induction. Nature. 511 (7509), 312-318 (2014).
- Daniel, M. G., Pereira, C. F., Lemischka, I. R., Moore, K. A. Making a Hematopoietic Stem Cell. Trends Cell Biol. , (2015).
- Pereira, C. F., et al. Induction of a hemogenic program in mouse fibroblasts. Cell Stem Cell. 13 (2), 205-218 (2013).
- Mader, S. L., Libal, N. L., Pritchett-Corning, K., Yang, R., Murphy, S. J. Refining timed pregnancies in two strains of genetically engineered mice. Lab Anim (NY. 38 (9), 305-310 (2009).
- Jain, K., Verma, P. J., Liu, J. Isolation and handling of mouse embryonic fibroblasts). Methods Mol Biol. 1194, 247-252 (2014).
- Bryja, V., Bonilla, S., Arenas, E. Derivation of mouse embryonic stem cells. Nat Protoc. 1 (4), 2082-2087 (2006).
- Carey, B. W., et al. Reprogramming of murine and human somatic cells using a single polycistronic vector. Proc Natl Acad Sci U S A. 106 (1), 157-162 (2009).
- Hockemeyer, D., et al. A drug-inducible system for direct reprogramming of human somatic cells to pluripotency. Cell Stem Cell. 3 (3), 346-353 (2008).
- 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).
- Gekas, C., K, E. R., H, K. A. M. Isolation and analysis of hematopoietic stem cells from the placenta. J Vis Exp. (16), (2008).
- Lewis, I. D., et al. Umbilical cord blood cells capable of engrafting in primary, secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system. Blood. 97 (11), 3441-3449 (2001).
- 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).
- Koh, C. M. Preparation of cells for microscopy using cytospin. Methods Enzymol. 533, 235-240 (2013).
- Orkin, S. H., Zon, L. I. Hematopoiesis: an evolving paradigm for stem cell biology. Cell. 132 (4), 631-644 (2008).
- Martinez-Agosto, J. A., Mikkola, H. K., Hartenstein, V., Banerjee, U. The hematopoietic stem cell and its niche: a comparative view. Genes Dev. 21 (23), 3044-3060 (2007).
- Butler, J. M., et al. Development of a vascular niche platform for expansion of repopulating human cord blood stem and progenitor cells. Blood. 120 (6), 1344-1347 (2012).
- Gori, J. L., et al. Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells. J Clin Invest. 125 (3), 1243-1254 (2015).
- Bar-Nur, O., et al. Lineage conversion induced by pluripotency factors involves transient passage through an iPSC stage. Nat Biotechnol. 33 (7), 761-768 (2015).
- Maza, I., et al. Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors. Nat Biotechnol. 33 (7), 769-774 (2015).
- Kim, K., et al. Epigenetic memory in induced pluripotent stem cells. Nature. 467 (7313), 285-290 (2010).
- Vaskova, E. A., Stekleneva, A. E., Medvedev, S. P., Zakian, S. M. “Epigenetic memory: phenomenon in induced pluripotent stem cells. Acta Naturae. 5 (4), 15-21 (2013).
- Elcheva, I., et al. Direct induction of haematoendothelial programs in human pluripotent stem cells by transcriptional regulators. Nat Commun. 5, 4372 (2014).
- Vereide, D. T., et al. An expandable, inducible hemangioblast state regulated by fibroblast growth factor. Stem Cell Reports. 3 (6), 1043-1057 (2014).
- Batta, K., Florkowska, M., Kouskoff, V., Lacaud, G. Direct reprogramming of murine fibroblasts to hematopoietic progenitor cells. Cell Rep. 9 (5), 1871-1884 (2014).
