Summary

对T细胞推导<em>在体外</em>从小鼠胚胎干细胞

Published: October 14, 2014
doi:

Summary

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

Abstract

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Introduction

A cell culture system has been established in which mouse embryonic stem cells (mESC) are differentiated to T cells in vitro.1 This system exploits the ability of Notch signaling to drive T cell differentiation.2 The OP9-DL1 cell line was created by transducing bone marrow-derived OP9 cells3 with a Notch ligand, Delta-like 1 (DL1).4 Activation of the Notch signaling cascade in vitro facilitates T cell development to the exclusion of other cell lineages. With the inclusion of appropriate cytokines, this system provides a cell culture “microenvironment” that supports the sequential advancement of mESC toward hematopoietic and ultimately T cell lineages. This system supports the flow cytometric identification of T cells at the various developmental stages seen during normal T cell ontogeny in the thymus. For investigating selected questions relating to T cell development, this procedure has become an attractive alternative to in vivo whole mouse models5 and in vitro fetal thymic organ culture methods used to elicit T cell development from mouse embryonic stem cell derived hematopoietic precursors.6 The major advantage of the OP9 co-culture system is that it involves standard and straightforward cell culture techniques and does not depend on the continual use of experimental animals.

We follow a detailed, previously published protocol in our experiments using this approach.7 We have utilized this technology to examine the hematopoietic differentiation products of non-manipulated mESC clones, high quality mESC clones handpicked to make chimeric embryos8 and stably-transfected ESC clones coming directly out of drug selection.9 We have noted that the temporal kinetics of initial in vitro differentiation from mESC to mesoderm-like colonies in this model can be variable among individual clones. The mESC-OP9 co-cultures can be visually assessed for progression to mesoderm. While this will usually be completed by the fifth day of co-culture, among individual clones, completion can be delayed for one or two days. Quantitative (~80-90%) mesoderm formation must be achieved prior to transfer in order to obtain optimal hematopoietic progenitor cell (HPC) formation and robust lymphopoiesis. Thus, when working with multiple mESC clones, this “day 5” passage is best delayed until all clones complete the transition to mesoderm-like colonies. This enables synchrony of subsequent development among the clones after their transfer into hematopoietic differentiation conditions. Three days after the passaging of the 80-90% mesodermal formations, HPCs are collected from the OP9 monolayers. HPCs can be seeded on new OP9 cells to allow differentiation of monocytic, erythroid and B cell lineages. Alternatively, HPCs can be seeded on OP9-DL1 cells and driven towards T cell development. All in vitro differentiation cultures are provided Flt-3L beginning at day 5, with further addition of IL-7 beginning at day 8. Flow cytometry analyses performed at various time points during the experiment enable monitoring of progress through the stages and lineages of hematopoietic differentiation and T cell development. CD4/CD8 double positive (DP) T cells begin emerging by day 16 of the co-culture, and both DP and CD8 single positive (SP) cells are abundant by day 20. The general outcome and robustness of co-culture is greatly dependent on the ability to visually ascertain the completion of the significant developmental turning points that occur. This protocol aims to be a guide to the recognition of these milestones, as well as the other critical parameters, that are key to successful differentiation.

Protocol

1,制备培养基,细胞因子和糊化板的用鹰的培养基(DMEM)的贝科的修改与高糖和丙酮酸钠准备胚胎干细胞培养基。添加20%ESC合格的胎牛血清(FBS),1%青霉素/链霉素,1%L-谷氨酰胺,1%HEPES缓冲液,1%非必需氨基酸,0.1%庆大霉素(50毫克/毫升)和0.1%( 55μM)β-巯基乙醇。经过滤消毒胚胎干细胞培养基。 通过根据制造商的说明制备1升阿尔法最低必需培养基(α-MEM)中从粉?…

Representative Results

当在LIF存在下生长在MEF中,mESCs分化可以保持在未分化状态。在理想条件下,它们显示为细胞通过一个闪亮卤素在相差显微镜包围的紧凑集落( 图1)。这些培养物必须每天监测。根据细胞的汇合,媒体可以被改变或细胞可以分裂。相邻卓制菌落不应该来彼此接触的点。未分化mESCs分化的正常,健康的文化是一个重要的起点, 在体外 T细胞分化。在起始的共培养,则建议的细胞?…

Discussion

在OP9-DL1共培养系统已经利用血细胞类型的干细胞在发育过程中,研究了不同的基因产物的作用。8,12,13这也证明研究的基因调节DNA的功能的有效模式9,14中的细胞分化。使用这种方法作为一种替代整个小鼠模型所用的时间和实验,解决在造血许多基本问题,成本得到相当大的节约。然而,该协议用于这些目的的适应性要求,将在这些过程中使用卓制克隆中的电位变化的认定。公布协…

Declarações

The authors have nothing to disclose.

Acknowledgements

We thank Joon Kim for expert flow cytometry assistance. Research in the authors’ labs is supported by the SCORE program of the National Institutes of Health (grant SC1-GM095402 to B.D.O) and the Canadian Institutes of Health Research (to J.C.Z.P.). J.C.Z.P. is supported by a Canada Research Chair in Developmental Immunology. The biomedical research infrastructure of Hunter College is supported in part by the NIH Research Centers in Minority Institutions (RCMI) program via grant MD007599. We also acknowledge the New York State Stem Cell Science Program (NYSTEM) for its support of the initiation of stem cell research at Hunter College via grant C023048.

Materials

MATERIALS COMPANY CATALOG NUMBER COMMENTS
DMEM Corning 15-013-CV
Stem cell qualified FBS Gemini 100-125 Heat inactivated
Penicillin/Streptomycin Corning 30-002-CI
L-alanyl L-glutamine Corning 25-015-CI
HEPES buffer  Millipore TMS-003-C
Non-Essential Amino Acids ThermoScientific SH30853.01
Gentamicin Regent Solution (50 mg/mL) Life Technologies  15750-060
β-mercaptoethanol (55 mM) in DPBS Life Technologies  21985-023
Filter Unit Millipore SCGPU05RE 0.22μm PES membrane
Cell Culture Grade Water Corning 25-055-CM
α-MEM  Life Technologies  12000-022 Powder, reconstitute per manufacture recommendation
Sodium bicarbonate  Sigma S5761-500G
FBS ThermoScientific SH 30396.03 Testing of individual lots required 
Dimethyl Sulphoxide  Sigma D2650
Recombinant Human Flt-3 Ligand R&D Systems 308-FK
Recombinant Murine IL-7 PeproTech 217-17
LIF  Millipore ESG1107
Utrapure water with 0.1% gelatin Millipore ES-006-B
MEFs mitomycin C treated Millipore PMEF-CF Any mitotically arresteded MEFs can be used
DPBS Corning 21-031-CV
Trypsin EDTA, 1X Corning 25-053-Cl
Cell strainer (40 μm) Fisher 22363547
Tissue culture dish 100 X 20 mm BD Falcon 353003
Multiwell 6-well BD Falcon 353046
1.5 ml microcentrifuge tubes USA Scientific 1615-5500
15 ml centrifuge tubes BD Falcon 352096
50 ml centrifuge tubes BD Falcon 352070
5 ml Polystyrene Round-Bottom Tube with Cell-Strainer Cap BD Falcon 352235 Tubes for FACS 
ES R1 cells ATCC SCRC-1011
OP9 cells Cells can be obtained from the Riken Laboratory Cell Repository (Japan).
OP9-DL1 cells      Cells can be requested from the Zúñiga-Pflücker laboratory.
FlowJo software  Tree Star FACS data analyses
Flow Cytometer BD FACScan, FACSCalibur and FACSVantage have been used in our lab

Referências

  1. Schmitt, T. M., et al. Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro. Nat Immunol. 5, 410-417 (2004).
  2. Pui, J. C., et al. Notch1 expression in early lymphopoiesis influences B versus T lineage determination. Immunity. 11, 299-308 (1999).
  3. Nakano, T., Kodama, H., Honjo, T. Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science. 265, 1098-1101 (1994).
  4. Schmitt, T. M., Zuniga-Pflucker, J. C. Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity. 17, 749-756 (2002).
  5. Chen, J., Lansford, R., Stewart, V., Young, F., Alt, F. W. RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development. Proceedings of the National Academy of Sciences of the United States of America. 90, 4528-4532 (1993).
  6. de Pooter, R. F., Cho, S. K., Carlyle, J. R., Zuniga-Pflucker, J. C. In vitro generation of T lymphocytes from embryonic stem cell-derived prehematopoietic progenitors. Blood. 102, 1649-1653 (2003).
  7. Holmes, R., Zuniga-Pflucker, J. C. The OP9-DL1 system: generation of T-lymphocytes from embryonic or hematopoietic stem cells in vitro. Cold Spring Harb Protoc. 2009, (2009).
  8. Arsov, I., et al. A role for autophagic protein beclin 1 early in lymphocyte development. J Immunol. 186, 2201-2209 (2011).
  9. Lahiji, A., et al. Complete TCR-alpha gene locus control region activity in T cells derived in vitro from embryonic stem cells. J Immunol. 191, 472-479 (2013).
  10. Hirashima, M., Kataoka, H., Nishikawa, S., Matsuyoshi, N., Nishikawa, S. Maturation of embryonic stem cells into endothelial cells in an in vitro model of vasculogenesis. Blood. 93, 1253-1263 (1999).
  11. Nishikawa, S. I., Nishikawa, S., Hirashima, M., Matsuyoshi, N., Kodama, H. Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages. Development. 125, 1747-1757 (1998).
  12. de Pooter, R. F., et al. Notch signaling requires GATA-2 to inhibit myelopoiesis from embryonic stem cells and primary hemopoietic progenitors. J Immunol. 176, 5267-5275 (2006).
  13. Watarai, H., et al. Generation of functional NKT cells in vitro from embryonic stem cells bearing rearranged invariant Valpha14-Jalpha18 TCRalpha. 115, 230-237 (2010).
  14. Pipkin, M. E., et al. Chromosome transfer activates and delineates a locus control region for perforin. Immunity. 26, 29-41 (2007).

Play Video

Citar este artigo
Kučerová-Levisohn, M., Lovett, J., Lahiji, A., Holmes, R., Zúñiga-Pflücker, J. C., Ortiz, B. D. Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells. J. Vis. Exp. (92), e52119, doi:10.3791/52119 (2014).

View Video