Summary

T 세포의 유도<em> 체외</em> 마우스 배아 줄기 세포에서

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

문화 미디어, 사이토 카인 및 젤라틴 판의 1 준비 높은 포도당과 피루브산 나트륨과 이글의 중간 (DMEM) 둘 베코의 변형 례를 사용하여 ES 세포 매체를 준비한다. 1 % HEPES 버퍼, 1 % 비 필수 아미노산, 0.1 % 겐타 마이신 (50 ㎎ / ㎖) 및 0.1 % (태아 혈청 (FBS)를 공인 20 % ESC, 1 % 페니실린 / 스트렙토 마이신, 1 % L-글루타민을 추가 55 μM) β-머 캅토 에탄올. 여과에 의해 ES 세포를 멸균 매체. 제조?…

Representative Results

LIF의 존재 MEFs에 성장하면 mESCs는 미분화 상태로 유지 될 수있다. 이상적인 조건에서, 그들은 위상차 현미경에 빛나는 후광에 둘러싸인 세포의 소형 식민지 (그림 1)로 나타납니다. 이러한 문화는 매일 모니터링해야합니다. 세포의 합류에 따라 미디어가 변경 될 수 있거나 세포를 분리 할 수​​있다. 이웃 MESC 식민지는 서로의 접점에 와서는 안된다. 미분화 mESCs의 정상적이고 건강한 ?…

Discussion

OP9-DL1 공동 배양 시스템은 줄기 세포에서 혈액 세포 유형의 개발시 각종 유전자 산물의 역할을 연구하기 위해 이용되어왔다. 8,12,13 또한 유전자 조절 DNA의 기능을 연구하기위한 효과적인 모델을 입증 세포 분화 과정. 9,14 시간 및 조혈 많은 기본적인 질문을 해결 실험에서 상당한 비용 절감 효과를 얻을 수있는 전체적인 마우스 모델의 대안으로이 방법을 사용. 그러나 이러한 목적…

Divulgaciones

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

Referencias

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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).

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