Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

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 ....

Protocol

1. Preparation of Culture Media, Cytokines and Gelatinized Plates

1. Prepare ES cell media by using Dulbecco’s Modification of Eagle’s Medium (DMEM) with high glucose and sodium pyruvate. Add 20% ESC Qualified Fetal Bovine Serum (FBS), 1% Penicillin/Streptomycin, 1% L-glutamine, 1% HEPES Buffer, 1% Non-Essential Amino Acids, 0.1% Gentamicin (50 mg/ml) and 0.1% (55 μM) β-mercaptoethanol. Sterilize ES cell media by filtration.
2. Prepare OP9 media by making 1 L of Alpha Minimum Essential Medium (α-MEM) from powder according to the manufacturer’s instructions. Add 20% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin....

Results

When grown on MEFs in the presence of LIF, mESCs can be maintained in an undifferentiated state. Under ideal conditions, they appear as compact colonies of cells surrounded by a shiny halo in phase contrast microscopy (Figure 1). These cultures must be monitored daily. Depending on the confluence of the cells, media can be changed or cells can be split. Neighboring mESC colonies should not come to the point of contact with one another. A normal, healthy culture of undifferentiated mESCs is an essential s.......

Discussion

The OP9-DL1 co-culture system has been utilized to study the role of various gene products during the development of blood cell types from stem cells.^8,12,13 It has also proven an effective model to study the function of gene regulatory DNA during cellular differentiation.^9,14 Using this approach as an alternative to whole mouse models can yield considerable savings in time and cost of experiments that address many basic questions in hematopoiesis. However, adaptation of this protocol for these purp.......

Materials

List of materials used in this article

Name	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	0.22 μm 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
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