We thank Joon Kim for expert flow cytometry assistance. Research in the authors&#8217; 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.

1. Preparation of Culture Media, Cytokines and Gelatinized Plates
<ol>
	<li>Prepare ES cell media by using Dulbecco&#8217;s Modification of Eagle&#8217;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 &#956;M) &#946;-mercaptoethanol. Sterilize ES cell media by filtration.</li>
	<li>Prepare OP9 media by making 1 L of Alpha Minimum Essenti...

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

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...

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 &#8220;microenvironment&#8221; 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 &#8220;day 5&#8221; 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.

<table border="0" cellpadding="0" cellspacing="0" width="1055">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:355px;">MATERIALS</td>
			<td style="width:127px;">COMPANY</td>
			<td style="width:141px;">CATALOG NUMBER</td>
			<td style="width:432px;">COMMENTS</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DMEM</td>
			<td>Corning</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Stem cell qualified FBS</td>
			<td>Gemini</td>
			<td>100-125</td>
			<td>Heat inactivated</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Penicillin/Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">L-alanyl L-glutamine</td>
			<td>Corning</td>
			<td>25-015-CI</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">HEPES buffer&#160;</td>
			<td>Millipore</td>
			<td>TMS-003-C</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Non-Essential Amino Acids</td>
			<td>ThermoScientific</td>
			<td>SH30853.01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Gentamicin Regent Solution (50 mg/mL)</td>
			<td>Life Technologies&#160;</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#946;-mercaptoethanol (55 mM) in DPBS</td>
			<td>Life Technologies&#160;</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Filter Unit</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td>0.22&#956;m PES membrane</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cell Culture Grade Water</td>
			<td>Corning</td>
			<td>25-055-CM</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#945;-MEM&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>12000-022</td>
			<td>Powder, reconstitute per manufacture recommendation</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sodium bicarbonate&#160;</td>
			<td>Sigma</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">FBS</td>
			<td>ThermoScientific</td>
			<td>SH 30396.03</td>
			<td>Testing of individual lots required&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dimethyl Sulphoxide&#160;</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Recombinant Human Flt-3 Ligand</td>
			<td>R&#38;D Systems</td>
			<td>308-FK</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Recombinant Murine IL-7</td>
			<td>PeproTech</td>
			<td>217-17</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">LIF&#160;</td>
			<td>Millipore</td>
			<td>ESG1107</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Utrapure water with 0.1% gelatin</td>
			<td>Millipore</td>
			<td>ES-006-B</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MEFs mitomycin C treated</td>
			<td>Millipore</td>
			<td>PMEF-CF</td>
			<td>Any mitotically arresteded MEFs can be used</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DPBS</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Trypsin EDTA, 1X</td>
			<td>Corning</td>
			<td>25-053-Cl</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cell strainer (40 &#956;m)</td>
			<td>Fisher</td>
			<td>22363547</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue culture dish 100 X 20 mm</td>
			<td>BD Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Multiwell 6-well</td>
			<td>BD Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1.5 ml microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">15 ml centrifuge tubes</td>
			<td>BD Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">50 ml centrifuge tubes</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">5 ml Polystyrene Round-Bottom Tube with Cell-Strainer Cap</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td>Tubes for FACS&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">ES R1 cells</td>
			<td>ATCC</td>
			<td>SCRC-1011</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">OP9 cells</td>
			<td></td>
			<td></td>
			<td>Cells can be obtained from the Riken Laboratory Cell Repository (Japan).</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">OP9-DL1 cells</td>
			<td>&#160;&#160;&#160;&#160;</td>
			<td></td>
			<td>Cells can be requested from the Z&#250;&#241;iga-Pfl&#252;cker laboratory.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">FlowJo software&#160;</td>
			<td>Tree Star</td>
			<td></td>
			<td>FACS data analyses</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Flow Cytometer</td>
			<td>BD</td>
			<td></td>
			<td>FACScan, FACSCalibur and FACSVantage have been used in our lab</td>
		</tr>
	</tbody>
</table>

derivation of t cells in vitro from mouse embryonic stem cells

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.

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...

Watch this Scientific Journal Video about Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells at JoVE.com

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

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.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

derivation-of-t-cells-in-vitro-from-mouse-embryonic-stem-cells

Research

JoVE Journal

Immunology and Infection

10.8K vistas.  City University of New York. 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.