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Embryonic stem cells (ESCs) are derived from the inner cell mass ofthe developing blastocyst and have the potential to give rise to all celltypes found in the adult organism. Thus, ESCs could potentially serveas a renewable source of transplantable tissue-specific stem cells. Therealization of this therapeutic potential requires the identification ofthe molecular cues necessary for the induction of the directed differ-entiation of ESCs toward specific cell lineages.

In the absence of external stimuli, ESCs cultured in vitro sponta-neously differentiate and rapidly proliferate to form cystic structurescalled embryoid bodies1, which consist of several semiorganized germlayer tissues, including mesoderm-derived hematopoietic cells thatundergo an early wave of erythromyeloid differentiation similar tothat in yolk sac blood islands2. Culture of ESCs on the bone marrowstromal cell line OP9 (ref. 3) selectively facilitates the development oflymphocyte lineages4,5. Despite these advances, attempts to transferESC-derived hematopoietic progenitors in vivo have been mostlyunsuccessful6, whereas undifferentiated ESCs develop into teratocar-cinomas after in vivo transplantation7. The latter finding emphasizesthe necessity of identifying the molecular pathways responsible forthe lineage-specific induction of ESC differentiation in vitro. Withoutthis understanding, the promise of ESCs as a renewable source oftransplantable lineage-specific stem cells may remain unrealized8.

Although ESCs can differentiate into most blood cell lineages invitro4,6, attempts to generate T cells from unmanipulated ESC-derived hematopoietic progenitor cells have failed. ESCs cultured on

OP9 stromal cells differentiate into mesoderm-derived prehe-matopoietic progenitors with limited potential to further differentiateinto T cells after transfer into a thymic microenvironment9. Giventhose results and findings demonstrating that OP9 cells ectopicallyexpressing the Notch ligand Delta-like 1 (OP9-DL1 cells) acquire theability to induce the full differentiation of T cells from hematopoieticstem cells (HSCs) in vitro10, we hypothesized that Notch signals deliv-ered by OP9-DL1 cells may facilitate the induction of ESC differenti-ation toward the T cell lineage.

The Notch signaling pathway is an essential factor in determiningwhether lymphocyte progenitors adopt a T cell or B cell fate11,12 andhas been linked to multiple stages of intrathymic T cell develop-ment13–18. In addition to Notch, many other transcription factorsinfluence T cell commitment and early differentiation, such as thebasic helix-loop-helix transcription factors E2A19 and HEB20, the Etsprotein PU.1 (ref. 21) and the zinc-finger transcription factor GATA-3(ref. 22). Given the central function of Notch during T cell develop-ment, it is likely that signals ‘downstream’ of Notch are involved in theregulatory interplay among these transcription factors.

Here we show that ESCs cultured on OP9-DL1 cells (ESC–OP9-DL1cultures) are able to differentiate into hematopoietic cells, commit tothe T cell lineage, undergo stage-specific proliferation and mature intoCD4+ and CD8+ T cells in vitro. The generation of T cells from ESCswas dependent on the expression of Delta-like 1 by the OP9 cells. T cellsgenerated from ESCs cultured on OP9-DL1 cells displayed a diverse

1Department of Immunology, University of Toronto, Sunnybrook and Women’s College Health Sciences Centre, 2075 Bayview Avenue, Toronto, Ontario, M4N 3M5Canada. 2Departments of Medical Biophysics and Immunology, Ontario Cancer Institute, Toronto, Ontario M5G 2M9, Canada. 3Present address: Division of BiologicalSciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA. Correspondence should be addressed to J.C.Z.-P.(jc.zuniga.pflucker@utoronto.ca).

Published online 21 March 2004; doi:10.1038/ni1055

Induction of T cell development and establishmentof T cell competence from embryonic stem cellsdifferentiated in vitroThomas M Schmitt1, Renée F de Pooter1, Matthew A Gronski2, Sarah K Cho1,3, Pamela S Ohashi2 & Juan Carlos Zúñiga-Pflücker1

Embryonic stem cells (ESCs) have the potential to serve as a renewable source of transplantable tissue-specific stem cells.However, the molecular cues necessary to direct the differentiation of ESCs toward specific cell lineages remain obscure. Herewe report the successful induction of ESC differentiation into mature functional T lymphocytes with a simple in vitro coculturesystem. The directed differentiation of ESCs into T cells required the engagement of Notch receptors by Delta-like 1 ligand (DL1) expressed on the OP9-DL1 stromal cell line. We found a normal program of T cell differentiation in ESC–OP9-DL1 cellcocultures. ESC-derived T cell progenitors effectively reconstituted the T cell compartment of immunodeficient mice, enablingan effective response to a viral infection. These findings provide a powerful tool for the molecular analysis of T cell developmentand open new avenues for the development of immunotherapeutic approaches using defined sources of stem cells.

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antigen receptor repertoire, and CD8+ T cellsproliferated and produced interferon-γ (IFN-γ) in response to T cell receptor (TCR) stimu-lation. Using T lineage progenitors derivedfrom ESC–OP9-DL1 cultures, we were able tosuccessfully reconstitute the T cell compart-ment of immunodeficient, recombinationactivation gene 2–deficient (Rag2–/–) mice.Furthermore, these ESC-derived peripheral T cells were capable of mounting an antigen-specific immune response to a viral challenge.

The use of a simple coculture system for the efficient differentiationof ESCs into T cells represents a powerful new approach for the eluci-dation of the molecular events governing lymphocyte development.This, along with the use of ESC-derived T cell progenitors for theimmune reconstitution of mice, brings us closer to the elusive goal ofusing human ESCs as an efficient and effective source of trans-plantable lineage-specific progenitor cells.

RESULTSGeneration of T cells from ESC–OP9-DL1 culturesMouse ESCs remain undifferentiated when cultured on embryonicfibroblasts in the presence of leukemia inhibitory factor23. Removal of

ESCs from these culture conditions activates a program of rapid pro-liferation and uncontrolled differentiation. To determine whetherESCs cultured on OP9 cells expressing Delta-like 1 (OP9-DL1) couldbe directed to differentiate into T lymphocytes, we placed undifferen-tiated ESCs on either OP9-control cells (ESC–OP9-control) or OP9-DL1 cells (ESC–OP9-DL1; Fig. 1a). We analyzed the differentiation ofESCs cultured on these two stromal cell lines in parallel with that ofbone marrow–derived HSCs (Fig. 1b). ESCs differentiated on OP9cells gave rise to an early wave of erythro-myeloid lineage cells, suchthat CD11b and Ter119 were the only lineage-specific markers seenon day 8 of both cultures (Fig. 1 and data not shown). However, theseerythro-myeloid cells steadily decreased in both control OP9 andOP9-DL1 cultures, as lymphoid cells became the predominant cells inthe cultures at later days of development.

On day 14, a small population of B cell progenitors began toemerge from the ESC–OL9-control cultures (Fig. 1a). In contrast,ESCs cultured on OP9-DL1 cells gave rise to a population of cellsexpressing CD25 and/or CD44 and hence probably to belonging to

a bFigure 1 T cell development from ESCs invitro. ESCs (a) and CD117+Sca-1+Lin– (CD4,CD8, CD11b, CD19, CD24 and NK1.1) bonemarrow–derived HSCs (b) were induced todifferentiate by culture on OP9-control orOP9-DL1 cells. Nonadherent hematopoieticcells were collected on days 8, 14 and 20 of culture and analyzed by flow cytometry.Myeloid progenitors were characterized byCD11b expression; B cell progenitors wereidentified by CD19 expression (from OP9-control cultures); early CD4 CD8 DN T cellprogenitors were distinguished by high CD25(IL-2Rα) expression (from OP9-DL1 cultures) and at later time points, CD4 CD8 DP T cells were evident. Data are representative of three independent experiments. Numbers in quadrants indicate percentages of eachpopulation.

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Figure 2 ESC-derived T cell development recapitulates thymic development invitro. (a) ESC-derived T cells undergo multiple Tcrb D-J gene rearrangements.DNA was isolated from fibroblasts, CD25+CD44– ESC-derived T cells and totalfetal thymus from day 14 and was analyzed by PCR for rearrangements at theTcrb locus. (b) ESCs cultured on OP9-DL1 give rise to T cells that display adiverse repertoire of T cell receptors. ESC-derived T cells were analyzed at day20 by flow cytometry for various TCR Vβ gene products. RCN, relative cellnumber. Numbers above bracketed lines indicate the percentage of cellsexpressing the TCR Vβ gene products in the gated subpopulations. (c) ESCsgive rise to both αβ and γδT cells when differentiated on OP9-DL1 cells. ESCswere cultured on OP9-DL1 cells for 20 d, and the resulting cell population was analyzed for TCRγδand TCRαβ expression by flow cytometry. Numbers inquadrants indicate percentages of each population. Data are representative ofat least two independent experiments.

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the T cell lineage. Thus, ESCs cultured on OP9-DL1 cells seemed tofollow a typical thymocyte developmental progression through theCD4 CD8 double-negative (DN) stage, as defined by CD44 andCD25 expression24. Furthermore, these cultures at day 14 containeda small population of CD4 and CD8 double-positive (DP) cells.

Consistent with previous findings5, we found only B cells by day 20from ESCs cultured on control OP9 cells. In contrast, ESCs culturedon OP9-DL1 cells failed to generate B cells; instead, these culturescontained a robust population of DP cells, which accounted for closeto half of the hematopoietic cells present in the cultures.

We also differentiated bone marrow–derived CD117+Sca-1hiLin–

HSCs on OP9-control or OP9-DL1 cells (Fig. 1b). Initially, the HSCsdeveloped with faster temporal kinetics than did the ESC cultures(Fig. 1a). As early as day 8 of culture, we found CD19+ B lineage cellsfrom OP9-control cultures, whereas we found a large population ofDN CD25+ cells in OP9-DL1 cultures. In comparison, the ESC cul-tures contained a proportion of CD44–CD25+ DN cells on day 14 ofculture as large as that from HSCs cultured for 8 days, yet these cul-tures contained a similar percentage of DP cells when analyzed on day20. These data indicate that the hematopoietic progenitor cells thatdevelop from ESCs cultured on OP9-DL1 cells develop faster thantheir adult counterparts. In keeping with this, fetal liver–derivedHSCs cultured on OP9-DL1 also require less than a week to developfrom the DN to the DP stage of T cell development10.

Tcrb rearrangements of ESC-derived T cellsRearrangement at the TCRβ locus (Tcrb) is a hallmark of T cell lineagecommitment and is essential for the progression of DN thymocytes tothe DP stage during normal αβ T cell development. To determinewhether the T cells that develop from ESCs cultured on OP9-DL1 cellsundergo normal rearrangement of the Tcrb locus, we used PCR toanalyze genomic DNA from CD44–CD25+ DN progenitors from an

ESC–OP9-DL1 culture at day 12. We also analyzed DNA from fetalthymus at day 14 and from embryonic fibroblasts as rearranged andgermline controls, respectively (Fig. 2a). ESC-derived T cells showed apattern of Tcrb rearrangement similar to that of ex vivo fetal thymo-cytes (Fig. 2a). Thus, ESCs cultured on OP9-DL1 cells gave rise to apopulation of T cells containing a potentially diverse set of TCR generearrangements.

To correlate the diverse repertoire of TCR gene rearrangements tothe functional Tcrb product expressed at the cell surface noted byPCR analysis, we used flow cytometry to analyze ESC-derived T cellsat day 21 of culture for surface expression of several commonly usedTCR Vβ gene segments (Fig. 2b). Compared with ex vivo thymocytes,ESC-derived T cells showed a similar pattern of TCR Vβ gene expres-sion, indicating that the T cells that develop in this culture systemhave the potential to generate a diverse TCR repertoire.

During normal thymocyte development, T cells bearing both αβand γδ TCRs develop in the thymus24. To determine whether bothpopulations of T cells develop from ESCs cultured on OP9-DL1 cells,we analyzed ESC-derived T cells for surface expression of αβ and γδTCR. Both populations of T cells developed from ESCs cultured onOP9-DL1 (Fig. 2c).

Analysis of gene expression by differentiating ESCsDuring hematopoietic development, HSCs undergo a program ofsequential lineage restriction that is thought to involve key transcrip-tional regulators that in turn mediate the coordinated expression oflineage-specific genes25. To elucidate at the molecular level the processby which ESCs differentiate on OP9 cells in the presence or absence ofDelta-like 1, we assessed the expression of developmentally regulatedgenes over time by semiquantitative RT-PCR analysis (Fig. 3). Genesencoding factors responsible for regulating hematopoiesis at multipledifferentiation steps, such as the Ets protein, PU.1 and the E2A family

Figure 3 Gene expression analysis of ESCs cultured in vitro. ESCs were cultured on OP9-control and OP9-DL1 cells and were collected at multiple timepoints (above gels), then mRNA was purified and cDNA was generated. For each time point, threefold serial dilutions of the cDNA were normalized to β-actinand then were analyzed with gene-specific primers to determine expression of developmentally regulated genes (products, left margin). Data presented areinverted images of ethidium bromide–stained gels.

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member E47, showed a dynamic pattern ofexpression throughout the time course. Inparticular, PU.1 was expressed throughoutthe ESC–OP9-control culture period, inkeeping with its known involvement in regu-lating early myelopoiesis and B cell differenti-ation21. In contrast, ESC–OP9-DL1 culturesshowed PU.1 transcripts only at early timepoints, consistent with the idea that PU.1expression is important for the initiation ofhematopoiesis, whereas downregulation ofPU.1 expression is then required for proper T cell differentiation to proceed26. E47, whichis involved in both B and T cell differentia-tion19,27,28, was expressed throughout the cul-ture period on both stromal cell lines (Fig. 3).

We also analyzed expression of the geneencoding the interleukin 7 (IL-7) receptor(Il7r)29 and Rag1 (ref. 30), which arerequired for the survival and proliferation of lymphocyte progenitors, as well as for both B cell and T cell antigen receptor gene rearrangement, respectively (Fig. 3). Wefound both Il7r and Rag1 transcripts at timepoints when B and T lymphopoiesis firstbecome apparent by flow cytometry (Fig. 1a).

Expression of Delta-like 1 by OP9 cells fundamentally changed thestromal environment from one that strongly induced B cell develop-ment to one that efficiently supported all aspects of T cell develop-ment, consistent with the involvement of Notch signaling in theregulation of B versus T lineage commitment (Fig. 1). To addresswhether the different programs of B and T cell gene expression aretightly and coordinately regulated in these culture conditions, weanalyzed several B cell– and T cell–specific genes. Transcripts encod-ing for λ5 and Igα, which are part of the pre-B cell receptor complexand are expressed during early B cell development, were present onlyin cells from ESC–OP9-control cultures (Fig. 3). Notably, transcriptsencoding CD3ε and pre-Tα, which are part of the pre-TCR complexand are expressed during early T cell development, were expressedonly in cells from the ESC–OP9-DL1 cultures. These data indicatethat the induction of lineage-specific gene expression in differentiat-ing ESCs was appropriately coordinated in these culture conditions.Thus, the temporal kinetics of this regulated gene expression serve asan indication of when B and T lineage commitment first occur withinthe ESC–OP9-control and ESC–OP9-DL1 cultures, respectively.

We also examined whether the expression of lineage-specific genescorrelated with the presence of transcription factors known to beimportant during T cell development, such as HEB20, HES-1 (ref. 31)and GATA-3 (ref. 22; Fig. 3). The basic helix-loop-helix transcriptionfactor HEB, which is essential for the transition from the DN to theDP stage20, was highly expressed at later time points in the ESC–OP9-DL1 cultures. HES-1, a basic helix-loop-helix transcription factor andtranscriptional repressor that is induced downstream of Notch signal-ing, was expressed at all time points analyzed in cells from theESC–OP9-DL1 cultures. We also detected HES-1 transcripts, albeit inlower abundance, from ESC–OP9-control cultures probably presentas a result of Notch signaling initiated by the Notch ligands Jagged-1and Jagged-2, which are both expressed by OP9 cells10. GATA-3,which is expressed by all developing T cells and is essential for theirgeneration22, was expressed throughout the culture period and athigh abundance after day 12 in the ESC–OP9-DL1 cultures.

Functional T cells generated in ESC–OP9-DL1 culturesTo determine whether ESC cultures contained mature CD4 or CD8single-positive (SP) T cells, we analyzed ESC–OP9-DL1 cultures atday 22 (Fig. 4a), which showed the presence of CD8 SP and DP Tcells but not CD4 SP T cells. For comparison, we also analyzed theexpression of CD4 and CD8 on thymocytes from an adult mouse,which showed a typical CD4 and CD8 expression pattern. TCR sur-face expression normally increases after thymocyte maturationfrom the DP to the SP stage. A subset of CD8 SP T cells (R1-gated)from ESC–OP9-DL1 cultures at day 22 displayed high TCR surfaceexpression similar to that of ex vivo CD8 SP thymocytes (Fig. 4a).However, most CD8 SP T cells from ESC–OP9-DL1 cultures at day22 were either TCR– or had low TCR expression and therefore prob-ably represented immature SP CD8 cells. The absence of CD4 SP T cells from ESC–OP9-DL1 cultures probably reflects the fact thatOP9 cells express major histocompatibility complex class I but notclass II molecules.

To address whether the CD8 SP T cells with high expression of TCRwere indeed functionally mature, we purified CD4–CD8+TCRhi cellsderived from ESCs by cell sorting and cultured them for 3 d in thepresence or absence of plate-bound antibody to CD3 (anti-CD3) andanti-CD28. ESC-derived CD8 SP T cells proliferated and producedIFN-γ in response to TCR engagement (Fig. 4b). Therefore, ESCs cul-tured on OP9-DL1 cells can fully differentiate into mature functionalSP T cells that show high TCR surface expression, undergo robustproliferation (stimulated cells showed an increase of ∼ 70-fold overthat of nonstimulated control cultures) and produce IFN-γ inresponse to antigen receptor stimulation.

Immune reconstitution by ESC-derived T cellsAlthough ESCs have enormous potential theoretically as an efficientand effective source of transplantable tissue-specific progenitor cells,experimentally this potential has not yet been demonstrated withgenetically unmodified ESCs8. To determine whether ESC-derived T cell progenitors could differentiate normally after transfer into an

Figure 4 Functionally mature CD8+ T cells develop from ESCs cultured on OP9-DL1 cells. (a) SP T cells with high TCRβ surface expression were generated from ESCs cultured on OP9-DL1 cells for 22 dand then analyzed by flow cytometry. Numbers in quadrants indicate percentages of each population;numbers above bracketed lines indicate the percentage of cells expressing TCRβ in the gatedsubpopulations. (b) CD8+TCRβhi T cells were purified from ESC–OP9-DL1 cultures at day 22 and thenwere stimulated for 3 d with plate-bound anti-CD3 (α-CD3) and anti-CD28 (α-CD28; 10 µg/ml each) orwere left unstimulated (left). Data are representative of at least two independent experiments. Numbersin outlined areas indicate percentages of each population.

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intact thymic micro-environment, we isolated CD25+CD44+ andCD25+CD44– DN T cell progenitors (CD45.2) by flow cytometriccell sorting from an ESC–OP9-DL1 culture at day 12. We then usedthese cells to seed CD45.1-congenic deoxyguanosine-treated fetalthymic organ cultures (FTOCs), which we analyzed by flow cytome-try after 10 d in culture. CD25+ DN T cell progenitors obtained froman ESC–OP9-DL1 culture at day 12 were capable of developing inFTOC (nearly 100% of thymocytes were of donor origin, CD45.2+;Fig. 5a) and generated both CD4 CD8 DP T cells and TCRhi CD4 andCD8 SP T cells.

These results showing that ESC-derived T cell progenitors canreadily reconstitute and develop normally in FTOC allowed us toaddress whether implantation of in vitro–reconstituted FTOCsmight provide the means by which differentiated ESCs could beintroduced into an adult host animal. Therefore, we obtainedCD25+CD44+ and CD25+CD44– DN T cell progenitors from anESC–OP9-DL1 culture at 2 weeks (Fig. 1a), transferred them intoCD45.1-congenic fetal thymic lobes and placed them in culture for 5d. We then implanted these reconstituted thymic lobes under theskin of sublethally irradiated Rag2–/– mice. Three weeks afterimplantation, we analyzed the spleens and lymph nodes of the recon-stituted mice by flow cytometry for the presence of TCR+CD45.2+

donor-derived T cells (Fig. 5b). As expected, nongrafted Rag2–/–

mice were devoid of TCR-bearing CD4+ or CD8+ T cells. In contrast,Rag2–/– mice implanted with FTOCs seeded with ESC-derived T cell

progenitors showed a robust reconstitution with both TCR+ CD4and TCR+ CD8 SP T cells present in the spleen and lymph nodes (8.7× 105 and 3.6 × 106 donor T cells, respectively). Notably, the effi-ciency and effectiveness of T cell reconstitution with ESC-derived T cell progenitors was similar to that in the spleen and lymph nodesof Rag2–/– mice implanted with control untreated fetal thymic lobes(8.6 × 105 and 5.8 × 106 donor T cells, respectively; Fig. 5b). Thus,these results demonstrate that ESCs induced to adopt a T lineage fateon OP9-DL1 cells are fully capable of reconstituting the T cell com-partment of a host mouse.

To directly test whether the Rag2–/– mice that had been reconsti-tuted with T cell progenitors derived from ESC–OP9-DL1 culturescould in fact mount a functional immune response, we infectedthese mice with the lymphocytic choriomeningitis virus (LCMV).After 8 d, we recovered splenocytes and assayed them for LCMV-specific cytotoxic T lymphocyte (CTL) activity in vitro. ESC-derived T cells were capable of generating an effective antigen-specific immune response after LCMV infection (Fig. 5c).Moreover, the LCMV-specific CTL response detected in Rag2–/–

mice reconstituted with ESC-derived T cells was equivalent to thatof normal C57BL/6 mice. In these experiments, the fetal thymiclobes that were seeded with the ESC-derived T cell progenitors werealso RAG-2 deficient, ensuring that any TCR-bearing cells present inthe reconstituted Rag2–/– mice could be derived only fromESC–OP9-DL1 cells.

Figure 5 ESC-derived T cells can reconstituteimmune function in immunodeficient hosts. (a) ESC-derived T cell progenitors can reconstituteFTOC. CD45.2+CD25+ ESC-derived T cellprogenitors (1 × 105) were seeded into CD45.1congenic, deoxyguanosine-treated fetal thymiclobes and were cultured for 10 d. Only T cells of donor origin (CD45.2) are found. Numbers inquadrants indicate percentages of each population;number above bracketed line indicates thepercentage of cells expressing CD3 in the gatedsubpopulations. (b) ESC-derived T cell progenitorscan reconstitute an immune-deficient host. ESC-derived T cell progenitors were seeded into fetalthymic lobes from day 14 from CD45 congenic B6mice and were cultured in FTOC. After 5 d, thereconstituted fetal thymic lobes or nondepletedfetal thymic lobes were implanted under the skin ofRAG2-deficient mice. After 3 weeks, the spleensand lymph nodes of recipient mice (center andright) as well as those of an unmanipulated Rag2–/–

mouse (left) were analyzed by flow cytometry forTCRβ+ donor-derived T cells. Numbers in quadrantsand near outlined areas indicate percentages ofeach population. (c) Adoptively transferred ESC-derived T cells can mount an antigen-specific CTLresponse. ESC-derived T cell progenitors werecultured in FTOC for 5 d in fetal thymic lobes fromRag2–/– mice. After 5 d, the reconstituted lobeswere implanted under the skin of Rag2–/– mice. At 2 weeks after transfer, recipient mice and wild-typeB6 control mice were infected intravenously with2,000 plaque-forming units of LCMV; 8 d afterinfection, splenocytes were isolated and assayed for CTL activity by 51Cr-release assay with EL4target cells prepulsed with 51Cr and the LCMVglycoprotein gp33 or a control adenovirus-derivedpeptide (AV). Data are representative of at least twoindependent experiments.

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DISCUSSIONHere we have described an efficient and simple system for the in vitrodifferentiation of ESCs into T lymphocytes capable of functionalimmune reconstitution. This finding has many experimental andclinical applications, as ESCs have enormous potential as a renewablesource of immune cell progenitors that could enable the treatment ofa multitude of immune disorders, including congenital and acquiredimmunodeficiencies, malignancies and autoimmunity. Moreover, theuse of gene-targeted ESCs in combination with this simple differenti-ation system should facilitate the elucidation of the molecular path-ways governing T cell development.

The ability to direct the differentiation of ESCs in vitro towardthe T cell lineage was dependent on Delta-like 1 expression by theOP9 cells, indicating that the engagement of Notch receptors ondeveloping progenitors in the context of an otherwise B cell–supportive environment is sufficient to coordinate the expressionof the multiple transcription factors that regulate T cell commit-ment and differentiation. This was seen from the shift in the pat-tern of expression from genes encoding B cell lineage–specificmolecules, such as Igα and λ5, to expression of genes that encode T cell lineage–specific proteins, such as CD3ε and pre-Tα. Notably,important transcription factors such as PU.1 and GATA-3 alsoshow temporal and lineage-specific regulation32, with PU.1decreasing and GATA-3 increasing in expression during T cell lineage development. Moreover, these findings establish Notch–Delta-like interactions as being pivotal for the induction of com-pletely de novo T cell lineage commitment.

The fact that ESCs seem to undergo a normal program of T celldevelopment on OP9-DL1 cells establishes the broad applicability ofthis model system. Both γδand αβ TCR–bearing T cells develop fromESC–OP9-DL1 cultures and analysis of the Tcrb locus showed multi-ple diversity (D) and joining (J) rearrangements, which together withthe surface expression of multiple TCR-Vβ chains by αβ T cells indi-cates that these T cells have a diverse antigen receptor repertoire.Furthermore, functional mature CD8+ T cells develop fromESC–OP9-DL1 cultures, which show high surface expression of TCRand produce IFN-γ in response to TCR stimulation.

The inability to efficiently generate functional T lymphocytes fromunmanipulated ESCs has constituted a substantial barrier to theimplementation of ESCs as a renewable source of transplantable T cellprogenitors for immune reconstitution. So far, attempts to engraftESC-derived hematopoietic progenitors have required the geneticmodification of ESCs, by forced expression of the homeoproteinHoxB4 and have yielded only limited T cell reconstitution33. Ourfindings overcome this impediment and alleviate the necessity forgenetic manipulation of ESCs to allow successful engraftment.Indeed, this versatility provides an ideal approach with which toexamine the molecular pathways responsible for the development ofspecific lymphoid lineages by allowing the differentiation of ESCsbearing either new or previously characterized gene-targeted dele-tions and, in particular, those genetic modifications resulting inembryonic lethality.

The experimental approach described here for the in vivoengraftment of ESC-derived T cells takes advantage of ESC-derivedT cell progenitors differentiated in vitro, which are first seeded intoFTOC before transfer into host mice. This rationale is based on thefact that ESC-derived pro-genitor T cells failed to show any sub-stantial ability to seed the thymus when injected intravenously intohost mice, which is probably because of an inability of these cells tohome to the host thymus. Therefore, we reasoned that as in vitroreconstitution of FTOC was effective, this approach would allow

ESC-derived progenitors to fully differentiate within an engraftedfetal thymus and then emerge into the periphery of the host mouse.The transfer of ESC-derived T cell progenitors into FTOC alsoemphasizes the fact that ESCs can indeed efficiently give rise to SP Tcells, indicating that the paucity of SP T cells seen in vitro fromESC–OP9-DL1 cultures is not due to an intrinsic peculiarity ofESC-derived precursors.

ESC-derived T cells generated in vivo in implanted fetal thymiclobes were fully capable of colonizing the peripheral lymphoid organsand mounting an effective antigen-specific CTL response after viralchallenge. The use of early T lineage–specific progenitors for in vivoreconstitution permitted ESC-derived cells to undergo selectionevents within the host thymus. Thus, the mature T cells that emergeinto the periphery are not only functional but also self-tolerant, thusavoiding the immunological incompatibility between host and donorthat might arise if T cells differentiated to maturity in vitro were used.Therefore, these results establish that ESC-derived T cell progenitorsgenerated in vitro can give rise to mature lymphocytes that are func-tional in vivo, bringing us closer to realizing the untapped therapeuticpotential of ESCs.

METHODSMice. ‘Timed-pregnant’ B6-Ly5.2 mice from day 14 were obtained from theNational Cancer Institute, Frederick Cancer Research and DevelopmentCenter. RAG2-deficient mice were bred and maintained in our animal facility.All animal procedures were approved by the Sunnybrook and Women’sCollege Health Science Centre Animal Care Committee.

Cell lines. The OP9-control and OP9-DL1 cell lines were generated byinfection of the bone marrow stromal cell line OP9 with the empty MigR1retroviral vector or with the MigR1 retroviral vector engineered to expressthe gene encoding Delta-like 1 5′ of the internal ribosomal entry site, allow-ing the bicistronic expression of Delta-like 1 and green fluorescent protein,as described10. OP9-control cells and OP9-DL1 cells were cultured asmonolayers in OP9 media (αMEM supplemented with 20% FCS(HyClone), 10 U/ml of penicillin, 100 µg/ml of streptomycin and 2.2 g/literof sodium bicarbonate).

The ESC line R1 was obtained from G. Caruana (Mount Sinai Hospital,Toronto, Canada). ESCs were maintained by culture in ESC media (DMEMsupplemented with 15% FCS, 10 U/ml of penicillin, 100 µg/ml of strepto-mycin, 100 µg/ml of gentamicin, 2 mM glutamine, 110 µg/ml of sodium pyru-vate, 50 µM 2-mercaptoethanol and 10 mM HEPES) containing 1 ng/ml ofleukemia inhibitory factor (R&D Systems) on irradiated mouse embryonicfibroblasts. Embryonic fibroblasts were generated from embryos at days 15–18as described34 and were cultured in ESC media.

Flow cytometry and cell sorting. Flow cytometry was done with aFACScalibur (BD Biosciences) instrument as described10. Fluorescein isoth-iocyante–conjugated CD3 (clone 145-2C11), CD4 (clone GK1.5), TCR Vβ5(clone MR9-4), TCR Vβ8 (clone MR5-2) and TCRγδ(clone GL3); recombi-nant-phycoerythrin-conjugated CD4 (clone GK1.5), CD11b (clone M1/70),CD44 (clone IM7), TCRβ (clone H57-597), TCR Vβ6 (clone RR4-7) andIFN-γ (clone XMG1.2); allophycocyanin-conjugated CD8 (clone 53-6.7),CD19 (clone 1D3) and CD25 (clone 7D4); and biotin-conjugated CD3(clone 145-2C11) and TCR Vβ3 (clone KJ25) monoclonal antibodies andstreptavidin-allophycocyanin were purchased from BD Biosciences. Foranalysis, live cells were gated based on forward and side scatter and lack ofpropidium iodide uptake. Cells were sorted with a FACSDiVa (BDBiosciences). Bulk sorted cells were ≥99% pure, as determined by analysisafter sorting.

OP9 cell cocultures. ESCs (5 × 104) were induced to differentiate by cultureon either OP9-control or OP9-DL1 cell monolayers in a 10-cm dish in theabsence of leukemia inhibitory factor or any other additional cytokines. Onday 5 of culture, when most ESC colonies were mesoderm-like in appearance,

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ESC–OP9 cocultures were disrupted by treatment with 0.25% trypsin(Gibco-BRL). The resulting single-cell suspensions were preplated for 30 minand nonadherent cells were then replated at a density of 6 × 105 cells per 10-cm dish containing fresh OP9 cells in OP9 media with the addition of Flt3ligand (5 ng/ml; Peprotech). On day 8 of culture and every 4 days thereafter,nonadherent ESC-derived hematopoietic cells were collected by vigorouspipetting, were filtered through a 40-µm nylon mesh and were transferredonto fresh OP9 monolayers in OP9 media. On day 8 of culture another 5 ng/ml of Flt3 ligand was added, and exogenous IL-7 (5 ng/ml; Peprotech)was added. Both cytokines were included at all subsequent passages.Typically, during the passage at day 8, 1 × 104 to 5 × 104 cells were recoveredfrom one 10-cm dish and were then transferred to one well of a 6-well platecontaining stromal cells. The method outlined above is similar to a publishedprotocol detailing the ESC-OP9 coculture system35. Several other ESC lineshave been used, including the E14 cell line, and these cell lines act the sameway as described here for the R1 cell line.

Purification of adult bone marrow. Bone marrow was purified from thefemurs of mice 4–6 weeks old and was disrupted by repetitive passage througha 25-gauge syringe needle. HSCs (CD117+Sca-1hi) were purified from bonemarrow–derived hematopoietic cells by flow cytometric cell sorting.HSC–OP9-DL1 cells were cultured as described10.

PCR and RT-PCR. Genomic DNA was purified with the EasyDNA kit(Invitrogen) from embryonic fibroblasts, from fetal thymus at day 14 andfrom CD117–CD44–CD25+ cells from ESC–OP9-DL1 cultures. DNA samples(100 ng of each) were amplified with a PTC-225 Peltier Thermal Cycler (MJResearch). Primers used for the Tcrb Dβ-Jβ rearrangement analysis have beendescribed36. Isolation of total RNA and reverse transcription reactions wasdone as described37. All semiquantitative PCR reactions used the same seriallydiluted cDNA batches shown for β-actin. The gene-specific primers, expectedproduct lengths and annealing temperatures are in Supplementary Table 1online. PCR products were separated by agarose gel electrophoresis and werevisualized by ethidium bromide staining. All PCR products shown correspondto expected molecular sizes.

T cell stimulation assay. The αβTCRhiCD4–CD8+ T cells were sorted fromESC–OP9-DL1 cultures at day 21, and 7.5 × 104 cells were stimulated byculture for 3 d with 10 µg/ml of plate-bound anti-CD3 (clone 145-2C11from BD Biosciences) and 5 µg/ml of plate-bound anti-CD28 (clone 37.51from BD Biosciences) in the presence of 5 ng/ml of IL-2 and 1 ng/ml of IL-15 (Peprotech). The cellularity of each of the samples was determined byflow cytometry. Intracellular staining for IFN-γ was done with theCytofix/Cytoperm with GolgiStop kit (BD Biosciences) according to themanufacturer’s instructions. Phycoerythrin-conjugated anti-IFNγ (cloneXMG1.2) was purchased from BD Biosciences.

FTOC. Lymphocyte-depleted fetal thymic lobes were prepared by culture ofthe thymic lobes from C57BL/6 (CD45.1) embryos at day 15 in medium con-taining 1.25 mM deoxyguanosine for 5 d as described9. CD25+CD45+ ESC-derived T cell progenitors (CD45.2+) were purified by flow cytometric cellsorting from ESC–OP9-DL1 cultures from days 11–12, which correspondsto the time when CD44+CD25+ T cell progenitors are initially detected.CD25 expression, which is characteristic of progenitor thymocytes38, wasused to isolate potential T cell pro-genitors, as CD44+CD25– cells wereunable to reconstitute FTOCs. Sorted CD25+CD44+ and CD25+CD44– cellswere seeded into lymphocyte-depleted thymic lobes (1 × 104 cells/pair oflobes) and were cultured for 10 d. Typically each reconstituted FTOC con-tained 6 × 104 to 8 × 104 cells per lobe pair, of which nearly all thymocyteswere of donor origin.

Adoptive transfer of ESC-reconstituted lobes. Fetal thymic lobes were recon-stituted with ESC-derived T cell progenitors as described above and were cul-tured in vitro for 6 d, followed by adoptive transfer under the skin of Rag2–/–

recipient mice. After 3 weeks, the host mice were killed and their spleens andlymph nodes were analyzed for ESC-derived T cells by flow cytometry (ESC-derived T cells expressed CD45.2 and TCRβ).

LCMV challenge and cytotoxicity assay. Rag2–/– mice were reconstituted withESC-derived T cell progenitors as described above, except that fetal thymiclobes from Rag2–/– mmouse fetuses were used. Two weeks after reconstitution,recipient mice and wild-type B6 control mice were infected intravenously with2,000 plaque-forming units of LCMV. Then, 8 d after infection, splenocyteswere isolated and the cytolytic activity of ESC-derived T cells was determinedby 51Cr-release assay. EL4 target cells were coated with the LCMV glycoproteinpeptide gp33 or a control adenovirus-derived peptide at a concentration of 1 µM and were labeled with 51Cr for 1.5 h. After being washed, 1 × 104 targetcells were mixed with ESC-derived T cells from reconstituted Rag2–/– mice atratios of 90:1, 30:1, 10:1 and 3:1 in 96-well round-bottomed plates. Cells wereincubated for 5 h and then supernatants were analyzed for 51Cr release associ-ated with cytolytic activity.

Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTSWe thank P. Poussier for critical review of the manuscript and G. Knowles forassistance in cell sorting. Supported by Canadian Institutes of Health Research(R.F.d.P. and J.C.Z.-P.), the National Cancer Institute of Canada and the CanadianCancer Society.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 15 December 2003; accepted 27 January 2004Published online at http://www.nature.com/natureimmunology/

1. Doetschman, T.C., Eistetter, H., Katz, M., Schmidt, W. & Kemler, R. The in vitro devel-opment of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac,blood islands and myocardium. J. Embryol. Exp. Morphol. 87, 27–45 (1985).

2. Kennedy, M. et al. A common precursor for primitive erythropoiesis and definitivehaematopoiesis. Nature 386, 488–493 (1997).

3. Nakano, T., Kodama, H. & Honjo, T. Generation of lymphohematopoietic cells fromembryonic stem cells in culture. Science 265, 1098–1101 (1994).

4. Nakano, T. Lymphohematopoietic development from embryonic stem cells in vitro.Semin. Immunol. 7, 197–203 (1995).

5. Cho, S.K. et al. Functional characterization of B lymphocytes generated in vitro fromembryonic stem cells. Proc. Natl. Acad. Sci. USA 96, 9797–9802 (1999).

6. Keller, G.M. In vitro differentiation of embryonic stem cells. Curr. Opin. Cell Biol. 7,862–869 (1995).

7. Kaufman, M.H., Robertson, E.J., Handyside, A.H. & Evans, M.J. Establishment ofpluripotential cell lines from haploid mouse embryos. J. Embryol. Exp. Morphol. 73,249–261 (1983).

8. Orkin, S.H. & Morrison, S.J. Stem-cell competition. Nature 418, 25–27 (2002).9. de Pooter, R.F., Cho, S.K., Carlyle, J.R. & Zúñiga-Pflücker, J.C. In vitro generation of

T lymphocytes from embryonic stem cell-derived prehematopoietic progenitors.Blood 102, 1649–1653 (2003).

10. Schmitt, T.M. & Zúñiga-Pflücker, J.C. Induction of T cell development fromhematopoietic progenitor cells by delta-like-1 in vitro. Immunity 17, 749–756 (2002).

11. Pui, J.C. et al. Notch1 expression in early lymphopoiesis influences B versus T lin-eage determination. Immunity 11, 299–308 (1999).

12. Radtke, F. et al. Deficient T cell fate specification in mice with an induced inactiva-tion of Notch1. Immunity 10, 547–558 (1999).

13. Wolfer, A., Wilson, A., Nemir, M., MacDonald, H.R. & Radtke, F. Inactivation ofNotch1 impairs VDJb rearrangement and allows pre-TCR-independent survival ofearly ab lineage thymocytes. Immunity 16, 869–879 (2002).

14. Washburn, T. et al. Notch activity influences the ab versus gd T cell lineage deci-sion. Cell 88, 833–843 (1997).

15. Robey, E. et al. An activated form of Notch influences the choice between CD4 andCD8 T cell lineages. Cell 87, 483–492 (1996).

16. Izon, D.J. et al. Notch1 regulates maturation of CD4+ and CD8+ thymocytes by mod-ulating TCR signal strength. Immunity 14, 253–264 (2001).

17. Fowlkes, B.J. & Robey, E.A. A reassessment of the effect of activated Notch1 onCD4 and CD8 T cell development. J. Immunol. 169, 1817–1821 (2002).

18. Deftos, M.L., Huang, E., Ojala, E.W., Forbush, K.A. & Bevan, M.J. Notch1 signaling pro-motes the maturation of CD4 and CD8 SP thymocytes. Immunity 13, 73–84 (2000).

19. Engel, I., Johns, C., Bain, G., Rivera, R.R. & Murre, C. Early thymocyte development isregulated by modulation of E2A protein activity. J. Exp. Med. 194, 733–745 (2001).

20. Barndt, R.J., Dai, M. & Zhuang, Y. Functions of E2A-HEB heterodimers in T-celldevelopment revealed by a dominant negative mutation of HEB. Mol. Cell. Biol. 20,6677–6685 (2000).

21. Scott, E.W., Simon, M.C., Anastasi, J. & Singh, H. Requirement of transcription fac-tor PU.1 in the development of multiple hematopoietic lineages. Science 265,1573–1577 (1994).

22. Ting, C.N., Olson, M.C., Barton, K.P. & Leiden, J.M. Transcription factor GATA3 isrequired for development of the T-cell lineage. Nature 384, 474–478 (1996).

23. Williams, R.L. et al. Myeloid leukaemia inhibitory factor maintains the developmen-

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

urei

mm

unol

ogy

A RT I C L E S

NATURE IMMUNOLOGY VOLUME 5 NUMBER 4 APRIL 2004 417

tal potential of embryonic stem cells. Nature 336, 684–687 (1988).24. Shortman, K. & Wu, L. Early T lymphocyte progenitors. Annu. Rev. Immunol. 14,

29–47 (1996).25. Rothenberg, E.V., Telfer, J.C. & Anderson, M.K. Transcriptional regulation of lym-

phocyte lineage commitment. Bioessays 21, 726–742 (1999).26. Anderson, M.K., Weiss, A.H., Hernandez-Hoyos, G., Dionne, C.J. & Rothenberg, E.V.

Constitutive expression of PU.1 in fetal hematopoietic progenitors blocks T celldevelopment at the pro-T cell stage. Immunity 16, 285–296 (2002).

27. Zhuang, Y., Soriano, P. & Weintraub, H. The helix-loop-helix gene E2A is required forB cell formation. Cell 79, 875–884 (1994).

28. Bain, G. et al. E2A proteins are required for proper B cell development and initiationof immunoglobulin gene rearrangements. Cell 79, 885–892 (1994).

29. Peschon, J.J. et al. Early lymphocyte expansion is severely impaired in interleukin 7receptor-deficient mice. J. Exp. Med. 180, 1955–1960 (1994).

30. Mombaerts, P. et al. RAG-1-deficient mice have no mature B and T lymphocytes.Cell 68, 869–877 (1992).

31. Tomita, K. et al. The bHLH gene Hes1 is essential for expansion of early T cell pre-cursors. Genes Dev. 13, 1203–1210 (1999).

32. Anderson, M.K. et al. Definition of regulatory network elements for T cell developmentby perturbation analysis with PU.1 and GATA3. Dev. Biol. 246, 103–121 (2002).

33. Kyba, M., Perlingeiro, R.C. & Daley, G.Q. HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoieticprogenitors. Cell 109, 29–37 (2002).

34. Robertson, E.J. Derivation and maintenance of embryonic stem cell cultures.Methods Mol. Biol. 75, 173–184 (1997).

35. Cho, S.K. & Zúñiga-Pflücker, J.C. Development of lymphoid lineages from embry-onic stem cells in vitro. Methods Enzymol. 365, 158–169 (2003).

36. Rodewald, H.-R., Kretzschmar, K., Takeda, S., Hohl, C. & Dessing, M. Identificationof pro-thymocytes in murine fetal blood: T lineage commitment can precede thymuscolonization. EMBO J. 13, 4229–4240 (1994).

37. Carlyle, J.R. & Zúñiga-Pflücker, J.C. Requirement for the thymus in ab T lymphocytelineage commitment. Immunity 9, 187–197 (1998).

38. Godfrey, D.I., Kennedy, J., Suda, T. & Zlotnik, A. A developmental pathway involvingfour phenotypically and functionally distinct subsets of CD3–CD4–CD8– triple nega-tive adult mouse thymocytes defined by CD44 and CD25 expression. J. Immunol.150, 4244–4252 (1993).

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