T lineage differentiation from humanembryonic stem cellsZoran Galic*†‡, Scott G. Kitchen*†, Amelia Kacena*, Aparna Subramanian*†, Bryan Burke†§, Ruth Cortado*,and Jerome A. Zack*†‡§¶

Departments of *Medicine and §Microbiology, Immunology, and Molecular Genetics, †UCLA AIDS Institute, and ‡Institute for Stem Cell Biology andMedicine, David Geffen School of Medicine, 11-934 Factor Building, 650 Charles Young Drive South, University of California, Los Angeles, CA 90095-1678

Communicated by Owen N. Witte, University of California, Los Angeles, CA, May 26, 2006 (received for review February 10, 2006)

Harnessing the ability of genetically manipulated human embry-onic stem cells (hESC) to differentiate into appropriate lineagescould revolutionize medical practice. These cells have the theoret-ical potential to develop into all mature cell types; however, theactual ability to develop into all hematopoietic lineages has notbeen demonstrated. Using sequential in vitro coculture on murinebone marrow stromal cells, and engraftment into human thymictissues in immunodeficient mice, we demonstrate that hESC candifferentiate through the T lymphoid lineage. Stable transgeneexpression was maintained at high levels throughout differentia-tion, suggesting that genetically manipulated hESC hold potentialto treat several T cell disorders.

SCID-hu mouse � T cell development � gene therapy � immunereconstitution � hematopoiesis

Human embryonic stem cells (hESC) show promise to rev-olutionize treatment strategies for many diseases because of

their potential to differentiate into all tissues and cell types in thebody. However, signals required for proper directed differenti-ation of these cells are not well defined. Regarding hematopoi-etic differentiation, these cells have been directed toward my-eloid and erythroid lineages by exposure to murine bone marrowstromal cells (1) or by induced formation of embryoid bodies inthe presence of various cytokines (2), both conditions resultingin differentiation into CD34� hematopoietic progenitor cellscapable of forming hematopoietic colonies in vitro. Similarly, Blineage differentiation has been achieved by sequential exposureof hESC to two different murine bone marrow stromal cell lines,OP9 and MS-5 (3). Recently, functional dendritic cells (4),natural killer cells (5), and macrophages (6) were derived fromhESC. Although T cells were successfully derived from murineembryonic stem (ES) cells (7), the differentiation toward the Tlymphoid lineage from hESC has not been reported. Herein weuse a combination of in vitro exposure to the OP9 stromal cellline, followed by implantation of the newly differentiated pre-cursors into human thymic tissues growing in immunodeficientmice, to induce T lymphoid differentiation of these cells in vivo.Costimulation of the resulting T lineage cells by CD3 and CD28resulted in expression of activation markers, indicating that thesecells are functional. Furthermore, a lentiviral vector expressingEGFP under the control of the elongation factor 1� (EF1�)promoter, introduced at the hESC stage, continued to expressthe reporter gene at high frequency throughout thymopoiesis.Our results suggest that genetically manipulated hESC may holdpromise for treatment of disorders of the T cell lineage.

ResultsTo obtain genetically marked ES cells, the hESC line H1 (8, 9)was transduced with the pSIN18.cPPT.hEF1�.EGFP.WPRElentiviral vector, which allows constitutive expression of theEGFP reporter gene under the control of the EF1� promoter inhESC (9). To obtain colonies expressing EGFP at high fre-quency, �20 passages of manually isolating GFP� cells wereperformed (Fig. 1b). However, these cells continued to express

markers of ES cells, including Oct-4, alkaline phosphatase (Fig.1 c and f ), and SSEA-4 (not shown), for many more passages andmaintained a normal karyotype past passage 120 (not shown),indicating that they remained undifferentiated. Aliquots of thesecells, passaged up to 40 times after transduction, were placed onthe OP9 murine bone marrow stromal cell line to direct differ-entiation toward hematopoietic progenitor cells. Before cultureon the stromal cells, the ES cells expressed markers of pluripo-tent stem cells (Fig. 1) and did not express hematopoietic lineagemarkers (Fig. 2a). As described in ref. 3, over time a subset ofthese transduced ES cells began expressing markers character-istic of hematopoietic cells, including CD34, CD133, and CD117,while retaining expression of EGFP (Fig. 2 a and b). Interest-ingly, only minimal expression of CD45 was observed. After 7–14days of culture on OP9, these hESC gained the ability to formmyeloid and erythroid colonies in methylcellulose in responseto erythropoietin, stem cell factor, granulocyte–macrophagecolony-stimulating factor, and IL-3. Unsorted EGFP� cellsformed colonies at a frequency of �0.5% (data not shown). Theresulting colonies retained the ability to express EGFP (Fig.2c). These results establish that hESC, differentiated towardhematopoietic lineages after coculture with OP9 cells, maintainthe expression of transgenes under the control of the EF1�promoter.

To provide a microenvironment for T lymphoid differentia-tion, in separate experiments 1 and 2, we used the SCID-hu(Thy�Liv) mouse, a chimeric model that is constructed byinsertion of small pieces of human fetal liver and thymus underthe renal capsule of severe combined immunodeficient (SCID)mice (10, 11). In this model, the human fetal liver provideshematopoietic stem cells and the thymus fragments providestromal elements necessary for T lymphoid differentiation in theconjoint Thy�Liv organ. We and others have shown previouslythat direct injection of exogenous CD34� human hematopoieticprogenitor cells into Thy�Liv implants in sublethally irradiatedSCID-hu mice results in engraftment and T lymphoid differen-tiation of the exogenous cells (12, 13). EGFP-transduced H1 cellswere cultured for 7–14 days on OP9 cells in these two indepen-dent experiments. Differentiated cells were selected for expres-sion of CD34 or CD133 in the absence of CD34 (CD133��CD34�; see Fig. 3a). Some of these cells were assessed forcolony-forming potential, the others were directly injected intoThy�Liv implants in SCID-hu mice previously sublethally irra-diated (300 rads) to partially deplete endogenous thymocytesand fetal liver-derived progenitor cells. Enriched CD34� cellsformed hematopoietic colonies at a frequency of �1%, whereasCD133� cells did not form colonies in these studies (not shown).

Three to 5 weeks after injection of hESC-derived cells intoThy�Liv implants, multicolor flow cytometric analysis was used

Conflict of interest statement: No conflicts declared.

Abbreviations: EF1�, elongation factor 1�; TCR, T cell antigen receptor.

¶To whom correspondence should be addressed. E-mail: jzack@ucla.edu.

© 2006 by The National Academy of Sciences of the USA

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to identify human (CD45�) cells that expressed EGFP. EGFP�

human cells were identified in Thy�Liv implants in animalsreceiving either CD34� or CD133� cells (Fig. 3 b and c).Engraftment was seen in 11 of 32 SCID animals and ranged from0.1% to 6.2% of human cells (Table 1). Whereas the frequencyof engraftment was low, no engraftment was seen in animals notpreviously irradiated (not shown). Analysis for CD3, CD4, andCD8 expression demonstrated that the EGFP-expressing cellscould be found in various stages of thymocyte maturation,including immature CD4��CD8� cells and mature CD4��CD8�

and CD8��CD4� cells. Successive biopsies of Thy�Liv implantsfrom SCID-hu animals established that engraftment was stablefor at least 5 weeks (Fig. 3c). We determined the frequency ofT lineage engraftment of hESC cocultured for various times onOP9 cells. Although our data set is limited, it appears that underthese conditions, 10 days of coculture on OP9 cells yielded thebest engraftment frequency (50%; Table 2).

In experiment 3, we attempted to improve engraftment byusing human Thy�Liv implants in RAG 2�/� mice, which are lesssensitive to irradiation than SCID mice. This mouse system thusallows a higher dose of irradiation (a total of 900 rads, delivered

by two 450-rad exposures, 6 h apart) to deplete endogenousthymocytes and fetal liver-derived progenitor cells before en-graftment of hESC. We reasoned that this treatment might allowa greater degree of reconstitution by ES-derived cells, becausethere would likely be less competition by renewed replicationand differentiation of endogenous cells present in the Thy�Livimplants. ES cells were cocultured on OP9 feeder cells for 10days, based on the initial studies shown above, and similarlysorted ES-derived progenitors were injected into these RAG-huanimals. These animals were also injected with 16-fold morehESC-derived CD34� cells than in experiments 1 and 2. Weagain saw engraftment of these cells, with a total of six of nineanimals containing EGFP-expressing cells (Table 2). TheseRAG-hu mice had generally smaller implants than did theSCID-hu mice, likely because of the higher irradiation dose.However, the overall percentage of human cells that expressedEGFP in these implants was considerably higher than inSCID-hu mice (Table 1; an example of this finding can also beseen in Fig. 4b). These results suggest that the modificationsmade in experiment 3 (increased irradiation and higher numbersof CD34 cells injected) together allowed for a greater percent of

Fig. 1. Genetic modification of undifferentiated hESC. The hESC line H1 was transduced with the EGFP expression lentiviral vectorpSIN18.cPPT.hEF1�.EGFP.WPRE and selected for homogenously green colonies. Upper and Lower illustrate the same colonies in each column. (a and b)Phase-contrast (a) and fluorescent (b) images of an EGFP-labeled H1 colony at passage 71. (c, e, and f ) The undifferentiated phenotype and transgene expressionwere maintained as shown by the expression of the early stem cell markers Oct-4 (c) and alkaline phosphatase ( f), as well as EGFP (e), at passage 122. (d) DAPIstain.

Fig. 2. In vitro differentiation of hESC toward hematopoietic lineage. (a) Changes in hematopoietic marker expression after in vitro culture of EGFP� H1 cellson OP9 cells. Cells were analyzed by flow cytometry and gated on EGFP� cells (which typically ranged between 70% and 90% of total live cells). Expression ofeach marker was determined at the indicated times. (b) Flow cytometric analysis of hematopoietic marker expression in the CD34� cell population (typically �5%of total live cells). (c) hESC-derived hematopoietic progenitor cells give rise to erythroid (Upper) and myeloid (Lower) colonies that express EGFP, as viewed byphase-contrast (Left) and fluorescent (Right) microscopy after 10 days of differentiation on OP9 cells and 14 days of growth in methylcellulose containingerythropoietin, stem cell factor, granulocyte–macrophage colony-stimulating factor, and IL-3.

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reconstitution by ES-derived precursor cells in the RAG-humice.

Additional phenotypic studies were performed on cells fromexperiments 2 and 3. Specifically, we noted that in comparisonto the endogenous fetal thymocytes, CD45 expression was dimon the majority of ES-derived thymocytes expressing EGFP (Fig.

4). In experiment 2, the fetal thymus used to generate theThy�Liv implant was derived from a donor who did not expressHLA-A2. Because H1 hESC express HLA-A2, thymocytesderived from these cells could be distinguished from endogenousfetal thymocytes on the basis of HLA-A2 staining (Fig. 4a).Levels of HLA-A2 on ES-derived CD45dim thymocytes weresimilar to those on HLA-A2� thymocytes derived from normalSCID-hu mice generated from HLA-A2� fetal donors (notshown) and were �10-fold higher than on HLA-A2� fetalliver-derived thymocytes within the same tissue (Fig. 4a). Inanimals in experiment 3, we assessed the expression of additionalthymopoietic markers on these cells. We found levels of CD1a,T cell antigen receptor (TCR), CD3, CD7, and CD127 similar to

Fig. 3. In vivo T lymphoid differentiation of H1-derived progenitor cellsexpressing EGFP. (a) Schematic representation of differentiation protocol. (band c) Flow cytometry profiles of cells derived from irradiated SCID-hu Thy�Livmice 3 weeks (b) (denoted week 3) and 5 weeks (c) (denoted week 5) aftertransplantation with no cells (Mock) (Upper Left), sorted CD34� progenitorcells (Upper Center), or sorted CD133� progenitor cells (Upper Right) thatwere derived from HI cells previously cultured on OP9 cells for 11 days(denoted by d11). SCID-hu mice were biopsied on two successive occasions,and thymocytes were analyzed for expression of EGFP, CD45, CD3, CD4, andCD8 by multicolor flow cytometry. Cells were analyzed by gating on live cells(forward vs. side scatter), then gating on CD45� to identify human hemato-poietic cells (not shown). (Upper) Live, CD45� cells were then analyzed for CD3and EGFP. Numbers in each corner represent the percentage of cells in eachquadrant, and the gate drawn through the upper and lower right quadrantsdenotes EGFP� cells, with the percentage of cells given within the gate.(Lower) CD4 vs. CD8 profiles of the EGFP� population, determined by gating.

Table 1. Engraftment of EGFP� cells in SCID-hu and RAG-huThy�Liv implants

Cells

Time incoculture,

days

% EGFP�

Week 3 Week 4 Week 5 Week 6

Experiment 1CD133 10 NT 0.44 NT NTCD133 10 NT 0.42 NT 5.6CD133 10 2.6 NT 0.16 NTCD34 10 2 NT 5.8 NTCD34 10 1 NT NT NTCD34 14 4 NT 0.6 NT

Experiment 2CD133 7 0.33 NT 6.2 NTCD133 11 0.1 1.8 2.89 NTCD133 11 NT 3.45 NT NTCD34 7 NT 0.47 NT NTCD34 11 0.14 NT 0.71 NT

Experiment 3CD133 10 NT 24.4 NT NTCD133 10 NT NT 23.1 NTCD133 10 NT NT 18.6 NTCD34 10 NT 3.1 NT NTCD34 10 NT NT 15.6 NTCD34 10 NT NT 8.7 NT

Thy�Liv implants in SCID-hu (experiments 1 and 2) and RAG-hu (experiment3) mice received direct injections of progenitor cells derived from EGFP-labeled H1 cells cocultured on OP9 cells for various times. Progenitor cells wereenriched for expression of CD34 or for CD133��CD34� phenotype beforeinjection as indicated. Implants were assessed for engraftment at the timesindicated by flow cytometric analysis first by gating on human CD45 andsubsequently by quantitating the percentage of CD45��GFP� cells. NT, nottested.

Table 2. Engraftment frequency of mice to which hESC-derivedCD34� or CD133� CD34� cells had been transferred

Cells Day 7 Day 10 Day 11 Day 14

Experiment 1CD133� NT 3�5 NT 0�3CD34� NT 2�5 NT 1�3

Experiment 2CD133� 1�3 NT 2�7 NTCD34� 1�4 NT 1�2 NT

Experiment 3CD133� NT 3�4 NT NTCD34� NT 3�5 NT NT

Two experiments with SCID-hu mice (experiments 1 and 2) and one exper-iment with RAG-hu mice (experiment 3) are shown. Those mice found positivefor engraftment were determined to have CD45�, EGFP� cells at the indicatedtime points. NT, not tested.

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those expressed on normal thymocytes (Fig. 4b). By gating onCD45dim HLA-2� cells in these implants that received ES-derived progenitor cells, we could further assess the relativepercentage of these cells that maintained the ability to expressthe EGFP transgene during thymopoiesis. In three animalsassessed in experiment 2, EGFP expression was 92%, 95%, and100% of ES-derived cells (Fig. 5 and data not shown). Thustransgene expression is maintained at high efficiency throughthymopoiesis.

We also determined whether the T lineage cells arising fromhESC were functional and able to respond to TCR-mediatedsignals. Thy�Liv cells from two animals in experiment 2 were

cultured ex vivo in control medium or under costimulatingconditions (plate-bound anti-CD3 plus soluble anti-CD28). Wehave shown previously that these costimulating conditions in-duce the expression of the high-affinity IL-2 receptor (CD25) onhuman thymocytes (14). As shown in Fig. 6, which illustratesresults from one of the two animals tested, costimulated hESC-derived EGFP� thymocytes similarly demonstrated dramaticincreases in CD25 expression in comparison with nonstimulatedcontrol cells cultured in parallel. Similar responses were seenwith cells obtained from the second animal tested (not shown).

Fig. 4. Phenotypic analysis of hESC-derived thymocytes. (a) To distinguish endogenous HLA-A2� and hESC-derived HLA-A2� cells, thymocytes from control mice(Left) and SCID-hu mice into which hESC-derived CD34� cells had been transferred (Right) were stained and gated based on the expression of CD45 and EGFP.MFI, mean fluorescence intensity. Cells within each gate were subsequently analyzed for the expression of HLA-A2. (b) CD45�, EGFP� cells from the Thy�Livimplants from the RAG-hu mice transferred with hESC-derived CD34� cells (Upper) and CD45� cells from the implants of the control animals (Lower) were gatedand analyzed for the presence of the indicated T cell surface markers. Region markers are based on isotype controls.

Fig. 5. hESC-derived thymocytes maintain expression of the reporter trans-gene in vivo. CD45dim HLA-A2� thymocytes from SCID-hu mice into whichhESC-derived CD34� cells had been transferred (Center) were analyzed for theexpression of EGFP (Right). (Left) Lack of gated population in control micereceiving no hESC-derived progenitors.

Fig. 6. Costimulation of EGFP� thymocytes after 5 weeks of differentiationin vivo. Cells were cultured in medium alone (unstimulated) or in the presenceof anti-CD3 and anti-CD28 monoclonal antibodies (costimulated), and EGFP�

cells were analyzed for expression of CD25 by flow cytometry.

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Thus the thymocytes resulting from transduced hESC are capa-ble of receiving TCR-mediated signals. Together with the phe-notypic data presented above, our studies indicate that normalhuman thymocytes developed from these transduced ES cells.

DiscussionOur results indicate that hESC can develop through the Tlymphoid lineage when provided an appropriate microenviron-ment for differentiation. We observed very low levels of thehematopoietic marker CD45 on the ES-derived precursors thatgenerated these thymocytes in the human thymic implants.However, it has previously been reported that hematopoieticprogenitors in early mouse and human embryos lack CD45expression (15, 16) and that CD45� precursors can differentiateinto various hematopoietic lineages in both human and mouseES systems in vitro (17, 18). Interestingly, hESC-derived thymo-cytes did express CD45; however, the levels of this marker werelower than those found on typical thymocytes derived from fetalliver progenitors in the SCID-hu mouse. This CD45 expressionmay reflect the embryonic rather than fetal origin of these cells.However, virtually every other T-lineage marker assessed wassimilar on thymocytes derived from ES cells and fetal liverprogenitors. In addition, ES-derived thymocytes responded nor-mally to costimulation, suggesting that they are functional.

The availability of SCID-hu mice constructed from HLA-A2�

donors allowed us to discriminate HLA-A2� ES-derived cellsfrom endogenous human thymocytes. This discrimination fur-ther allowed us to determine the relative level of ES-derivedthymocytes that retained the ability to express the EGFP trans-gene while undergoing differentiation in the human thymus.Importantly, we found that transgene expression was generallyhigh, with �90% of ES-derived cells expressing detectable levelsof EGFP under the control of the EF1� promoter. This findingsuggests that genetic manipulations of these cells are feasible.With further optimization to increase efficiency and�or incor-porate inducible promoters, this system could prove useful inproviding a ready source of primary human T lymphocytesexpressing genes of interest for basic mechanistic studies. Sim-ilarly, human T cells expressing inhibitory RNAs could begenerated without manipulation of the T cell itself, to provide a‘‘knockdown’’ phenotype. This type of strategy could prove quiteuseful, for example, for mechanistic studies of quiescent T cellsor of T cell activation, because stimulation or perturbation of theT cell to introduce the vector would not be required. Thus,effects of the transgenes could be accurately assessed becauseresponses of these cells to experimental stimuli would be oth-erwise similar to those of true quiescent cells.

Further studies are required to define the exact molecularsignals needed to induce the appropriate hematopoietic differ-entiation pathway. However, these results have important im-plications for hematopoietic stem cell gene therapy approaches.Genetically manipulated hESC with well characterized vectorintegration sites could be expanded to large numbers. If thesemanipulated cells were intended to be used to therapeuticallytarget the T lineage, our results suggest that they need only bedifferentiated as far as hematopoietic progenitor cells in vitroand that the thymus would then complete the differentiationprogram to CD4� and CD8� T cells in vivo. This type ofapproach might lead to improved therapeutic strategies to treatgenetic hematopoietic disorders, such as X-linked SCID (19)or infectious diseases such as acquired immunodeficiencysyndrome (20).

Materials and MethodsCell Culture. The hESC line H1 was cultured on mouse embryonicfibroblast feeders in D-MEM�F12 medium containing 20%serum replacer (Invitrogen), 2 mM L-glutamine, a 100 �Mconcentration of each nonessential amino acid, and 8 ng�ml basic

fibroblast growth factor. The cells were passaged on a weeklybasis, and passages 88, 90, and 108 were used for the differen-tiation studies. All work with hESC was approved by theUniversity of California Los Angeles Institutional ReviewBoard. The mouse bone marrow stromal cell line OP9 (providedby Owen Witte, Howard Hughes Medical Institute, University ofCalifornia, Los Angeles) was maintained in �-modified mini-mum essential medium (�-MEM) containing 20% FBS.

Differentiation Protocol. For the differentiation studies, a protocolfrom Vodyanik et al. (3) was adapted. Briefly, gelatinized six-wellplates were seeded with 2 � 104 OP9 cells per well and grown for5 days in OP9 medium, with the medium change on day 4.Undifferentiated H1-GFP hESC were harvested with collage-nase IV (1 mg�ml) and dispersed into small clumps by scrapingand pipetting. The resulting clumps were added to the OP9layers, and the OP9�H1 cocultures were maintained for 5–14days in �-modified minimum essential medium supplementedwith 10% non-heat-inactivated defined FBS (HyClone) and 100�M monothioglycerol (Sigma), with half-medium changes everysecond day. To obtain a single-cell suspension, the cultures weretreated with collagenase IV for 20 min at 37°C, followed bytreatment with trypsin�EDTA (0.05%) for 15 min at 37°C. Thecell clumps were further disrupted by pipetting, and the resultingsuspension was washed twice and filtered through a 70-�mstrainer. The cells were then moved into T162 flasks (Corning)and incubated at 37°C for 1 h to allow OP9 cells to adhere toenrich for hESC-derived cells. This step removes �90% of OP9cells. Nonadhered cells were collected and used for flow-basedphenotypic analysis and further purification steps.

Virus Production. The virus was produced in 293T cells by cotrans-fection of three plasmids: (i) pSIN18.cPPT.hEF1�.EGFP.WPRE,a vector expressing EGFP under control of the EF1� promoter (9),(a gift from M. Gropp and B. E. Reubinoff, Hadassah UniversityHospital, Jerusalem, Israel), (ii) the vesicular stomatitis virus G(VSVG) expression plasmid pHCMVG, and (iii) the packagingplasmid pCMV�R8.2DVPR. In our protocol, 5 � 106 293FThuman fibroblasts, plated on a 100-mm2 dish the previous night,were transfected with 5 �g of pSIN18.cPPT.hEF1�.EGFP.WPRE,5 �g of pCMV�R8.2DVPR, and 2 �g of pHCMVG, by usingLipofectamine 2000 (Invitrogen) according to the manufacturer’sinstructions. Culture supernatants that contained virus were col-lected on days 2 and 3, centrifuged to remove floating cells, passedthrough 0.45-�m filters, and subjected to centrifugation at 17,000rpm for 90 min at 4°C in an L8-M ultracentrifuge (Beckman) toconcentrate the virus. The viral pellets were resuspended in 1� PBSovernight at 4°C. Concentrated virus was titrated on the 293Tfibroblast cell line.

Transduction of H1 hESC. At the time of infection, undifferentiatedH1 colonies were mechanically disrupted into smaller clumpsand mixed with the virus in 200-�l final volume consisting of PBSand HES medium (1:1 ratio) in the presence of 4 �g�mlPolybrene (Sigma). The mixture was incubated for 2 h at 37°Cwith constant shaking, then washed and plated on fresh mousefibroblast feeders. Newly grown colonies consisting of bothtransduced and nontransduced cells were analyzed under UVmicroscopy, and GFP-positive regions were selectively excisedand passaged. After several such passages, H1 colonies wereenriched for GFP expression to near-complete homogeneity.

Immunohistochemistry. H1-GFP cells were grown on coverslips ona feeder layer of mouse embryonic fibroblasts. For alkalinephosphatase (AP) staining, cells were first fixed in 0.2% form-aldehyde for 1 h at 37°C, then washed with 1� PBS. Staining forAP was performed with the Vector Red alkaline phosphatasesubstrate kit I (Vector Laboratories), following the manufac-

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turer’s instructions. For Oct-4 staining, cells were first fixed incold methanol for 15 min followed by permeabilization inPBS�0.1% Triton X-100 for 20 min at room temperature. Thecoverslips were blocked in 0.5% goat serum (Calbiochem) and0.3% BSA (Sigma) for 1 h followed by incubation with Oct-3�4antibody (Santa Cruz Biotechnology). Secondary staining wasperformed with goat anti-mouse IgG2b-phycoerythrin (SantaCruz Biotechnology). Isotype control antibody was purchasedfrom Serotec. The coverslips were mounted on a glass slide inVECTASHIELD mounting medium with DAPI (Vector Lab-oratories) and observed under bright and fluorescent fields.

In Vivo T Cell Development. SCID-hu mice were generated asdescribed in ref. 21. Briefly, small pieces of human fetal liver andthymus were inserted beneath the renal capsule of SCID miceand allowed to develop into a thymus-like organoid called aThy�Liv implant. RAG-2�/� mice were used in experiment 3instead of SCID mice. All immunodeficient mouse work wasapproved by the University of California Los Angeles AnimalResearch Committee. SCID-hu mice were irradiated with 300rads and injected with either 5 � 104 purified CD34� or 106

purified CD133� CD34� cells directly into Thy�Liv implants.RAG-hu mice were exposed to a total of 900 rads, given in twodoses of 450 rads administered 6 h apart, and were reconstitutedwith 2 � 106 mouse bone marrow cells. The implants of theseanimals were injected with either 8 � 105 purified CD34� or 106

purified CD133� CD34� hESC-derived cells. The Thy�Liv im-plant biopsies were performed at various time points as specifiedin the text. Single-cell suspensions were obtained, and the cellswere analyzed for the expression of EGFP and T cell develop-mental markers and used in the functional assay.

Cell Sorting and Flow Cytometry. H1-GFP hESC cultured in vitroand cells derived from Thy�Liv implants were stained withmonoclonal antibodies to CD3, CD4, CD8, CD19, CD25, CD34,

CD45, CD56, CD127 (Coulter); CD1a, CD7, CD117 (Bio-science, San Diego, CA); SSEA-4 (R & D Systems); HLA-A2(Serotec); TCR (Pharmingen); and CD133 (Miltenyi Biotec,Auburn, CA), conjugated to phycoerythrin-cyanin 7, electron-coupled dye, allophycocyanin, or PC7. Cells were analyzed forfluorochrome and EGFP expression by flow cytometry with aCoulter FC500 flow cytometer and FLOJO (Tree Star, Ashland,OR). At the indicated times after differentiation in vitro, H1-GFP hESC were sorted by magnetic cell sorting (MACS). Cellswere initially magnetically labeled with anti-CD34 microbeads(Miltenyi Biotec), and the positive fraction was collected aftertwo-column sorting (POSSEL�D2 program) on an AutoMACS cellsorter (Miltenyi Biotec). The CD34� fraction was then labeledwith anti-CD133 microbeads (Miltenyi Biotec), and the CD133�

fraction was collected after another AutoMACS sort (POSSEL�D2program). Typically, there was �85% enrichment for the re-spective CD34� and CD133� populations as determined bypostsort f low cytometry analysis.

Cell Stimulation. Cells derived from biopsied Thy�Liv implantswere cultured in RPMI medium 1640 containing penicillin (100units�ml), streptomycin (100 �g�ml) (Sigma), and 10% humanAB serum (Gemini Bioproducts, Sacramento, CA). A fraction ofthe cells were costimulated with anti-CD3 (1 �g�ml) (OrthoBiotech, Bridgewater, NJ) and cross-linked to the plate with goatanti-mouse IgG and soluble anti-CD28 (100 ng�ml) (Coulter).Cells were then removed after 3 days of costimulation andassessed for phenotype by flow cytometry for CD25 expression.

We thank Ken Dorshkind for critically reviewing this manuscript, Drs.M. Gropp and B. E. Reubinoff for providing us with thepSIN18.cPPT.hEF1�.EGFP.WPRE lentiviral vector, Greg Bristol andLianying Gao for technical help, and Dimitrios Vatakis for assistancewith preparation of this manuscript. This work was supported byNational Institutes of Health Grant AI036554.

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