Immunogenicity and Engraftment of Mouse Embryonic Stem Cells in Allogeneic Recip - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:01 |只看该作者 |倒序浏览 |打印

作者：Sabrina Bondea, Nicholas Zavazavaa,b作者单位：aRoy J. and Lucille A. Carver College of Medicine, University of Iowa and Department of Veterans Affairs Medical Center;
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      【摘要】
   Embryonic stem cells (ESCs) are pluripotent and therefore able to differentiate both in vitro and in vivo into specialized tissues under appropriate conditions, a property that could be exploited for cellular therapies. However, the immunological nature of these cells in vivo has not been well understood. In vitro, mouse-derived ESCs fail to stimulate T cells, but they abrogate ongoing alloresponses by a process that requires cell-cell contact. We further show that despite a high expression of the NKG2D ligand retinoic acid early inducible-1 by mouse ESCs, they remain resistant to natural killer cell lysis. In vivo, allogeneic mouse ESCs populate the thymus, spleen, and liver of sublethally irradiated allogeneic host mice, inducing apoptosis to T cells and establishing multilineage mixed chimerism that significantly inhibits alloresponses to donor major histocompatibility complex antigens. Immunohistochemical imaging revealed a significant percentage of ESC-derived cells in the splenic marginal zones, but not in the follicles. Taken together, the data presented here reveal that nondifferentiated mouse embryonic stem cells are non-immunogenic and appear to populate lymphoid tissues in vivo, leading to T-cell deletion by apoptosis. # E4 Y( O1 o) x. |0 G# m& U3 x' m
      【关键词】 Embryonic stem cells Engraftment Apoptosis Mixed chimerism Retinoic acid early inducible-
   INTRODUCTION
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ESCs are pluripotent, a unique property that makes them ideal candidates for the treatment of degenerative diseases in humans. In general, ESCs are generated by harvesting blastocysts and cultivating them in vitro. The inner cell mass outgrows into a mixture of ESCs and trophoblast cell debris . Both techniques are clearly more ethically acceptable than the conventional method of extracting ESCs from blastocysts and would take out an important argument out of the current debate. However, additional studies are required to determine the pluripotency of these cells.

Typical phenotypic characteristics of ESCs include stage-specific-embryonic antigen (SSEA) expression, Oct-3/4, and Nanog-1, which are transcription factors required for ESC self-renewal. SSEA-1 is defined in mice by a monoclonal antibody .

Data on preimplantation stage stem cells . The mechanism by which these cells become immunogenic in vivo is unclear. It is possible that under inflammation, such as during a myocardial infarction as seen in the two studies cited above, inflammatory cytokines may stimulate ESCs to upregulate MHC antigens. Here, we further studied the susceptibility of ESCs to alloreactive cytotoxic T cell (CTL) and natural killer (NK) lysis and show that these cells show limited susceptibility to killing by either cell type.
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EXPERIMENTAL PROCEDURES5 b/ M2 }; B' {0 {

Cell Lines and Mice

The mouse 129/SvJ RW-4 and the 129SvJ R1 (both H-2b) cell lines were originally described by Hug et al. . The culture medium was changed every day, and the cells were passaged every 2 or 3 days to avoid overgrowth and differentiation.( N2 |; r* O  L

Phenotyping of ESCs by Flow Cytometry and by Immunohistochemistry
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An important characteristic of mouse ESCs is their low expression of MHC class I and lack of expression of MHC class II antigens. ESCs possess the property of quickly differentiating in culture. Thus, to ensure that nondifferentiated cells were used in our studies, ESC expression of SSEA-1 and MHC expression were measured before each experiment. The SSEA-1 antibody was purchased from the University of Iowa Hybridoma Facility, and the MHC antibodies were purchased from BD Pharmingen (San Diego, http://www.bdbiosciences.com/pharmingen). Only cultures with >80% SSEA-1 and 2 z0 z, w5 j7 F2 _

To determine alkaline phosphatase expression, the AEC kit (DakoCytomation, Carpinteria, CA, http://www.dakocytomation.com) was used according to the manufacturer's instructions. Upon oxidation, AEC forms a red end product. Oct-4 expression was determined by the use of the anti-Oct-4 antibody, purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, http://www.scbt.com), using the BCIP/NBT stain (DakoCytomation) as instructed by the manufacturer.& I# y% ]; }& ?1 U" ?
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Transplantation of ESCs

We previously infused rat ESCs in rats via the portal vein . This is technically challenging and accompanied with a high mortality and bleeding complication rate. Thus, to establish an easier and more reliable technique for the infusion of ESCs, we established the i.v. infusion of mouse ESCs via the supraorbital vein. Cell numbers of 106, 2 x 106, and 5 x 106, of all three cell lines, were infused in 6¨C8-week-old MRL mice (n = 10), respectively. To enhance ESC engraftment, recipient mice were sublethally irradiated (550 cGy) 24 hours prior to ESC infusion. Multilineage mixed chimerism was studied by flow cytometry. Venous blood was drawn from the supraorbital vein and stained with fluorescein-conjugated antibodies against CD3, CD19, and CD16/32 (granulocytes and monocytes). All antibodies were purchased from BD Pharmingen. The cells were washed and further incubated by a lysis buffer containing ammonium chloride to lyse the erythrocytes. After 10 minutes, the cells were washed in PBS, and cell fluorescence was measured using a flow cytometer (Becton, Dickinson and Company, Mountain View, CA, http://www.bd.com). The animals were either left untreated, irradiated with 500 cGy prior ESC infusion, or injected 15 mg/kg cyclosporine A once at the time of ESC infusion (n = 10 in each group).0 ?: F8 Y3 e3 K' N' ~; l' h& d
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ESC Susceptibility to NK Killing

To determine mouse ESC susceptibility to NK cells, ESCs were used as target cells in a 4-hour 51Cr-release assay. The cells were labeled with 51Na2CrO4 (GE Healthcare, Little Chalfont, Buckinghamshire, U.K., http://www.gehealthcare.com/) and incubated with splenocytes separated from MRL mice. ESCs were incubated with NK cells at a serial dilution of target:effector cell ratios. 51Cr release was measured in a gamma counter. To activate NK cells in vivo before use, MRL mice were infused 200 µl of tilorone dihydrochloride, 95% (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) 24 hours before the NK assay. This reagent strongly induces interferon- (IFN-) production, thus enhancing NK activity.

ESC Susceptibility to CTL Killing
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To determine whether mouse ESCs are susceptible to CTL killing, cytotoxic T cells against 129/SvJ were generated according to the standard protocol, described in this laboratory previously . After 2¨C3 restimulations, the CTL were used as effector cells against ESCs. Positive target cell controls were Con A-derived blast cells from the two donor strains C57BL/6 and 129/SvJ. Third-party controls were BALB/c (H-2d)-derived Con A blast cells. We hypothesized that lack of killing of the ESCs by CTL could be due to low MHC expression by ESCs. Therefore, ESCs were treated with 100 µg of recombinant IFN- 24 hours prior to the cytotoxicity assay to boost MHC expression and then used as target cells as well. Syngeneic and BALB/c-derived third party Con A blast cells were used as controls.

Mixed Lymphocyte Cultures
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Mixed lymphocyte cultures were performed as we have previously described in great detail .
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Detection of Donor-Derived Cells and Apoptosis in Peripheral Organs' m: M+ W/ h+ z5 h2 B# F5 T
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Chimeric animals were sacrificed on day 7 post-ESC infusion, and the thymus, spleen, and liver were harvested (n = 6). The organs were cut in half; one-half was frozen for immunohistochemistry and the other half was minced and a free suspension of lymphocytes prepared by Ficoll gradient separation. The cells were stained with the anti-H-2b antibody and measured by flow cytometry to determine the percentage of donor-derived cells. Frozen sections of the thymus, liver, and spleen were prepared and stained with the anti-H-2b antibody or with an anti-CD3 antibody to determine donor cells or T cells, respectively. To determine apoptotic cells in the spleen, thymus, and liver, frozen sections of chimeric animals were stained using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay (Trevigen, Inc., Gaithersburg, MD, http://www.trevigen.com) according to the manufacturer's instructions. Control tissue was also stained by the TUNEL assay to determine background apoptosis. To determine the involvement of Fas/FasL in apoptosis induction, Con A blast cells were co-incubated with ESCs. Apoptosis was measured using the TUNEL assay. Furthermore, to determine the involvement of the Fas/FasL system, apoptosis was neutralized by an anti-FasL antibody (BD Biosciences, San Diego, http://www.bdbiosciences.com) in a concentration-dependent manner.

Immunomodulation of alloresponses in chimeric animals was determined using the interleukin 2 (IL-2) enzyme-linked immunospot assay (BD Biosciences) as instructed by the manufacturer. Statistical analysis was performed using the Graphpad-Prism software package, version 3.0 (Graphpad Software, San Diego, http://www.graphpad.com/ecommerce/buynow.htm).

Karyotyping/ b4 i2 T1 R/ v/ T; `1 _

Karyotyping is now well-established and was performed according to standard procedure. The cells were preplated on slides, lysed, fixed, and stained with the Giemsa stain. The number of chromosomes was counted under a light microscope.5 X8 L3 o( L& M5 B
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RESULTS  J6 I, a6 G# ?( |9 v$ c
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Characterization of Mouse Embryonic Stem Cells1 C4 A, y/ Y9 @4 W0 {

Three mouse ESC lines, 129SvJ R1, 129SvJ RW-4, and C57BL/6, were adapted to grow in feeder cell-free medium to avoid contamination with inactivated mouse fibroblast cells traditionally used as feeder cells. The medium was based on the protocol by Keller et al. , ESCs express high levels of FasL (Fig. 1C). To determine whether ESC-derived lineage-specific cells continue to express FasL, ESCs were differentiated into beating cardiomyocytes and stained for FasL. Indeed, cardiomyocytes were positive, although to a lower degree than that by ESCs. Expression of SSEA-1 was measured by flow cytometry (Fig. 1E), whereas alkaline phosphatase was determined by immunhistochemistry (Fig. 1F). Finally, the karyotype of the ESCs was determined to be diploid, as expected (Fig. 1G). Over 80% of the culture was diploid, with a few cells showing tetraploidy or chromosomal aberrations of fewer chromosomes than expected.
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Figure 1. Characterization of ESCs. (A): ESCs grow as clusters in vitro in feeder-cell-free medium. Characteristically, ESCs express Oct-4 (B) and FasL (C). Interestingly, ESC-derived cardiomyocytes were shown to express low levels of FasL (D). Stage-specific-embryonic antigen-1 expression was measured by flow cytometry (E), and alkaline phosphatase was measured by immunohistochemistry (F). On karyotyping, ESCs were found to be predominantly diploid (G). Magnification, x200., P" g& X& C9 b1 u$ ?

Mouse ESCs Show Limited Susceptibility to NK and Alloreactive CTL Killing& ?! {8 q( y$ _5 |1 O
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Previous results from this laboratory and colleagues suggested that preimplantation stage stem cells are immune-privileged and could be used to induce stable mixed chimerism in allogeneic recipients without immunosuppression . These RAE-1 molecules are predominantly expressed in embryonic tissues and certain tumors, but hardly in adult healthy tissues. We therefore wondered whether ESCs express RAE-1 proteins, making them susceptible to NK cells. Indeed, flow cytometric analysis of ESCs showed high expression of RAE-1 but a lack of expression on splenocytes (Fig. 2A).% Z1 ?" i- }8 o

Figure 2. ESCs express RAE-1, but lack major histocompatibility complex (MHC) antigens. (A): ESCs were stained by anti-class I and anti-class II antibodies, respectively. MHC class I expression was negative in 129SvJ RW-4 ESCs, shown as the broken line overlay on the gray-shaded isotype control. After stimulation with IFN-, ESCs strongly upregulated MHC class I expression (open histogram), whereas this stimulation failed to upregulate MHC class II expression. As expected, splenocytes express class I, including a subpopulation that expresses class II antigens. Interestingly, ESCs, but not splenocytes, express RAE-1 molecules (open histogram). (B): To determine ESC susceptibility to NK cells, MRL-derived splenocytes were used as effector cells against ESC target cells in a 4-hour 51chromium release assay. Cytolysis of ESCs was nondetectable. However, when the MRL mice had been pretreated with an IFN- inducer to activate NK cells, ESCs were modestly lysed in a concentration-dependent manner. Yac-1 target cells were lysed by both activated and nonactivated NK cells. (C): In vitro generated alloreactive CTL against H-2b were used as effector cells against ESCs, IFN--activated ESCs, or 129SvJ-derived Con A blast cells. Unstimulated ESCs were hardly lysed, but were modestly lysed after IFN- stimulation, indicating that ESCs are susceptible to alloreactive T-cell killing after upregulation of MHC class I antigens by IFN- stimulation. The 129SvJ-derived, but not third party Balb/c-derived Con A blast cells, were lysed as expected. (D): ESCs immunogenicity was determined by a mixed lymphocyte reaction against MRL responder cells. As expected, 129SvJ splenocytes strongly stimulated MRL responder cells. ESCs used as stimulator cells failed to stimulate T-cell response and abrogated an ongoing response to 129SvJ stimulator cells. Self-MRL-stimulator cells were used as controls. (E): To determine whether abrogation of the alloreactive T-cell response requires cell-cell contact between the ESCs and T cells, transwells were set up. As expected from the experiment shown in (D), the alloresponse to splenocyte stimulator cells was normal, but it was completely abrogated by addition of ESCs. Empty TWs used as controls over the mixed lymphocyte cultures had no effect, nor did TWs containing ESCs. Thus, cell-cell contact is required for abrogation of T-cell responses by ESCs. Abbreviations: ESC, embryonic stem cell; FITC, fluorescein isothiocyanate; IFN-, interferon-'3b NK, natural killer; RAE, retinoic acid early inducible; TW, transwell.2 v9 v, p+ j, u' r9 N7 U0 K% z; c
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Based on these data, lack of class I and high expression of RAE-1, we determined whether ESCs are susceptible to NK-cell killing, using the standard cytotoxicity test. Splenocytes from C57BL/6 and MRL mice, respectively, were used in a 4-hour 51Cr-release assay as effector cells against 129SvJ RW-4 ESC target cells. ESCs were not lysed (Fig. 2B). In addition, expression of nonclassical MHC class I molecules (Qa-2) was ruled out by flow cytometry, as a possible inhibitor of NK killing. We surmised that ESCs might, however, be sensitive to activated NK cells. Mice were therefore treated with the IFN- inducer tilorone, intraperitoneally, 24 hours prior to the NK assay. This reagent, augments NK killing in rodents. ESCs were now more susceptible to NK killing (Fig. 2B), although to a much lesser extent than the Yac-1 target cells. The poor susceptibility of ESCs to NK killing despite high expression of Rae-1 allows speculation on whether ESCs induced apoptosis to NK cells based on their high expression of FasL.4 U' r( {- y8 C" R" b

Finally, to determine whether mouse ESCs are susceptible to CTL killing, anti-H-2b CTL were generated by coculturing irradiated 129SvJ splenocytes (H-2b), with MRL splenocytes (H-2k) as responders. Con A blast cells of the 129SvJ or third-party BALB/c-derived splenocytes and the 129SvJ RW-4 ESCs were used as target cells in a 4-hour 51Cr release assay. The 129SvJ Con A blast cells were lysed as expected (Fig. 2C), but not the BALB/c-derived Con A blast cells. Interestingly, the 129SvJRW-4 ESCs were not lysed by the CTL, presumably due to a lack of MHC expression. Thus, ESCs were stimulated by IFN- to upregulate class I expression. Stimulated ESCs were more susceptible to CTL killing than nonstimulated ESCs, suggesting that under stimulation or inflammatory conditions, as might be the case after allograft transplantation, ESCs could be rejected." I2 N; o& I7 W; S# h; y& @

The immunogenicity of ESCs was further investigated in vitro using mixed lymphocyte reaction assays. Irradiated 129SvJ RW-4 ESCs or 129SvJ-derived splenocytes were used as stimulator cells and MRL-derived splenocytes as responder cells in a 72-hour mixed lymphocyte reaction. Splenocyte-stimulated responder cells responded strongly; however, ESCs failed to induce T-cell proliferation (Fig. 2D). To determine whether ESCs abrogate an ongoing allostimulation, ESCs were added to a mixture of MRL and irradiated 129SvJ splenocytes. The alloresponse was completely abrogated, suggesting a strong downregulatory capacity of alloresponses by ESCs (Fig. 2D). This effect was shown to require cell-cell contact since transwell cultures, where ESCs were cultivated in the upper chambers above mixed lymphocyte cultures, had no effect on T-cell proliferation (Fig. 2E). Although transforming growth factor-ß has been shown to be secreted by ESCs and could potentially downregulate T-cell proliferation, these experiments appear to suggest that soluble molecules may not be involved, but that there is a requirement for cell-cell contact, which agrees with recent data in a xenogeneic model of human embryonic stem cells .% l% |" A& J2 f$ x4 `. r1 @2 k6 d5 i
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ESCs Induce Mixed Chimerism in Allogeneic Recipients! C  v+ k, W2 n. @$ n

It has been established that the homing of hematopoietic stem cells to the liver and the degree of established engraftment is ideal after intraportal infusion of hematopoietic stem cells rather than after i.v. infusion .# x9 L3 D! z' B! `, P9 C- ^
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Figure 3. Embryonic stem cells (ESCs) induce multilineage mixed chimerism after sublethal irradiation. (A, B): 2 x 106 ESCs were infused intravenously in MRL recipient mice, and mixed chimerism was monitored in peripheral blood by flow cytometry. The amount of donor cells detected in nontreated mice was approximately 2%¨C5% (A). Donor cells were lymphoid, and no donor myeloid cells were detected in these animals. In contrast, mixed chimerism was over 20%¨C30% in sublethally irradiated mice (B) and was multilineage. Representative profiles of donor-derived CD3 , CD19  B, and CD16/32  cells are shown 14 days after ESC infusion. The cells were doubly stained with an anti-donor major histocompatibility complex class I antibody. Abbreviation: FITC, fluorescein isothiocyanate.& X$ O, K9 e. d  a" |
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ESCs were only infused after prior measurement of SSEA-1 and MHC class I and II antigens to ensure that the cells were nondifferentiated. However, another explanation my be the fact that once we determined lack of establishment of mixed chimerism, the animals were euthanized. The time period for tumor formation might have been too short. Our interest was not to study teratoma formation, but to establish whether mixed chimerism can be established without aggressive host immunosuppressive treatment. Of interest is the observation that animals monitored to over 100 days with less than 2% mixed chimerism did not develop teratomas either within that observation period (n = 10).

ESCs Populate the Thymus, Spleen, and Liver After Intravenous Infusion in Allogeneic Recipients
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To track mouse ESC-derived cells in lymphoid organs, recipient animals were sublethally irradiated to create "space," and ESCs were infused. Chimeric animals were sacrificed 14 days after ESC infusion to track ESC progenitors. For histological studies, frozen sections of the thymus, liver, and spleen were stained for donor MHC class I antigens (H-2b), whereas minced samples were analyzed by flow cytometry after Ficoll gradient separation. A high proportion of donor-derived cells were identified in the splenic marginal zones (Fig. 4A). These cells were confirmed by the use of an antibody that stains marginal zone macrophages (MOMA; BD Biosciences; data not shown). Flow cytometric analysis of splenocytes indicated 11.7% donor-derived cells (Fig. 4B). Interestingly, ESC-derived cells in the thymus were more uniformly distributed within the organ (Fig. 4B). Only 3.0% of thymic cells were donor-derived (Fig. 4D). However, in the liver, most donor-derived cells were detected around the large blood vessels (Fig. 4E) and had a high proportion of approximately 25% of donor cells as detected by flow cytometry (Fig. 4F). Tissues from nonchimeric animals were used at all times as controls and remained negative. The immunohistochemical stains in Figure 4 were all based on donor class I staining. We therefore wondered whether more definitive proof could be attained by a double stain strategy. Here, enhanced yellow-fluorescent protein (E-YFP)-transduced ESCs were infused in recipient mice, and the animals were sacrificed after 14 days for use in staining for both class I and class II antigens. Our data indicate that we obtained both class I and class II staining cells which were E-YFP-positive (Fig. 4G). Thus, clearly these were donor-derived cells that had differentiated in thymic tissue into class I- and class II-expressing cells.
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Figure 4. Population pattern of embryonic stem cell (ESC)-derived cells in the spleen, thymus, and liver of recipient mice. To detect ESC-derived cells in lymphoid organs and the liver, chimeric animals were sacrificed on day 14; the thymus, spleen, and liver were harvested and shock-frozen; and sections were stained for donor H-2b antigen. Characteristically, donor cells were located in the perifollicular T-cell region of the spleens (A), which had 11.7% donor cells as determined by flow cytometry (B). In the thymus, however, ESC-derived cells were more homogenously distributed in the organ (C) and constituted only 3.0% of the total hematopoietic cell population (D). In the liver, donor cells were detected around the large blood vessels (E), constituting 25.1% of all cells (F). Thus, these results reveal different homing patterns of ESCs in these organs, suggesting a possible influence of the microenvironment in each organ. Control sections were negative for donor H-2b. Magnification, x200. To confirm that the class I-expressing cells represented viable donor-derived cells, enhanced yellow fluorescent protein (E-YFP)-transduced ESCs (green) were infused, and thymic histological sections stained for either class I or class II molecules 14 days later. In both cases, the E-YFP positive cells overlapped with the MHC stains, confirming that these cells were ESC-derived and differentiated into hematopoietic cells (G).. Z8 X% j6 S/ G
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To further verify this homing pattern, E-YFP-transduced 129SvJ R1 ESCs (a kind gift by Dr. Nagy, Toronto, ON, Canada) were infused in recipient mice and studied by multiphoton microscopy. The animals were anaesthetized and the spleen exposed to allow examination using the Bio-Rad Radience 2100 MP confocal/multiphoton microscope (Bio-Rad, Hercules, CA, http://www.bio-rad.com). Together with the ESCs, beads (Fluoresbrite Carboxy BB 1.75-micron microspheres; Polysciences Inc., Warrington, PA, http://www.polysciences.com) were infused, to allow tissue contrast. ESCs located around the follicles, with hardly any cells visible in the splenic medulla (data not shown). Thus, the unique compartmentalization of ESC-derived cells in the splenic marginal zones could additionally be confirmed using intravital imaging.
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ESCs Induce Apoptosis in T Cells of Lymphoid Organs, Regulating Alloresponses- _. @3 {# E4 Q' f

The high expression of FasL by ESCs is an interesting phenotypic characteristic. We determined whether ESCs induce apoptosis to T cells in lymphoid organs, protecting themselves from T-cell-mediated rejection. Frozen sections obtained 14 days after ESC infusion in irradiated allogeneic MRL mice were stained for apoptotic cells using the TUNEL assay. In the spleens, apoptosis was prominent in the T-cell region of the marginal zones (Fig. 5A), following the pattern of distribution of ESC-derived cells detected in Figure 4 above. This finding suggested that ESC-induced apoptosis to T cells, as confirmed by costaining with an anti-CD3 antibody (Fig. 5C). Similarly, the thymus was stained for apoptotic cells (Fig. 5B) and then costained for T cells (Fig. 5D, 5E). The inset of Figure 5E, at higher magnification, clearly indicates that the apoptotic cells are doubly stained for CD3. These results suggested that ESCs may delete activated alloreactive T cells. Splenic histologic sections from control irradiated, but nonchimeric animals showed low levels of apoptosis, as expected (Fig. 5F, 5G). These control experiments are summarized in Figure 5H, which indicates that the number of apoptotic cells in thymuses of chimeric animals 14 days after ESC infusion was clearly much higher than in control animals. To further determine whether the apoptotic cells were a result of the FasL expression by ESCs, we coincubated Con A blast cells with ESCs and measured the number of apoptotic cells using the TUNEL assay. At a ratio as low as 1:100 (ESCs:blast cells), a high proportion of the cells was apoptotic (Fig. 5I). Apoptosis was blocked by an anti-FasL antibody, clearly confirming that the FasL expressed by ESCs was a powerful apoptosis inducer.) [- N! K; ~+ \$ c

Figure 5. ESCs induce apoptosis in T cells in vivo. (A): For the detection of apoptosi, in situ, frozen sections of the spleen and thymus were stained for apoptosis using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, and apoptotic cells were visualized either by immunohistochemistry or by immune fluorescence. In the spleen, apoptotic cells were detected in the marginal zones of the follicles (A), whereas they were homogenously distributed in the thymus (B), as stained by 3,3'-diaminobenzidine tetrahydrochloride immunohistochemistry. These results were further confirmed by double staining for apoptosis and CD3 to determine whether T cells were apoptotic. Apoptotic cells are shown in red, and the CD3  T cells are shown in green in both the spleen (C) and thymus (D). Inset shows the double stained apoptotic thymic cells at higher magnification (x600) (E). Control sections showed no apoptosis in the spleen (F) and very little in the thymus (G). This result for the thymus is set up as a bar graph (H) to show that the number of background apoptotic cells in the thymus was relatively small. Con A blast cells were co-incubated with ESCs, and the cell mixture stained for apoptotic cells using the TUNEL assay. High numbers of apoptotic cells were measured by the TUNEL assay, which was neutralized by a neutralizing anti-FasL antibody in a concentration-dependent manner (I). This experiment confirmed that the ESCs-induced apoptosis was Fas/FasL-mediated. Magnification, x400 unless otherwise indicated. Abbreviation: ESC, embryonic stem cell.

Finally, to determine the extent of immunomodulation in chimeric animals, we used the ELISPOT to measure IL-2 secretion after T-cell stimulation with alloantigen. Splenocytes derived from the chimeric animals and nonchimeric controls were used as responder cells to irradiated 129SvJ-derived splenocytes. Cells derived from control nonchimeric animals showed strong release of IL-2 as compared with that in splenocytes from chimeric animals that was significantly abrogated (p ' [9 w2 ?( b* [, `( q

Figure 6. The modulation of the alloresponse by embryonic stem cell-induced mixed chimerism was tested by an interleukin 2 (IL-2) enzyme-linked immunospot assay. Splenocytes derived from chimeric animals showed significant reduction in the amount of IL-2 released on stimulation by donor-type irradiated splenocytes (A). Response to third-party alloantigen was the same in both chimeric and nonchimeric animals. (B): Summary of results in graph form.
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DISCUSSION

ESCs are unique because of their pluripotency and low immunogenicity. Here, we adapted mouse-derived ESCs to grow in feeder-cell-free medium using the modified protocol previously published by Keller et al. . Exposure to human serum containing antibodies against Neu5Gc led to antibody binding and complement activation leading to cell death, a scenario that potentially could occur in vivo, compromising the engraftment of ESCs during clinical treatment.
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Consistent with previously published data by us and others, ESCs express low MHC class I and no class II MHC antigens . Furthermore, ESCs blocked alloresponses in vitro by a process that required cell-cell contact as detected in transwell experiments.
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The results so far therefore suggested that ESCs could be protected from rejection in vivo. Subsequently, we tested whether ESCs engraft in naïve allogeneic mice, without any preconditioning. Indeed, the ESCs engrafted differentiating into lymphoid, but not myeloid cells at a low percentage of donor-derived cells, less than 5%. In contrast, after sublethal irradiation of recipient mice, ESC-derived cells were abundantly detected in the major lymphoid organs, spleen, thymus, and liver. In the spleen, ESC-derived cells were detected in the splenic marginal zones, in contrast with the pattern of donor cells in recipient mice after bone marrow transplantation, which were uniformly distributed within the spleen (not shown). The presence of ESC-derived cells in the marginal zones was further confirmed by intravital imaging, which indicated ESCs homing in the splenic subcapsular region, in particular around the follicles. The multiphoton microscopy allows tracking cells in vivo in real time. In both the thymus and liver, ESC-derived cells were more homogenously distributed in the tissues. The presence of ESC-derived cells in the thymus is significant, since the thymus is the site of T-cell education and maturation. Furthermore, the identification of ESC-induced apoptotic T cells in the thymus, spleen, and liver suggests possible clonal deletion of alloreactive T cells. This is confirmed by our observations that engraftment of ESCs in thymectomized mice was not successful (data not shown).0 l8 ?( z6 n  E' A

Our recently published data indicate that ESCs fail to engraft in Fas-deficient mice, suggesting that the constitutively expressed FasL by ESCs contributes to their ability to engraft in allogeneic recipients . Sufficient numbers of cells engrafted to correct factor IX deficiency in mice. These findings show the enormous potential for the application of ESCs in cellular transplants, although there may be tissue-specific differences, such as those observed in the induction of hematopoietic mixed chimerism.% P, S6 j0 `( i$ q6 o" C* v0 E

DISCLOSURES

The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS
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This work was made possible by Grant R01 HLO73015 (NIH/NHLBI), a Department of Veterans Affairs Merit Review Award, a grant from the Roche Organ Transplantation Research Foundation, and Grant Award 0455585Z from the American Heart Association. We are indebted to Dr. Nagy (Toronto, ON, Canada) for the E-YFP-transduced embryonic stem cells.
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