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First published online June 5, 2008
Stem Cells Vol. 26 No. 8 August 2008, pp. 1939 -1950
doi:10.1634/stemcells.2008-0078; www.StemCells.com
© 2008 AlphaMed Press

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EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS

Embryonic Stem Cells and Their Differentiated Derivatives Have a Fragile Immune Privilege but Still Represent Novel Targets of Immune Attack

Douglas C. Wu, Ashleigh Susan Boyd, Kathryn J. Wood

Transplantation Research Immunology Group, Nuffield Department of Surgery, University of Oxford, Oxford, United Kingdom

Key Words. Cell transplantation • Embryonic stem cells • Immune escape • Immunobiology • Differentiation

Correspondence: Kathryn J. Wood, D.Phil., Transplantation Research Immunology Group, Nuffield Department of Surgery, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom. Telephone: 00-44-(0)-1865-222508; Fax: 00-44-(0)-1865-768876; e-mail: kathryn.wood{at}nds.ox.ac.uk

Received February 27, 2008; accepted for publication April 30, 2008.
First published online in STEM CELLS EXPRESS   June 5, 2008.


   ABSTRACT
Top
 Footnotes
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Embryonic stem cells (ESCs) offer an attractive potential in cell replacement therapy and regenerative medicine because of their inherent plasticity and ability to self-renew. However, the immunological response against transplanted ESC-derived allografts requires further evaluation. In this study, we showed that ESCs expressing the major histocompatibility complex class I molecule H2Kb escape immune recognition by H2Kb-reactive CD8+ T cells, irrespective of H2Kb expression levels. In the face of more robust immunological challenge, however, evidence of ESC allograft rejection becomes apparent. We further assessed the adaptive immune response against terminally differentiated insulin-producing tissue derived from an ESC source to examine the potential future applicability of this tissue as a β-cell replacement therapy for type 1 diabetes mellitus. The functional ESC-derived insulin-producing tissue was infiltrated by alloreactive T cells and rejected in immunocompetent hosts. Hence, although ESCs and their terminally differentiated derivatives may possess a fragile immune privilege, they still represent novel targets of attack by elements of the immune system and are rejected. These findings provide insight into the mechanisms of adaptive immunity toward ESCs and their derivatives.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Footnotes
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Mouse embryonic stem cells (ESCs) were first isolated in 1981 [1], followed 17 years later by the derivation of pluripotent human ESCs [2]. ESCs are derived from the inner cell mass of the preimplantation blastocyst and have the potential to generate all the tissues of the body. In addition to this remarkable plasticity, these unique cells are able to undergo a process termed self-renewal, allowing them to passage indefinitely in vitro under nondifferentiating culture conditions. Mouse ESCs can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF) [3, 4]. When implanted back into the mouse embryo, they are able to contribute to all the tissue types of the fetus [5]. Human ESCs have also shown this spontaneous ability to generate all three embryonic germ layers [6]. The maintenance of human ESC pluripotency, however, differs from that of mouse ESCs in that LIF does not appear to be the key factor. Although currently not completely defined, human ESC pluripotency seems to involve signaling pathways mediated by the transforming growth factor β family cytokines [7]. Because of their plasticity and inherent ability to self-renew, ESCs may have future potential in cell replacement therapy and regenerative medicine [8].

An obstacle that remains to be fully addressed before ESC technology can be applied to the clinic is the potential immune response that may be elicited by an ESC-derived allograft [9]. ESCs have been reported to express low levels of major histocompatibility complex (MHC) class I molecules and to be deficient in MHC class II expression [10]. As a result, their ability to provoke a proliferative response among naïve allogeneic T cells in vitro has been questioned, perpetuating the belief that they may enjoy some degree of immune privilege [11, 12]. In support of this hypothesis, Ménard et al. reported the engraftment and function without immunosuppression of a mouse ESC-derived population of cardiomyocytes into the infarcted hearts of sheep [13]. In addition, Drukker et al. used a humanized mouse model to assess the immunogenicity of human ESCs in vivo and found that their ability to provoke an allogeneic immune response was considerably weaker than that of normal adult allografts [14]. A recent study by Bonde et al. also suggested that mouse ESCs were nonimmunogenic when transplanted into allogeneic recipients [15]. On the other hand, studies have shown that when ESCs differentiate into embryoid bodies (EBs) in vitro or into teratomas in vivo, the level of MHC expression increases 4-fold and 10-fold, respectively [10]. Further evidence in vivo suggests that when ESCs are transplanted into mouse myocardium, they provoke an allogeneic immune response and are rejected [16, 17].

In this study, we used T cell receptor-transgenic (TCR-tg) T cells (BM3), which recognize the MHC class I molecule H2Kb via the direct pathway of allorecognition [18], to assess the adaptive immunological response against mouse ESCs that express the mouse MHC class I molecule H2Kb. Furthermore, we analyze the alloresponse against mouse ESCs that were terminally differentiated into insulin-producing cell clusters. We show that both undifferentiated mouse ESCs and their terminally differentiated derivatives appear to have reduced immunogenicity in vivo compared with H2Kb+ islet allografts but that both are ultimately susceptible to rejection by immunocompetent wild-type recipients.


   MATERIALS AND METHODS
Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Animals
CBA.Ca RAG-1 knockout (CBA-Rag–/–) mice were a generous gift of Dr. D. Kioussis (Mill Hill, London). BM3 TCR-transgenic mice (BM3; H2k) were provided by A.L. Mellor (Institute of Molecular Medicine and Genetics, Augusta, GA). BM3 mice were crossed onto a CBA-Rag–/– background for use in these studies. CBA.Ca (CBA; H2k) and C57BL/10 (B10; H2b) mice were originally purchased from Harlan, Blackthorn, Bicester, U.K. (http://www.harlan.com). All mice were bred and housed by Biomedical Services, John Radcliffe Hospital, in accordance with the Animals (Scientific Procedure) Act 1986 of the U.K. All experiments were performed with mice between ages 6 and 8 weeks at the time of first procedure.

ESC Lines
All ESC lines were a generous gift from Professor Sir Richard Gardner of the Department of Zoology, University of Oxford. ESF122 was derived from a CBA (H2k) background [19], ESF134 was derived from a C57BL/6 (H2b) background, and ESF166 was derived from a transgenic CBK (H2k+H2Kb) background. The ESC lines used in these studies are summarized in supplemental online Table 1.

Maintenance Culture of ESCs
ESCs (1 x 106) were plated on top of a feeder layer of primary embryonic fibroblasts (PEF) in a 25-ml culture flask in knockout Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, http://www.invitrogen.com) supplemented with 15% fetal calf serum (FCS; PAA Laboratories, Linz, Austria, http://www.paa.at), 1% 100 µM L-glutamine (PAA Laboratories), 1% 100 µM penicillin-streptomycin (PAA Laboratories), 1% nonessential amino acids (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 100 µM β-mercaptoethanol (Sigma-Aldrich), and 100 µl/10 ml medium 10 µg/ml LIF (Chemicon, Temecula, CA, http://www.chemicon.com). Upon reaching 80%–90% confluence, cells were passaged under maintenance culture conditions or used in further experimentation.

Interferon-{gamma} Culture Stimulation
ESCs (1 x 106) were harvested from 80%–90% confluent maintenance cultures and then plated onto a fresh feeder layer of PEF in a 25-ml culture flask. Standard maintenance medium was supplemented with 1,000 U/ml mouse recombinant interferon (IFN)-{gamma} (Peprotech, Rocky Hill, NJ, http://www.peprotech.com). Cells were cultured for 48 hours and then harvested for flow cytometric analysis.

In Vitro Differentiation Culture
ESCs (1 x 106) were harvested from 80%–90% confluent maintenance cultures and then plated into a fresh 25-ml culture flask in the absence of PEF feeder cells. Using standard maintenance medium, but without supplementation with LIF, cells were then cultured for 48 hours before flow cytometric analysis.

Directed Differentiation of ESCs into Insulin-Producing Cell Clusters
ESCs were differentiated into insulin-producing cell clusters (IPCCs) as previously described [20]. Briefly, undifferentiated ESCs were subjected to hanging drop culture for 48 hours to promote the formation of embryoid bodies. Embryoid bodies were then plated and cultivated for 3 days in maintenance medium lacking LIF before transfer onto 60-mm culture dishes coated with gelatin. After further expansion for 8 days, ESC clusters were then transferred to fresh 60 mm culture dishes coated with poly-L-ornithine (Sigma-Aldrich) and laminin (Sigma-Aldrich). Pancreatic differentiation was completed by an additional 19-day culture period in final-stage directed differentiation medium. This medium consisted of DMEM:Ham's F-12 medium, N2 supplement, B27 supplement, 10 mM nicotinamide, laminin (100 µl/100 ml; Sigma-Aldrich), L-glutamine, and insulin (200 µl/100 ml; Sigma-Aldrich) [21, 22]. End-stage IPCCs were either harvested for molecular characterization or transplanted into streptozocin (STZ)-induced diabetic mice.

Molecular Analysis of End-Stage IPCCs: Reverse Transcriptase-Polymerase Chain Reaction
Total cellular RNA was isolated from IPCCs using the Stratagene Absolutely RNA kit (Stratagene, La Jolla, CA, http://www.stratagene.com). Two micrograms of RNA, 2 µl of oligo DT, and a topping volume of deionized H2O (ddH2O) were added to thin-walled polyethylene polymerase chain reaction (PCR) tubes (Elkay Laboratory Products Ltd., Basingstoke, Hampshire, U.K., http://www.elkay-uk.co.uk). Following 10 minutes at 75°C, 15.5 µl of reaction master mix was added to the tubes on ice. The master mix consisted of 8 µl of 5x first-strand buffer, 4 µl of dithiothreitol, 2 µl of 10 mM dNTP, 1 µl of DNase (Ambion, Austin, TX, http://www.ambion.com), and 0.5 µl of RNasin (Promega, Madison, WI, http://www.promega.com). The tubes were then incubated for 30 minutes at 37°C followed by 5 minutes at 80°C. Finally, 1 µl of Moloney murine leukemia virus reverse transcriptase (RT; Invitrogen) and 1 µl of RNasin were added on ice, and the reaction proceeded at 42°C for 1 hour, 95°C for 5 minutes, and 4°C for 10 minutes. Total volume of the RT reaction was 40 µl. For the PCR, 1 µl of cDNA was amplified on a gradient cycler using gene-specific oligonucleotide primers for insulin-1, insulin-2, glucagon, and pancreatic amylase. The PCR master mix consisted of 0.3 µl of Taq polymerase (Bioline, London, http://www.bioline.com), 1 µl of 20 µM dNTP, 1.5 µl of MgCl2 (Bioline), 2.5 µl of 10x reaction buffer (Bioline), 1.55 µl of dimethyl sulfoxide, 15.15 µl of ddH2O, and 1 µl each of sense and antisense primers (10 µM stock; MWG Biotech, Ebersberg, Germany, http://www.mwg-biotech.com) for a total reaction volume of 25 µl (including the initial 1 µl of cDNA). PCR products were resolved via 2% agarose gel electrophoresis and visualized with 10 µg/ml ethidium bromide.

Immunofluorescence
Samples were collected and frozen in Tissue-Tek OCT Compound (Sakura Finetek, Torrance, CA, http://www.sakura.com) embedding medium prior to sectioning. Using a cryostat, embedded samples were sectioned at a thickness of 8–10 µm. The primary antibodies used were guinea pig anti-swine insulin (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com; 1:300 dilution) and rabbit anti-human c-peptide (Biogenesis Ltd., Kidlington, U.K., http://www.biogensis.co.uk; 1:150 dilution). All sections were fixed in 100% acetone and then blocked with 4% goat serum in phosphate-buffered saline (PBS) for 30 minutes at room temperature. After washing, primary antibody was applied for an overnight incubation at 4°C. The next day, the sections were washed, and secondary antibody was applied. Each secondary antibody was applied separately for 1 hour at room temperature. The secondary antibodies used were rabbit anti-guinea pig fluorescein isothiocyanate (FITC)-conjugated (DakoCytomation; 1:150 dilution) and goat anti-rabbit Alexa Fluor 594-conjugated (Molecular Probes, Eugene, OR, http://probes.invitrogen.com; 1:150 dilution). Following washing, specimens were mounted with Vectashield containing 4,6-diamidino-2-phenylindole nuclear counterstain (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) and analyzed under fluorescence microscopy using Openlab 4.0.3 (Improvision, Coventry, U.K., http://www.improvision.com).

Preparation of BM3-Rag–/– CD8+ T Cells
A single-cell suspension was made from spleens harvested from BM3-Rag–/– TCR-tg mice. A small aliquot of the total cell suspension was then taken for flow cytometric analysis, whereupon BM3 T cells were identified on the basis of the coexpression of CD8-allophycocyanin (APC) (BD Biosciences, San Diego, http://www.bdbiosciences.com) and transgenic TCR, as revealed by the monoclonal antibody Ti98-biotin. The Ti98 hybridoma was a generous gift of Andrew Mellor. Ti98 hybridoma was grown and the purified antibody biotinylated in the laboratory. The Ti98-biotin monoclonal antibody was developed with streptavidin-PerCP (BD Biosciences). Using this analysis, the proportion of BM3 T cells in total splenocytes was assessed, and total numbers of BM3 T cells for adoptive transfer was determined. As BM3 mice were bred on a Rag–/– background, no CD4+ T cells contaminated the cell preparations.

Carboxyfluorescein Succinimidyl Ester Labeling
Single-cell suspensions were incubated for 10 minutes at 37°C with 10 µM carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes), washed twice in ice-cold RPMI 1640 (Invitrogen), and resuspended in PBS ready for i.v. injection.

Adoptive Transfer of Cells
BM3 T cells (1 x 105, as enumerated by flow cytometric analysis) were intravenously injected either unlabeled or after CFSE labeling into CBA-Rag–/– mice. For alloantigen priming of BM3 T cells in vivo, 107 H2Kb+ splenocytes were injected intravenously 1 day after adoptive transfer of T cells. After 8 days to allow for alloantigen clearance, primed animals were either analyzed by flow cytometry or transplanted with ESCs.

Preparation of Purified Mature Dendritic Cells
Bone marrow was flushed from the femurs of C57BL/6 mice, and a single-cell suspension was plated onto 10-cm Petri dishes (BD Falcon 1029, BD, Oxford, U.K., http://www.bdeurope.com). Cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10% FCS (PAA Laboratories), 1% L-glutamine (PAA Laboratories), 1% penicillin-streptomycin (PAA Laboratories), β-mercaptoethanol (Sigma-Aldrich), and 20 ng/ml recombinant mouse granulocyte macrophage–colony-stimulating factor (GM-CSF; Peprotech). Cells that remained in suspension were cultivated for a further 8 days in the continuous presence of GM-CSF. Purity and maturity of these dendritic cells (DC) was then ascertained via flow cytometric analysis.

Islet Isolation
Donor mice were sacrificed by cervical dislocation. Liberase RI (0.25 mg/ml; Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) was then injected into the pancreas via the common bile duct. Following sufficient distension, the pancreas was retrieved and digested by 10-minute incubation at 37°C. Undigested pancreas was filtered through a nylon mesh, and the resultant digest was applied onto a discontinuous Ficoll gradient (1.108, 1.096, 1.037 g/l). Purified islets were then collected at the 1.096:1.037 interface and counted via dithizone staining.

Induction of Diabetes
CBA and CBA-Rag–/– mice were rendered diabetic by a single i.v. injection of 200 mg/kg STZ (Sigma-Aldrich). C57BL/10 mice were rendered diabetic by a single i.v. injection of 150 mg/kg STZ. Dosages of STZ were titrated to give a diabetic level of a roughly 20 mM blood glucose concentration at the time of transplantation.

Islet and Embryonic Stem Cell Transplantation
After sufficient anesthesia and analgesia was achieved, a 1.5-cm incision was made through the skin and peritoneum across the left flank of recipient mice. The left kidney was then extracorporealized, and 1 x 106 ESCs, 400 pancreatic islets, or 1,000 IPCCs were implanted beneath the kidney capsule. Muscle and skin layers were closed with a 4.0 Vicryl running suture. All animals were monitored closely during the postoperative period.

Immunohistochemistry
At the endpoint of the observation period, ESC grafts were removed and frozen in OCT embedding medium prior to cryostat sectioning at a thickness of 8–10 µm. After drying, sections were fixed in 100% acetone. All sections were then blocked with 10% bovine serum albumin at room temperature for 30 minutes. Primary antibodies rat anti-mouse CD8 (BD Biosciences), mouse anti-mouse H2Kb (BD Biosciences), rat anti-mouse CD4 (BD Biosciences), and rat anti-mouse CD11b (BD Biosciences) were then applied. Following several washes, horseradish peroxidase-conjugated rabbit anti-mouse secondary antibody (DakoCytomation) was applied, and sections were visualized with diaminobenzidine (Sigma-Aldrich).

Flow Cytometric Analysis
Single-cell suspensions were either harvested from culture flasks or generated from whole spleens. BM3 CD8+ T cells were identified on the basis of the coexpression of CD8-APC (BD Biosciences), TCRβ-phycoerythrin (PE) (BD Biosciences), CD3-FITC (BD Biosciences), and Ti98-biotin (developed with streptavidin-PerCP, BD Biosciences). When cells were labeled with CFSE, the CD3-FITC was omitted. CD44-PE (BD Biosciences) was used as a marker of antigen experience. ESC MHC expression was assessed with H2Kb-biotin (BD Biosciences; developed with streptavidin-APC, eBioscience) and polyclonal MHC class II-FITC (BD Biosciences). DC were characterized on the basis of the expression of CD11c-FITC (BD Biosciences), H2Kb-biotin (developed with streptavidin-APC), and polyclonal MHC class II-PE (BD Biosciences). All data were acquired using the BD FACSAria multicolor flow cytometer (BD Biosciences) and analyzed using BD FACSDiva software (BD Biosciences) and WinMDI, version 2.8 (The Scripps Research Institute, La Jolla, CA, http://www.scripps.edu).

Statistical Analysis
Statistical evaluations were performed using a one-way analysis of variance test for groups with unknown and potentially disparate variances.


   RESULTS
Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
ESCs Have Impaired MHC Expression Levels
Because ESCs have previously been reported to express very low levels of MHC molecules [10], we sought to characterize the expression of MHC class I and MHC class II molecules in the ESC lines used in our studies. ESF134 expressed low levels of H2Kb (2% expression above the isotype control, Fig. 1A) and no MHC class II (Fig. 1B). Upon allowing ESF134 to spontaneously differentiate in vitro for 48 hours via the removal of all sources of LIF from the culture conditions, MHC expression levels remained unchanged compared with cells maintained under nondifferentiating culture conditions (Fig. 1C, 1D). On the other hand, 48-hour stimulation of ESF134 with 1,000 U/ml of mouse recombinant IFN-{gamma} resulted in a dramatic upregulation of H2Kb (Fig. 1E). MHC class II expression remained low in the presence of IFN-{gamma} stimulation (Fig. 1F). We next characterized the MHC expression levels of ESF166, which expresses H2Kb ectopically under a transgene promoter. Undifferentiated and unstimulated ESF166 expressed high levels of H2Kb, similar to that found after IFN-{gamma} stimulation of ESF134 (Fig. 1G). Again, MHC class II expression was undetectable (Fig. 1H). In comparison, and as a positive control, splenocytes were isolated from C57BL/6 mice and assayed for both H2Kb and MHC class II expression. As expected, nearly all C57BL/6 splenocytes expressed the MHC class I molecule H2Kb, and a large percentage expressed the MHC class II molecules H2A (Fig. 1I, 1J).


Figure 1
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  		Figure 1. Characterization of the major histocompatibility complex (MHC) expression levels of embryonic stem cells. (A): ESF134 (H2b) expresses the MHC class I molecule H2Kb at only 2% above isotype control. (B): MHC class II (H2Ab) expression is undetectable above the level of staining by an isotype control. (C): After 48 hours of in vitro spontaneous differentiation, H2Kb expression by ESF134 remains unchanged compared with undifferentiated controls. The colored histogram represents the H2Kb expression by undifferentiated ESF134, whereas the overlay is the H2Kb expression after 48 hours spontaneous in vitro differentiation. No difference was detectable. (D): MHC class II (H2Ab) expression remained absent after 48 hours spontaneous in vitro differentiation. (E): After 48 hours of in vitro stimulation by 1,000 U/ml mouse recombinant interferon (IFN)-{gamma}, ESF134 exhibited a dramatic upregulation of H2Kb expression compared with the isotype control. (F): Despite IFN-{gamma} stimulation, expression of MHC class II remained absent. (G): Transgenic ESF166 expresses high levels of H2Kb in an undifferentiated and unstimulated state, similar to that of ESF134 after 48 hours of IFN-{gamma} stimulation. (H): MHC class II expression is also undetectable in transgenic ESF166. (I): Virtually all C57BL/6 splenocytes express H2Kb, in striking contrast to ESF134. (J): Similarly, a large percentage of C57BL/6 splenocytes express H2Ab MHC class II molecule. All overlays are in direct comparison with matched isotype controls (colored), except in (C) and (D) as noted above. Flow cytometric analysis was performed on three separate cultures for each experimental condition, and representative histograms are shown.

 
BM3 T Cells Do Not Recognize an Allogeneic ESC Graft
Although ESF134 exhibits deficient MHC expression under nondifferentiating culture conditions (Fig. 1), we hypothesized that transplantation in vivo would induce an upregulation of H2Kb and therefore contribute to the immunogenicity of the ESC graft as previously suggested [10]. Four days after transplantation of ESF134 into the renal subcapsular space of T and B cell-deficient CBA-Rag–/– (H2k) mice, H2Kb expression by the ESCs could be easily detected by immunoperoxidase staining (Fig. 2A). However, despite the dramatic upregulation of H2Kb, the allogeneic ESC graft did not elicit a proliferative response from naïve H2Kb-specific BM3 T cells above the background homeostatic proliferation observed when a syngeneic ESC graft was implanted (ESF-122 (H2k); Fig. 2B). Subsequent immunohistochemical analysis confirmed that the BM3 T cells were not activated and failed to infiltrate H2Kb+ ESC grafts (Fig. 2C). We next hypothesized that the initial low expression of H2Kb by ESF134 may contribute to its lack of immunogenicity. However, even when ESF166—which expressed constitutively higher levels of H2Kb (Fig. 1G)—was transplanted into the renal subcapsular space, no infiltrating BM3 T cells could be detected (Fig. 2D). In striking contrast, an H2Kb+ adult pancreatic islet graft transplanted beneath the renal subcapsular space began to be infiltrated by BM3 T cells by day 5 and was rejected by day 10 (Fig. 2E).


Figure 2
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  		Figure 2. Embryonic stem cells do not elicit an allogeneic response from BM3 T-cells. (A): In contrast to their basal undifferentiated state, ESF134 dramatically upregulates expression of H2Kb after 4 days of spontaneous in vivo differentiation, as revealed by H2Kb+ immunohistochemistry. (B): Despite upregulation of H2Kb, an ESF134 allograft does not provoke a proliferative response from BM3 H2Kb-reactive CD8+ TCR transgenic T cells in the spleen. The CFSE division profiling compares the response of BM3 T cells against a syngeneic ESF122 (H2k) graft and an allogeneic ESF134 (H2b) graft at two different time points (days 5 and 10) post-transplantation. Calculating the number of cells at each division and comparing across three replicates, no significant difference could be detected. BM3 T cells were gated on coexpression of transgenic TCR (as revealed by the monoclonal antibody Ti98), CD8, and TCRβ chain. Dot plots are representative of n = 3 animals analyzed at each time point. (C): BM3 T cells are incapable of recognizing and infiltrating ESF134 (H2b) allografts at either day 5 (top panel) or day 10 (bottom panel) time points post-transplantation, as revealed by lack of CD8+ staining by immunohistochemistry. Photomicrographs are representative of n = 3 grafts analyzed at each time point. (D): ESF166 (H2k+Kb) has constitutive expression of H2Kb (top panel) but still remains free of BM3 T-cell infiltration at day 10 (bottom panel). Photomicrographs are representative of n = 3 grafts analyzed at day 10. (E): An adult H2Kb+ islet allograft is infiltrated by day 5 (top panel) and obliterated by day 10 (bottom panel) by BM3 T cells, as revealed by CD8+ immunohistochemistry. Photomicrographs are representative of n = 2 grafts analyzed at each time point. All photomicrographs were taken with a Nikon Coolpix 995 digital camera (Nikon, Tokyo, http://www.nikon.com) attached to a Zeiss Axiovert 25 inverted microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com) using a x10 objective lens. Abbreviation: CFSE, carboxyfluorescein succinimidyl ester.

 
Passenger APC Potentiate the Immunogenicity of an ESC Graft
A unique feature of ESC-derived tissue grafts is their inherent lack of donor-derived APC, which, in the case of adult organ allografts, have the potential to migrate to secondary lymphoid organs and mediate a robust acute rejection response via direct presentation of alloantigen [23]. We therefore hypothesized that provision of immunostimulatory passenger APC would increase the immunogenicity of the ESC graft and allow for BM3 T-cell recognition of the MHC class I molecules they express. Simultaneous, but separated, transplantation of an H2Kb+ pancreatic islet graft along with ESF134 (H2b) into the renal subcapsular space (islets into the upper pole, ESF134 into the lower pole) allowed us to simulate the provision of donor-derived APC in a normal transplantation setting and to monitor the fates of both allografts concurrently in situ. Under these circumstances, BM3 T cells began to acquire the ability to infiltrate H2Kb+ ESC grafts (Fig. 3A, right panel). However, the degree of infiltration remained markedly less than that observed in the simultaneously transplanted H2b+ islet allograft (compare Fig. 3A, center and right panels). In control grafts, where syngeneic islets (H2k) rather than allogeneic islets were cotransplanted, the ESF134 ESC graft remained free of infiltration (Fig. 3A, left panel).


Figure 3
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  		Figure 3. Passenger APC potentiate the response of H2Kb-reactive BM3 CD8+ T cells against an embryonic stem cell allograft. (A): Left panel: When a syngeneic CBA (H2k) islet graft was transplanted into the upper pole of the renal subcapsular space, and ESF134 (H2b) into the lower pole of a T and B cell-deficient CBA.rag–/– (H2k) mouse that had been reconstituted with BM3 T cells, the embryonic stem (ES) cell allograft remained free of BM3 T-cell infiltration, as revealed by lack of CD8+ immunohistochemistry. Center panel: H2Kb+ islets were heavily infiltrated and destroyed by BM3 T cells. Right panel: Simultaneous transplantation of an H2Kb+ islet allograft into the upper pole of the renal subcapsular space and ESF134 into the lower pole allowed for BM3 T-cell infiltration into the ESC allograft, as revealed by CD8+ immunohistochemistry. Photomicrographs are representative of n = 3 grafts under each experimental condition at day 10 post-transplantation. (B): Flow cytometric analysis of BM3 T cells at day 10 post-islet/ESC cotransplantation revealed the priming effect of the cotransplanted islet allograft. BM3 T cells, gated on the coexpression of CD3, CD8, and transgenic TCR, were analyzed for CD44 expression, a marker of T-cell antigen experience and memory. Priming of BM3 T cells by the islet allograft resulted in a modest upregulation of CD44 (right panel). Syngeneic islet allografts did not induce upregulation of CD44 on BM3 T cells (left panel). Dot plots are representative of n = 3 animals analyzed under each experimental condition. (C): Purified matured DC derived from the bone marrow of C57BL/6 mice expressed high levels of both H2Kb major histocompatibility complex (MHC) class I (left panel) and H2Ab MHC class II (right panel) molecules. Colored histograms are isotype controls, with the overlay representing the expression levels of DC. (D): Cotransplantation of mature DC along with ESF134 (H2b) enabled BM3 T-cell infiltration at days 5 (left panel) and 10 (right panel), as revealed by CD8+ immunohistochemistry. Photomicrographs are representative of n = 3 grafts for each time point. (E): Baseline expression of CD44 by BM3 T cells (far left panel) was unchanged by the transplantation of mature DC alone (left center panel) or the transplantation of ESF134 (H2b) alone (right center panel) into the renal subcapsular space. Modest upregulation of CD44 was detectable only with the cotransplantation of both mature DC and an ESF134 allograft (far right panel). Dot plots are representative of n = 3 animals analyzed at day 10 post-transplantation. All photomicrographs were taken with a Nikon Coolpix 995 digital camera attached to a Zeiss Axiovert 25 inverted microscope using a x10 objective lens. Abbreviation: DC, dendritic cells.

 
Further flow cytometric analysis of BM3 T cells revealed a modest increase in activation state induced by the H2Kb+ islet allograft as measured by an increased CD44 expression compared with controls (Fig. 3B). Using a more direct approach, we cotransplanted a purified, matured population of DC derived from the bone marrow of C57BL/6 (H2b) mice along with ESF134 cells (H2b) into the same pole of the recipient kidney. These purified matured DC expressed high levels of both MHC class I (H2Kb) and MHC class II (H2Ab) molecules (Fig. 3C). With the addition of these passenger APC, the ESF134 graft began to be infiltrated by BM3 T cells by day 5, and the infiltration was increased by day 10 (Fig. 3D, left and right panels, respectively). Again, modest activation of BM3 T cells was apparent as a result of the cotransplantation of DC (Fig. 3E, right panel compared with the other three controls). The priming of naïve BM3 T cells by passenger APC either derived naturally from a normal adult allograft or added artificially along with the ESC allograft partially overcomes the apparent immune privilege of ESF134. Furthermore, the modest degree of priming and activation required to initiate a productive response against the ESF134 allograft indicates that the immune privilege of ESCs is fragile at best and is unlikely to remain in the face of robust immunological challenge.

Activated BM3 T Cells Destroy an H2KB+ ES Cell Graft
The above data suggested that the provision of additional signals, such as passenger APC stimulation, to BM3 T cells could enable them to recognize an ESC allograft. Another mechanism by which CD8+ T-cell responses may be facilitated is via the provision of so-called T-cell help mediated by CD4+ T cells. In many situations, CD8+ T cells require these helper signals to generate full activation and cytotoxic effector function. To further investigate the role of T-cell help in the generation of a productive BM3 response against an ESC allograft, we stimulated BM3 T cells with allogeneic splenocytes and then examined the ability of these activated T cells to recognize and respond to an ESC allograft (ES134 H2b). Alloantigen-primed BM3 T cells demonstrated a dramatic upregulation of CD44 expression compared with the level of CD44 expressed by naïve BM3 T cells (Fig. 4A). These antigen-experienced BM3 T cells mounted a vigorous rejection response against an ESF134 allograft, which was marked by heavy infiltration by day 8 (Fig. 4B) and complete obliteration of the graft by day 10 (Fig. 4C). Thus, the upregulation of MHC class I molecules by ESCs upon in vivo differentiation combined with the presence of effector CD8+ T cells specific for alloantigen is sufficient to induce the complete rejection of an ESC allograft.


Figure 4
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  		Figure 4. Preprimed CD8+ H2Kb-reactive BM3 T cells infiltrate and destroy an allogeneic embryonic stem cell graft. (A): Dramatic priming of BM3 T cells in vivo could be accomplished by the i.v. administration of H2Kb+ splenocytes 1 day after adoptive transfer of BM3 T cells into CBA-rag–/– recipients. Overwhelming upregulation of CD44 was evident by day 8 post-i.v. injection of splenocytes (bottom panel). Animals that did not receive i.v. splenocytes maintained baseline expression of CD44 by BM3 T cells (top panel). (B): Transplantation of ESF134 (H2b) into preprimed recipients resulted in heavy BM3 T-cell infiltration at day 8, as revealed by CD8+ immunohistochemistry (bottom panel). ESF134 transplanted under identical circumstances, except without prepriming, exhibited no graft infiltration by BM3 T cells (top panel). Photomicrographs are representative of n = 3 grafts analyzed under each experimental condition. (C): The ESF134 (H2b) allograft was completely rejected by day 10 by preprimed BM3 T cells such that it was no longer even visible (bottom panel). Control grafts exhibited typical teratoma formation (top panel). Photographs are representative of n = 3 grafts analyzed under each experimental condition. All photomicrographs were taken with a Nikon Coolpix 995 digital camera attached to a Zeiss Axiovert 25 inverted microscope using a x10 objective lens.

 
ESCs Are Rejected by Fully Allogeneic Wild-Type Recipients
Having established that ESC allografts can be rejected under the correct circumstances, we next tested their fate when transplanted into immunocompetent wild-type fully MHC-mismatched recipients (CBA; H2k). By day 5 post-transplantation, MHC-mismatched ESC grafts (ESF134; H2b) were infiltrated by CD11b+ macrophages (Fig. 5A, left panel). CD4+ T-cell infiltration could also be detected (Fig. 5A, center panel); CD8+ T-cell infiltration was absent (Fig. 5A, right panel). By day 10, CD4+ and CD8+ T cells had infiltrated the ESC allograft and macrophage infiltration persisted (Fig. 5B). In contrast, syngeneic ESC grafts (ESF122; H2k) were completely free of cellular infiltration at day 10 post-transplantation (Fig. 5C). Taken together, the data from our studies indicate that although ESCs may have lower immunostimulatory potential compared with normal adult allografts such as islet allografts, ultimately they do not escape immune recognition and the ensuing rejection following transplantation into immunocompetent recipients.


Figure 5
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  		Figure 5. Allogeneic embryonic stem (ES) cell grafts are recognized and infiltrated when transplanted into fully major histocompatibility complex-mismatched wild-type recipients. Allogeneic (ESF134; H2b) or syngeneic (ESF122; H2k), cells were transplanted under the kidney capsule of immunocompetent fully allogeneic CBA (H2k) recipient mice. Kidneys were removed at various times after transplantation (days 5 and 10) and analyzed by immunohistochemistry. (A): Graft infiltration began as early as day 5, with CD11b+ macrophage infiltration throughout the graft (left panel) and CD4+ T cells appearing at the border between ESC graft and kidney (center panel). CD8+ T cells were absent at this time point (right panel). Photomicrographs are representative of n = 3 grafts analyzed at day 5 post-transplantation. (B): At day 10, a response from macrophages (left panel), CD4+ T cells (center panel), and CD8+ T cells (right panel) was evident. Photomicrographs are representative of n = 3 grafts analyzed at day 10 post-transplantation. (C): Syngeneic ESC grafts are completely free of cellular infiltration at day 10. Photomicrographs are representative of n = 3 grafts analyzed at day 10 post-transplantation. All photomicrographs were taken with a Nikon Coolpix 995 digital camera attached to a Zeiss Axiovert 25 inverted microscope using a x10 objective lens.

 
Islet-Like Tissue Can Be Derived from Undifferentiated ESCs
A critical factor currently hampering the widespread applicability of cell replacement therapy is the paucity of suitable donor tissue. ESCs, because of their inherent plasticity, represent an attractive source of potential replacement or supplementary tissue [8]. However, a challenge that remains is to develop an effective protocol whereby undifferentiated ESCs may be directed toward desired cell type fates. In addition, the immune response against such terminally differentiated tissue remains to be elucidated. Here we applied a multistage in vitro culture protocol as previously described [20] to direct undifferentiated ESCs into IPCCs. These clusters phenotypically resembled adult pancreatic islet tissue, with the expression of insulin-1, insulin-2, glucagon, and pancreatic amylase transcript (Fig. 6A). Furthermore, de novo insulin production was confirmed by immunofluorescence detection of both insulin and c-peptide (Fig. 6B). Hence, terminally differentiated islet-like tissue derived from an ESC source could then be used in further in vivo immunological studies.


Figure 6
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  		Figure 6. End-stage Ins-producing cell clusters exhibit a mature islet-like phenotype. (A): Embryonic stem cells were subjected to a multistage in vitro differentiation protocol. Terminally differentiated cells exhibited an islet-like phenotype, expressing Ins-1, Ins-2, glucagon, and pancreatic amylase transcript, as revealed by RT-polymerase chain reaction. The data shown are from four random samples of differentiated end-stage products (samples 1–4) and are representative of n = 12 total independent differentiation experiments performed. (B–D): Ins expression (green) (B), c-peptide expression (red) (C), and coexpression (merged) (D) were observed in terminally differentiated cells. Blue is 4,6-diamidino-2-phenylindole counterstain. Images are representative of n = 12 independent differentiation experiments. All images were taken at a magnification of x40 with identical camera settings. (E): Terminally differentiated cells were positive for c-peptide single staining, confirming de novo production of Ins and ruling out the possibility of antibody cross-reactivity in double-stained images. The image is representative of n = 12 independent differentiation experiments. All images were taken at a magnification of x40 with identical camera settings. Abbreviations: Ins, insulin; RT, reverse transcriptase.

 
Immune Recognition of Allogeneic IPCCs
Having evaluated the potential adaptive immune response to undifferentiated ESCs, we next sought to analyze the response against a terminally differentiated functional allograft derived from an ESC source. In keeping with our previous experiments, we first tested the ability of BM3 T cells to mount a productive response against an IPCC allograft derived from an H2Kb+ ESC source (ESF134). CFSE division profiling revealed that the allogeneic IPCC graft did not elicit a greater proliferative response from BM3 T cells than a syngeneic ESF122-derived IPCC graft (Fig. 7A). Furthermore, the allogeneic IPCC graft remained free of BM3 T-cell infiltration, despite strong immunoperoxidase detection of the MHC class I molecule H2Kb (Fig. 7B, left panel; data not shown). However, when H2b IPCCs were transplanted into fully MHC-mismatched wild-type CBA (H2k) recipients, dramatic CD8+ T-cell infiltration was observed by day 10 post-transplant (Fig. 7B, right panel).


Figure 7
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  		Figure 7. IPCCs represent novel targets of immune attack. (A): Terminally differentiated allogeneic IPCCs derived from ESF134 (H2b) did not provoke a proliferative response from BM3 T cells compared with that seen in syngeneic IPCCs (ESF122 [H2k]-derived) controls. Proliferation of BM3 T cells was assessed by CFSE division profiling in the spleen 10 days post-transplantation. BM3 T cells were gated on the coexpression of CD8, transgenic TCR, and TCRβ chain. Dot plots are representative of n = 3 animals analyzed at day 10. (B): Although allogeneic IPCCs were not recognized by BM3 T cells (left panel), they were heavily infiltrated by CD8+ T cells when transplanted into wild-type fully major histocompatibility complex (MHC)-mismatched recipients (right panel) by day 10 post-transplantation. Photomicrographs are representative of n = 3 grafts analyzed at day 10 post-transplantation. (C): ESF122 (H2k)-derived IPCCs transplanted into the renal subcapsular space of streptozocin (STZ)-induced diabetic CBA.rag–/– (H2k) mice were capable of establishing short-term normoglycemia (14 ± 3 days; mean ± SEM). A blood glucose reading of 14.5 mM served as the cutoff between nondiabetic and diabetic states. Initial diabetic level of recipient mice was roughly 20 mM after STZ induction. (D): Transplantation of IPCCs into immunocompetent recipients resulted in impaired functionality, even under syngeneic conditions (ESF122 (H2k) IPCCs transplanted into CBA (H2k) wild-type recipients). Functionality was dramatically abolished when transplanted across full MHC disparity (ESF122 IPCCs transplanted into C57BL/10 recipients). Data are represented as mean ± SEM, and p value was determined by one-way analysis of variance. Abbreviations: CFSE, carboxyfluorescein succinimidyl ester; IPCC, insulin-producing cell cluster; txp, transplant.

 
IPCCs Represent Novel Targets of Immune Attack
The functional capacity of the in vitro-generated IPCCs was tested in a STZ-induced mouse diabetes model. In these experiments, ESF122 (CBA, H2k) was directed toward an IPCC fate and then transplanted into STZ-induced diabetic mice. Short-term rescue from STZ-induced diabetes could be achieved by the transplantation of large numbers of IPCCs into the renal subcapsular space of CBA-Rag–/– mice (Fig. 7C, 14 ± 3 days, mean ± SEM). However, when transplanted into immunocompetent hosts, IPCC functionality was dramatically impaired, even under syngeneic conditions (Fig. 7D). Taken together, these data suggest that both innate and adaptive immune systems may work in concert to affect the ultimate fate of ESC-derived allografts that could potentially be used in cell replacement therapy and regenerative medicine.


   DISCUSSION
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 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
In this report, we assess the immune response directed against allogeneic ESCs and their functional derivatives in an in vivo transplantation setting. In accordance with established data, the ESCs used here expressed extremely low levels of MHC molecules in their undifferentiated state. Furthermore, despite the dramatic upregulation of H2Kb upon transplantation and differentiation in vivo, the ESC graft did not elicit a response from naïve CD8+ T cells (BM3) with the capacity to recognize ESC allogeneic MHC class I molecules and remained less immunogenic in direct comparison with an H2Kb+ pancreatic islet allograft. Interestingly, ESCs transgenic for H2Kb (ESF166), expressing constitutively high levels of H2Kb, also escaped BM3 T-cell recognition. On the other hand, in the presence of APC stimulation or alloantigen priming, effector BM3 T cells mounted a productive response against the ESC graft. Importantly, the immunogenicity of the ESC graft was sufficient to elicit a rejection response after transplantation into a wild-type fully MHC-mismatched recipient. Similarly, end-stage differentiated functional IPCCs that were capable of reversing STZ-induced diabetes in vivo were heavily infiltrated by T cells when transplanted across MHC barriers and were rapidly rejected by immunocompetent hosts.

By using the direct pathway of allorecognition BM3 TCR transgenic model, we established that ESCs are incapable of stimulating a direct alloresponse after transplantation in the absence of other modes of T-cell activation. This fragile state of so-called immune privilege could be overcome by the artificial provision of "passenger" APC. Indeed, a unique feature of ESC-derived tissue grafts is their natural lack of donor-derived APC. Early work showed that these APC have the potential to elicit a strong immune response from recipient T cells and trigger acute rejection of mismatched kidney allografts in rats [24, 25]. More recently, Rutzky et al. subjected pancreatic islets to culture periods postisolation and observed the gradual depletion of graft-resident dendritic cells via transmission electron microscopy. These cultured islets were then shown to maintain optimal function over a period of 100 days after transplantation into allogeneic recipients [26]. Hence, although in some settings donor-derived APC can have a beneficial effect on establishing recipient tolerance to an organ allograft [27], their presence often potentiates graft immunogenicity. Here we show that the inherent lack of passenger APC within ESC grafts contributes to their low immunostimulatory potential in vivo. However, this low immunogenicity is of a fragile nature, as modest priming of BM3 T cells via cotransplantation of a population of matured allogeneic dendritic cells enabled BM3 T cells to reject an H2Kb+ ESC graft.

The data shown here further suggest that CD8+ T cells are unable to mount a response against an ESC graft without the provision of T-cell help. There remains much debate over the role of such help in the generation of CD8+ T-cell responses. In the absence of CD4+ T cells, CD8+ T cells have been shown to produce effective primary responses, but the generation and maintenance of CD8+ T-cell memory generally appears reliant on CD4 help [28]. In a transplantation setting, however, CD8+ T cells appear to play an important role independent of CD4+ T cells. For example, CD8+ T cells can prevent tolerance induction by costimulatory blockade, despite such treatment being able to control CD4+ T-cell-mediated rejection [29, 30]. Furthermore, recent work from our group using the BM3 system demonstrated that these CD8+ T cells could develop effector function in vivo, mediate allogeneic skin graft rejection, and produce memory responses all independently of CD4+ T cells [31]. However, despite the ability of BM3 T cells to acutely reject skin, heart, and islet allografts [32], alone, they are unable to mount a productive response against an H2Kb+ ESC allograft. In this respect, the ESC graft is less immunogenic than conventional adult allografts, such as islet allografts. This may be due to the inability of the ESC allografts to stimulate a direct alloresponse. The indirect pathway of allorecognition may therefore play a relatively larger role in the immune response against ESC-derived allografts.

Upon priming of BM3 T cells against H2Kb alloantigen, the apparent immune privilege of the H2Kb+ ESC graft was abolished. This finding was evident even when only a very modest priming of BM3 T cells to the H2Kb alloantigen could be detected. Therefore, although allogeneic ESC-derived tissue grafts may indeed possess features that result in a lesser immunogenic potential in comparison with adult organ allografts, their ultimate avoidance of immune recognition and subsequent acceptance after transplantation without additional immunomodulatory therapy is unlikely.

The data presented here further demonstrate that terminally differentiated tissue derived from an embryonic stem cell source represents a novel target of immune attack when transplanted into a foreign host. IPCC functionality in vivo was dramatically hampered when transplanted into immunocompetent recipients or across major histocompatibility barriers. The graft infiltration by alloreactive T cells observed at later time points gives a clear indication of a classic T-cell-mediated rejection response. However, the failure of IPCC grafts when transplanted into syngeneic but immunocompetent recipients, as opposed to Rag–/– recipients, suggests that additional mechanisms of early graft damage may be at play. Very early graft damage can be mediated by recipient macrophages and neutrophils that infiltrate the graft hours after transplantation [33, 34]. The involvement of neutrophils and macrophages after transplantation of ESC-derived tissue warrants further study.

One possibility to explain the unique immunogenicity of IPCC grafts in syngeneic immunocompetent hosts is via the expression of novel immunogenic epitopes. Embryonic tissue expresses a variety of antigenic cell surface determinants that disappear at later stages of development and are absent in normal adult tissue [35]. Although the IPCCs were differentiated in culture, it was impossible to exclude the persistence of embryonic precursors even in the end-stage products. Furthermore, the artificial manipulations associated with in vitro directed differentiation may also lead to the cell surface expression of novel immunogenic molecules [36, 37]. These antigenic determinants may represent unique targets of immunological attack. Recognition of these novel epitopes by T cells, B cells, or natural killer (NK) T cells—three main effector cell populations absent in the Rag–/– but present in the wild-type recipient—potentially contributed to the acute graft failure observed here. For example, embryonic antigen derived from the IPCC graft and subsequently presented by recipient DC may prime a T-cell-mediated anti-IPCC rejection response even under syngeneic conditions. Furthermore, a possible role for humoral mediated rejection cannot be excluded. Hyperacute and acute antibody-mediated rejection (AMR) can arise from naturally occurring recipient antibodies that cross-react against foreign donor-derived epitopes [38–40]. The most spectacular manifestation of hyperacute AMR is illustrated when unmodified xenogeneic pig organs are engrafted into Old World monkeys. Old World monkeys (and humans) do not express the oligosaccharide Gal{alpha}1–3Galβ1–4GlcNAc (Gal), which is constitutively found on pig vascular endothelium, and have naturally high titers of circulating antibody against this determinant. This preformed antibody cross-reacts onto Gal and fixes the complement, inducing an extremely rapid destruction of pig xenografts that also lack complement regulatory proteins, such as human decay accelerating factor, which would normally control such a reaction [41, 42]. This setting could be analogous to the presence of embryonic antigen or culture-induced antigen expressed on IPCCs but absent in normal adult tissue [36]. Recently, Koch et al. demonstrated that ESC-derived tissue may be susceptible to complement mediated destruction, further suggesting a possible role for antibody in the rejection of IPCC grafts [43]. Finally, NK T cells may also be able to mediate early graft damage even in a syngeneic setting [44], and they appear to play a role in the rejection of pancreatic islet allografts transplanted into the liver [45]. However, the mechanisms of NK T action in transplantation settings are currently ill-defined.

Recently, the derivation of pluripotent cells from adult somatic cells has raised intriguing possibilities for future cell replacement therapy. Using defined transcriptional regulators such as Oct4 and Sox2, investigators were able to reprogram human dermal fibroblasts into cells that shared many characteristics of human embryonic stem cells, including the ability to differentiate into all three germ layers in vitro and into teratomas in vivo [46–48]. These induced pluripotent stem cells (iPS) could theoretically be derived in a patient-specific manner, thus potentially alleviating the immunological pitfalls inherent in transplantation across foreign human leukocyte antigen barriers. Further characterization of these iPS may be a promising way forward in regenerative cellular therapies.


   CONCLUSION
Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
Disclosure of Potential...
 Acknowledgments
 References
 
The immunogenicity of ESC-derived tissue grafts requires further evaluation before strategies can be developed to maximize their potential in cell replacement therapy and regenerative medicine. On the one hand, our data support the hypothesis that ESCs and their derivatives may enjoy a certain immune privilege over conventional adult allografts. However, the ESC allografts and their terminally differentiated derivatives were recognized and rejected under the correct circumstances, demonstrating that their immunogenicity must still be taken into account. Even so, the potentially low immunostimulatory capacity of ESC-derived allografts may still enable unique strategies in dealing with their rejection, perhaps allowing a window of opportunity to apply therapeutics before an overwhelming immunological response is initiated. Continued research on the immunogenicity of ESCs, as well as their potential to differentiate into useful replacement tissue, is a promising pathway toward future applications in cell replacement therapy and regenerative medicine.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
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 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
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 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank the staff of the BMS-JR for expert care of the mice used in this study, Nick Jones for careful reading of the manuscript and helpful comments, and other members of Transplantation Research Immunology Group for helpful discussions throughout the project. Funding for this project was provided by grants from the Medical Research Council (MRC) U.K. and Becton Dickinson Preanalytical Systems (Oxford, U.K.) as part of an MRC Collaborative Studentship (to A.S.B.) and by the Wellcome Trust. The Transplantation Research Immunology Group is a member of the U.K. SCIP Consortium. D.C.W. and A.S.B. contributed equally to this work. D.C.W. holds a Clarendon Scholarship, University of Oxford. A.S.B. held an MRC Collaborative Studentship in association with Becton Dickinson. A.S.B. is currently affiliated with the Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom. K.J.W. holds a Royal Society Wolfson Research Merit Award.


   FOOTNOTES
 
Author contributions: D.C.W.: conception and design, provision of study material, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; A.S.B.: conception and design, manuscript writing, final approval of manuscript; K.J.W.: conception and design, financial support, administrative support, data analysis and interpretation, manuscript writing, final approval of manuscript.


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 Abstract
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 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 

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