Novel approaches for immune reconstitution and adaptive immune modeling with human pluripotent stem cells
© Green and Snoeck; licensee BioMed Central Ltd. 2011
Received: 31 March 2011
Accepted: 10 May 2011
Published: 10 May 2011
Pluripotent stem cells have the capacity to generate all cell lineages, and substantial progress has been made in realizing this potential. One fascinating but as yet unrealized possibility is the differentiation of pluripotent stem cells into thymic epithelial cells. The thymus is a primary lymphoid organ essential for naïve T-cell generation. T cells play an important role in adaptive immunity, and their loss or dysfunction underlies in a wide range of autoimmune and infectious diseases. T cells are generated and selected through interaction with thymic epithelial cells, the functionally essential element of thymus. The ability to generate functional thymic epithelial cells from pluripotent stem cells would have applications in modeling human immune responses in mice, in tissue transplantation, and in modulating autoimmune and infectious disease.
Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst and can be maintained in a pluripotent state in defined conditions in both human and mouse . Theoretically, ES cells can differentiate into every somatic and germ cell type. The recent discovery that adult somatic cells can be reprogrammed into a pluripotent state (induced pluripotent stem (iPS) cells) [2, 3] opens the way for the generation of patient-specific PS cells, which would overcome rejection problems associated with transplantation of ES cell-derived tissues. The development of appropriate conditions to differentiate ES and iPS cells into a variety of cell types and tissues therefore holds major promise for future cell-replacement therapy. A major challenge in this field is the directed differentiation of pluripotent cells into functional mature tissue [1, 4].
A relatively underinvestigated area is the generation of cells of the thymus from ES and iPS cells. The thymus is the site of development of T cells, cells essential for adaptive immunity. In the thymus, a self-tolerant T-cell repertoire is established through positive and negative selection [5, 6]. Very early hematopoietic precursors seed the thymus from the bone marrow to initiate T cell development . Hence, the thymus consists of a hematopoietic, an epithelial and a mesenchymal component. The hematopoietic component includes developing T cells and mature dendritic cells [5–7]. The epithelial component includes thymic epithelial cells (TECs), which have two subtypes: cortical (cTEC) and medullary (mTEC). These two types of TECs form a three-dimensional structure together with endothelial cells, poorly defined mesenchymal cells, neural elements, neural crest-derived pericytes, and adipocytes [5, 6]. Only TECs seem to be essential, as purified undifferentiated TECs at embryonic day (E) 12.5 can reconstitute a functional thymus after aggregation and transplantation under the kidney capsule [8–11]. During T-cell maturation, early T-cell precursors enter the thymus and undergo expansion, followed by cTEC-dependent positive selection for high antigen affinity, and then mTEC- and dendritic cell-dependent negative selection against strong autoantigen recognition .
Rationale for the generation of TECS from pluripotent stem cells
A second potential reason is the attenuation of immunological aging (Figure 1b). It is widely hypothesized that improving thymic function in the older person will increase health, and perhaps extend life span [15–24]. Some rather old reports suggest that transplantation of multiple thymi into aged animals is beneficial for immune function, and increases mean, but not maximal life span, although there is evidence for significant mouse strain-dependent variation [22–24].
Congenital diseases in which a thymus is lacking are good candidates for thymus replacement therapy (Figure 1c). In these diseases, correction of or functional compensation for the genetic defect during in vitro differentiation may allow transplantation of autologous, iPS-derived thymic tissue (Figure 1c). Loss of FOXN1, an essential gene for thymic development, causes the rare 'nude/severe combined immune deficiency (SCID)' syndrome in which patients have no hair or thymus . In DiGeorge syndrome (DGS), the most frequently diagnosed microdeletion in humans (1 in 4000 people), a region of chromosome 22 is monoallelically deleted. This region includes a crucial transcription factor required for the early stages of thymic development, TBX1. The syndrome (DGS/velofaciocardial syndrome (VFCS)) is characterized by absent or hypoplastic thymus and parathyroids, in addition to a variety of cardiac and facial malformations [26, 27]. In both syndromes, allogeneic thymic transplantation has been beneficial [28–30]. Obvious problems include the allogeneic nature of the graft and the scarcity of donors. In the particular case of thymus transplantation, this problem is exacerbated by the fact that thymi from donors that would otherwise be considered young have already undergone severe age-related thymic atrophy.
A final clinical rationale is the treatment of autoimmune disease (Figure 1d). TECs derived from patient-specific iPS cells could be genetically modified to influence the process of positive and negative selection in the engrafted thymic tissue. For example, expression of a culprit autoantigen in the putative medullary cells of the graft might induce tolerance to these antigens by mediating negative selection of the immature T cells recognizing these antigens .
TECs derived from ES or iPS cells could also improve humanized mouse models (Figure 1e). A major challenge in immunology is the establishment of mouse models of the human immune system. Currently the best models are immunodeficient Rag1 -/- ilr2g -/- or non-obese diabetic (NOD)-SCIDilr2g -/- mice engrafted neonatally with human cord-blood hematopoietic stem and progenitor cells. In such mice, all major hematopoietic lineages are reconstituted, and even the structure of secondary lymphoid organs seems 'human'. However, human T-cell responses are weak except for alloreactivity, and peripheral T-cell homeostasis is abnormal [31–34]. An improvement of this model with more robust T-cell activity is the bone marrow/liver/thymus (BLT) mouse [35, 36]. This is a NOD-SCIDilr2g -/- mouse transplanted with a human fetal thymus and liver under the kidney capsule, and subsequently transplanted with fetal liver CD34+ progenitor cells. In these mice, both innate and major histocompatibility complex I and II-restricted, T-cell-dependent immune responses were seen. Interestingly, all T-cell development occurred in the grafted human thymus, not in the endogenous mouse thymus. These data suggest that the presence of human thymic tissue may be crucial to develop a humanized mouse. It follows that as TECs are the essential functional component of the thymus, ES or iPS-derived TECs could also be used to construct an improved humanized mouse model. In addition, by using patient-specific iPS cells, it is possible to capture in a mouse model some of the genetic diversity in disease susceptibility and immune responses among humans. Finally, iPS technology will allow cotransplantion of syngeneic human tissues (Figure 1e). In such a mouse, organ or tissue-specific immune responses in the context of autoimmunity or infection can be studied, and vaccines can be tested. The development of such a mouse may revolutionize translational research.
Generating pluripotent stem cell-derived TECs
To date, it has not been possible to generate TECs from human ES or iPS cells, but important steps towards this goal have been made. TECs are derived from the anterior region of the third pharyngeal pouch, and are therefore of endodermal origin . Subsequently, the tissue descends to the precordial region. The presumptive thymic epithelium seems largely uniform in the mouse until E12. How the different subtypes of TECs are generated is unclear, but it is well established that the E12 thymic anlage contains cells that can clonally generate both cTECs and mTECs. As fetal TEC progenitors can reconstitute a fully functional thymus [8–11], directed differentiation of ES/iPS cells to an early fetal developmental stage might suffice. This goal of generating an early fetal progenitor stands in sharp contrast to efforts to generate other more mature cell types, such a cardiomyocytes, hepatocytes and pancreatic islet cells [1, 4].
The success thus far in quantitative differentiation of ES cells into pancreatic β cells, cardiomyocytes, neurons and other lineages relies on mimicking the sequential developmental states in vivo in ES cells in vitro [1, 4, 37–39]. As developmental cell states are induced by morphogen gradients, directed differentiation relies on the application of these same state-specific morphogens to ES cultures. Surprisingly, a recent report claimed generation of TECs from mouse ES cells through the direct application of terminal thymic maturation signals directly to pluripotent cultures . The efficiency of this protocol was not reported, nor was the presence of alternative lineages assessed, and we have not been able to reproduce this approach in human pluripotent cells. Theoretically, directed differentiation of TECs should proceed by generation of definitive endoderm, followed by patterning into pharyngeal or anterior foregut endoderm . Subsequently, third pharyngeal pouch and then TEC cell fates should be specified. Although definitive endoderm has been generated [42–44], subsequent specification of this germ layer to anterior and pharyngeal fates has remained a challenge. We have recently shown that dual inhibition of transforming growth factor-β/bone morphogenetic protein signaling of human ES and iPS-derived definitive endoderm led to the quantitative generation of anterior foregut endoderm, which could subsequently differentiate into cells expressing markers of parathyroids and lung . It is likely that this strategy will serve as a useful platform for the generation of tissues derived from anterior foregut endoderm, including but not limited to the thymus.
While the potential clinical and scientific benefits of TECs derived from human pluripotent are great, it has not yet been possible to generate TECs from human ES or iPS cells. Given recent progress in the generation of derivatives of definitive endoderm, however, we anticipate significant progress in this area in the near future.
Our work in this field was supported by NY State grant NO8G-422 to HWS. MG was supported by a Training grant in Cancer Biology (5T32CA078207).
- Murry CE, Keller G: Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell. 2008, 132: 661-680. 10.1016/j.cell.2008.02.008.View ArticlePubMedGoogle Scholar
- Yamanaka S: A fresh look at iPS cells. Cell. 2009, 137: 13-17. 10.1016/j.cell.2009.03.034.View ArticlePubMedGoogle Scholar
- Takahashi K, Okita K, Nakagawa M, Yamanaka S: Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc. 2007, 2: 3081-3089. 10.1038/nprot.2007.418.View ArticlePubMedGoogle Scholar
- Irion S, Nostro MC, Kattman SJ, Keller GM: Directed differentiation of pluripotent stem cells: from developmental biology to therapeutic applications. Cold Spring Harb Symp Quant Biol. 2008, 73: 101-110. 10.1101/sqb.2008.73.065.View ArticlePubMedGoogle Scholar
- Rodewald HR: Thymus organogenesis. Annu Rev Immunol. 26: 355-388.
- Ladi E, Yin X, Chtanova T, Robey EA: Thymic microenvironment for T cell differentiation and selection. Nat Immunol. 2006, 7: 337-343. 10.1038/nrg1838.View ArticleGoogle Scholar
- Bhandoola A, Sambandam A: From stem cell to T cell: one route or many?. Nat Rev Immunol. 2006, 6: 117-126. 10.1038/nri1778.View ArticlePubMedGoogle Scholar
- Rossi SW, Jenkinson WE, Anderson G, Jenkinson EJ: Clonal analysis reveals a common progenitor for thymic cortical and medullary epithelium. Nature. 2006, 441: 988-891. 10.1038/nature04813.View ArticlePubMedGoogle Scholar
- Gill J, Malin M, Holländer GA, Boyd R: Generation of a complete thymic microenvironment by MTS24+ thymic epithelial cells. Nat Immunol. 2002, 3: 635-641.View ArticlePubMedGoogle Scholar
- Farley A, Blair NF, Gordon J, Sharp L, Blackburn CC: Identification and characterization of thymic epithelial progenitor cells. Immunity. 2002, 16: 803-814. 10.1016/S1074-7613(02)00321-7.View ArticlePubMedGoogle Scholar
- Rossi SW, Chidgey AP, Parnell SM, Jenkinson WE, Scott HS, Boyd RL, Jenkinson EJ, Anderson G: Redefining epithelial progenitor potential in the developing thymus. Eur J Immunol. 2007, 37: 2411-2418. 10.1002/eji.200737275.View ArticlePubMedGoogle Scholar
- Van den Brink MRM, Alpdogan O, Boyd RL: Strategies to enhance T-cell reconstitution in immunocompromised patients. Nat Rev Immunol. 2004, 4: 856-867. 10.1038/nri1484.View ArticlePubMedGoogle Scholar
- Storek J, Saxon A: Immunity of patients surviving 20 to 30 years after allogeneic or syngeneic bone marrow transplantation. Blood. 2001, 98: 3505-3512. 10.1182/blood.V98.13.3505.View ArticlePubMedGoogle Scholar
- Shlomchik W: Graft-versus-host disease. Nat Rev Immunol. 2007, 7: 340-352. 10.1038/nri2000.View ArticlePubMedGoogle Scholar
- Linton PJ, Dorshkind K: Age-related changes in lymphocyte development and function. Nat Immunol. 2004, 5: 133-139.View ArticlePubMedGoogle Scholar
- Montecino-Rodriquez E, Min H, Dorshkind K: Reevaluating current models of thymic involution. Semin Immunol. 2005, 5: 356-361.View ArticleGoogle Scholar
- Aspinall R, Andrew D: Thymic involution in aging. J Clin Immunol. 2000, 20: 250-256. 10.1023/A:1006611518223.View ArticlePubMedGoogle Scholar
- Franceschi C, Bonafè M, Valensin S: Human immunosenescence: the prevailing of innate immunity, the failing of clonotypic immunity, and the filling of immunological space. Vaccine. 2000, 18: 1717-1720. 10.1016/S0264-410X(99)00513-7.View ArticlePubMedGoogle Scholar
- Shanker A: Is the thymus redundant after adulthood?. Immunol Lett. 2004, 791: 79-89.View ArticleGoogle Scholar
- Aw D, Silva AB, Palmer DB: Immunosenescence: emerging challenges for an ageing population. Immunology. 2007, 120: 435-446. 10.1111/j.1365-2567.2007.02555.x.View ArticlePubMedPubMed CentralGoogle Scholar
- Franceschi C, Bonafe M: Centenarians as a model for healthy aging. Biochem Soc Trans. 2003, 31: 457-461. 10.1042/BST0310457.View ArticlePubMedGoogle Scholar
- Hirokawa K, Ustuysama M: The effect of sequential multiple grafting of syngeneic newborm thymus on the immune functions and life expectancy of aging mice. Mech Aging Dev. 1984, 28: 111-121. 10.1016/0047-6374(84)90157-X.View ArticlePubMedGoogle Scholar
- Hitokawa K, Utsuyama M: Combined grafting of bone marrow and thymus, and sequential multiple thymus graftings in various strains of mice. The effect on immune functions and life span Mech Aging Dev. 1989, 49: 49-60.Google Scholar
- Metcalf D: Multiple thymus graft in aged mice. Nature. 1965, 208: 87-89. 10.1038/208087a0.View ArticlePubMedGoogle Scholar
- Frank J, Pignata C, Panteleyev AA, Prowse DM, Baden H, Weiner L, Gaetaniello L, Ahmad W, Pozzi N, Cserhalmi-Friedman PB, Aita VM, Uyttendaele H, Gordon D, Ott J, Brissette JL, Christiano AM: Exposing the human nude phenotype. Nature. 1999, 398: 473-474. 10.1038/18997.View ArticlePubMedGoogle Scholar
- Sullivan KE: Chromosome 22q11.2 Deletion syndrome: DiGeorge syndrome/velocardiofacial syndrome. Immunol Allergy Clin North Am. 2008, 28: 353-366. 10.1016/j.iac.2008.01.003.View ArticlePubMedGoogle Scholar
- Kobrynski LJ, Sullivan KE: Velocardiofacial syndrome, DiGeorge syndrome: the chromosome 22q11.2 deletion syndromes. Lancet. 2007, 370: 1443-1452. 10.1016/S0140-6736(07)61601-8.View ArticlePubMedGoogle Scholar
- Markert ML, Boeck A, Hale LP, Kloster AL, McLaughlin TM, Batchvarova MN, Douek DC, Koup RA, Kostyu DD, Ward FE, Rice HE, Mahaffey SM, Schiff SE, Buckley RH, Haynes BF: Transplantation of thymus tissue in complete DiGeorge syndrome. N Engl J Med. 1999, 341: 1180-1189. 10.1056/NEJM199910143411603.View ArticlePubMedGoogle Scholar
- Markert ML, Sarzotti M, Ozaki DA, Sempowski GD, Rhein ME, Hale LP, Le Deist F, Alexieff MJ, Li J, Hauser ER, Haynes BF, Rice HE, Skinner MA, Mahaffey SM, Jaggers J, Stein LD, Mill MR: Thymus transplantation in complete DiGeorge syndrome: immunologic and safety evaluations in 12 patients. Blood. 2003, 102: 1121-1130. 10.1182/blood-2002-08-2545.View ArticlePubMedGoogle Scholar
- Markert ML, Marques JG, Neven B, Devlin BH, McCarthy EA, Chinn IK, Albuquerque AS, Silva SL, Pignata C, de Saint Basile G, Victorino RM, Picard C, Debre M, Mahlaoui N, Fischer A, Sousa AE: First use of thymus transplantation therapy for FOXN1 deficiency (nude/SCID): a report of 2 cases. Blood. 2011, 117: 688-696. 10.1182/blood-2010-06-292490.View ArticlePubMedPubMed CentralGoogle Scholar
- Manz MG: Human-hemato-lymphoid-system mice: opportunities and challenges. Immunity. 2007, 26: 537-541. 10.1016/j.immuni.2007.05.001.View ArticlePubMedGoogle Scholar
- Traggiai E, Chicha L, Mazzucchelli L, Bronz L, Piffaretti JC, Lanzavecchia A, Manz MG: Development of a human adaptive immune system in cord blood cell-transplanted mice. Science. 2004, 304: 104-107. 10.1126/science.1093933.View ArticlePubMedGoogle Scholar
- Legrand N, Weijer K, Spits H: Experimental model for the study of the human immune system: production and monitoring of "human immune system" Rag2-/-gamma c-/- mice. Methods Mol Biol. 2008, 415: 65-82. 10.1007/978-1-59745-570-1_4.PubMedGoogle Scholar
- Gimeno R, Weijer K, Voordouw A, Uittenbogaart CH, Legrand N, Alves NL, Wijnands E, Blom B, Spits H: Monitoring the effect of gene silencing by RNA interference in human CD34+ cells injected into newborn RAG2-/- gammac-/- mice: functional inactivation of p53 in developing T cells. Blood. 2004, 104: 3886-3893. 10.1182/blood-2004-02-0656.View ArticlePubMedGoogle Scholar
- Lan P, Tonomura N, Shimizu A, Wang S, Yang YG: Reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and CD34+ cell transplantation. Blood. 2006, 108: 487-492. 10.1182/blood-2005-11-4388.View ArticlePubMedGoogle Scholar
- Melkus MW, Estes JD, Padgett-Thomas A, Gatlin J, Denton PW, Othieno FA, Wege AK, Haase AT, Garcia JV: Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1. Nat Med. 2006, 12: 1316-1322. 10.1038/nm1431.View ArticlePubMedGoogle Scholar
- Gouon-Evans V, Boussemart L, Gadue P, Nierhoff D, Koehler CI, Kubo A, Shafritz DA, Keller G: BMP-4 is required for hepatic specification of mouse embryonic stem cell-derived definitive endoderm. Nat Biotechnol. 2006, 24: 1402-1411. 10.1038/nbt1258.View ArticlePubMedGoogle Scholar
- Yang L, Soonpaa MH, Adler ED, Roepke TK, Kattman SJ, Kennedy M, Henckaerts E, Bonham K, Abbott GW, Linden RM, Field LJ, Keller GM: Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature. 2008, 453: 524-528. 10.1038/nature06894.View ArticlePubMedGoogle Scholar
- Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L: Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009, 27: 275-280. 10.1038/nbt.1529.View ArticlePubMedPubMed CentralGoogle Scholar
- Lai L, Jin J: Generation of thymic epithelial cell progenitors by mouse embryonic stem cells. Stem Cells. 2009, 27: 3012-3020.PubMedGoogle Scholar
- Zorn AM, Wells JM: Vertebrate endoderm development and organ formation. Annu Rev Cell Dev Biol. 2009, 25: 221-251. 10.1146/annurev.cellbio.042308.113344.View ArticlePubMedPubMed CentralGoogle Scholar
- Gadue P, Huber TL, Paddison PJ, Keller GM: Wnt and TGF-betasignaling are required for the induction of an in vitro model ofprimitive streak formation using embryonic stem cells. Proc Natl AcadSci USA. 2006, 103: 16806-16811. 10.1073/pnas.0603916103.View ArticleGoogle Scholar
- Kubo A, Shinozaki K, Shannon JM, Kouskoff V, Kennedy M, Woo S, Fehling HJ, Keller G: Development of definitive endoderm from embryonic stem cells in culture. Development. 2004, 131: 1651-1662. 10.1242/dev.01044.View ArticlePubMedGoogle Scholar
- D'Amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, Baetge EE: Efficient differentiation of human embryonic stem cells to definitiveendoderm. Nat Biotechnol. 2005, 23: 1534-1541. 10.1038/nbt1163.View ArticlePubMedGoogle Scholar
- Green M, Nostro MC, d'Souza S, Schaniel C, Lemischka IR, Gouon-Evans V, Keller G, Snoeck HW: Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells. Nat Biotechnol. 2011, 29: 267-272. 10.1038/nbt.1788.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/9/51/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.