The Rejection Barrier to Induced Pluripotent Stem Cells : Journal of the American Society of Nephrology

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The Rejection Barrier to Induced Pluripotent Stem Cells

Balasubramanian, Savithri*; Kota, Satya K.; Valerius, M. Todd‡,§

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Journal of the American Society of Nephrology 22(9):p 1583-1586, September 2011. | DOI: 10.1681/ASN.2011070707
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Tissue regeneration and organ development from patient-induced pluripotent stem cells (iPSCs), created through cellular reprogramming,1,2 are new approaches for resolving the inadequate number of organs available for transplantation, including the kidney. Because iPSCs are derived from the patient, self-tolerance of the tissues or organs made from these cells is expected. However, recent work by Zhao et al.3 reveals some previously unappreciated wrinkles regarding potential immunogenicity of iPSCs that raise issues regarding future use of iPSCs in the clinical setting.

Since the first successful kidney transplantation in 1954, the field of organ transplantation has prolonged many lives worldwide. However, despite the technical improvements in organ transplantation and the coordinated efforts to maximize organ donation, more than 89,000 Americans await kidney transplantation due to the paucity of available organs.4 New technologies such as tissue regeneration and organ development from stem cells (from embryonic or adult sources) offer promise not only to meet the demand for organs but also to aid the repair of injured organ tissues.59

Patient-specific pluripotent stem cells may be one solution for innumerable medical conditions requiring cell- or tissue-based replacement therapies. Unfortunately, embryonic pluripotent stem cells (ESCs) can be derived only during a specific window of time during early development, and further, to obtain such cells without affecting the embryo has not yet been proven in humans and raises ethical concerns. Takahashi and Yamanaka, and others, have shown that expression of a transcription factor cocktail consisting of Oct4, Sox2, Klf4, and Myc can revert terminally differentiated cells to a pluripotent state, both in mice10 and humans,11,12 a technique called reprogramming, therefore permitting adult tissues to serve as a stem-cell source.

In the mouse, these iPSCs are similar to ESCs in gene expression profile, the ability to form all three germ layers in teratoma assays, and the ability to contribute to germline-competent chimeric mice.13 Similarly, human iPSCs derived from diverse tissues also give rise to teratomas in nude mice, demonstrating the pluripotency of human iPSCs.14 Although original reprogramming methods used viral transduction to deliver the four factors, recent work has led to nonviral alternatives,15,16 including nonintegrating gene expression episomes, direct mRNA17 or miRNA18 delivery, and reducing the need for the oncogene Myc.19 The range of techniques available and the ease of applying them raise hopes for developing histocompatible therapeutics from self-derived iPSCs isolated from patients. Furthermore, iPSCs can be used to model transitions of disease pathologies of various disorders in vitro2025 and may help in the development of personalized pharmaceutical interventions.26

Until now, the self-tolerance of iPSCs, although assumed, has not been comprehensively tested. Zhao et al.3 examine this very important question by comparing ESCs and iPSCs derived from mouse fibroblasts by transplantation into recipients of identical genetic strains, using teratoma formation to assay for tolerance. ESCs derived from the blastocysts of B6 mice were able to form teratomas in B6 mice, demonstrating tolerance for ESCs from a syngeneic source. However, iPSCs obtained by viral-mediated delivery of reprogramming factors into B6 mouse embryonic fibroblasts failed to form detectable teratomas. Although the ESCs and iPSCs are derived from the same genetic background, can both contribute to chimeric mice and form teratomas in nude mice, the iPSCs are rejected by the syngeneic host. The authors observed that the inability of iPSCs to sustainably form teratomas in the syngeneic mice was due to an immune response to the teratomas, characterized by massive infiltration of T lymphocytes.

To test if the immune response was due to the viral delivery method or due to persistent ectopic expression of stably integrated pluripotency transgenes, the authors used an episomal-mediated gene delivery approach wherein the pluripotency transgene cassette is removed from the undifferentiated iPSCs to prevent the reactivation of these four genes during differentiation. Similar to ViPSCs (viral-mediated iPSCs), the EiPSC (episome-mediated iPSCs) lines were also immunogenic in syngeneic host mice, with the teratoma tissues heavily infiltrated by T lymphocytes. One important difference is that, unlike ViPS cells, the teratomas formed by a majority of EiPSCs reached the maximum size allowed in the assay. This suggests the severity of the immune response is due in part to the components or effects of the viral delivery system, such as random integration into the genome. However, an immune response to iPSCs still occurs with the alternative EiPSC approach, suggesting there are other factors contributing to the observed immunogenicity of iPSCs.

To elucidate the reasons for the apparent immune rejection of iPSCs, but not ESCs, the authors conducted gene expression analysis of ESC-derived teratomas, as well as EiPSC-derived teratomas, and identified several differentially expressed genes. Nine such genes enriched in iPSC teratomas, compared with ESC teratomas, were tested for inducing immunogenicity by transgenic overexpression in ESCs and assayed for teratoma formation. Not surprisingly, the syngeneic hosts acutely rejected these transgenic ESC clones. Furthermore, specific T cell immune reactivity toward two abnormally expressed iPSC-specific antigens (Hormad1, Zg16) was found, unambiguously proving that the added expression of self neoantigens indeed leads to recognition by the immune system. Complementary loss-of-function studies by specific deletion or gene knockdown experiments of these genes in iPSCs could further validate their role in breaking self-tolerance.

The findings from this study add to the growing evidence that iPSCs are not as similar to ESCs as previously thought and, importantly here, these subtle differences can lead to immunogenicity of the iPSCs, raising obstacles to their eventual use in the clinic. For example, several recent studies have uncovered epigenetic disparities between these two types of cells in both human and mouse,2729 and a recent study reporting the acquisition of deleterious somatic mutations in protein-coding regions in the iPSCs derived from a variety of sources.30 Collectively, these findings raise concerns regarding the ultimate use of iPSCs in treating human disorders.

Whether the differences in promoter DNA methylation, as well as histone modifications, are indeed responsible for the misexpression of neoantigens described in Zhao et al.,3 and what stages of reprogramming or transplantation these gene expression changes arise, remains unknown and needs investigation. It will also be interesting to see what involvement the innate immune system has during various stages of the tissue engraftment. Furthermore, roles of host factors and chemokines in exacerbating the altered gene expression profiles should also be considered. Moreover, all of the iPSCs reprogrammed in this study came from fetal fibroblasts, not the likely source of iPSCs for future therapies. It is worthwhile to examine the rejection response to overexpressed antigens in iPSCs derived from a range of source tissues in order to look for common themes. Recently, novel reprogramming cocktails using only RNAs or microRNAs have been developed. Whether these alternative reprogramming methods or alternative culture conditions will have any impact on tolerance needs further study. A majority of proposed clinical applications involve differentiated cells or in vitro engineered tissues from stem cells. Whether the iPSC-derived differentiated cells of all lineages will show similar transplant rejections as reported by Zhao et al.3 is presently unclear.

While high throughput sequencing efforts are underway to have a full understanding of the similarities and differences between ESCs and iPSCs, both at the genetic and epigenetic levels, the Zhao et al. study3 demonstrates the need for an in-depth understanding of immune tolerance of ESCs/iPSCs for future applications. Since the self-derived iPSCs (the preferred source) are MHC identical, the immune response against the peptide antigens misexpressed by these cells, as reported in the Zhao study, should be easily muted by simple co-stimulation blockade regimens.31 While it is imperative to dissect the root cause of the differences between iPSCs and ESCs, short-term immunosuppression therapies may address the immunogenicity of iPSCs and allow the their promise to be fulfilled. Moreover, a better understanding of host responses toward reprogrammed cells derived from self will hasten the development of novel targeted gene therapy strategies32 that specifically replace or correct defective mutations in in vitro reprogrammed patient derived cells with inherited disorders.



The authors thank Terry B. Strom for the stimulating discussions and suggestions.

Published online ahead of print. Publication date available at


1. Zhou T, Benda C, Duzinger S, Huang Y, Li X, Li Y, Guo X, Cao G, Chen S, Hao L, Chan YC, Ng KM, Cy Ho J, Wieser M, Wu J, Redl H, Tse HF, Grillari J, Grillari-Voglauer R, Pei D, Esteban MA: Generation of induced pluripotent stem cells from urine. J Am Soc Nephrol 22: 1221–1228, 2011
2. Song B, Niclis JC, Alikhan MA, Sakkal S, Sylvain A, Kerr PG, Laslett AL, Bernard CA, Ricardo SD: Generation of induced pluripotent stem cells from human kidney mesangial cells. J Am Soc Nephrol 22: 1213–1220, 2011
3. Zhao T, Zhang ZN, Rong Z, Xu Y: Immunogenicity of induced pluripotent stem cells. Nature 474: 212–215, 2011
4. United Network for Organ Sharing Web site. Accessed June 2011.
5. LeBleu V, Sugimoto H, Mundel TM, Gerami-Naini B, Finan E, Miller CA, Gattone VH 2nd, Lu L, Shield CF 3rd, Folkman J, Kalluri R: Stem cell therapies benefit Alport syndrome. J Am Soc Nephrol 20: 2359–2370, 2009
6. Ross EA, Williams MJ, Hamazaki T, Terada N, Clapp WL, Adin C, Ellison GW, Jorgensen M, Batich CD: Embryonic stem cells proliferate and differentiate when seeded into kidney scaffolds. J Am Soc Nephrol 20: 2338–2347, 2009
    7. Bruno S, Grange C, Deregibus MC, Calogero RA, Saviozzi S, Collino F, Morando L, Busca A, Falda M, Bussolati B, Tetta C, Camussi G: Mesenchymal stem cell-derived microvesicles protect against acute tubular injury. J Am Soc Nephrol 20: 1053–1067, 2009
      8. Oliver JA, Klinakis A, Cheema FH, Friedlander J, Sampogna RV, Martens TP, Liu C, Efstratiadis A, Al-Awqati Q: Proliferation and migration of label-retaining cells of the kidney papilla. J Am Soc Nephrol 20: 2315–2327, 2009
        9. Langworthy M, Zhou B, de Caestecker M, Moeckel G, Baldwin HS: NFATc1 identifies a population of proximal tubule cell progenitors. J Am Soc Nephrol 20: 311–321, 2009
        10. Takahashi K, Yamanaka S: Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663–676, 2006
        11. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S: Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131: 861–872, 2007
        12. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA: Induced pluripotent stem cell lines derived from human somatic cells. Science 318: 1917–1920, 2007
        13. Okita K, Ichisaka T, Yamanaka S: Generation of germline-competent induced pluripotent stem cells. Nature 448: 313–317, 2007
        14. Park IH, Lerou PH, Zhao R, Huo H, Daley GQ: Generation of human-induced pluripotent stem cells. Nat Protoc 3: 1180–1186, 2008
        15. Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S: Generation of mouse induced pluripotent stem cells without viral vectors. Science 322: 949–953, 2008
        16. Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K: Induced pluripotent stem cells generated without viral integration. Science 322: 945–949, 2008
        17. Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, Ebina W, Mandal PK, Smith ZD, Meissner A, Daley GQ, Brack AS, Collins JJ, Cowan C, Schlaeger TM, Rossi DJ: Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7: 618–630, 2010
        18. Anokye-Danso F, Trivedi CM, Juhr D, Gupta M, Cui Z, Tian Y, Zhang Y, Yang W, Gruber PJ, Epstein JA, Morrisey EE: Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell 8: 376–388, 2011
        19. Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S: Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 26: 101–106, 2008
        20. Hargus G, Cooper O, Deleidi M, Levy A, Lee K, Marlow E, Yow A, Soldner F, Hockemeyer D, Hallett PJ, Osborn T, Jaenisch R, Isacson O: Differentiated Parkinson patient-derived induced pluripotent stem cells grow in the adult rodent brain and reduce motor asymmetry in Parkinsonian rats. Proc Natl Acad Sci U S A 107: 15921–15926, 2010
        21. Liu GH, Barkho BZ, Ruiz S, Diep D, Qu J, Yang SL, Panopoulos AD, Suzuki K, Kurian L, Walsh C, Thompson J, Boue S, Fung HL, Sancho-Martinez I, Zhang K, Yates J 3rd, Izpisua Belmonte JC: Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome. Nature 472: 221–225, 2011
          22. Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, Lensch MW, Cowan C, Hochedlinger K, Daley GQ: Disease-specific induced pluripotent stem cells. Cell 134: 877–886, 2008
            23. Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, Croft GF, Saphier G, Leibel R, Goland R, Wichterle H, Henderson CE, Eggan K: Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321: 1218–1221, 2008
              24. Carvajal-Vergara X, Sevilla A, D'Souza SL, Ang YS, Schaniel C, Lee DF, Yang L, Kaplan AD, Adler ED, Rozov R, Ge Y, Cohen N, Edelmann LJ, Chang B, Waghray A, Su J, Pardo S, Lichtenbelt KD, Tartaglia M, Gelb BD, Lemischka IR: Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome. Nature 465: 808–812, 2010
                25. Wu SM, Hochedlinger K: Harnessing the potential of induced pluripotent stem cells for regenerative medicine. Nat Cell Biol 13: 734, 2011
                26. Inoue H, Yamanaka S: The use of induced pluripotent stem cells in drug development. Clin Pharmacol Ther 89: 655–661, 2011
                27. Lister R, Pelizzola M, Kida YS, Hawkins RD, Nery JR, Hon G, Antosiewicz-Bourget J, O'Malley R, Castanon R, Klugman S, Downes M, Yu R, Stewart R, Ren B, Thomson JA, Evans RM, Ecker JR: Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 471: 68–73, 2011
                28. Kim K, Doi A, Wen B, Ng K, Zhao R, Cahan P, Kim J, Aryee MJ, Ji H, Ehrlich LI, Yabuuchi A, Takeuchi A, Cunniff KC, Hongguang H, McKinney-Freeman S, Naveiras O, Yoon TJ, Irizarry RA, Jung N, Seita J, Hanna J, Murakami P, Jaenisch R, Weissleder R, Orkin SH, Weissman IL, Feinberg AP, Daley GQ: Epigenetic memory in induced pluripotent stem cells. Nature 467: 285–290, 2010
                  29. Stadtfeld M, Apostolou E, Akutsu H, Fukuda A, Follett P, Natesan S, Kono T, Shioda T, Hochedlinger K: Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature 465: 175–181, 2010
                  30. Gore A, Li Z, Fung HL, Young JE, Agarwal S, Antosiewicz-Bourget J, Canto I, Giorgetti A, Israel MA, Kiskinis E, Lee JH, Loh YH, Manos PD, Montserrat N, Panopoulos AD, Ruiz S, Wilbert ML, Yu J, Kirkness EF, Izpisua Belmonte JC, Rossi DJ, Thomson JA, Eggan K, Daley GQ, Goldstein LS, Zhang K: Somatic coding mutations in human induced pluripotent stem cells. Nature 471: 63–67, 2011
                  31. Pearl JI, Lee AS, Leveson-Gower DB, Sun N, Ghosh Z, Lan F, Ransohoff J, Negrin RS, Davis MM, Wu JC: Short-term immunosuppression promotes engraftment of embryonic and induced pluripotent stem cells. Cell Stem Cell 8: 309–317, 2011
                  32. Li H, Haurigot V, Doyon Y, Li T, Wong SY, Bhagwat AS, Malani N, Anguela XM, Sharma R, Ivanciu L, Murphy SL, Finn JD, Khazi FR, Zhou S, Paschon DE, Rebar EJ, Bushman FD, Gregory PD, Holmes MC, High KA: In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475: 217–221, 2011
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