The Rejection Barrier to Induced Pluripotent Stem Cells

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.^5–9

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 vitro^20–25 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,^27–29 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.

Disclosures

None.

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