Inducible pluripotent stem cells (iPSCs) hold great promise as therapeutic mediators of organ and tissue repair. In addition to their capacity to produce unlimited progeny that can be differentiated into a cell type of choice,1 iPSCs are generated from fibroblasts and therefore do not raise the ethical issues associated with embryonic stem cells.2,3 In the context of transplantation, differentiated iPSCs have the potential to promote the repair of injuries sustained through ischemia/reperfusion injury, fibrosis, or tissue atrophy, thereby enhancing the function and long-term success of an allograft. Alternatively, these cells could be used as a first-line treatment for tissue injury as a means of reducing the demand for donor organs.4
For transplantation, an advantage of iPSC-derived tissues is that they may be produced from autologous cells. Nevertheless, there is debate as to whether autologous iPSCs may acquire a degree of immunogenicity resulting from residual expression of embryonic antigens, genetic alterations following plasmid uptake, or from epigenetic changes during the culturing period.5 Notwithstanding this challenge, the extensive process of generating and differentiating autologous cells to obtain the desired type is highly impractical for treating acute conditions.1 In these situations, pre-expanded allogeneic iPSC-derived cells may be helpful but carry the additional drawback of triggering an alloresponse. In xenotransplantation, producing genetically edited hypoimmunogenic organs has revived hopes in the field.6 Engineering iPSCs and their derivatives to have no immunogenic potential may indeed represent a most relevant milestone.
Deuse and coworkers show that the overexpression of CD47, in combination with inhibition of MHC Class I and II expression, enables iPSCs to avoid immune clearance with great efficacy and thereby to achieve long-term survival in allogeneic hosts.1 To achieve this, murine iPSCs (miPSCs) with knockouts of B2m and Ciita, the respective genes encoding MHC Class I and II, were engineered using CRISPR-Cas9. CD47, a transmembrane protein with a key role in preventing innate responses against self-peptides, is already known as an inhibitor of phagocytosis by macrophages and dendritic cells.7 In accordance with the “missing self” theory, which proposes that natural killer (NK) cells respond against aberrant or absent expression of MHC Class I molecules,8 CD47 was transduced into B2m−/−Ciita−/− iPSCs to compensate for the loss of MHC through inhibition of the NK cell cytotoxic response. In transplanted iPSCs, abolition of MHC Class I and II with simultaneous overexpression of CD47 led to the absence of detectable IFN-γ or antibody responses in allogeneic mice. B2m−/−Ciita−/−Cd47 miPSCs showed evidence of teratoma formation in vivo, which, along with expression of pluripotency genes, verified the ability of transgenic miPSCs to maintain their capacity for differentiation. The role of CD47 in protection against NK cells was confirmed in experiments by blocking CD47 resulting in NK cell-mediated killing of transgenic stem cells and their derivatives.1 The mechanism by which CD47 prevents NK cell cytotoxicity remains to be determined but will be of value to future studies in transplantation.
iPSCs were subsequently differentiated into the following 3 derivative lines: cardiomyocytes, endothelial cells, and smooth muscle cells. All transgenic B2m−/−Ciita−/−Cd47-derived cells displayed the standard surface markers, morphology, and gene expression of their mature target cell lines. After transplantation into allogeneic mice, 100% of transgenic cells survived the observation period (50 days) in the absence of immunosuppression. Moreover, virtually no immune cell infiltration or cytokine upregulation was observed in the transplanted cell environment.
In further experiments, human iPSCs (hiPSCs) were engineered from CD34+ cord blood with CRISPR-Cas9-targeted removal of B2M and CIITA, followed by lentiviral transduction with CD47; hiPSCs were subsequently differentiated into endothelial-like cells (hiECs) and cardiomyocyte-like cells (hiCMs).1 As with transgenic miPSCs, the differentiated cells exhibited downregulation of HLA I and II, and upregulation of CD47. The functionality and immunogenicity of derivatives were then assessed in allogeneic humanized NSG-SGM3 mice that rapidly rejected unmanipulated hiPSC derivatives. Conversely, B2M−/−CIITA−/− CD47 derivatives not only survived the observation time but also failed to provoke a cytokine or antibody response. Of major importance, the transgenic hiECs were reported to gradually form primitive three-dimensional structures resembling vasculature, a small number of which contained erythrocytes. The transgenic hiCMs developed into polarized structures in keeping with the role of mature cardiomyocytes in heart contraction.
Together, these observations suggest that derivatives of iPSCs can mediate structural biogenesis of specific tissue types in vivo (Figure 1).
The authors raised a fundamental issue surrounding immunologically invisible cell lines, namely the potential for malignancies and viral infections to develop unchecked by host defenses. Many previous studies have explored the use of safety switches or suicide genes to improve the safety of cell therapies. This strategy would allow external intervention to prevent disease progression, and therefore presents a possible solution to this problem.2,9 Another safety consideration relates to the tissue-specific functionality of differentiated iPSCs. For example, results from the literature indicate that the electrical properties of iPSC-derived cardiomyocytes are present but immature, and improvements need to be made before they are suitable for therapeutic applications.10 While this should not be seen as an impenetrable barrier for stem cell research, it highlights the wider importance of assessing and optimizing the functionality of iPSC-derived cells of various types to ensure that they perform to the standards of adult somatic cells. It would be particularly interesting to determine whether hypoimmunogenic iPSC derivatives can repair existing organ damage in humanized models. Through powerful approaches to modify cellular immunogenicity, stem cell therapy is slowly maturing.
1. Deuse T, Hu X, Gravina A, et al. Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients. Nat Biotechnol. 2019; 37:252–258
2. Sułkowski M, Konieczny P, Chiebanoska P, et al. Introduction of exogenous HSV-TK suicide gene increases safety of keratinocyte-derived induced pluripotent stem cells by providing genetic “emergency exit” switch. Int J Mol Sci. 2018; 19:E197
3. Akram KM, Patel N, Spiteri MA, et al. Lung regeneration: endogenous and exogenous stem cell mediated therapeutic approaches. Int J Mol Sci. 2016; 17:E128
4. Xu H, Yi BA, Chien KR. Shortcuts to making cardiomyocytes. Nat Cell Biol. 2011; 13:191–193
5. Wood KJ, Issa F, Hester J. Understanding stem cell immunogenicity in therapeutic applications. Trends Immunol. 2016; 37:5–16
6. Cooper DKC, Ezzelarab M, Iwase H, et al. Perspectives on the optimal genetically engineered pig in 2018 for initial clinical trials of kidney or heart xenotransplantation. Transplantation. 2018; 102:1974–1982
7. Mohme M, Riethdorf S, Pantel K. Circulating and disseminated tumour cells - mechanisms of immune surveillance and escape. Nat Rev Clin Oncol. 2017; 14:155–167
8. Malmberg KJ, Carlsten M, Björklund A, et al. Natural killer cell-mediated immunosurveillance of human cancer. Semin Immunol. 2017; 31:20–29
9. Ivics Z. Self-destruct genetic switch to safeguard ips cells. Mol Ther. 2015; 23:1417–1420
10. Poon E, Kong CW, Li RA. Human pluripotent stem cell-based approaches for myocardial repair: from the electrophysiological perspective. Mol Pharm. 2011; 8:1495–1504
Engineering iPSCs and their derivatives to have no immunogenic potential may indeed represent a most relevant milestone.