Induced pluripotent stem cell-derived gamete-associated proteins incite rejection of induced pluripotent stem cells in syngeneic mice

Abstract

The safety of induced pluripotent stem cells (iPSCs) in autologous recipients has been questioned after iPSCs, but not embryonic stem cells (ESCs), were reported to be rejected in syngeneic mice. This important topic has remained controversial because there has not been a mechanistic explanation for this phenomenon. Here, we hypothesize that iPSCs, but not ESCs, readily differentiate into gamete-forming cells that express meiotic antigens normally found in immune-privileged gonads. Because peripheral blood T cells are not tolerized to these antigens in the thymus, gamete-associated-proteins (GAPs) sensitize T cells leading to rejection. Here, we provide evidence that GAPs expressed in iPSC teratomas, but not in ESC teratomas, are responsible for the immunological rejection of iPSCs. Furthermore, silencing the expression of Stra8, 'the master regulator of meiosis', in iPSCs, using short hairpin RNA led to significant abrogation of the rejection of iPSCs, supporting our central hypothesis that GAPs expressed after initiation of meiosis in iPSCs were responsible for rejection. In contrast to iPSCs, iPSC-derivatives, such as haematopoietic progenitor cells, are able to engraft long-term into syngeneic recipients because they no longer express GAPs. Our findings, for the first time, provide a unifying explanation of why iPSCs, but not ESCs, are rejected in syngeneic recipients, ending the current controversy on the safety of iPSCs and their derivatives.

Keywords: CD4+ T cells; gamete-associated proteins; rejection of induced pluripotent stem cells.

Figure 1

Induced pluripotent stem cells (iPSCs) are rejected by CD4⁺ T cells. (a) To determine whether iPSCs are rejected in syngeneic mice, luciferase‐expressing 129x1/SvJ iPS or embryonic stem cells (ESCs) were injected into 129x1/SvJ mice, n = 6. Mice were imaged regularly to determine the engraftment of the cells. iPSCs could not be detected after 14 days. (b) ESCs () were not rejected in syngeneic mice over the 40 days of observation. In contrast iPSCs () were rejected after a mean of 12 days. Furthermore, mice challenged for a second time with iPSCs () rejected those iPSCs within 5–6 days. For statistical analysis, the Log rank test was used. *P < 0·05, **P < 0·01. (c) To prove that both iPSCs and ESCs were pluripotent, the teratoma assay was performed in NOD‐SCID mice. In both cases, large teratomas developed. This is a representative result for the 129SvJ cells. (d) To determine the mechanism of iPSC rejection, splenocytes of mice that had rejected iPSCs were collected and CD4⁺ and CD8⁺ cells were sorted. The cells were exposed to iPS‐embryoid body (EB) cells in a proliferation assay. iPSCs, but not ESCs, stimulated CD4⁺ T cells derived from animals that had rejected iPSCs. In contrast, CD8⁺ T cells minimally proliferated to stimulation by iPS‐EB cells (e). Bothe CD4⁺ and CD8⁺ T cells from naive animals proliferated minimally. iPS‐EB cells induce T‐cell stimulation much more than ES‐EBs. These experiments were performed in triplicates in three mice and repeated twice. **P < 0·01 and *P < 0·05. (f) iPSCs are pluripotent and iPS‐EB cells poorly express MHC I and MHC II molecules. iPS‐EB cells do not express MHC antigens. EBs were harvested on day 7 and the cells were used to measure MHC class I and class II expression. 129x1/SvJ splenocytes were used as controls. EB cells hardly express any class I or class II antigens. Open histograms indicate class I or class II positive population and filled histograms indicate isotype control staining. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Meiotic and spermatogenetic genes expressing induced pluripotent stem cells (iPSC) embryoid bodies (EBs) are rejected in syngeneic mice. (a) To determine, whether iPSC derivatives are rejected in syngeneic mice like iPSCs, we differentiated the 129x1/SvJ iPSCs into haematopoietic progenitor cells (HPCs). These iPSC‐derived HPCs express sca‐1⁺/c‐kit⁺, which are haematopoietic progenitor markers, indicating that those iPSCs are successfully differentiated into HPCs. (b) To determine whether iPSC‐derived HPCs engraft in syngeneic mice, 129x1/SvJ mice were sublethally irradiated and transplanted with the 129x1/SvJ iPS‐HPCs. After that, these mice were subsequently injected with 129x1/SvJ iPSCs, embryonic stem cells (ESCs) or with both, subcutaneously. The luminescence emitted by the teratomas (21 days post transplantation) are shown if they are not rejected (circles denote original injection sites). Mice transplanted with luciferase‐expressing iPSCs either singly or together with luciferase‐expressing ESCs rejected iPSCs, but not ESCs (Each group of mice comprised 10 mice, and these experiments were repeated four times). However, GFP‐expressing iPS‐HPCs were detected in chimeric mice long term in the peripheral blood system (6 weeks after iPSC transplantations) (c). To identify the genes that are differently expressed, we performed quantitative PCR analysis expression between iPSCs and ESCs. (d) iPS‐EBs but not ES‐EBs strongly expresses genes involved in meiosis and spermatogenesis (top row). In particular, Stra8, Vasa and Mga were strongly expressed in iPS‐EBs compared with in ES‐EBs. The genes displayed in the lower row either remained unchanged or were down‐regulated. These quantitative PCR experiments were repeated at least four times in triplicates. ***P < 0·001, **P < 0·01 and *P < 0·05. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Silencing of Stra8 abrogates rejection of induced pluripotent stem cells (iPSCs). (a) Embryonic stem cells (ESCs) express Stra8 under treatment with retinoic acid (RA) and testosterone (TT). To demonstrate that ESCs are capable of undergoing meiosis, ESCs were allowed to form embryoid bodies (EBs) with or without treatment with both RA and TT. The iPSCs were treated the same way. Indeed ES‐EBs only expressed Stra8 under treatment with TT and RA but not when left untreated. In contrast iPS‐EBs expressed Stra8 without further treatment with RA and TT. However, Stra8 expression was stronger in the treated iPSCs. (b) Stra8 protein expression is silenced in Stra8‐shRNA‐iPSCs. The control iPSCs (scRNA‐iPSCs) and Stra8‐silenced iPSCs (Stra8‐shRNA‐iPSCs) were either left alone or treated with RA. scRNA‐iPSCs responded to RA treatment by expression of Stra8, whereas Stra8‐shRNA‐iPSCs do not express Stra8 as expected. (c) Sensitized CD4⁺ T cells with iPSCs highly proliferated to dendritic cells pulsed with Stra8 overexpressed cell lysates. In contrast, sensitized CD8⁺ T cells showed similar level of naive T‐cell responses (***P < 0·001). (d) Silencing of Stra8 leads to down‐regulation of the expression of several critical genes in meiosis. To determine whether meiotic gene expression is regulated by Stra8, Stra8‐shRNA‐iPS cell‐EBs and scRNA‐iPS cell‐EBs were formed and used to extract RNA for quantitative PCR. As expected, Stra8 gene expression was abrogated in the Stra8‐shRNA‐iPS cell‐EBs but not in the scRNA‐iPS cell‐EBs. In addition, Dazl, Stella, Vasa and Scyp3 were significantly down‐regulated. Ct values were first normalized within the sample to the housekeeping gene GAPDH before comparison across samples. ***P < 0·001, **P < 0·01 and *P < 0·05. (e) Rejection of iPSCs is after silencing Stra8. To determine the impact of the down‐regulation of the meiotic programme on iPS rejection, 129x1/SvJ mice were transplanted subcutaneously with either shRNA‐iPSCs (n = 10) or Stra8‐shRNA‐iPSCs (n = 10). ESCs were used as controls (n = 10). scRNA‐iPSCs were rejected in normal fashion, the mean of the rejection day was day 12. As expected, ESCs were not rejected. However, Stra8‐shRNA‐iPS cell showed delayed rejection suggesting that abrogation of meiosis delays rejection of iPSCs. The difference in the rejection mean times was highly significant, For statistical analysis, Log rank test was used (P = 0·02).