The utility of autologous induced pluripotent stem cell (iPSC) therapies for tissue regeneration depends on reliable production of immunologically silent functional iPSC derivatives. However, rejection of autologous iPSC-derived cells has been reported, although the mechanism underlying rejection is largely unknown. We hypothesized that de novo mutations in mitochondrial DNA (mtDNA), which has far less reliable repair mechanisms than chromosomal DNA, might produce neoantigens capable of eliciting immune recognition and rejection. Here we present evidence in mice and humans that nonsynonymous mtDNA mutations can arise and become enriched during reprogramming to the iPSC stage, long-term culture and differentiation into target cells. These mtDNA mutations encode neoantigens that provoke an immune response that is highly specific and dependent on the host major histocompatibility complex genotype. Our results reveal that autologous iPSCs and their derivatives are not inherently immunologically inert for autologous transplantation and suggest that iPSC-derived products should be screened for mtDNA mutations.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $20.83 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
All epitope sequences and 20-residue oligomer sequences are presented in the paper. All data supporting the findings of this study are available in the paper and its supplementary information files. Sequencing data are available from the Sequence Read Archive under accession code PRJNA544330.
de Almeida, P. E. et al. Transplanted terminally differentiated induced pluripotent stem cells are accepted by immune mechanisms similar to self-tolerance. Nat. Commun. 5, 3903 (2014).
Zhao, T., Zhang, Z. N., Rong, Z. & Xu, Y. Immunogenicity of induced pluripotent stem cells. Nature 474, 212–215 (2011).
Araki, R. et al. Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells. Nature 494, 100–104 (2013).
Guha, P., Morgan, J. W., Mostoslavsky, G., Rodrigues, N. P. & Boyd, A. S. Lack of immune response to differentiated cells derived from syngeneic induced pluripotent stem cells. Cell Stem Cell 12, 407–412 (2013).
Zhao, T. et al. Humanized mice reveal differential immunogenicity of cells derived from autologous induced pluripotent stem cells. Cell Stem Cell 17, 353–359 (2015).
Ji, J. et al. Elevated coding mutation rate during the reprogramming of human somatic cells into induced pluripotent stem cells. Stem Cells 30, 435–440 (2012).
Brown, W. M., George, M. Jr. & Wilson, A. C. Rapid evolution of animal mitochondrial DNA. Proc. Natl Acad. Sci. USA 76, 1967–1971 (1979).
Yakes, F. M. & Van Houten, B. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proc. Natl Acad. Sci. USA 94, 514–519 (1997).
Zastawny, T. H. et al. Comparison of oxidative base damage in mitochondrial and nuclear DNA. Free Radic. Biol. Med. 24, 722–725 (1998).
He, Y. et al. Heteroplasmic mitochondrial DNA mutations in normal and tumour cells. Nature 464, 610–614 (2010).
Kang, E. et al. Age-related accumulation of somatic mitochondrial DNA mutations in adult-derived human iPSCs. Cell Stem Cell 18, 625–636 (2016).
Loveland, B., Wang, C. R., Yonekawa, H., Hermel, E. & Lindahl, K. F. Maternally transmitted histocompatibility antigen of mice: a hydrophobic peptide of a mitochondrially encoded protein. Cell 60, 971–980 (1990).
Hanekamp, J. S. et al. Cytoplasmic inheritance of transplantation antigens in animals produced by nuclear transfer. Transplantation 88, 30–37 (2009).
Deuse, T. et al. SCNT-derived ESCs with mismatched mitochondria trigger an immune response in allogeneic hosts. Cell Stem Cell 16, 33–38 (2015).
Yates, A. et al. Ensembl 2016. Nucleic Acids Res. 44, D710–716 (2016).
Lee, A. S. et al. Effects of cell number on teratoma formation by human embryonic stem cells. Cell Cycle 8, 2608–2612 (2009).
Mercer, T. R. et al. The human mitochondrial transcriptome. Cell 146, 645–658 (2011).
Au-Yeung, B. B. et al. A sharp T-cell antigen receptor signaling threshold for T-cell proliferation. Proc. Natl Acad. Sci. USA 111, E3679–3688 (2014).
Shakiba, N. et al. Cell competition during reprogramming gives rise to dominant clones. Science 364, eaan0925 (2019).
Young, M. A. et al. Background mutations in parental cells account for most of the genetic heterogeneity of induced pluripotent stem cells. Cell Stem Cell 10, 570–582 (2012).
Quinlan, A. R. et al. Genome sequencing of mouse induced pluripotent stem cells reveals retroelement stability and infrequent DNA rearrangement during reprogramming. Cell Stem Cell 9, 366–373 (2011).
Laurent, L. C. et al. Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture. Cell Stem Cell 8, 106–118 (2011).
Sugiura, M. et al. Induced pluripotent stem cell generation-associated point mutations arise during the initial stages of the conversion of these cells. Stem Cell Reports 2, 52–63 (2014).
Diecke, S. et al. Novel codon-optimized mini-intronic plasmid for efficient, inexpensive, and xeno-free induction of pluripotency. Sci. Rep. 5, 8081 (2015).
Deuse, T. et al. Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients. Nat. Biotechnol. 37, 252–258 (2019).
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
Andrews, R. M. et al. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat. Genet. 23, 147 (1999).
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Koboldt, D. C. et al. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res. 22, 568–576 (2012).
Wang, K., Li, M. & Hakonarson, H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164 (2010).
The UniProt Consortium UniProt: the universal protein knowledgebase. Nucleic Acids Res. 45, D158–D169 (2017).
Fleri, W. et al. The immune epitope database and analysis resource in epitope discovery and synthetic vaccine design. Front. Immunol. 8, 278 (2017).
Kim, Y. et al. Immune epitope database analysis resource. Nucleic Acids Res. 40, W525–530 (2012).
We thank C. Pahrmann for cell culture work, assistance with experiments and immunoblots; G. Tediashvili for assistance with the in vivo experiments; and M.S. Mahadevan for computer assistance. Special thanks to R. Jaenisch (MIT, Department of Biology, MIT, Cambridge, MA) for providing the NT-ESCs and to J. Wu (Stanford Cardiovascular Institute, Stanford, CA) and P.-L. So (Stem Cell Core, Gladstone Institutes, San Francsico, CA) for providing the mouse and human iPSCs. Special thanks to D. Sun and X. Wu for their help in developing the targeted mtDNA sequencing and droplet digital PCR assays. We thank R. Nelson and K. Copeland for their support with in vivo imaging and M. Lewis for his support with the Xcelligence experiments. S.S. received research grants from the Deutsche Forschungsgemeinschaft (DFG; grants SCHR992/3-1, SCHR992/4-1), the Fondation Leducq (CDA 2013-2015) and the DZHK (German Center for Cardiovascular Research; grant FKZ 81Z2710105). D.W. was supported by the Max-Kade-Foundation (DFG). M.H.S. received an NIH shared instrumentation grant (1S10OD018040-01). B.P. and Z.K.-Y. were supported by the NIH (grant 1R21AI134127-01). S.A.-E., A. M., Y. Y. and H. V. were supported by National Heart Lung and Blood Institute (NHLBI) Intramural Research Program.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
a-b, B/c splenocytes from animals receiving syngeneic fibroblasts overexpressing syngeneic B/c or allogeneic B6 Co3 (a) or Cytb proteins (b) were harvested and analyzed by mass cytometry (representative pictures of 6 animals per group). Statistical scaffold analysis was performed on the resulting data to identify immune cell populations of differing frequencies. Immune cell subsets that are significantly increased or decreased in animals receiving B6-overexpressing cells are shown in red and blue, respectively. Landmark cell populations manually identified in the data are shown in black and facilitate interpretation of the map.
Supplementary Figure 2 Animals challenged with allogeneic proteins have expanded effector / memory T cell populations.
a-d, Animals were immunized with B/c or B6 Co3 proteins (a, c) or Cytb (b, d). Mass cytometry data were analyzed for the frequency of effector / memory CD4 (a, b) and CD8 (c, d) T cell subsets in the spleen (mean ± s.d., 6 animals per group, two-tailed Student’s t-test). Frequencies of total antigen experienced (CD44+), short-lived effector (CD45+CD27−), effector memory (CD45+CD27+CD62L−), and central memory (CD45+CD27+CD62L+) CD4 or CD8 T cells are shown.
Supplementary Figure 3 There is no cross-reactivity between fetal nuclear gene-derived antigens and the Co3 and Cytb 20mers.
B/c mice were immunized with B/c ESCs, which were suspended in saline and injected into the thigh muscle. After 5 days, ELISpot assays were performed with recipient splenocytes to test reactivity against 20mers carrying the B6-specific (blue) or B/c-specific (red) amino acid of the SNP at position 6 or 15 (mean ± s.d., quadruplicates of 4 animals per protein, two-tailed Student’s t-test).
a, Total DNA was extracted from donor tissue and recipient blood. mtDNA was enriched and amplified and mtDNA libraries were created. After sequencing, raw reads underwent quality control and map reads were aligned to human rCRS. After removal of duplicates and pileup of variants, either heteroplasmic or homoplasmic donor-recipient SNP variants were identified. b, Of 15 patient pairs, the number of homoplasmic nonsynonymous mtDNA SNPs is shown. c, Nonsynonymous homoplasmic SNPs were found at different frequencies in 12 mitochondrial proteins. d, The number of nonsynonymous mtDNA SNPs in donor-recipient pairs by ethnic background is shown.
Blood was drawn from three organ recipients 3 weeks after transplantation. All patients were on a regimen including tacrolimus, mycophenolate mofetil, and steroids with therapeutic tacrolimus trough levels (mean ± s.d., n = 3 patients). PBMCs were isolated using Ficoll gradient centrifugation and one fraction was immediately tested for their immune responsiveness (0 h). Another fraction of PBMCs was washed and cultured in the presence of anti-CD3 and anti-CD28 for 24 h before being tested (24 h). PBMCs were unspecifically stimulated with PMA and ionomycin and their IL2 expression was quantified by PCR (mean ± s.d., n = 3 patients, two-tailed Student’s t-test) and their IFNγ release by ELISpot assays (mean ± s.d., quadruplicates of n = 3 patients, two-tailed Student’s t-test).
Five more patients are displayed from the human transplant study outlined in Figure 2 (mean ± s.d., quadruplicates per 20mer and patient, two-tailed Student’s t-test). Blue shades correspond to donor, red shades to recipient 20mers. ** P < 0.01.
Six patients with 4-5 nonsynonymous mtDNA SNPs to their donor were chosen for this study. Blood was drawn 6 months after liver or kidney transplantation and PBMCs underwent in vitro reactivation. In Elispot assays, PBMCs were challenged with autologous (red) or two sets of allogeneic 20mers reflecting mtDNA SNPs of their donor (blue) or another unconnected donor (green; mean ± s.d., quadruplicates per 20mer and patient, two-tailed Student’s t-test). Among the depicted SNPs, yellow asterisks mark SNPs that are identical between the patient’s donor and the unconnected donor. ** P < 0.01.
a, Blood was drawn from two volunteers for 4-digit HLA typing. Using in silico prediction models, the top 5 peptides with the highest predicted immunogenicity and bottom 5 SNPs with the lowest predicted immunogenicity from the annotated human SNP library were identified for each volunteer. b, 20mers were generated to cover either the mutant or autologous SNP and used in extended naïve in vitro immunization Elispot assays with volunteer PBMCs (Volunteer 1: mean ± s.d., triplicates per 20mer, two-tailed Student’s t-test; Volunteer 2: mean ± s.d., quadruplicates per 20mer, two-tailed Student’s t-test). Blue shades correspond to autologous, yellow shades to mutant 20mers. c, In silico predictions for B6 MHC binding was performed for the 3 neoantigenic SNP candidates identified in P37 iPSCs. d, B6 mice were immunized with autologous P7 or P37 iPSCs and after 5 days, splenocytes were incubated in Elispot assays with 20mers from the 2 neoantigen candidate SNPs or the B6 reference SNPs. e, Elispot assays from mice immunized with P7 iPSCs (mean ± s.d., quadruplicates of 6 animals per 20mer, two-tailed Student’s t-test). f, Elispot assays from mice immunized with P37 iPSCs (mean ± s.d., quadruplicates of 6 animals per 20mer, two-tailed Student’s t-test). g, FVB mice were immunized with autologous P13 or P38 iPSCs and after 5 days, splenocytes were incubated in Elispot assays with 20mers from the neoantigen candidate SNP or the FVB reference SNP. h, Elispot assays from mice immunized with P13 iPSCs (mean ± s.d., quadruplicates of 6 animals per 20mer, two-tailed Student’s t-test). i, Elispot assays from mice immunized with P38 iPSCs (mean ± s.d., quadruplicates of 6 animals per 20mer, two-tailed Student’s t-test). Blue shades correspond to the B6 reference 20mer, green to the FVB reference 20mer, and yellow shades to the neoantigenic 20mer. ** P < 0.01.
a, FVB mice received different cell amounts of autologous low-passage (P13) or high-passage (P38) iPSCs grafts injected into the thigh muscle and were followed for immune response and teratoma development. b, After 5 days, splenocytes were recovered for Elispot assays (mean ± s.d., quadruplicates of 6 animals per group, two-tailed Student’s t-test). c, Teratoma growth is depicted for every animal and the percentage of teratoma formation for each group is shown in a separate bar graph (n = 6 per cell amount and iPSC group).
a, B6 iPSC grafts from P7 and P37 were injected into the thigh muscle of immunodeficient SCID-beige mice. b, Teratoma growth of iPSCs P7 and P37 was followed and the percentage of teratoma formation for each group is shown in a separate bar graph (Individual animals are shown, n = 4 per group). c, In vitro growth of P7 and P37 iPSC cultures was captured with time-laps life cell imaging and the confluency at 0 min and 24 h is shown (representative pictures of two independent experiments). d, The calculated cell proliferation after 24 h is shown (mean ± s.d., 5 replicates per group, two-tailed Student’s t-test).
a, iPSC grafts from P7 and P37 were injected below the kidney capsule of syngeneic B6 recipients. b-d, Infiltration of P7 and P37 iPSC grafts with CD3+ lymphocytes (b), CD335+ natural killer cells (c), and F4/80+ macrophages (d) by immunofluorescence staining is shown. Scale bar 25 μm. e, P37 iPSC grafts at two low cell amounts, which were completely or mostly rejected in immunocompetent B6 mice in Figure 3, were injected into the thigh muscle of B6 recipients immunosuppressed with tacrolimus. f, Elispot assays with splenocytes recovered after 5 days are shown (mean ± s.d., quadruplicates of 5 animals per group). g, Teratoma growth is depicted for every animal and the percentage of teratoma formation for each group is shown in a separate bar graph (n = 5 animals per group). h, P37 iPSC grafts at the same two low cell amounts were then transplanted into immunocompromized CD4 knockout recipients on B6 background. i, Elispot assays with splenocytes recovered after 5 days are shown (mean ± s.d., quadruplicates of 5 animals per group). j, Teratoma growth is depicted for every animal and the percentage of teratoma formation for each group is shown in a separate bar graph (n = 5 animals per group).
a, iPSCs (P7) underwent CRISPR-Cas9 inactivation of the B2m and Ciita genes to generate double MHC knockout (dKO) iPSCs and iEC (dKO) were differentiated. b, The knockouts of B2m and Ciita in iEC (dKO) were confirmed by PCR (representative gel of two independent experiments). c, Surface expression of MHC class I and II was assessed by flow cytometry (mean ± s.d., 4 independent experiments per group, two-tailed Student’s t-test). d, B/6 mice were immunized with either iEC (P7) or iEC (dKO) overexpressing B/c (red) or B6 (blue) Co3 and Cytb. The splenocyte response after 5 days against the same overexpressed proteins in either iEC (P7) or iEC (dKO) was assessed in Elispot assays to determine the mechanistic role of MHC. e, Elispot assays of B/6 mice immunized with iEC (P7) or iEC (dKO) overexpressing syngeneic B/6 proteins are shown (mean ± s.d., quadruplicates of 3 animals per group, two-tailed Student’s t-test). f, Elispot assays of B/6 mice immunized with iEC (P7) or iEC (dKO) overexpressing allogeneic B/c proteins are shown. Unstimulated responder splenocytes served a control (mean ± s.d., quadruplicates of 3 animals per group, two-tailed Student’s t-test).
PBMCs were repeatedly isolated from 6 volunteers over a minimum of 6 months and mtDNA sequencing was performed. SNPs in the non-coding D-loop are marked in red. SNPs with < 1% heteroplasmy in the first specimen that increased to > 1% in subsequent specimens were considered de novo mutations and are marked in green. SNPs that have been present in the first PBMC specimen and changed their heteroplasmy >1% over time are marked in black. None of the volunteers developed candidate SNPs for neoantigens in vivo.
Supplementary Figs. 1–13 and Supplementary Notes 1–5
Supplementary Tables 1–18
The proliferation of B6 P7 and P37 iPSCs in vitro was recorded over 24 h and then quantified.