Mitochondria are essential organelles for eukaryotes that perform vital functions, such as ATP-induced energy production, the synthesis of nucleotides, calcium homeostasis, inflammation, and apoptosis (1, 2). Human mtDNA is double-stranded and encodes 13 proteins, two rRNAs, and 22 tRNAs (3). Defects in mtDNA are associated with inherited and acquired degenerative diseases in humans (4-6). Kearns-Sayre syndrome, chronic progressive external ophthalmoplegia, and Pearson syndrome (PS) are known to be caused by large-scale mtDNA deletions (7-9).

Nuclear genes, such as POLG and SSBP1, are essential in mtDNA replication and proofreading (10, 11). The defect in mtDNA replication could lead to single or multiple mutations in mtDNA and result in mtDNA deletions (12). Co-deficient expression of POLG and SSBP1 genes, caused by nuclear mutations and hypermethylation, could induce large mtDNA deletions (13).

Screening for genetic defects in the cells for clinical application should be involved in selecting intact cells. Mutations in mtDNA could occur randomly in individual cells (14), and the heteroplasmic level of the same mutation could be different in each cell (15, 16). Therefore, cells with a low or absent number of mtDNA mutations could be selected among pooled cells for therapeutic application. Furthermore, previous studies have demonstrated that the heteroplasmic level of mtDNA deletion in iPSCs could be reduced during expanded culture or differentiation, even the original tissue of the cells harbored high heteroplasmy in humans (17, 18). However, the presence of nuclear mutations was not analyzed in those studies.

In our previous study, the PS patient harbored nuclear mutations in POLG and SSBP1 genes, which could induce deficient gene expression. Finally, large mtDNA deletions occurred in the multiple organs of the patient (13). Based on these results, we attempted to examine whether iPSC clones in this patient could maintain the deletion level during cell differentiation. Therefore, we generated single-cell derived iPSC clones in PS patient, selected iPSC clones with or without mtDNA deletion, and analyzed the deletion levels during in vitro and in vivo differentiation to examine the potential for autologous cell therapy (Fig. 1A).

Screening of mtDNA deletions in iPSCs derived from blood and skin fibroblasts in PS patient

In a previous study, we identified large mtDNA deletion in PS patient, 4,395 base pairs (bp) located between the ATPase6 (mt8921) and ND5 (mt13316) genes, in skin tissue with 9% heteroplasmy and 24% with blood (Fig. 1B) (13). We generated iPSC clones from skin fibroblasts (e.g., skin-derived iPSCs, SiPSCs) and peripheral blood mononuclear cells (PBMCs) of blood cells (blood-derived iPSCs, BiPSCs). SiPSCs and BiPSCs showed typical morphology of human iPSCs (19) (Fig. 1C).

Thirteen SiPS clones were randomly selected and analyzed for patient-specific mtDNA deletion (4,395 bp deletion). Three SiPS clones (SiPS1, 9, and 12) did not exhibit mtDNA deletion, whereas the levels in the other 10 SiPS clones ranged from 7 to 53% (Fig. 2A). Furthermore, 10 BiPS clones were analyzed in patient-specific mtDNA deletion, resulting in no mtDNA deletion in all BiPS clones (Fig. 2B). The average heteroplasmy of mtDNA deletion in 13 SiPS clones was 19% in SiPSCs, whereas there was no mtDNA deletion in any of the BiPS clones. (Fig. 2C).

We also analyzed the presence of other mtDNA deletions, including the common deletion of 4,977 bp in BiPS clones (20). Among the 10 BiPS clones, five (BiPS1, 3, 4, 8, and 9) exhibited no deletion, while the other five clones showed 1-7% heteroplasmy (Fig. 2D).

In the expansion culture of iPS clones, the high heteroplasmy of mtDNA deletion, such as 53% heteroplasmy in SiPS2, did not survive until passage 5, while SiPS10 clones with 27% heteroplasmy survived (Supplementary Fig. 1A).

In summary, some BiPS clones had no patient-specific and other deletions, including common mtDNA deletion. These clones could have an advantage for clinical treatment regarding the safety of the deletion-free iPSC clones. For further study, we used the deletion-free BiPS clones (BiPS1, 3, and 8) and SiPS10 with 27% heteroplasmic deletion (red boxes in Fig. 2A, B) to induce differentiation in vitro and in vivo.

Pluripotency of the deletion-free iPSC clones

Before differentiation in vitro and in vivo, the deletion-free iPSC clones (BiPS1, 3, and 8) were selected and analyzed for pluripotent status. Pluripotent stem cell markers, including OCT4, SOX2, and NANOG (21), were analyzed by qRT-PCR, resulting in the gene expression levels being significantly increased in iPSC clones compared to original cells (Supplementary Fig. 2A). Furthermore, OCT4, SSEA4, and TRA-1–60 proteins, as markers of pluripotent stem cells (22), were detected in all BiPSC clones (Supplementary Fig. 2B). Based on the results, pluripotency was confirmed in the deletion-free BiPS clones.

Maintenance of deletion-free during in vitro and in vivo differentiation

The deletion-free iPSC clones, BiPS1, 3, and 8, were performed the differentiation in vitro. BiPS clones were aggregated in 3D culture dishes and differentiated to EB, which were expressed as three germ layer markers (Fig. 3A, B and Supplementary Fig. 3A) (22, 23). PS patient-specific mtDNA deletion was examined in differentiated EBs, and no deletion was found in any of the EBs (Supplementary Fig. 1B). Furthermore, different deletions, including 4,977 bp common deletion, were not detected in all EBs (Supplementary Fig. 1B).

These deletion-free BiPSC clones (BiPS1, 3, and 8) were injected into immunosuppressed mice to analyze the maintenance of the deletion-free status during in vivo differentiation. Subsequently, teratomas were formed, and three germ layers were observed (Fig. 3C, D and Supplementary Fig. 3B) (22, 24). All teratomas derived from BiPS clones were neither detected with PS patient-specific mtDNA deletion nor other deletions (Supplementary Fig. 1C).

The SiPS10 with 27% heteroplasmy was examined for the formation of EB and teratoma. The EB was formed, and three germ layer markers were expressed significantly higher in EB than in the original iPS clone (Fig. 3E, F). However, the relative mRNA expressions of three germ layer markers in EB were lower than those in BiPS clones (Fig. 3B). Nevertheless, the heteroplasmy of EB was 25% similar to the original iPS clone (Supplementary Fig. 1B). Teratomas were formed and observed three germ layers with 45% heteroplasmy, which was increased than original iPS clone (Fig. 3G, H and Supplementary Fig. 1C). These results demonstrated that mtDNA deletion was not regenerated during in vitro and in vivo differentiation in the deletion-free iPS clones, while the deletion level was retained or increased after differentiation when original iPSCs harbored deletion.

The PS patient with nuclear mutations in the POLG and SSBP1 genes harbored patient-specific mtDNA deletion (mt8921– 13316 deletion) in skin tissue and blood, and multiple iPSC clones were generated from the skin fibroblasts and PBMCs. The iPSC clones were screened for mtDNA deletion level, and the deletion-free iPSC clones were selected for differentiation. The deletion-free iPSC clones induced EB formation in vitro and teratoma formation in vivo, resulting in non-patient-specific deletion status after differentiation in vitro and in vivo. We further analyzed other mtDNA deletions, including the 4,977 bp common deletion, which was not detected after in vitro and in vivo differentiation. These results demonstrated that mtDNA deletion in iPSCs was not newly generated during differentiation, suggesting that deletion-free iPSC clones could be a valuable source for autologous cell therapy in patients.

Autologous cell therapy in patients has the advantage of eliminating the risk of immune rejection and reducing the need for immunosuppressant treatment (25-27). However, the application of patient-derived cells in autologous cell therapy may be limited in the presence of potential gene mutations that could induce cellular malfunction and systemic disease (28). The PS patient in our study presented with a nuclear mutation in both the POLG and SSBP1 genes, which could affect the induction of large mtDNA deletion in multiple organs (13). Further, mtDNA deletion is associated with mitochondrial dysfunction. The previous reports demonstrated that the fibroblasts and iPSCs of PS patients with mtDNA deletion displayed lower mitochondrial metabolism than cells with no deletion (13, 17). In addition, the SiPS clones with the highest heteroplasmic mtDNA mutations could not survive after freezing and thawing, which could be also associated with mitochondrial dysfunction (17).

Thus, it was imperative to investigate whether mtDNA deletions could re-appeared during differentiation for clinical applications. To address this concern, the deletion-free iPSC clones of the PS patient should be confirmed to not re-appear deletion status following in vitro and in vivo differentiation. Further, the nuclear mutation in PS patients could produce other deletions including the 4,977 bp common deletion, which could be also investigated. Our results demonstrated that mtDNA deletions were not generated during in vitro and in vivo differentiation in iPSCs, suggesting that deletion-free iPSC clones of PS patients could be a safe candidate for cell therapy.

In this study, all BiPS clones had no identified mtDNA deletion even though the original PBMC was 27% heteroplasmy. The previous study reported that heteroplasmy levels of the 4,977 bp common mtDNA deletion were various among the type of blood cells (29). Of them, monocytes and B lymphocytes had fewer deletion levels than T lymphocytes and granulocytes. Probably, our BiPS clones could be established from monocytes (30). Several studies reported that the level of mtDNA deletion in iPSCs could be reduced during in vitro expanded culture or differentiation (17, 18). On the other hand, the deletion levels were increased after teratoma formation in vivo (31).

The present study demonstrated that strategies, such as the screening of mtDNA deletion in iPSCs and the selection of the deletion-free iPSC clones, could be suitable to select the cell sources for clinical application in patients harboring systemic mtDNA deletion induced by nuclear mutation. Further, an opportunity for autologous transplantation for patients with nuclear mutations may be provided by these strategies.

Ethics

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the Asan Medical Center (IRB number 2017-0260). Informed consent was obtained from the subject involved in the study.

PBMC isolation

PBMC isolation was performed as previously described (14). PBMCs were isolated from whole blood using Lymphoprep (STEMCELL Technologies) according to the manufacturer’s protocol. Briefly, the diluted blood with an equal volume of PBS supplemented with 2% fetal bovine serum (FBS) was centrifuged in a density gradient medium at 1,200 x g for 15 min at room temperature. Then, a white buffy coat layer containing PBMC was collected.

Skin fibroblast culture

Isolated fibroblasts from skin tissues were cultured as previously described (13). Fibroblasts were cultured using Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12 medium, Life Technologies), supplemented with 10% FBS (Life Technologies) and 4 ng/ml of basic fibroblast growth factor (bFGF; Sigma).

Generation of iPSCs

PBMCs or skin fibroblasts were transduced with a CytoTune-iPS Reprogramming Kit (Life Technologies) according to the manufacturer’s protocol. One day before transduction, 1 × 10⁵ cells per well were plated in 6-well culture dishes containing DMEM/F12 with 10% FBS. The cells were transduced with reprogramming vectors (at a multiplicity of infection of 5) diluted with 1 ml of medium per well. One week after transduction, cells were replated onto 100 mm dishes containing feeder layers of mitomycin C-inactivated mouse embryonic fibroblasts. iPSC colonies appeared 3–4 weeks after transduction. Colonies were randomly picked, manually propagated and plated onto 6-well culture dishes coated with 5 μg/ml vitronectin (Life Technologies) in StemMACS medium (Miltenyi Biotec).

Quantitative PCR for the detection of patient-specific mtDNA deletion

Real-time PCR to detect patient-specific mtDNA deletion was performed, as previously described (13). DNA was extracted using the PicoPure DNA Extraction Kit (Qiagen). Regions of the mtDNA, including patient-specific mtDNA deletion (mt8921–13316), were amplified by PCR using the following primer set: F-8822 CTATAAAC-CTAGCCATGGCC and R-13436 GGTATGGTTTTGAGTAGTCC. Wild mtDNA was amplified using PCR with the following primer set: F-16483 GTGAACTG-TATCCGACATCTG and R-100 CAGCGTCTCGCAATGCTATC. PCR reactions were performed under the following conditions: 1 cycle at 95°C for 10 min, then 40 cycles at 95°C for 15 s, 60°C for 30 s and 72°C for 30 s, and finally one cycle at 72°C for 5 min. Heteroplasmy for each deletion was calculated as follows: heteroplasmy of deletion (%) = quantity of deletion mtDNA/quantity of wild mtDNA × 100.

Quantitative PCR for the detection of mtDNA deletions, including common deletion

Real-time PCR to detect mtDNA deletions, including common deletion, was performed as previously described with minor modifications (32). Briefly, real-time PCR was performed using a probe-based, single-tube multiplex qPCR assay on a QuantStudio 6 Flex Real-Time PCR System (Life Technologies). PCR reactions were performed under the following conditions: 95°C for 10 min, 40 cycles of 95°C for 15 s, 55°C for 15 s, and 60°C for 1 min.

Immunocytochemistry

Immunocytochemistry was performed as previously described (22). Fixed iPSCs with 4% formaldehyde were permeabilized with 0.1% Triton X-100 in 0.1% PBS/Tween for 30 min at room temperature and blocked using a blocking solution consisting of 0.1% PBS/Tween with 3% BSA for 30 min at room temperature. After blocking, the cells were incubated with primary antibody (TRA-1-60, SSEA4, and OCT4, Stemgent) solution diluted with 0.1% PBS/Tween (1:100) in 4°C overnight. Cells were washed three times with 0.1% PBS/Tween and incubated for 1 hour at room temperature with a secondary antibody solution (1:1,000). Samples were imaged and captured using a Carl Zeiss inverted microscope.

EB formation

EB formation was performed as described previously, with minor modifications (22). Dissociated iPSCs were seeded onto a StemFIT 3D culture dish (MicroFIT) and cultured in Essential 6 (Gibco) with 100 U/ml penicillin, 100 mg/ml streptomycin (GE Life Science), and 10 μM fasudil (Adooq) for two days. Aggregated cells were transferred to Petri dishes and cultured for six days using the same medium.

Quantitative RT-PCR (RT-qPCR)

Total RNAs were isolated using RNeasy Mini Kits (Qiagen, Valencia, CA, USA), and reverse transcription was performed with cDNA synthesis kits (PCR Biosystems). qRT-PCR was performed according to the previously described (13) with minor modifications using the following gene-specific primers: OCT4 F-GACAGGGGGAGGGGAGGAGCTAGG, R-CTTCCCTCCAACCAGTTGCCCCAAA; SOX2 F-AGCTACAGCATGATGCAGGA, R-GGTCATGGAGTTGTACTGCA; NANOG F-TGAACCTCAGCTACAAACAG, R-TGGTGGTAGGAAGAGTAAAG; PAX6 F-TCCGTTGGAACTGATGGAGT, R-GTTGGTATCCGGGGACTTC; TBXT F-ACCCAGTTCATAGCGGTGAC, R-CCATTGGGAGTACCCAGGTT; SOX17 CAAGGGCGAGTCCCGTAT, R-CGACTTGCCCAGCATCTT; GAPDH F-CCCATGTTCGTCATGGGTGT, R-TGGTCATGAGTCCTTCCACGATA. The expression levels for each gene were measured with qRT-PCR using Power SYBR Green PCR Master Mix (Applied Biosystems). The reaction of qRT-PCR was as follows: one cycle at 95°C for 10 min; followed by 40 cycles at 95°C for 30 s, 56°C for 30 s, and 72°C for 30 s; and a final elongation step at 72°C for 5 min. Gene expression levels were expressed as relative 2^−∆∆Ct values.

Teratoma formation

Teratoma formation was performed as previously described (22). iPSCs were injected into the femoral region of 7-week-old NOD-SCID Gamma mice (Jackson Laboratory) using a 1 ml syringe (Korea Vaccine Co.). Mice with tumors were euthanized, and isolated teratomas were sectioned and stained with hematoxylin and eosin. Further, mtDNA deletion in teratomas was also analyzed.

Statistical analysis

Statistical comparisons between the two groups were made using the Student’s two-tailed t-test. Statistical analyses were performed using Prism 8.0.1 software (GraphPad). Data are reported as the mean ± standard error of the mean (SEM).

The authors have no conflicting interests.