Establishment of iPS cells
This study followed the tenets of the Declaration of Helsinki and was approved by the Institutional Ethics Committee of RIKEN Center for Biosystems Dynamics Research (authorization number: KOBE-IRB-13-23) and Kobe University (authorization number: 1624). Two LHON patients (“LHON 1” and “LHON 2”) with the mtDNA G11778A mutation and a healthy volunteer (Control 1) participated in the study. LHON 2 also has psychiatric disorder and type 2 diabetes mellitus and is thus deemed to have LHON plus, i.e., a LHON patient with systemic diseases [22].
LHON 1, LHON 2, and Control 1 each provided 40 ml of peripheral blood in Kobe University Hospital. Each iPS line was established by delivering episomal vectors (Center for iPS Cell Research and Application, Kyoto University) into peripheral blood mononuclear cells isolated from whole blood via electroporation. Patient-derived iPS cells were established from LHON 1 and LHON 2's samples, as previously reported [21]. Control iPS cells were derived from Control 1's sample using the same method. Another control sample (Control 2) was also established from PBMCs (iPS Portal, Kyoto, Japan) as previously described [23]. The LHON 1 iPS cells were deposited to RIKEN Bioresource Center (HPS1900, https://cellbank.brc.riken.jp/cell_bank/CellInfo/?cellNo=HPS1900). Lastly, the 201B7 cells were obtained (Center for iPS Cell Research and Application, Kyoto University).
Differentiation of three-dimensional retinal organoids
The iPSCs in this study were maintained and differentiated into 3D retinal organoids as described [19, 24]. Briefly, 3D retinal organoids were differentiated from iPS cells and maintained for 6 or 7 days. The iPS cells were dissociated and disseminated onto 96-well V-bottom plates at a density of 12,000 cells/well (Sumitomo Bakelite, Tokyo, Japan) in a differentiation medium (1:1 mixture of Iscove Modified Dulbecco Medium and F12 medium) (Gibco) supplemented with 10% knockout serum replacement (KSR), 1-thioglycerol, and lipid emulsion. On DD 6 or 7, 1.5 nM human BMP4 (R&D Systems, Minneapolis, MN, USA) were added to each well. Every 3 days, half the medium was replaced. On DD18 or 19, retinal organoids were harvested and removed to a 90-mm low-cell binding dish (Sumitomo Bakelite, Tokyo, Japan) in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12; Life Technologies) supplemented with 1% of N2 supplement (GIBCO), VEGFR/FGFR inhibitor (SU5402; Sigma-Aldrich Corp.), and 3 mM GSK-3 inhibitor (CHIR99021; Stemgent, Cambridge, MA, USA). On DD21, the medium was changed to DMEM/F12 supplemented with 10% fetal bovine serum, 1% N2 supplement, 100 nM retinoic acid (Sigma-Aldrich Corp), and 50 µM taurine (Sigma-Aldrich Corp).
Immunohistochemistry
The 3D-retinal organoids were fixed with 3% paraformaldehyde/phosphate-buffered saline (PFA/PBS) with 7.5% sucrose for 60 min, washed in 7.5% sucrose/PBS for 15 min, cryoprotected in 15% sucrose, and then in 30% sucrose, embedded with an OCT compound (Sakura Finetek, Tokyo, Japan), and cryopreserved.
Frozen sections with a thickness of 10 µm were heated in retrieval solution (0.1M citric acid, pH6.0) to improve antibody detection efficiency. After 1.5 h of blocking in 5% horse serum in 0.1% triton-X-100/PBS, the sections were incubated with the primary antibody against Crx (Takara Bio, 1:500) or Brn3b (Santa Cruz Biotechnology, 1:500) in 1% horse serum overnight, and then incubated with secondary antibodies Alexa Fluor 488 or 546, respectively. Next, the sections were counterstained with 4'6-diamidino-2-phenylindole (DAPI) and imaged using a confocal microscope (LSM700; Carl Zeiss, Japan, Germany).
Exposing retinal organoids to hypoxia
The retinal organoids harvested on around DD35 were, on designated Day 0, exposed to low oxygen concentration at 5% for 3 days, and then returned to normal oxygen concentration at 20%. The session of hypoxic exposure was repeated three times.
Measurement of the relative mtDNA copy number
On days 0, 3, 6, 9, 12, 15, and 18, 3 retinal organoids derived from each research participant were collected. Whole-genome DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen) according to the manufacture's protocol. The extracted whole-genome DNA was quantified, and its purity was evaluated by the A260/280 absorbance ratios using an ultraviolet spectrometer (NanoDrop, Thermo Fisher Scientific, Massachusetts, USA).
An mtDNA copy number relative to nuclear DNA (nDNA) was measured using real-time PCR with a human mtDNA detection primer set (Human Mitochondrial DNA (mtDNA) Monitoring Primer Set, Takara Bio Inc., Kusatsu, Japan). Briefly, the extracted genomic DNA was mixed with four primer pairs, including mtDNA primer pairs ND1 and ND5 and nDNA primer pairs SLCO2B1 and SERPINA1, to detect mtDNA and nDNA. PCR was performed at 95℃ for 30 sec, 95℃ for 5 sec for 40 cycles, and at 60℃ for 30 sec. The difference between the Ct values of the ND1 and SLCO2B1 reactions was defined as ΔCt1, and the difference between the Ct values of the ND5 and SERPINA1 reactions was defined as ΔCt2. MtDNA copy number was calculated as the average of 2ΔCt1 and 2ΔCt2.
Statistical Analysis
All statistical analysis were performed by Med Calc (version 19.6.4; MedCalc Software, Mariakerte, Belgium) that statistical significance was set at P < 0.05.