Pcxwb ebna1

Plasmids used in this study were: 1) pAC_SV40ori, a derivative of pEco3’Δ, supplied by C. Schildkraut [23 (link)], obtained after removal of the EcoRI-XhoI 1,680 bp fragment corresponding to the FR of the EBV’s oriP region and insertion of the AvaI 1,437 bp fragment from pBR18 into the AvaI site to obtain a plasmid of 6,126 bp; 2) pAC_EBVoriP, a derivative of pHEBo, supplied by J. Yates [24 (link)] obtained after removal of the SalI-SspI 842 bp fragment containing no relevant elements; 3) pEco3’Δ; and 4) pCXWB-EBNA1 (Addgene number: #37624) containing the coding region of EBNA1.

The human induced pluripotent stem cell (hiPSC) line FF-PB-3AB4 was established from a healthy donor’s peripheral blood mononuclear cells (PBMCs), as described previously^{46 (link),47 (link)}. In brief, PBMCs were electroporated with the episomal vectors pCXLE-hOCT3/4-shp53 (#27077; Addgene, Cambridge, MA, USA), pCXLE-hSK (#27078; Addgene), pCXLE-hUL (#27080; Addgene), and pCXWB-EBNA1 (Addgene; #37824) using the Nucleofector IIb device (Lonza, Basel, Switzerland) and plated on iMatrix-511-coated cell culture plates (Nippi, Inc., Tokyo, Japan). The iPSCs were induced by changing the medium to StemFit medium (Ajinomoto, Tokyo, Japan). Twenty-nine days after electroporation, colonies were isolated and expanded for validation.
The institutional review board of Kobe University Graduate School of Medicine approved this study (No. 1722), and informed consent was obtained from the donor. FF-PB-3B4 showed typical human embryonic stem cell-like morphology (Fig. S1A), expressed pluripotent stem cell markers OCT3/4 and NANOG (Fig. S1B) and had the ability to differentiate into cells comprising all three germ layers in vitro (Fig. S1C). In addition, we performed a “pluritest” and confirmed that the generated clone was pluripotent and similar to validated normal PSCs (Fig. S1D). Karyotype of FF-PB-3AB4 was normal (data not shown).

pCXLE-hOCT3/4-shp53-F, pCXLE-hUL, pCXLE-hSK, pCXLE-hGLIS1, and pCXWB-EBNA1 were purchased from Addgene. pCXLE-hTET-1 was constructed. iMATRIX-511 was purchased from Nippi^® (Tokyo, Japan). Fibroblasts (1.0 × 10⁶) from each tissue sample were prepared, then gene transfection for iPS cell reprogramming was performed with the electroporation device Nucleofector 2b (Lonza Japan^®, Tokyo, Japan) using the U-020 protocol, VPI-1002 transfection kit (Lonza Japan^®), 1.2 µg each of pCXLE-hOCT3/4-shp53-F, pCXLE-hUL, pCXLE-hSK, pCXLE-hGLIS1, and pCXLE-hTET-1, and 1.0 µg of pCXWB-EBNA1. To reduce the possibility of the induction efficiency being decreased by the entry of a nick when using the stocked vectors, we irradiated the vectors before use. After electroporation, the cells were seeded on 100-mm dishes that had previously been coated with collagen and 0.125 µg/cm² of iMATRIX-511. In protocol 1, after day 1 post-transfection, they were cultured in an incubator set at 37 °C with 5% O₂. In protocol 2, from day 1 to day 5 post-transfection, the cells were cultured in an incubator set at 37 °C with 20% O₂; after day 5 post-transfection, they were cultured in an incubator set at 37 °C with 5% O₂.

A skin biopsy was obtained from a 12‐year‐old female patient carrying a compound heterozygous variant c.2548G>A p.(Gly850Ser) and c.4006‐10A>G in the CRB1 gene and a healthy control (WT). Human dermal fibroblasts were reprogrammed using integration‐free episomal vectors: pCXLE‐hOCT3/4‐shp53‐F (Addgene, Watertown, MA, USA; Catalogue No. 27077), pCXLE‐Hsk (Addgene; Catalogue No. 27078), pCXLE‐Hul (Addgene; Catalogue No. 27080), and pCXWB‐EBNA1 (Addgene; Catalogue No. 37624) as previously described [48 (link)]. hiPSC were differentiated towards a retinal lineage [49 (link)]; briefly, hiPSCs were grown to 80–90% confluency (day 0), mTesR™ Plus was replaced by Essential 6™ (Thermo Fisher Scientific; Catalogue No. A1516401). From day 2 to day 28, differentiating cells were subjected to a neural induction medium Essential 6™, 1% N2 supplement (Stemcell Technologies, Cambridge, UK; Catalogue No. 17502048) and 0.1% Pen/Strep. By 3–4 weeks, neural retina‐like structures emerged from the cell layer and were manually dissected at day 28 using a 21G needle and further cultured until day 35 in maturation media [DMEM/F12 (Thermo Fisher Scientific; Catalogue No. 21331020), 1% MEM NEAA (Thermo Fisher Scientific; Catalogue No. 10370021), 2% B27 supplement (Thermo Fisher Scientific; Catalogue No. 12587010), 10 ng/ml FGF2 (Thermo Fisher Scientific; Catalogue No. 100‐18B), and 0.1% Pen/Strep].

A human iPSC line previously established from peripheral mononuclear cells obtained from a healthy subject employing episomal vectors (pCXLE-hOCT3/4-shp53-F, Cat.#. 27077; pCXLE-hSK, Cat.#. 27078; pCXLE-hUL, Cat.#. 27080; and pCXWB-EBNA1, Cat.#. 37624, Addgene, Watertown, MA, United states) was used in this study. Human iPSCs were maintained on iMatrix-511 (Cat.#. 892021, Matrixome, Osaka, Japan)-coated 6-well plates (Cat.#. 3516, Corning, Glendale, Arizona, United States) in StemFit AK02N (Cat.#. AK02N, Ajinomoto, Tokyo, Japan), and were passaged every 7 days.