Stemspan medium

Cord blood mononuclear cells (CBMCs) were directly obtained from the Cord Blood Bank of Seoul St. Mary’s Hospital. The Institutional Review Board (IRB) of the Catholic University of Korea, Seoul St. Mary’s Hospital approved this study. The reprogramming of CBMCs into iPSCs was induced using the Cytotune-iPS Sendai Reprogramming Kit (Life Technologies, Carlsbad, CA, USA). Briefly, CBMCs were seeded in a 24-well plate (3 × 10⁵ cells/well) with StemSpan™ medium (STEMCELL Technologies, Seattle, WA, USA). After addition of the viral components, the plate was centrifuged at 1160×g at 25 °C for 30 min and then incubated at 37 °C in 5% CO₂. On the next day, the cells were transferred to a 12-well plate coated with vitronectin (Life Technologies). The plate was centrifuged at 1160×g at 25 °C for 10 min, and Essential 8™ medium (Thermo Fisher Scientific, Waltham, MA, USA) was added at a 1:1 ratio. The cells were maintained in E8 medium until iPSC colonies were generated. The colonies were maintained in E8 medium (Thermo Fisher Scientific) on vitronectin-coated culture dishes. iPSCs were passaged every 3–4 days using Accutase Cell Detachment Solution (Global Cell Solutions, North Garden, VA) with Y-27632 dihydrochloride (R&D Systems, Minneapolis, MN, 10 µM). The medium was changed every day.

LSK48–150+, LSK depleted from CD48–150+ cell sorting and transduction were performed as previously described¹² . Briefly BM cells from bones of hind legs, spines and sterna of naïve, female B6 mice were crushed and stained with antibodies against Sca-1, c-Kit, CD48, CD150, CD3ε, Gr1, Ter119 and B220. Cells were sorted using MoFlo XDP and MoFlo Astrios (Beckman Coulter) cell sorters and prestimulated in StemSpan medium (STEMCELL Technologies) supplemented with 300 ng/ml stem cell factor (SCF), 1 ng/ml Flt3 ligand (Amgen), and 20 ng/ml IL-11 (R&R Systems) for 24 h. Cells were transduced with viral supernatant containing barcoded MIEV or 633-miR125a vectors in RetroNectin-covered plates (Takara Bio Inc.) in the presence of 2 µg/ml polybrene (Sigma-Aldrich).

Electroporation was performed using Lonza 4D Nucleofector (V4XP-3032 for 20-μl Nucleocuvette Strips or V4XP-3024 for 100-μl Nucleocuvette Strips) according to the manufacturer’s instructions. The modified synthetic sgRNA (2′-O-methyl 3′ phosphorothioate modifications in the first and last three nucleotides) were purchased from Synthego and BE mRNA was obtained through in vitro transcription using mMESSAGE mMACHINE^TM T3 Transcription kit (Invitrogen). A total of 2 × 10⁵ HSPCs from healthy donor were resuspended in 20 µL P3 solution and electroporated in 20-µL Nucleocuvette wells using program EO-100 with increasing concentration of BE mRNA and sgRNA (3 µg of BE mRNA and 3.2 µg sgRNA for HD CB cells and 6 µg of BE mRNA and 6.4 µg sgRNA for HD mPB cells). For 100-µL cuvette electroporation, 1 × 10⁶ HSPCs were resuspended in 100 µL P3 solution and electroporated using 30 µg of BE mRNA and 32 µg of sgRNA with program EO-100. FA Lineage negative cells were electroporated using similar conditions. Electroporated cells were resuspended in StemSpan medium (StemCell Technologies) with corresponding cytokines. Then, 24 h later, cells were used for transplant or maintained in culture for 5 days for DNA extraction and Sanger/NGS analysis to evaluate basal gene editing.