Stem cellbanker

Germ cells were isolated from Hy-line Brown layer embryos heterozygote for an RFP reporter gene (36 (link)) at Hamburger–Hamilton (HH) stage 16⁺ and cultured in vitro (37 (link)). Briefly, 1 μL of embryonic blood was aspirated from the dorsal aorta of embryos and placed in FAOT (FGF, Activin, ovotransferrin) culture medium (37 (link)). Expanded germ cell populations (3 wk) were cotransfected with 1.5 μg of high fidelity CRISPR-Cas9 vector (HF-PX459 V2.0) which included a targeting guide (single guide RNA [sgRNA]) for the DMRT1 locus and two single-stranded donor oligonucleotides (ssODNs) (5 pmol of each) (SI Appendix, Table S1) using Lipofectamine 2000 (Thermo Fisher Scientific) (21 (link)). Twenty-four hours after transfection, PGCs were treated with puromycin (at 400 ng/mL) for 48 h to select for edited cells. Following puromycin treatment, PGCs were sorted into single wells of 96-well plates using a FACSAria III (BD Biosciences) at one PGC per well in 110 μL of FAOT to produce clonal populations. PGCs were expanded in culture, DNA was extracted for analysis, and then clonal PGCs were cryopreserved in STEM-CELLBANKER (AMSBIO).

After collection, the blood samples were kept at room temperature (RT) before mononuclear cell isolation within 24h. Peripheral blood mononuclear cells (PBMCs) were isolated in a biosafety level 2+ cell laboratory under constant airflow in a laminar flow hood. All reagents used were devoid of animal-derived products. Human recombinant albumin (HAS) was used in place of fetal bovine serum to avoid any trigger of monocyte activation. All procedures were performed at RT. According to the manufacturer’s instructions, PBMCs were isolated using SepMate tubes (StemCell Technologies, UK) containing Lymphoprep (Serumwerk, Germany) by density gradient centrifugation. PBMCs were frozen in StemCellBanker (Amsbio, UK) at −80 °C. The next day, samples were cryopreserved in liquid nitrogen (−170°C) or −155°C freezer for long-term storage until further analysis.

Adipose tissue samples were obtained from one healthy donor during routine abdominoplasty with the patient’s informed consent and were isolated, cultured, and characterized at Histocell’s advanced cell therapy factory in Bilbao. Cells were cultured in DMEM+ 1% penicillin/streptomycin (Sigma–Aldrich, St. Louis, MO, USA) +2.5% platelet lysate (UltraGro, AventaCell BioMedical Corp. Ltd., Atlanta, GA, USA) culture medium, changing medium every 4 days, performing the necessary passages until a homogeneous MSC population was obtained (usually at passage 3 so that, upon thawing of the cells, they remain at passage 4). After culture, cells were frozen on a freezing ramp of −0.5 °C/min to −80 °C in freezing medium consisting of 10% StemCellBanker (Amsbio, Abingdon, UK), following protocols previously described by Nieto-Aguilar et al. in 2011 [27 (link)] and Carriel et al. in 2013 [28 (link)]. They were grown to passage 4 and seeded in six-well plates at a density of 50,000 cells/well. An automatic counter, BioRad TC-20 (BioRad, Hercules, CA, USA) was used for counting.