Stem cellbanker

Ten southland cells were stored at −80 °C in STEM CELLBANKER^® (Takara Bio Inc.) until use. The cells were washed with PBS and nuclei were extracted. The extracted nuclei were resuspended in 50 μl of transposition mix (100 nM TED1 (Illumina), 0.01% digitonin, and 0.1% Tween-20, in TD buffer (Illumina)) and incubated at 37 °C for 30 min with 1000 RPM mixing. DNA was extracted from the reaction mixture with DNA Clean and Concentrator (Zymo Research, Irvine, CA, USA). DNA library was prepared using NEBNext^® Ultra™ DNA Library Prep Kit for Illumina^® (New England BioLabs) with five cycles of pre-amplification and three to seven cycles of PCR amplification. The amplified DNA library was purified with Zymo DNA Clean and Concentrator (Zymo Research), followed by two size-selection steps with SPRIselect (1:0.6 and 1:0.2 sample vol. to beads vol.; Beckman Coulter, CA, USA). The Omni-ATAC-seq libraries were sequenced using 50 bp single-end reads on the HiSeq 2500 (Illumina). The obtained sequence reads were mapped to the human hg19 genome by bowtie2. Reads mapped to the mitochondrial genome and duplicated reads were removed using removeChrom.py (Harvard ATAC-seq module) and samtools, respectively. Peak calling was performed using macs2 with a 10⁻⁵ cutoff P value. The Omni-ATAC-seq was performed in two biological replicates.

The NE cells from monkey ESCs were detached with TrypLE Select (Thermo Fisher Scientific) and suspended in Dulbecco’s Modified Eagle Medium (DMEM)/F12 (Thermo Fisher Scientific) supplemented with 20 ng/mL FGF2, 200 ng/mL SHH, 20 ng/mL PDGF-AA (R&D Systems), 1 × B-27 Supplement without vitamin A (Thermo Fisher Scientific), 1 × N-2 Supplement (Thermo Fisher Scientific), 100 units/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich), and were then allowed to grow in suspension as spheres for 44 days. The medium was changed twice weekly. The spheres were treated with 1 × Accutase (Thermo Fisher Scientific) for 5 min and centrifuged. The cell pellet was resuspended with the same medium and dissociated into single cells by pipetting. The cells were transferred to plates coated with 0.01% poly-L-ornithine (Sigma-Aldrich) and 10 μg/mL laminin (Sigma-Aldrich) (poly-L-ornithine/laminin) and cultured for 7 days. The medium was changed twice weekly. The cells were detached with Accutase and centrifuged. The cells were suspended in STEM-CELLBANKER (Takara Bio) and cryopreserved in -80°C refrigerator.

CB-MSCs were isolated and cultured according to previously published protocols.^{10 (link),11 (link)} In brief, C57BL/6J mice were sacrificed with an overdose of pentobarbital. The femurs and tibiae were excised. Epiphyses were cut, and bone marrow was flushed out using a 27-gauge needle and syringe. Bone tissues were transferred into phosphate-buffered saline [PBS (-); FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan], cut into 1–2 mm fragments with scissors, and incubated with 0.25% collagenase (FUJIFILM Wako Pure Chemical Corporation) for 45 min. The culture medium containing cells was collected into another centrifuge tube through a 40 μm cell strainer.
The cells were cultured in α-minimum essential medium (FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% fetal bovine serum (FBS), 1% penicillin–streptomycin–amphotericin solution, and 10 ng/mL recombinant human bFGF (PeproTech, Rocky Hill, NJ, USA), which was used as a basic culture medium in this study. When the adherent cells (representing CB-MSCs) reached 80% confluence, the cells were trypsinized and resuspended in freeze-preserving medium (STEM-CELLBANKER; Takara Bio, Inc., Tokyo, Japan) until use.