Fibroblast growth factor 2

The human iPSC-derived lt-NESCs were produced from human skin fibroblasts as previously described [9 (link)]. Briefly, human fibroblasts were subjected to retroviral transduction with plasmids encoding for the viral glycoprotein VSV-G and the reprogramming factors Oct4, Sox2, KLF4, and c-MYC and split into plates with mouse embryonic fibroblasts. Colonies were then picked and expanded to establish human iPSC lines. Those lines were induced to differentiate to neural phenotype as previously described [10 (link)] through an embryoid body-production step. Neural rosettes were generated and carefully picked and grown in the presence of 10 ng/ml fibroblast growth factor (FGF) 2, 10 ng/ml epidermal growth factor (EGF; both from R&D Systems, Inc., Minneapolis, MN, USA), and 1 μl/ml B27 (Invitrogen, Carlsbad, CA, USA). The human iPSC-derived lt-NES cell line was routinely cultured and expanded on 0.1 mg/ml poly-L-ornithine- and 10 μg/ml laminin- (both from Sigma-Aldrich, St. Louis, MO, USA) coated plates in the same media supplemented with FGF, EGF and B27. The lt-NESCs were passaged at a ratio of 1:2 to 1:3 every second to third day using trypsin (Sigma-Aldrich).

To generate MD-iPSCs, patient fibroblasts were infected with four retroviruses and then plated onto mitomycin C-treated (MMC; A.G. Scientific, San Diego, CA, USA) MEF feeder layers at a density of 10³ cells/cm². This study using patient fibroblasts was approved by the Institutional Review Board of Asan Medical Center, and written informed consent was obtained from their parents. Infected cells were cultured in human embryonic stem cell (ESC) medium at 37 °C, 5 % CO₂ in air for 2 to 3 weeks. The human ESC medium consists of DMEM/F12 (Invitrogen) supplemented with 20 % Knockout SR (Invitrogen), 1 % nonessential amino acids (Invitrogen), 1 % penicillin-streptomycin, 0.1 mM β-mercaptoethanol (Sigma, St. Louis, MO, USA), and 10 ng/ml fibroblast growth factor (FGF)2 (R&D systems, Minneapolis, MN, USA). Respective human ESC-like colonies were mechanically transferred onto new MMC-treated MEF feeders and subcultured for stabilization for 10 to 20 passages. iPSC characteristics were analyzed by the expression of human ESC markers, karyotypes, methylation states on promoters of human ESC marker genes, and teratoma formation. WT-iPSCs derived from foreskin fibroblasts [23 (link)] were used as a control group (Additional file 1: Figure S1).

Human bone marrow-derived MSCs were isolated with Institutional Review Board approval (University of Washington, Seattle, WA) from the femoral heads of patients undergoing hip arthroplasty using a standard plastic adhesion protocol. Cells flushed from the bone marrow were pelleted and resuspended in isolation medium (ISM) (α-minimum essential medium (MEM) + 1× antibiotic-antimycotic + 10% MSC qualified fetal bovine serum (FBS; all Gibco/Thermo Fisher Scientific, Waltham, MA, USA) + 1 ng/ml fibroblast growth factor (FGF)2 (R&D Systems, Minneapolis, MN)), seeded into T150 flasks (Corning Inc., Corning, NY, USA) and incubated for 3–4 days at 37 °C under 5% CO₂, then washed twice with phosphate-buffered saline (PBS; pH 7.4) and incubated in fresh isolated medium. When colonies reached 80% confluence, cells were harvested with trypsin/EDTA and resuspended at 1 × 10⁶ cells/T150 flask in ISM. Medium was changed every 3–4 days when cells reached 80–90% confluency, at which time cells were either frozen or passaged. MSC isolates were routinely characterized with respect to multilineage differentiation capabilities as described previously [30 (link)]. Multiple donor MSCs were pooled (Additional file 1: Table S1) and used in this study.