α mem basal medium

The normal hiPSCs (DYR0100) were purchased from ATCC. HA-iPSCs [32 (link)] and T-7-iPSCs [33 (link)] were generated previously. Briefly, HA-iPSCs were reprogrammed by urine cells from a severe HA patient with F8 intron 22 inversion. T-7-iPSCs were obtained by site-specific integration of CMV-driven BDDF8 at the rDNA locus in HA-iPSCs. All hiPSCs were routinely cultured (37 °C, 5% CO₂) on Matrigel- (BD Biosciences, Franklin Lakes, NJ, USA) coated 12-well plates (Corning, New York, NY, USA) in mTesR Plus medium (StemCell Technologies, Vancouver, BC, Canada).
The iMSCs were routinely cultured (37 °C, 5% CO₂) on 0.1% gelatin (StemCell Technologies, Vancouver, BC, Canada) coated 10-cm dishes (Corning, New York, NY, USA) in αMEM basal medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% FBS (Gibco, New York, NY, USA), 2 mM GlutaMAX^TM (Gibco, New York, NY, USA), and 0.1% bFGF (Gibco, New York, NY, USA).

BM from the femoral head was scooped into a sterile pot and transferred into a sterile Falcon tube containing 5 mL 0.5 M EDTA, pH = 8.0, and maintained at 4℃. Samples were immediately washed 3 times with DPBS, passed through a 70 μm strainer to remove bone fragments and cell clumps, and centrifuged at 300 g for 15 min at 25℃. Cells were resuspended in DPBS, then stratified on Ficoll Histopaque 1077 (Sigma-Aldrich) and centrifuged at 300 g for 45 min at 25℃ (with acceleration set at 1 and without deceleration). BM mononuclear cells layered at the interphase were collected and washed with DPBS. Cell viability was assessed by trypan blue staining. A total of 1 × 10⁷ cells were seeded into flasks in αMEM basal medium (ThermoFisher Scientific) supplemented with 20% (v/v) FBS and cultured for 48 h at 37℃, 5% CO₂. At this point, adherent cells were considered BM mesenchymal stem cells (BM-MSCs) and further expanded in αMEM + 20% (v/v) FBS.