Fibronectin coated dishes

Peripheral blood samples were obtained from patients before and three months after MSC injection. EPCs were isolated from samples using Ficoll-Paque and five million cells were seeded on 6-well fibronectin-coated dishes (BD biosciences) in CFU-Hill medium (stem cell technologies, cat#05900) (Hill et al., 2003 (link), Solomon et al., 2012 (link)). The non-adherent cells were collected 48 h later and one million cells were seeded on 24-well fibronectin-coated dishes. On day five, EPC-CFUs were counted in five wells and the average was obtained.

The peripheral blood of patients with NEC and healthy donors were subjected to peripheral blood mononuclear cells (PBMCs) isolation using a gradient with Ficoll-Paque (Amersham Biosciences, Piscataway, NJ). The PBMCs were further undertaken positive selection for CD14+ monocytes with anti-CD14 Dynabeads (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Cells were then cultured for 48 hours in fibronectin-coated dishes (BD Biosciences, San Jose, CA) in Dulbecco's modified Eagle's medium (Life Technologies, Gaithersburg, MD) supplemented with 20% fetal bovine serum (FBS; Invitrogen). Nonadherent cells were then removed by gentle aspiration, and they were cultured for another 14 days. Some of sample was further subjected to flow cytometry (FCM) analysis to determine the fibrocytes using specific fibrocyte markers. Isolated cultured fibrocytes were treated with 10 lg/mL of lipopolysaccharide (LPS, InvivoGen, San Diego) for 12 hours.

Human iPSCs were differentiated into MSCs as previously described (Choi et al., 2017b ; Mahmood et al., 2010 (link)). Briefly, human iPSC colonies were dissected into small clumps (approximately 0.5 mm × 0.5 mm in size). These clumps were transferred to uncoated Petri dishes (SPL Lifesciences, Pocheon, Korea) and incubated in EB medium for 2 days. The EB medium consisted of DMEM/F12 (Invitrogen, Carlsbad, CA) with 10% Knockout SR (Invitrogen), 1% nonessential amino acids (Invitrogen), 1.2 mg/mL sodium carbohydrate, and 0.1 mM β-mercaptoethanol (Sigma, St. Louis, MO). In this medium, the clumps spontaneously aggregated to form embryoid bodies (EBs). These EBs were cultured in the same medium supplemented with 10 μM SB431542 (Cayman Chemical, Ann Arbor, MI) for 8 d with media changes every other day. After 10 d of incubation, the EBs were attached to fibronectin-coated dishes (BD Biosciences, Franklin Lakes, NJ) and then further cultured in DMEM/F12 supplemented with 1 μM SB431542, 1% insulin plus transferrin liquid supplement (Sigma), 2% B27 supplement (Invitrogen), and 1% CD lipid (Invitrogen) for 8–10 additional days with medium changes every other day. For MSC maturation, the cells were then cultured in α-MEM (Invitrogen) containing 10% FBS for 14 days with medium changes once every 3 days.

The green fluorescent protein (GFP)-induced male Sprague-Dawley rats (SD-Tg(CAG-EGFP); Sankyo Lab Inc., Tokyo, Japan) were used. The heart was isolated under anesthesia and rinsed with phosphate-buffered saline (PBS; Wako, Tokyo, Japan) containing heparin sodium to wash out the blood. The atrium of the heart was diced into small pieces and was subjected to 10-min digestion with 0.05% trypsin-EDTA (Sigma-Aldrich, Tokyo, Japan). These explants were plated onto fibronectin-coated dishes (BD Biosciences, Tokyo, Japan) in Iscove’s modified Eagle medium (IMDM; Life Technologies, Tokyo, Japan) supplemented with 20% fetal bovine serum (FBS; Thermo Scientific, Yokohama, Japan), 1% penicillin-streptomycin (Life Technologies), and 2 mM l-glutamine (Life Technologies). Two weeks later, a primary outgrowth of adherent cells grew out radially in monolayer from the cardiac tissue. The outgrowth cells were then harvested to continue to culture to second passage. The cells were stored at −80°C and transferred to liquid nitrogen until thawed 1 week before injection.