Recombinant human leukemia inhibitory factor

Mouse iPSCs were generated in our laboratory using a similar approach as described previously 20, 40. Mouse iPSCs were cultured in gelatin‐coated flasks (phosphate buffer saline [PBS] containing 0.02% gelatin from bovine skin; Sigma‐Aldrich, SIGMA; England, UK http://www.sigmaaldrich.com/united-kingdom.html) in Dulbecco's modified Eagle's medium (ATCC) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 μg/ml streptomycin (Thermo Fisher Scientific), 10 ng/ml recombinant human leukemia inhibitory factor (Millipore); and 0.1 mM 2‐mercaptoethanol (Invitrogen) in a humidified incubator supplemented with 5% CO₂. The cells were passaged every 2 days at a ratio of 1:6. Differentiation of iPSCs was induced by seeding the cells on type IV mouse collagen (5 μg/ml)‐coated dishes in differentiation media (DM) containing α‐modified Eagle's medium supplemented with 10% FBS (Invitrogen), 0.05 mM 2‐mercaptoethanol, 100 units/ml penicillin, and 100 μg/ml streptomycin in the presence of 25 ng/ml VEGF for the time points indicated.

ASCs were isolated from mouse subcutaneous, abdominal, and perivascular adipose tissue, as described previously (Gu et al., 2019 (link)). Briefly, adipose tissue was cut into 1-mm³ small pieces, which was then digested with 2 mg/mL collagenase type I (Gibco) and 1 mg/mL Dispase II (Sigma) in Hanks’ balanced salt solution (HBSS) at 37°C for 30–45 min with occasional vortex. The digestion was stopped by DMEM/F12 with 10% FBS and subsequently passed through a 100-μm filter followed by a centrifugation at 300 g for 5 min. Cell precipitates were resuspended in a stem cell culture medium as the following: Minimal Essential Medium-α (Gibco) with 15% fetal bovine serum (Gibco), 10 ng/mL recombinant human leukemia inhibitory factor (Sigma), 5 ng/mL recombinant human FGF (R&D), 2 mmol/L L-glutamine (Sigma), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco); they were then incubated in a 5% CO₂ incubator. The cells were passaged at a ratio of 1:3 every 2 or 3 days. The medium was refreshed every 2 days.

Periaorta adipose tissues surrounding thoracic aorta (from the aortic arch to the aortic hiatus) of five 8-week-old C57BL/6J mice were pooled for each primary culture. Finely minced adipose tissue was washed with PBS once and then digested with 2 mg/mL collagenase type I (Life Tech, 17018–029) for 30 minutes at 37°C in a shaker with a speed set at 100 rpm. The pellet was resuspended in stem cell culture medium (α- minimal essential medium [MEM] with 15% embryomax [Millipore, ES-009-B], 0.1 mmol/L 2-mercaptoethanol [Sigma], 10 ng/mL recombinant human leukemia inhibitory factor [Chemicon, Temecula, CA], 5 ng/mL bFGF [basic fibroblast growth factor; R&D systems], 2 mmol/L L-glutamine [Sigma], 100 U/mL penicillin, and 100 mg/mL streptomycin [GIBCO, Grand Island, NY]) and placed in a 5% Co₂ incubator. ADSCs from miR-378a knockout mice were isolated from the inguinal subcutaneous adipose tissue and cultured in the same manner. Cells within 10 passages were used.

Sca1⁺‐VPCs were isolated from the outgrowth of adventitial tissues of mouse arterial vessels, as previously described.12, 13 Briefly, the arterial vessels were harvested from C57BL/6J mice (Charles River, Margate, Kent, UK) or Hd7‐7sFLAG transgenic mice and cut into 2‐mm rings after the removal of the intima and media; the pieces were placed in gelatin‐coated flasks and incubated at 37°C in a humidified incubator supplemented with 5% CO₂ for 6 hours. Stem cell culture medium ([Dulbecco's modified Eagle medium (DMEM); ATCC, Rockville, Maryland] supplemented with 10 ng/mL recombinant human leukemia inhibitory factor [Chemicon, Temecula, California], 10% fetal bovine serum [FBS, ATCC], 0.1 mmol/L 2‐mercaptoethanol, 100 U/mL penicillin, and 100 U/mL streptomycin) was added and refreshed every other day until the cells reached 80% confluence. The cells were expanded and subjected to Sca‐1⁺ cell purification using anti‐Sca‐1 immunomagnetic microbeads Miltenyi Biotec (Bergisch Gladbach, Germany). The purity of isolated Sca‐1⁺ cells was confirmed to around 85% using flow cytometry.12, 13, 14 The Sca‐1⁺‐VPCs were maintained in stem cell culture medium and split every other day. Cells passaged up to 30 times were used in this study, and Sca1‐selection was performed every five passages.

Primary RB cells were isolated as previously described with modifications (14 (link)). In brief, fresh human RB tumor samples were washed three times in ice-cold phosphate buffer saline (PBS). Dissociation into single cells was achieved by incubation in 0.25% trypsin-0.01% EDTA (Invitrogen, ThermoFisher Scientific) at 37°C for 10 min and gentle pipetting, then trypsin was neutralized with addition of fetal bovine serum (FBS, Gibco ThermoFisher Scientific). Dissociated cells were filtered with 70 μm cell strainer (Corning) and centrifuged at 1,000 rpm for 5 min, and followed by resuspension in serum free Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (Gibco, ThermoFisher Scientific) supplemented with 20 ng/mL recombinant human leukemia inhibitory factor (Chemicon). Isolated cells were maintained at 37°C, 5% CO₂ under a humidified atmosphere.