Differentiation of hESCs and iPSCs to VSMCs was essentially conducted as described by Xie et al. (2007 ) with minor modifications. Briefly, EBs were generated as described above and cultured in suspension in low cluster plates. After 7 days, EBs were plated on 0.1% gelatin‐coated plates for 3–5 days. EB outgrowths were trypsinized and cells were replated on Matrigel in Medium 231 (with growth supplement, Life Technologies). To differentiate VSMCs, cells were plated on gelatin‐coated six‐well plates and grown in VSMC differentiation medium (Medium 231, differentiation supplement, Life Technologies) for 7 days and characterized at each passage.
Medium 231
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
The Medium 231 is a general-purpose laboratory equipment designed for a variety of applications. It functions as a controlled environment for incubating, culturing, or storing samples at a set temperature and humidity. The device's core purpose is to provide a stable and consistent environment to support the growth or preservation of biological materials.
Automatically generated - may contain errors
Lab products found in correlation
Smooth muscle growth supplement, by Thermo Fisher Scientific (12 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Hasmc, by Thermo Fisher Scientific (4 mentions)
Hcasmc, by Thermo Fisher Scientific (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Smooth muscle growth supplement smgs, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Harvard Bioscience (2 mentions)
Collagenase, by Worthington (2 mentions)
Smooth muscle differentiation supplement, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Human umbilical vein endothelial cells (huvec), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Sodium l ascorbate, by Merck Group (1 mentions)
Collagenase type 2, by Worthington (1 mentions)
Dihydrotestosterone dht, by Merck Group (1 mentions)
Flutamide, by Selleck Chemicals (1 mentions)
Recombinant mouse tgf β, by R&D Systems (1 mentions)
39 protocols using medium 231
1
Differentiation of hESCs/iPSCs to VSMCs
2
Smooth Muscle Cell Differentiation
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To measure the expression level of lncRNAs and their neighboring protein-coding genes during differentiation of smooth muscle cells, we cultured HCASM cells (Gibco) according to the manufacturer’s protocol. These cells were maintained in Medium 231 (Gibco) supplemented with Smooth Muscle Growth Supplement (Gibco), and harvested for the sample of synthetic phenotype. To obtain the sample of contractile or differentiated phenotype, HCASM cells stabilized in the growth media were cultured in Medium 231 supplemented with Smooth Muscle Differentiation Supplement (Gibco) for three days, and then collected. We used TRIzol Reagent (Invitrogen) to extract total RNA, performed reverse transcription using RevertAid Reverse Transcriptase (Thermo Scientific) with Random Hexamers (Invitrogen). Quantitative real time polymerase chain reaction (PCR) was performed using SYBR Green PCR Master Mix (Applied biosystems) in Rotor-Gene Q (Qiagen). The primer sequences for PCR were included in the supplementary table. The differentiation of these cells was confirmed by measuring the level of Calponin 1 (CNN1) and Smooth muscle protein 22-alpha (SM22α). For normalization, the expression level of Glyceraldehyde 3-phosphate dehydrogenase (GADPH) was used.
3
COL6A1 silencing in human aortic vascular smooth muscle cells
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T/G Human aortic vascular smooth muscle cells (T/G HA-VSMC; HA-VSMC thereafter; cat. no. CL-0452; Procell Life Science & Technology Co., Ltd.) were cultured in Medium 231 (Life Technologies; Thermo Fisher Scientific, Inc.) supplemented with 5% smooth muscle growth supplement (SMGS; Thermo Fisher Scientific, Inc.) at 37°C under 5% CO2. Fresh culture medium was changed every two days. HA-VSMCs (1×105 cells) were then transfected with 0.25 µg siRNA specific for COL6A1 (si-COL6A1) or siRNA-control (EV; both purchased from Shanghai GenePharma Co., Ltd.) using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol, the cells were named si-COL6A1 and EV cells thereafter. The sequences of si-COL6A1 were: Forward, 5′-CCCACCUGAAGGAGAAUAAUU-3′ and reverse, 5′-UUAUUCUCCUUCAGGUGGGUU-3′. The sequences of EV were: Forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAATT-3′. Cells without transfection were designated as the control group (Cntl). Transfection efficiency was determined by measuring COL6A1 mRNA and protein levels after 48 h of transfection.
4
Culturing Human Aortic Smooth Muscle Cells
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HASMCs were cultured in Medium 231 (Life Technologies, Carlsbad, CA, USA) supplemented with a Smooth Muscle Growth Supplement (SMGS, Life Technologies, Carlsbad, CA, USA) and 1% of 100 units/mL penicillin and 100 mg/mL streptomycin (Sigma Aldrich, St. Louis, MO, USA) in tissue culture flasks at 37 °C in a humidified atmosphere and 5% CO2, as previously described [36 (link),37 (link)]. Cells were split 1:2 twice a week and used until passage 18.
5
Aortic Smooth Muscle Cell Culture
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Primary human aortic smooth muscle cells were purchased from the American Type Culture Collection (ATCC; PCS-100-012) and cultured in Medium 231 (Life Technologies), 500 ml of which was supplemented with 25 ml of growth supplement (smooth muscle growth supplement; Life Technologies) and sodium l -ascorbate (50 μg/ml; Sigma-Aldrich) for extracellular matrix secretion. The cells were seeded at a density of 1.25 × 104 per ml and used within the first three passages. The glass and PDMS surfaces were coated with collagen before seeding. The PDMS surfaces were oxygen plasma cleaned at 200 W for 10 min immediately before seeding collagen to make the surface hydrophilic.
6
Aortic Tissue Sampling in Marfan Syndrome
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Aortic tissue samples were collected in the surgery room from the dilated and non-dilated zone of MFS patients’ aortic aneurysm. From the same cohort of patients, and plasma samples were collected before the surgery procedure. The healthy control (HC) samples were provided by Treviso Tissue Bank Foundation. All MFS patients gave written informed consent for tissue collection and the procedure was approved by ASST FBF-Sacco Ethics Committee (Prot. N° 39138/2016, Milan, Italy). Aortic samples were stored in serum-free Medium 231 (Life Technologies, Carlsbad, CA, USA) and then divided into fragments dedicated to histology and molecular studies.
7
Generating iPSC-Derived Smooth Muscle Cells
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Human undifferentiated iPSCs reprogrammed from skin fibroblasts of a WBS patient (WS1-iPSC line C; refs. 65 (link), 66 (link)), an ELN-mutant nonsyndromic SVAS patient (ELN1; ref. 66 (link)), and a control human (CT2; ref. 66 (link)) were used to generate iPSC-SMC progenitor cell lines via an embryoid body stage (65 (link), 66 (link)). The age and sex of iPSC donors are provided in Supplemental Table 2 . As described previously (67 (link)), the SMC progenitor cells were expanded in Matrigel-coated 6-well plates in smooth muscle growth medium (Medium 231, growth supplement) (Life Technologies). Cells were passaged when they reached 80% to 90% confluence. To induce differentiation, the iPSC-SMC progenitors were trypsinized and plated in smooth muscle differentiation medium (Medium 231, differentiation supplement) (Life Technologies) on 0.1% gelatin–coated 6-well plates for 6 days. RNA was isolated before and after SMC differentiation and utilized for qRT-PCR analysis of SMC markers. Protein lysates from the differentiated iPSC-SMCs were collected and utilized for Western blotting.
8
Culturing Stem Cells and Fibroblasts
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H9 hESCs and iPSCs were maintained on Matrigel in E8 medium (DMEM/F12 supplemented with l ‐ascorbic acid, sodium selenite, bFGF, TGF‐β1, sodium bicarbonate, holo‐transferrin, and gentamycin). Hutchinson–Gilford progeria syndrome and normal patient fibroblasts were obtained from Progeria Foundation and Coriell, and were maintained in fibroblast medium (DMEM supplemented with 10% FBS, glutamax, and gentamycin). VSMCs were maintained in Medium 231 (Life Technologies, Canada) supplemented with VSMC growth supplement (Life Technologies).
9
Overexpression of key signaling proteins in human coronary artery smooth muscle cells
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Smooth muscle cells from the human coronary artery (hCASMC, lot 1130140, Gibco, Life Technologies) were cultured in 6 well plates (Nunclon, Thermo Scientific) containing Medium 231 (Life Technologies) with 5% growth supplement (SMGS, Life Technologies) and 50 U/50 μg/ml penicillin/streptomycin (Biochrom, A 2212) in a water-jacketed incubator with an atmosphere of 5% CO2 in air (37 °C). Cells were passaged by trypsin treatment and used between passage 3 and 8. Full length h-MAML2, the intracellular domain of NOTCH2 (nucleotides 5107–7425 of Notch2 cDNA sequence AF308601), a C-terminal fragment of h-FRYL and full length JAG1, all under control of the CMV promoter, were overexpressed using custom-made human adenoviruses Type 5 (dE1/E3) from Vector Biolabs. Ad-CMV-null at the same multiplicity of infection (MOI) was used as control for all transductions. Virus was added 24 h after seeding and maintained for 96 h. For harvest, cells were washed with ice-cold PBS and lysed in Qiazol (RNA) or Laemmli sample buffer (protein).
10
Aortic Smooth Muscle Cell Isolation
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Samples from aortic tunica media, stored in serum-free Medium 231 (Life Technologies, Carlsbad, CA, USA), were washed with Phosphate Buffer Saline (PBS), minced and digested O/N at 37 °C in a solution of 2 mg/mL collagenase type II (Worthington Biochemical Corporation, Lakewood, NJ, USA) in complete Medium 231, supplemented with Smooth Muscle Growth Supplement (SMGS, Life Technologies, Carlsbad, CA, USA). Then, the result of tissue digestion was filtered with 100 μm cell strainer, pelleted and plated in complete Medium 231. To improve cell growth, the day after isolation the medium was changed to remove the residual erythrocytes. Regarding MFS samples, cells used for the in vitro experiments were those isolated only by non-dilated zone.
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