Human adipose-derived mesenchymal stem cells (hMSCs) were purchased from Steminent Biotherapeutics Inc. (Taipei, Taiwan). hMSCs were maintained in expansion medium (MesenPRO RS™, Invitrogen, Grand Island, NY, USA) supplemented with 100 units/mL of penicillin, 1000 units/mL of streptomycin, and 2 mmol/L l -glutamine (Sigma-Aldrich, St. Louis, MO, USA), and incubated at 37 °C, 5% CO2 and 95% relative humidity. To induce osteogenic differentiation, hMSCs were cultured with osteogenic induction medium (OIM) consisting of Iscove’s Modified Dulbecco’s medium (Gibco, Grand Island, NY, USA) supplemented with 10 mM β-glycerol phosphate (Sigma-Aldrich, St. Louis, MO, USA), 0.1 µM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), and 0.2 mM ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), and the medium was changed every three days.
Hmscs
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The HMSCs are a line of high-performance laboratory centrifuges designed for a variety of applications. They feature advanced technology to provide consistent and reliable performance.
Automatically generated - may contain errors
Lab products found in correlation
Hmscs, by Lonza (13 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions)
α mem, by Thermo Fisher Scientific (4 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
β glycerol phosphate, by Merck Group (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Hmscs, by Cambrex (2 mentions)
Egm 2, by Lonza (2 mentions)
Huvecs, by Lonza (2 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions)
Low glucose dmem, by Thermo Fisher Scientific (2 mentions)
Hmscs, by Merck Group (2 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Iscove s modified dulbecco s medium, by Thermo Fisher Scientific (1 mentions)
Mesenpro rs, by Thermo Fisher Scientific (1 mentions)
33 protocols using hmscs
1
Osteogenic Differentiation of hMSCs
2
Expansion and Culture of Human Mesenchymal Stem Cells
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hMSCs (Lonza, Walkersville,
MD) were expanded prior to the study in media consisting of DMEM (Life
Technologies, Frederick, MD) supplemented with 10% fetal bovine serum
(Life Technologies), 1.0% v/v penicillin/streptomycin (Life Technologies),
0.1 mM nonessential amino acids (Life Technologies), and 4 mMl -glutamine (Life Technologies) using protocols set forth by
the manufacturer and previously described.16 ,23 (link) hMSCs were expanded on tissue culture polystyrene flasks with medium
changes every 3 days according to the manufacturer’s specifications.
hMSCs at passage 4 were used for all experiments. Cell cultures were
incubated at 37 °C, 5% CO2, and 80% humidity.
MD) were expanded prior to the study in media consisting of DMEM (Life
Technologies, Frederick, MD) supplemented with 10% fetal bovine serum
(Life Technologies), 1.0% v/v penicillin/streptomycin (Life Technologies),
0.1 mM nonessential amino acids (Life Technologies), and 4 mM
the manufacturer and previously described.16 ,23 (link) hMSCs were expanded on tissue culture polystyrene flasks with medium
changes every 3 days according to the manufacturer’s specifications.
hMSCs at passage 4 were used for all experiments. Cell cultures were
incubated at 37 °C, 5% CO2, and 80% humidity.
3
Chondrogenesis Induction in hMSCs
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hMSCs were purchased from Lonza and Thermo Scientific. hMSCs were cultured in α-MEM supplemented with 20% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin B. To induce chondrogenesis, 2.5 × 105 hMSCs were centrifuged to form a pellet in α-MEM supplemented with 20% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin B. After 3 days, the medium was changed to chondrogenic medium consisting of DMEM/F-12 supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, 250 ng/ml amphotericin B, 1.25 mg/ml BSA, 1% insulin–transferrin–selenium, 1 mM sodium pyruvate, 50 μM l -aspartic acid, 50 μM l -proline, 100 nM dexamethasone, and 10 ng/ml of TGF-β1 with or without indicated drugs. On day 21 (for drug treatment) or day 28 (for siRNA treatment), cells were harvested and subjected to Alcian blue/Fast red staining. RNA-Seq data of human MSCs (GSE10950347 (link)) during chondrogenesis at days 0, 1, 3, 7, 14, and 21 were obtained from gene expression omnibus repository at the National Center for Biotechnology Information (NCBI). Differential expression analysis was conducted by using the DESeq2 R package.
4
Culturing Adult Bone Marrow-Derived hMSCs
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5
Expansion and Seeding of HUVECs and hMSCs
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Human umbilical vein endothelial cells
(HUVECs, Lonza) were expanded and seeded in endothelial growth medium-2
(EGM-2, Lonza) according to the manufacturer’s instructions.
hMSCs were used between passages 4 and 6, and HUVECs were used between
passages 5 and 10. For 6 h experiments, hMSCs were seeded at a density
of 20 000 viable cells/cm2 and HUVECs were seeded
at a density of 30 000 viable cells/cm2, as determined
by Trypan Blue exclusion. For 48 h experiments, hMSCs and HUVECs were
seeded at different densities on flat and nN substrates to avoid confluent
overgrowth. For hMSCs, cells were seeded at 2500 and 8375 cells/cm2 for flat and nN substrates, respectively. For HUVECs, cells
were seeded at 3000 and 12 500 cells/cm2 for flat
and nN substrates, respectively.
Human mesenchymal stem cells
(hMSCs, Lonza Ltd., Basel, Switzerland) were expanded in serum-free,
chemically defined medium (MSCGM-CD) with supplements (TheraPEAK,
Lonza), as per the manufacturer’s instructions. When ∼80%
confluent, hMSCs were detached with 0.05% v/v trypsin-EDTA, reseeded
at a density of 100–500 cell/cm2, and cultured for
7–14 days before reaching confluence. For interfacing with
nN or flat substrates, hMSCs were seeded in minimum essential medium
alpha (αMEM, Gibco ThermoFisher Scientific, Paisley, United
Kingdom) with 10% v/v MSC-qualified fetal bovine serum (FBS, Gibco)
and 1% v/v penicillin/streptomycin (P/S, Gibco).
(HUVECs, Lonza) were expanded and seeded in endothelial growth medium-2
(EGM-2, Lonza) according to the manufacturer’s instructions.
hMSCs were used between passages 4 and 6, and HUVECs were used between
passages 5 and 10. For 6 h experiments, hMSCs were seeded at a density
of 20 000 viable cells/cm2 and HUVECs were seeded
at a density of 30 000 viable cells/cm2, as determined
by Trypan Blue exclusion. For 48 h experiments, hMSCs and HUVECs were
seeded at different densities on flat and nN substrates to avoid confluent
overgrowth. For hMSCs, cells were seeded at 2500 and 8375 cells/cm2 for flat and nN substrates, respectively. For HUVECs, cells
were seeded at 3000 and 12 500 cells/cm2 for flat
and nN substrates, respectively.
Human mesenchymal stem cells
(hMSCs, Lonza Ltd., Basel, Switzerland) were expanded in serum-free,
chemically defined medium (MSCGM-CD) with supplements (TheraPEAK,
Lonza), as per the manufacturer’s instructions. When ∼80%
confluent, hMSCs were detached with 0.05% v/v trypsin-EDTA, reseeded
at a density of 100–500 cell/cm2, and cultured for
7–14 days before reaching confluence. For interfacing with
nN or flat substrates, hMSCs were seeded in minimum essential medium
alpha (αMEM, Gibco ThermoFisher Scientific, Paisley, United
Kingdom) with 10% v/v MSC-qualified fetal bovine serum (FBS, Gibco)
and 1% v/v penicillin/streptomycin (P/S, Gibco).
6
Feeder-based culture of human iPSCs
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Human adult bone marrow mesenchymal stem cells (hMSCs) (from Stem Cells Technology Research Center, Tehran, Iran. Passage 5) which applied as a feeder was plated onto gelatin-coated dishes in DMEM (Gibco, 12491-015) supplemented with 15% FBS (Gibco, 10270106). When cells reach to 60–70% confluency, they inactivated via mitomycin-C treatment for 3 h (10μg/ml). After washing with PBS, undifferentiated human iPSCs (a gift from Stem Cells Technology Research Center, Tehran, Iran. The passage number of cells was used from 8 to 14)17 (link) were cultured on the hMSCs, in DMEM/F12 (Gibco, 31330038) supplemented with 20% knockout serum replacement (Gibco, 10828), 1mM L-glutamin (Gibco, 25030), 1% non-essential amino acids (PAA, M11-003), 0.1mM β-mercaptoethanol, 100U penicillin/streptomycin (Gibco, 15140-122) and 10 ng/ml basic fibroblast growth factor (Peprotech, 100-18B). Then, cells were incubated under standard condition at 95% humidity and 5% CO2 at 37°C. HiPSCs colonies for embryoid body (EB) formation or transferred onto new hMSCs were detached with collagenase IV (Gibco, 15140-122) every 5-7 days.
7
Culturing Cryopreserved Adult hMSCs
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Cryopreserved adult bone marrow-derived hMSCs were purchased from Cambrex Bioscience (Walkersville, MD, USA). The hMSCs (passages 4–10) were cultured in Dulbecco's modified Eagle's medium (DMEM)-low glucose containing 10% fetal bovine serum (FBS) (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C with 5% CO2.
8
Mitochondrial Visualization in Cell Lines
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HepG2 and H9c2 were purchased from American Type Culture Collection (ATCC). Human mesenchymal stem cell (hMSCs) was purchased from Gibco (Carlsbad, CA, USA). HepG2 and R-HepG2 were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin antibiotics mixture (PS), except that R-HepG2 cells were supplemented with 1.2 μM of DOX in culture medium. H9c2 and hMSCs were cultured in DMEM and alpha-MEM supplemented with 10% FBS and 1% PS, respectively. hMSCs were assayed within four passages. To generate stable cell lines constitutively expressing the mitochondrial-RFP tag for mitochondria visualization, a construct pDsRed2-Mito (Clontech, CA, USA) carrying a human cytochrome C oxidase subunit VIII mitochondrial targeting sequence was transfected to cells using lipofectamine-3000 reagent according to manufacturer’s protocol. After 48 h of incubation, untransfected cells were eliminated by 1 mg/mL of Geneticin (InvivoGen, San Diego, CA, USA). To ensure clonal purity, transfected cells were sorted to a single clone by a cell sorter (FACSMelody, BD Biosciences, San Jose, CA, USA). Cells carrying the mitochondrial-RFP tag were expanded from a single clone. Fluorescence of the RFP-labelled mitochondria was verified by flow cytometry (FACSVerse, BD Biosciences, San Jose, CA, USA) and confocal microscopy (SP8, Leica, Wetzlar, Germany).
9
Culturing Primary Human Mesenchymal Stromal Cells
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Primary human bone marrow‐derived mesenchymal stromal cells (hMSCs) were obtained from the Institute for Regenerative Medicine, Texas A&M Health Science Center. hMSCs were cultured in MEMα (Gibco, Gaithersburg, MD, USA) supplemented with 20% FBS, and used at passages 5–6. Control mesenchymal cells were human dermal fibroblasts.
10
Fluorescent Stem Cell Tracking in Micropillars
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Human mesenchymal stem cells (hMSCs), human foreskin fibroblasts (HFFs), and human umbilical vein endothelial cells (HUVECs) were purchased from Cell Bank, Type Culture Collection, Chinese Academy of Sciences. (Note: The uppercase or lowercase of “H” follows the tradition for the pertinent cell types.) The hMSCs were cultured in minimum essential medium Eagle - alpha modification (MEM-α, Gibco). The HFFs and HUVECs were cultured in high-glucose Dulbecco's modified Eagle medium (DMEM, Gibco). All culture media were supplemented with 10% fetal bovine serum (FBS, Gibco) and 100 U mL−1 penicillin/streptomycin (Gibco). After reaching about 90% confluence, cells were detached using 0.25% trypsin/ethylenediaminetetraacetic acid (Gibco) and passed to the next generation. The media were refreshed every two days.
LifeAct-RFP labeled human mesenchymal stem cells (RFP-hMSC) enable the track of live cells (red fluorescence) in micropillar arrays (phase-contrast) under a fluorescence microscope. RFP-tagged LifeAct was transfected into the hMSCs using pCMV-LifeAct-RFP (Ibidi) through the construction of LifeAct-RFP-overexpressed lentivirus vector pH5674-LifeAct-RFP. The transfection principle and some results are presented inFig. S3 .
LifeAct-RFP labeled human mesenchymal stem cells (RFP-hMSC) enable the track of live cells (red fluorescence) in micropillar arrays (phase-contrast) under a fluorescence microscope. RFP-tagged LifeAct was transfected into the hMSCs using pCMV-LifeAct-RFP (Ibidi) through the construction of LifeAct-RFP-overexpressed lentivirus vector pH5674-LifeAct-RFP. The transfection principle and some results are presented in
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