All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol (10-02214) was approved by the Ethics Committee of the UCSF Medical Center (CA, USA). Several stem cell lines, including hiPSC UEFhfiPS1.4 [32 (link)], hESC CCTL12 [13 (link),33 ,34 (link)] and hiPSC UB47 [12 (link),33 ], were maintained as colonies on mitotically inactivated mouse embryo fibroblast (MEF) feeder in HES medium with 10 ng/mL human FGF2 or single cells on Matrigel hES-qualified (Corning, Corning, NY, USA, ref: 354277) as previously described [12 (link)]. Briefly, the colonies were manually dissected using a needle and passaged every 4–6 days whereas the single cells were enzymatically dissociated using TrypLE enzyme (Gibco, Waltham, MA, USA, ref: 12604-013) and passaged every 4 days.
Hes qualified matrigel
Manufactured by Corning
Sourced in United States
HES-qualified Matrigel is a well-defined extracellular matrix preparation derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. It is a soluble basement membrane extract that polymerizes into a solid gel at physiological temperatures, providing a structural and functional support for cell growth and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Relesr, by STEMCELL (3 mentions)
Essential 8 flex medium, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Xpenicillin streptomycin, by Corning (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Hek293t 17 cells, by American Type Culture Collection (2 mentions)
Mtesr1, by STEMCELL (2 mentions)
Mtesr1 medium, by STEMCELL (2 mentions)
Tryple enzyme, by Thermo Fisher Scientific (1 mentions)
Psc cardiomyocyte differentiation kit, by Thermo Fisher Scientific (1 mentions)
Gentle cell dissociation reagent (gcdr), by STEMCELL (1 mentions)
Mtesr1 plus, by STEMCELL (1 mentions)
Collagenase, by Thermo Fisher Scientific (1 mentions)
Stempro accutase, by Thermo Fisher Scientific (1 mentions)
Pluris tem dispase 2, by Merck Group (1 mentions)
Mfresr, by STEMCELL (1 mentions)
Noggin, by R&D Systems (1 mentions)
8 protocols using hes qualified matrigel
1
Maintaining Pluripotent Stem Cell Lines
2
Feeder-free human iPSC culture
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GM23280 human iPS cells were purchased from Coriell Institute for Medical Research. Human iPS cells were maintained and propagated as colonies in feeder-free conditions in Essential 8 Flex Medium (Thermo Fisher Scientific, A2858501) on Matrigel hES qualified (Corning, 354277) coated plates.
Colonies were passaged as aggregates every 5-7 days. Briefly, iPSC colonies were treated with Gentle Dissociation Reagent (GDR, StemCell technologies, 07174) for 7 minutes at room temperature. GDR was then replaced with E-8 flex media and colonies were gently triturated by pipetting in order to obtain a suspension of c.a. 100 μm aggregates. Small colonies were plated in freshly coated plates, and the media was refreshed every other day.
Colonies were passaged as aggregates every 5-7 days. Briefly, iPSC colonies were treated with Gentle Dissociation Reagent (GDR, StemCell technologies, 07174) for 7 minutes at room temperature. GDR was then replaced with E-8 flex media and colonies were gently triturated by pipetting in order to obtain a suspension of c.a. 100 μm aggregates. Small colonies were plated in freshly coated plates, and the media was refreshed every other day.
3
Targeted CRISPR Repression in hESCs
4
Cardiomyocyte Differentiation from Human iPSCs
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To induce cardiomyocyte differentiation we utilized the PSC Cardiomyocyte Differentiation Kit (ThermoFisher, Grand Island, NY). Briefly, human iPSCs were grown in feeder-free conditions using hES qualified Matrigel (Corning, Auburn, MI) and mTeSR1 medium (Stem Cell Technologies, Vancouver, BC, Canada). iPSC colonies were dissociated from one 35 mm dish using Gentle Cell Dissociation Reagent (Stem Cell Technologies) for eight minutes at 37°C to make a single cell suspension. The cells were divided equally among the wells of a 12-well plate coated with Matrigel using mTeSR1 medium and ROCK inhibitor (10 uM final concentration for the first 24 hours). Medium was changed daily with mTeSR1 until the iPSCs formed a monolayer of approximately 80% confluency. To induce mesoderm differentiation, Cardiomyocyte Differentiation Medium A was added for 48 hours (Days 0-2). For cardiac mesoderm specification, Cardiomyocyte Differentiation Medium B was added for the next 48 hours (Days 2-4). For cardiomyocyte maturation, cells were maintained in Cardiomyocyte Maintenance Medium for the duration of culture (Day 4+), replacing medium every other day. Spontaneous cell contraction began on day 10.
5
Differentiated Sensory Neuron Co-culture
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hiPS cell line was generated from 2-year-old male foreskin fibroblasts ordered from Coriell institute (AG07095). hiPS cell line was maintained with daily media change of mTeSR1 plus (StemCell technologies) and passaging every 4-5 days using ReLeSR (StemCell technologies). hES-qualified Matrigel (Corning) was used as a matrix. Calu3 cells and HEK293T cells were cultured using DMEM, 10% FBS, L-Glutamine, Pen/Strep and non-essential amino acids. All cells were cultured at 37°C with 5% of CO2.
For the co-cultures, HEK293T cells were resuspended in Neuro maturation media (BrainPhys media, N2 supplement (1X), B27 supplement (1X), BDNF (10ng/ml), GDNF (10ng/ml), NT3 (10ng/ml), Pen/Strep and non-essential amino acids) and added to terminally differentiated sensory neurons. Co-cultures were analyzed or infected after 48h at 37°C.
For the co-cultures, HEK293T cells were resuspended in Neuro maturation media (BrainPhys media, N2 supplement (1X), B27 supplement (1X), BDNF (10ng/ml), GDNF (10ng/ml), NT3 (10ng/ml), Pen/Strep and non-essential amino acids) and added to terminally differentiated sensory neurons. Co-cultures were analyzed or infected after 48h at 37°C.
6
Culturing Human Embryonic Stem Cells
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The hESC line employed was H1 (WiCell Research Institute Inc., Madison, WI, USA), which was grown on hES-qualified Matrigel (Corning®, Tewksbury, MA, USA) coated plates with mTeSR1 medium (STEMCELL Technologies, Vancouver, Canada) as previously described [29 (link),30 (link)]. The cells were incubated at 37°C in a humidified atmosphere that contained 5% CO2 and were passaged every 5 days.
7
Targeted CRISPR Repression in hESCs
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H1 hESCs (WiCell, WA-01) were maintained in mTeSR1 (StemCell
Technologies, Inc., 05850) on hES-qualified Matrigel (Corning, 354277)
coated plates. Cells were fed daily and split with ReLeSR (StemCell
Technologies, Inc., 05872) every 4–5 days in mTeSR1. H1 hESCs were
used to generate the targeted H1-AAVS1-TetOn-dCas9-KRAB hES cell line used
in this study. H1-AAVS1-TetOn-dCas9-KRAB hESCs were maintained similarly to
H1 hESCs.
HEK293T/17 cells (ATCC, CRL-11268) were maintained in DMEM (Thermo,
11965) supplemented with 10% FBS (Thermo, 10437), 1X GlutaMAX (Thermo,
35050), 1X non-essential amino acids (NEAA; Thermo, 11140), and 1X
Penicillin-Streptomycin (Corning, 30–002-CI). Cells were split with
0.25% Trypsin (Thermo, 25200) every 3–5 days.
Technologies, Inc., 05850) on hES-qualified Matrigel (Corning, 354277)
coated plates. Cells were fed daily and split with ReLeSR (StemCell
Technologies, Inc., 05872) every 4–5 days in mTeSR1. H1 hESCs were
used to generate the targeted H1-AAVS1-TetOn-dCas9-KRAB hES cell line used
in this study. H1-AAVS1-TetOn-dCas9-KRAB hESCs were maintained similarly to
H1 hESCs.
HEK293T/17 cells (ATCC, CRL-11268) were maintained in DMEM (Thermo,
11965) supplemented with 10% FBS (Thermo, 10437), 1X GlutaMAX (Thermo,
35050), 1X non-essential amino acids (NEAA; Thermo, 11140), and 1X
Penicillin-Streptomycin (Corning, 30–002-CI). Cells were split with
0.25% Trypsin (Thermo, 25200) every 3–5 days.
8
Cortical Differentiation of Human ESCs
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Cortical differentiation from human ESC was performed using previously described protocol (Espuny-camacho et al., 2013) with slight modifications. At differentiation day -8, the hES cells were dissociated using PluriSTEM Dispase-II (Merck)/ Collagenase (Thermo Fisher Scientific) solution and plated on Growth factor-reduced matrigel (Corning) in Essential 8 Flex medium (Thermo Fisher Scientific). At differentiation day -2, the cells were dissociated using Stem-Pro Accutase (Thermo Fisher Scientific) and plated at low confluency (5,000-10,000 cells/cm 2 ) on hES qualified matrigel (Corning)
using Essential 8 Flex medium supplemented with 10 µM ROCK inhibitor (Y-27632; Merck). At differentiation day 0, the medium was changed to DDM (Gaspard et al., 2008) supplemented with B27 (Thermo Fisher Scientific) and 100 ng/mL Noggin (R&D systems), and the medium was replenished every day. After 16 days of differentiation, the medium was changed to DDM, supplemented with B27 (DDM/B27), and changed every day. At day 25, the progenitors were dissociated using Accutase and frozen using mFreSR (Stemcell technologies). Cortical progenitors were thawed on matrigelcoated coverslips 12-well using DDM/B27 supplemented with 10 µM ROCK inhibitor and fixed after 6 days using 4% paraformaldehyde for 20 min at 4° or ice-cold methanol for 6 min and then rinsed 3 times with PBS before immunostaining.
using Essential 8 Flex medium supplemented with 10 µM ROCK inhibitor (Y-27632; Merck). At differentiation day 0, the medium was changed to DDM (Gaspard et al., 2008) supplemented with B27 (Thermo Fisher Scientific) and 100 ng/mL Noggin (R&D systems), and the medium was replenished every day. After 16 days of differentiation, the medium was changed to DDM, supplemented with B27 (DDM/B27), and changed every day. At day 25, the progenitors were dissociated using Accutase and frozen using mFreSR (Stemcell technologies). Cortical progenitors were thawed on matrigelcoated coverslips 12-well using DDM/B27 supplemented with 10 µM ROCK inhibitor and fixed after 6 days using 4% paraformaldehyde for 20 min at 4° or ice-cold methanol for 6 min and then rinsed 3 times with PBS before immunostaining.
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