hESCs were maintained in the feeder-free cell culture medium mTeSR1 (STEMCELL Technologies) with daily media changes. For passaging, cells were dissociated en bloc with ReLeSR (STEMCELL Technologies) following the manufacturer’s protocol, and detached ES cell clumps were broken into smaller pieces (10–20 cells) by tapping the plate or gently pipetting several times with a wide-bore P1000 micropipette (Corning). Cells were passaged at a 1:12 split ratio onto Matrigel-coated (Corning) plates. Immediately following passage, cells were maintained in mTeSR1 supplemented with 10 µM ROCK inhibitor Y-27632 (STEMCELL Technologies) for 24 h before returning to culture in mTeSR1 alone.
Mtesr1
Manufactured by STEMCELL
Sourced in Canada, United States, France, Germany, Japan, United Kingdom
MTeSR1 is a complete serum-free culture medium formulated for the maintenance of undifferentiated human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) in feeder-free conditions.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by Corning (135 mentions)
Matrigel, by BD (72 mentions)
Relesr, by STEMCELL (51 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (39 mentions)
Accutase, by STEMCELL (38 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (33 mentions)
Geltrex, by Thermo Fisher Scientific (32 mentions)
Dmem f12, by Thermo Fisher Scientific (27 mentions)
Accutase, by Merck Group (26 mentions)
Glutamax, by Thermo Fisher Scientific (26 mentions)
Dispase, by STEMCELL (21 mentions)
Accutase, by Thermo Fisher Scientific (20 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (17 mentions)
Tryple express, by Thermo Fisher Scientific (15 mentions)
L glutamine, by Thermo Fisher Scientific (15 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (15 mentions)
Gentle cell dissociation reagent (gcdr), by STEMCELL (14 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (14 mentions)
Y 27632, by STEMCELL (13 mentions)
Tryple, by Thermo Fisher Scientific (13 mentions)
551 protocols using mtesr1
1
Feeder-free hESCs Culture and Passage
2
Feeder-free Expansion of Human iPSCs
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Hepatic fibroblast derived iPS line ATCC-HYS0103 Human Induced Pluripotent Stem (IPS) Cells were purchased directly from the vendor. hESC-Qualified Matrigel (Corning 354277) Matrigel was used coat culture dishes for feeder-free expansion of induced pluripotent stem cells and were routinely cultured and maintained in mTeSR™1 (STEMCELL Technologies, 85850) in a humidified incubator at 37 °C with 5% CO2. Cells were passaged once a week using selective dissociation reagent ReLeSR™ (STEMCELL Technologies 05872) and seeded using the cell aggregate counting method described in 'Plating Human ES and iPS Cells Using the Cell Aggregate Count Method' (Appendix 1) from the STEMCELL Technologies-'Maintenance of Human Pluripotent Stem Cells in mTeSR™1' technical handbook to assess the size of aggregates and seed them in low, medium or high densities, as described. All cryopreservation of cells was performed in CryoStor® CS10 (STEMCELL Technologies 07930) freezing media.
For the glass controls in the experiments -sterile rh-Vitronectin (Gibco, Life Technologies, A14700) was used to coat glass cover slips. In case of all experiments, cells were dissociated into a single cell suspension using StemPro™ Accutase™ (Gibco A1110501) cell dissociation reagent to facilitate cell counting. In case of single cell dissociation, cells were always seeded with 10uM Rock inhibitor Y27632 (ATCC® ACS-3030™).
For the glass controls in the experiments -sterile rh-Vitronectin (Gibco, Life Technologies, A14700) was used to coat glass cover slips. In case of all experiments, cells were dissociated into a single cell suspension using StemPro™ Accutase™ (Gibco A1110501) cell dissociation reagent to facilitate cell counting. In case of single cell dissociation, cells were always seeded with 10uM Rock inhibitor Y27632 (ATCC® ACS-3030™).
3
Stem Cell Culture Optimization
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The cell culture dishes were monitored for confluency before starting the experiment. All experiments were performed using in mTeSR1 (STEMCELL Technologies, 85850). The cells were dissociated using warm StemPro Accutase (Gibco A1110501) cell dissociation reagent for 6–7 min. Cells were counted using a hemocytometer and seeded in mTeSR1 (STEMCELL Technologies, 85850) and a density of 5 × 105 cells mL−1 as a 1 mL per well solution in a 12‐well culture dish with 10 µm Rock inhibitor Y27632 (ATCC ACS‐3030). The medium was replaced with media without Y‐27632 at 24 h for 10 and 100 kPa substrates, and 5 µm Y‐27632 in case of 1 kPa substrates (This was completely removed at 36 h). The cells were allowed to grow on the 4 substrate groups for 48 h.
For BMP4 induction experiments, same seeding method was used, fresh media supplemented with rhBMP4 (STEMCELL Technologies, 78211) at a final concentration of 50 ng mL−1 on the 4 substrate groups for 48 h.
For small molecule and protein inhibitor treatments— cell cultures were supplemented with the following concentration of each agent at 6 h of seeding—CHIR99021 (3 µm ), SB431542 (5 µm ), IWP‐2 (2.5 µm ), DKK1 (500 ng mL−1), sFRP1 (5 µg mL−1), Peptide17 (100 nm ).
For BMP4 induction experiments, same seeding method was used, fresh media supplemented with rhBMP4 (STEMCELL Technologies, 78211) at a final concentration of 50 ng mL−1 on the 4 substrate groups for 48 h.
For small molecule and protein inhibitor treatments— cell cultures were supplemented with the following concentration of each agent at 6 h of seeding—CHIR99021 (3 µ
4
Feeder-Free Expansion of Human Induced Pluripotent Stem Cells
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Hepatic fibroblast derived iPS line ATCC‐HYS0103 Human Induced Pluripotent Stem (IPS) Cells were purchased directly from the vendor. hESC‐Qualified Matrigel (Corning 354277) was used coat culture dishes for feeder‐free expansion of induced pluripotent stem cells and were routinely cultured and maintained in mTeSR1 (STEMCELL Technologies, 85850) in a humidified incubator at 37 °C with 5% CO2. Cells were passaged once a week using selective dissociation reagent ReLeSR (STEMCELL Technologies 05872) and seeded using the cell aggregate counting method described in “Plating Human ES and iPS Cells Using the Cell Aggregate Count Method” (Appendix 1) from the STEMCELL Technologies—“Maintenance of Human Pluripotent Stem Cells in mTeSR1” technical handbook to assess the size of aggregates and seed them in low, medium or high densities, as described. All cryopreservation of cells was performed in CryoStor CS10 (STEMCELL Technologies 07930) freezing media.
For the glass controls in the experiments—sterile rh‐Vitronectin (Gibco, Life Technologies, A14700) was used to coat glass cover slips. In case of all experiments, cells were dissociated into a single cell suspension using StemPro Accutase (Gibco A1110501) cell dissociation reagent to facilitate cell counting. In case of single cell dissociation, cells were always seeded with 10 µm Rock inhibitor Y27632 (ATCC ACS‐3030).
For the glass controls in the experiments—sterile rh‐Vitronectin (Gibco, Life Technologies, A14700) was used to coat glass cover slips. In case of all experiments, cells were dissociated into a single cell suspension using StemPro Accutase (Gibco A1110501) cell dissociation reagent to facilitate cell counting. In case of single cell dissociation, cells were always seeded with 10 µ
5
Kidney Organoid Differentiation Protocol
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Work with iPS and ES cells was conducted under the approval and auspices of the University of Washington Embryonic Stem Cell Research Oversight Committee. Specific cell lines used in this study are described below and were sourced from commercially available iPS and ES cell lines obtained with informed consent. Stem cell stocks were maintained in mTeSR1 media with daily media changes and passaging using Accutase (STEMCELL Technologies). One thousand to six thousand cells per well were placed in each 24-well plate precoated with 300 μL of DMEM-F12 containing 0.2 mg/mL Matrigel and sandwiched the following day with 0.2 mg/mL Matrigel in mTeSR1 (STEMCELL Technologies) to produce scattered, isolated spheroid colonies. Forty-eight hours after sandwiching, spheroids were treated with 12 μM CHIR99021 (Tocris Bioscience) for 36 hours, then changed to RB (Advanced RPMI + 1× Glutamax + 1× B27 Supplement, all from Thermo Fisher Scientific) and replaced every 3 days thereafter. Organoids were differentiated for 21 days from the time of plating, at which time tubular structures had formed. Gene-edited PKD2−/− organoids were picked from the adherent plate at day 21, placed in suspension culture with RB media replaced every 3 days until day 30 when cyst growth was prominent (37 (link)).
6
Generation and Culture of Human iPSCs
7
Directed Differentiation of hPSCs into Spheroid Colonies
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Work with hPSC was performed under the approval and auspices of the University of Washington Embryonic Stem Cell Research Oversight Committee. Specific cell lines used in this study are described below and are sourced from commercially available hPSC obtained with informed consent. hPSC stocks were maintained in mTeSR1 media with daily media changes and weekly passaging using Accutase or ReLeSR (STEMCELL Technologies, Vancouver). 5,000–20,000 hPSCs per well were placed in each 24-well plate pre-coated with 300 µL of DMEM-F12 containing 0.2 mg/mL Matrigel and sandwiched the following day with 0.2 mg/mL Matrigel in mTeSR1 (STEMCELL Technologies, Vancouver) to produce scattered, isolated spheroid colonies. 48 hrs after sandwiching, hPSC spheroids were treated with 12μM CHIR99021 (Tocris Bioscience) for 36 h, then changed to RB (Advanced RPMI + 1X Glutamax + 1X B27 Supplement, all from Thermo Fisher Scientific) after 48 h, and replaced with fresh RB every 3 days thereafter.
8
Efficient Genome Editing in SMA-iPSCs
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The SMA-iPSCs used in this study were described previously [20 (link)]. They were dissociated via TrypLE Select (Life Technologies, Grand Island, NY, USA) at 37 °C for 5 min. Single cells were counted and resuspended in 100 µL of the reagent from Human Stem Cell Nucleofector Kit 2 (Lonza, Alpharetta, GA, USA) for nucleofection by using Nucleofector II (Lonza, Alpharetta, GA, USA) set at B016 program. Then, 6 µg of pCMV-PE2 plasmid and 2 µg of pegRNA plasmid (1527N or 1327N) were used to transfect 1 × 106 cells. The transfected cells were cultured on Matrigel (Corning, NY, USA)-coated wells in mTeSR1 (STEMCELL Technologies, Vancouver, Canada). After 2 days, the cells were dissociated into single cells and counted. Subsequently, 500 cells were seeded in a 60 mm culture dish with an mTeSR1 medium supplemented with 10% CloneR (STEMCELL Technologies, Vancouver, Canada). After 14 days, clones were mechanically selected, expanded, and identified through PCR and Sanger sequencing of the modified targeted genome sequences.
9
iPSC UBQLN2 Gene Editing
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The sgRNA for UBQLN2 was designed using the CRISPR design tool (http://chopchop.cbu.uib.no . accessed on 8 November 2018), then the sgRNA oligonucleotides were annealed and ligated into lentiCRISPR v2 plasmids (Addgene, 52961, Cambridge, MA, USA), and sequenced via Sanger sequencing to confirm that no errors were introduced. After TrypLETM Select (Gibco, Grand Island, NY, USA) was used to detach WT iPSCs into a single cell, 1 × 106 cells, 1 μL of 40 μM ssODN, and 5 μg of leti-UBQLN2-sgRNA-cas9 plasmid were added to 18 μL Supplement 1 and 82 μL Solution 2 of Human Stem Cell Nucleofector Kit 2 (Lonza). The transfer procedure was B-016. The cells were seeded on Matrigel (BD Biosciences)-coated 6-well plates in mTeSR1 (STEMCELL Technologies, Vancouver, BC, Canada), containing 10 μm Y27632 for 24 h, then selected using mTeSR1 with a final concentration of 0.3 μg/mL puromycin. When the confluence of cells reached 70%, the cells were detached with TrypLETM Select. One thousand cells were seeded on a Matrigel-coated 6 cm dish in mTeSR1 containing 10% clone R (Stem cell) for four days and then replaced with mTeSR1 without clone R every day until clones were picked.
10
Culturing hESCs and HEK293FT Cells
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Female H9 (WA09; RRID: CVCL_9773 ) hESCs were obtained from WiCell and cultured in either mTeSR1 (Stem Cell Technologies 85850) for at least one passage before differentiation into CNCCs or mTeSR Plus (Stem Cell Technologies 100–0276) for gene editing, single-cell cloning, expansion and maintenance. hESCs were grown on Matrigel growth factor reduced basement membrane matrix (Corning 354230) at 37 °C. hESCs were fed every day for mTeSR1 or every 2 days for mTeSR Plus and passaged every 5–6 days using ReLeSR (Stem Cell Technologies 05872).
HEK293FT cells were obtained from Invitrogen (R70007 ) and cultured in complete medium (DMEM-HG (GE Healthcare Life Science SH30243.01), 10% FBS, 1× Non-essential amino acids (Gibco 1114-0050), 1× GlutaMAX (Gibco 4109-0036), 1× antibiotic–antimycotic (Gibco 1524-0062)). Cells were fed every other day and passaged every 2–3 days using trypsin–EDTA (Gibco 25200072).
HEK293FT cells were obtained from Invitrogen (
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