HTECs were generated from human tracheal epithelial brushings obtained from patients undergoing bronchoscopy at the Stanford Pulmonary and Cystic Fibrosis Center. HNECs were generated from human sinonasal epithelial brushings or from tissue obtained from patients undergoing endoscopic sinus surgery at Stanford Hospital. Cells from brushings were dissociated by incubation in a 1:1 mixture of 0.5% trypsin (Thermo Fisher) and Nonenzymatic Cell Dissociation Solution (Sigma-Aldrich) for 5 minutes at 37°C. Cells from tissue pieces were dissociated by overnight digestion in 1.5 mg/ml pronase (Roche) at 4°C. The isolated epithelial cells were cultured on Transwell filters (Corning) following the MTEC protocol (22 (link)). Y-27632 ROCK inhibitor (Abcam) was added to the proliferation medium at 10 μM for cultures derived from limited material (58 (link)). Healthy HNECs and HTECs reach maturity with 75%–90% MCCs with variable timing due to donor differences, but all cultures complete MCC differentiation by ALI+30 days. Cells were treated with human IL-1β, human IL-13, human TNF-α (all from Peprotech), or 1 μM DAPT in differentiation medium for various lengths of time.
Rock inhibitor y 27632
Manufactured by Abcam
Sourced in United States
ROCK inhibitor Y-27632 is a small molecule that inhibits the activity of Rho-associated, coiled-coil containing protein kinases (ROCK) I and II. It is commonly used in cell culture applications to modulate cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Accutase, by STEMCELL (2 mentions)
Pluronic f 127, by Merck Group (2 mentions)
Tryple select, by Thermo Fisher Scientific (2 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (2 mentions)
Human tnf α, by Thermo Fisher Scientific (1 mentions)
Non enzymatic cell dissociation solution, by Merck Group (1 mentions)
Transwell filter, by Corning (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Human il 1β, by Thermo Fisher Scientific (1 mentions)
Pronase, by Roche (1 mentions)
Human il 13, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Corning (1 mentions)
Sgrnas, by Synthego (1 mentions)
Knockout dmem, by Thermo Fisher Scientific (1 mentions)
Stemmacs ips brew xf, by Miltenyi Biotec (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Carl Roth (1 mentions)
Dmem f12, by Corning (1 mentions)
Matrigel solution, by Corning (1 mentions)
Kyou dxr0109b, by American Type Culture Collection (1 mentions)
10 protocols using rock inhibitor y 27632
1
Generating Human Airway Epithelial Cultures
2
CRISPR Knockout Generation in iPSCs
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Knockout generation was done by RNP electroporation as described (56 (link)). Briefly, sgRNAs were obtained from Synthego Corporation and gRNA sequences are given in Supplementary Table S1 . Electroporation was done using Lonza 4D nucleocuvettes (V4XP-3024, 100 μl) as described (56 (link)). Modified synthetic gRNAs (with first and last three nucleotides having 2′-O-methyl-3′-phosphorothrioate modification) and 3xNLS-SpCas9 were mixed in equimolar concentrations and incubated for 15 min at RT. iPSCs were derived to single cell suspension using Accutase (Stem Cell Technologies) and resuspended in P3 solution immediately before electroporation. 50 μM RNPs were mixed with 1 M iPSCs and electroporation was done with program CA-137. Cells were maintained in E8 media supplemented with Y-27632 ROCK inhibitor (Abcam) for in vitro culture on Matrigel (Corning) matrix.
3
Episomal and Sendai-Derived iPSC Lines
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Five human iPSC-lines were generated from mesenchymal stromal cells (iPSC-102, iPSC-104, and iPSC-106) or blood (PT4-WT4, PT18-WT18; Toledo et al., 2020) by reprogramming with episomal plasmids or sendai virus, respectively. The study was approved by the local committee and all samples were taken after written consent (EK206/09). The iPSC lines were cultured on tissue culture plastic coated with vitronectin (0.5 µg/cm 2 ) in StemMACS iPS-Brew XF (Miltenyi Biotec). Pluripotency was validated by three lineage differentiation potential and Epi-Pluri-Score analysis, as described in our previous work (Lenz et al., 2015) . For intermittent calcium depletion, cell culture medium was aspirated and iPSCs were incubated in 2 mM EGTA (Carl Roth) solution in PBS for 20 mins followed by incubation in culture medium containing Y-27632 ROCK inhibitor (Abcam). Control samples received knockout DMEM (Gibco) for the amount of same time.
4
Reprogramming Adult Cells to Induced Pluripotent Stem Cells
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iPS-KYOU, has been obtained in the Shinya Yamanaka laboratory (Kyoto University, Japan) by retroviral reprogramming of adult female skin fibroblasts. The iPS-KYOU cell line was purchased from the ATCC cell bank (KYOU-DXR0109B, ACS-1023™, ATCC®, United States).
iPS-AFS17, human Induced Pluripotent Stem Cells, obtained by lentiviral reprogramming of amniotic fluid stem cells by Dashinimaev et al. (2017) (link).
iPS-DP human Induced Pluripotent Stem Cells, obtained by lentiviral reprogramming of dermal papilla cells (Muchkaeva et al., 2014 (link)).
iPS-DYP0730, human Induced Pluripotent Stem Cells, derived from dermal fibroblasts obtained from ATCC CCL-54 Detroit 532, a human Down syndrome cell line. The iPS-DYP0730 cell line was purchased from the ATCC cell bank (ACS-1003™, ATCC®, United States).
For all pluripotent stem cell passaging, we used ACCUTASE™ сell detachment solution (Stem Cell Technologies) and Rock-inhibitor Y-27632 (5 μM; Abcam), the plastic surface being pre-coated with Matrigel solution (1/40 in DMEM/F12) (Corning). The cells were cultured in mTeSR™1 medium (Stem Cell Technologies) at 37°C in a CO2-incubator with 5% CO2 and 100% humidity.
iPS-AFS17, human Induced Pluripotent Stem Cells, obtained by lentiviral reprogramming of amniotic fluid stem cells by Dashinimaev et al. (2017) (link).
iPS-DP human Induced Pluripotent Stem Cells, obtained by lentiviral reprogramming of dermal papilla cells (Muchkaeva et al., 2014 (link)).
iPS-DYP0730, human Induced Pluripotent Stem Cells, derived from dermal fibroblasts obtained from ATCC CCL-54 Detroit 532, a human Down syndrome cell line. The iPS-DYP0730 cell line was purchased from the ATCC cell bank (ACS-1003™, ATCC®, United States).
For all pluripotent stem cell passaging, we used ACCUTASE™ сell detachment solution (Stem Cell Technologies) and Rock-inhibitor Y-27632 (5 μM; Abcam), the plastic surface being pre-coated with Matrigel solution (1/40 in DMEM/F12) (Corning). The cells were cultured in mTeSR™1 medium (Stem Cell Technologies) at 37°C in a CO2-incubator with 5% CO2 and 100% humidity.
5
Maintenance and Passage of hPSC Cultures
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WT hESCs (HuES629 (link) background, obtained from Harvard University), WT hiPSCs (F1 background, fetal liver fibroblast-derived21 (link)), and their genetically modified derivatives were maintained on 6-well dishes coated with 1 ml/well 1:75 diluted Matrigel™ HC (Corning #354263), in defined FTDA medium30 (link). FTDA was composed of DMEM/F12, 1 × PenStrep/Glutamine, 1 × defined lipids (Thermo), 1 × ITS (Corning), 0.1% human serum albumin (Biological Industries), 10 ng/ml FGF2 (PeproTech #100-18B), 0.2 ng/ml TGFβ1 (eBioscience #34-8348-82), 50 nM Dorsomorphin (Santa Cruz), and 5 ng/ml Activin A (eBioscience #34-8993-85). Fully confluent hPSC cultures were harvested by a 15–20 min incubation with Accutase™ (Sigma) containing 10 µM ROCK inhibitor Y-27632 (abcamBiochemicals) and seeded out for passaging into new 6-well plates at 400,000–500,000 cells per well, in FTDA + ROCKi. Cells were split every 3–4 days and kept in culture for a maximum of 30 passages. Short-term signaling stimulation experiments were carried out using semiconfluent cultures.
6
Generating Feeder-Independent Human and Chimpanzee iPSCs
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All chimpanzee and Caucasian (CAU) iPSCs were reprogrammed from fibroblasts with episomal vectors (Gallego Romero et al., 2015 (link); Burrows et al., 2016 (link)), while the Yoruba (YRI) iPSCs were similarly reprogrammed from lymphoblastoid cell lines (LCLs) (Banovich et al., 20162018 (link)). It has been shown, however, that cell-type of origin does not affect iPSC DNA methylation or gene expression patterns (Burrows et al., 2016 (link)). After reprogramming, iPSC colonies were cultured on a Mouse Embryonic Fibroblast feeder layer for 12–15 passages prior to conversion to feeder-free growth for 6–26 passages. Three new iPSC lines (H20682, H21792, H28815) were generated as previously described for H20961 (referred to as Ind1 F-iPSC in Burrows et al.), H28126 (Ind3 F-iPSC) and H21194 (Ind4 F-iPSC) (Burrows et al., 2016 (link)).
Human and chimpanzee feeder-independent stem cell lines were maintained at 70% confluence on Matrigel hESC-qualified Matrix (354277, Corning, Bedford, MA) at a 1:00 dilution. Cells were cultured in Essential 8 Medium (A1517001, ThermoFisher Scientific, Waltham, MA) at 37° C with 5% (vol/vol) CO2 with daily media changes. Cells were passaged by enzyme-free dissociation (0.5 mM EDTA, 300 mM NaCl in PBS), and seeded with ROCK inhibitor Y-27632 (ab120129, Abcam, Cambridge, MA). All cell lines tested negative for mycoplasma contamination.
Human and chimpanzee feeder-independent stem cell lines were maintained at 70% confluence on Matrigel hESC-qualified Matrix (354277, Corning, Bedford, MA) at a 1:00 dilution. Cells were cultured in Essential 8 Medium (A1517001, ThermoFisher Scientific, Waltham, MA) at 37
7
hESC Patterned Differentiation Assay
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Prefabricated glass coverslips (Arena A, CYTOO) containing 500 µm disk patterns were coated with 10 µg/ml recombinant human laminin 521 (BioLamina) diluted in pre-warmed DPBS (Thermo Fisher Scientific) for 3 hours at 37°C. Laminin was then serially washed with DPBS and coverslips were stored in a 35 mm tissue culture plastic submerged in the solution to prevent drying. hESCs were rinsed with DPBS -Mg/-Ca (Thermo Fisher Scientific) and dissociated to single cells with Accutase (STEMCELL Technologies). 8x10 5 cells were seeded on each coverslip in a defined volume of MEF-CM supplemented with 20ng/ml bFGF (R&D Systems), 10 µM ROCK inhibitor (Y-27632, Abcam), 1X Penicillin-streptomycin (Thermo Fisher Scientific), 100 µg/ml Normocin (Invivogen) and left unperturbed for 10 minutes to ensure homogenous distribution across the patterns. ROCK inhibitor was removed from the medium 3 hours after seeding and cells were induced the following day with 50 ng/ml BMP4 (R&D Systems), 2 µM IWP2 (Stemgent), 10 µM SB431542 (Stemgent), 100 ng/ml WNT3a (R&D Systems), 6 µM CHIR99021 (EMD Millipore) and/or 100 ng/ml ACTIVIN (R&D Systems). Fixed samples were analyzed by immunofluorescence 1, 24 or 48 hours following induction.
8
Maintenance and Differentiation of Human Pluripotent Stem Cells
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All hPSC cultures were maintained at 37°C, 5% CO2 and grown on a monolayer of irradiated CF1 mouse embryonic fibroblasts (MEFs; GlobalStem) in hPSC medium consisting of KO-DMEM (Invitrogen), 10% KOSR (Invitrogen), 1% GlutaMAX, nonessential amino acids, penicillin/streptomycin, and 0.1% β-mercaptoethanol supplemented with 10 ng/ml bFGF (Invitrogen). Medium was changed every 24 h, and lines were passaged using TrypLE (Invitrogen). Before differentiation experiments, cells were expanded for at least two passages on Matrigel (BD) in mTeSR (STEMCELL Technologies) with 10 µM of ROCK inhibitor Y-27632 (Abcam).
The HUES cell lines (HUES6, HUES8, HUES49, and H9; Cowan et al., 2004 (link); Osafune et al., 2008 (link)) and the iPSC line (iPSC11b; Boulting et al., 2011 (link)) were used in this study.
The HUES cell lines (HUES6, HUES8, HUES49, and H9; Cowan et al., 2004 (link); Osafune et al., 2008 (link)) and the iPSC line (iPSC11b; Boulting et al., 2011 (link)) were used in this study.
9
Pancreatic Progenitor Organoid Cultivation
10
Pancreatic Progenitor Organoid Cultivation
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Before cell seeding, microwell chips were coated with 10% Pluronic F-127 (Sigma) overnight and sterilized for 30 min using 254 nm UV light (NK-Optik). On the next day, microwell chips were washed twice with Dulbecco’s Phosphate Buffered Saline (PBS) (Gibco) and once with DMEM:F12 (Gibco). For ductal differentiation on microwell chips, pancreatic progenitors were washed with PBS, then incubated for 3-8 min with TrypLE Select (Gibco) at 37°C for detachment. pancreatic progenitors were centrifuged at 200 g for 5 min and then seeded in 35 μL PDLO medium per array. The surface tension on top of the arrays allowed an equal distribution of the cell-suspension. After cell settling at 37°C for 45 min, additional 660 μL PDLO medium was carefully added to the side of the microwell chip. For preliminary determination of ideal cell numbers for organoid formation, hiPSCs were seeded on the microwell chip in mTeSR1 medium, supplemented with 10 μM ROCK inhibitor Y-27632 (abcam) during the first 24h
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