iPSCs (DF19-19-9-11T) purchased from WiCell Technologies were maintained on vitronectin-coated plates in Essential 8 medium (ThermoFisher Scientific) and passaged when the colonies reached ~75-80% confluence. iPSCs were differentiated into CD34+ iPSC-EPs following an established low-serum medium protocol [39 (link)]. Five days after CHIR induction (LC Technologies), cells were purified by methods we have described previously [35 (link)]. Briefly, the resulting confluent monolayer of differentiated cells was dispersed into a single-cell suspension with Accutase, tagged with a CD34-PE antibody (Miltenyi Biotec), and purified on a fluorescence-activated cell sorting (FACS) instrument (S3e Cell Sorter, Bio-Rad). After sorting, CD34+ iPSC-EPs were encapsulated at 2.5 million cells/mL in collagen-only or IPN hydrogels, as described in Section 2.4 (Table S1 ). After encapsulation, cells were cultured in EGM-2 (PromoCell) supplemented with 10 uM Y-27632, 1X Penicillin-Streptomycin, and 50 ng/mL vascular endothelial growth factor (VEGF) for 24 hours. The medium was then refreshed daily with EGM-2 supplemented with 50 ng/mL VEGF.
Accutase
Manufactured by Miltenyi Biotec
Sourced in Germany
Accutase is a cell detachment solution used for gently dissociating adherent cells from cell culture surfaces. It is a mixture of proteolytic and collagenolytic enzymes that can be used as an alternative to trypsin for cell passaging or harvesting.
Automatically generated - may contain errors
Lab products found in correlation
Essential 8 medium, by Thermo Fisher Scientific (1 mentions)
Cd34 pe antibody, by Miltenyi Biotec (1 mentions)
S3e cell sorter, by Bio-Rad (1 mentions)
Egm 2, by PromoCell (1 mentions)
Human fibronectin, by Merck Group (1 mentions)
Automacs pro, by Miltenyi Biotec (1 mentions)
Vascular endothelial growth factor (vegf), by Merck Group (1 mentions)
Vascular endothelial growth factor (vegf), by Thermo Fisher Scientific (1 mentions)
Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (1 mentions)
Egm 2 bulletkit, by Lonza (1 mentions)
Matrigel, by BD (1 mentions)
Accutase, by STEMCELL (1 mentions)
Cd144 microbeads, by Miltenyi Biotec (1 mentions)
Ncsc microbeads, by Miltenyi Biotec (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Live dead near ir, by Thermo Fisher Scientific (1 mentions)
Anti pdgfrα, by Miltenyi Biotec (1 mentions)
Kaluza software, by Beckman Coulter (1 mentions)
4 protocols using accutase
1
iPSC-derived Endothelial Progenitor Cell Encapsulation
2
Endothelial Cell Differentiation from iPSCs
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Endothelial differentiation was performed as described in Patsch et al.14 (link). Briefly, IPSCs were detached using Accutase (Stem Cell Technologies) and seeded on growth factor-reduced Matrigel (BD Bioscience) in the corresponding stem cell medium (mTesR1 for hIPSCs, MT medium for cIPSCs) supplemented with 10 μM Rock Inhibitor Y-27632. After 24 hours, the cells were washed with PBS and covered with priming medium consisting of N2B27 supplemented with 25 ng/ml BMP4 (Gibco) and CP21 (Roche) at the indicated concentrations. After the priming period (between 3–4 days), the cells were washed, and the medium was switched to induction medium consisting of StemPro34-SFM complete (Life Technologies) supplemented with 200 ng/ml VEGF (Peprotech) and 2 μM forskolin (Roche). The induction medium was changed every other day.
At day 6 of differentiation, ECs were dissociated with Accutase or 0.05% trypsin-EDTA and MACS-separated using CD144 MicroBeads (Miltenyi Biotec) and an autoMACSpro (Miltenyi Biotec). Positive-sorted cells were replated on human fibronectin (Sigma-Aldrich)-coated dishes in EC expansion medium consisting of StemPro-34 SFM supplemented with 50 ng/ml VEGF. For functional assays, the cells were cultured in EBM medium supplemented with the EGM2 bullet kit (Lonza).
At day 6 of differentiation, ECs were dissociated with Accutase or 0.05% trypsin-EDTA and MACS-separated using CD144 MicroBeads (Miltenyi Biotec) and an autoMACSpro (Miltenyi Biotec). Positive-sorted cells were replated on human fibronectin (Sigma-Aldrich)-coated dishes in EC expansion medium consisting of StemPro-34 SFM supplemented with 50 ng/ml VEGF. For functional assays, the cells were cultured in EBM medium supplemented with the EGM2 bullet kit (Lonza).
3
Isolation and Purification of NCSCs
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At D15 of E6-CSFD treatment, cells were dissociated using Accutase and labeled with NCSC microbeads (20 μl per 107 cells; Miltenyi), FcR blocking reagent (20 μl per 107 cells), and MACS buffer [60 μl per 107 cells; 0.5% bovine serum albumin + 2 mM EDTA in sterile phosphate-buffered saline (PBS) without Ca2+/Mg2+] at 4°C for 15 min. Cells were washed in MACS buffer and resuspended in 500 μl of MACS buffer per 2 × 107 cells. Cells were sorted through two LS columns (Miltenyi Biotec) according to manufacturer instructions and resuspended in E6-CSFD + 10 μM Y27632 to appropriate density for specific NCSC lineage differentiations as described below.
4
OPC Expansion with bFGF Modulation
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Primary cultures of OPCs derived from neonatal rats were expanded and reseeded 500,000cells/well in a 48-well plate. Settled cells were cultured with 10 µM EdU for 72h in media containing Neurobrew, N2 and PDGF-BB as described above. In addition, media was supplemented with full (1/1), half (1/2) of absent (0) of bFGF concentration referred to the concentration described above. Cells were harvested with Accutase, labeled with anti-PDGFRα (Milteny Biotec, Bergisch, Germany). Dead cells were excluded using near IR Live/Dead (Thermo Fisher Scientific, Waltham, MA). Samples were analyzed with a 3-laser Beckman Coulter Gallios using Kaluza Software (Beckman Coulter, Brea, CA).
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