HUVECs were a gift from Dr. Sandra Davidge (University of Alberta). HUVECs were prepared and maintained in culture as previously described [23 ]. To generate iPSCs without the use of mouse embryonic feeder layer, HUVECs (passages 2–3) were seeded on BD Matrigel hESC-qualified Matrix that had been optimized to support iPSC growth for up to 25 passages [24 (link),25 (link)]. Twenty-four hours (h) after seeding, cells were approximately 70% confluent, and were transduced with 5MOI of six lentiviral vectors expressing the genes Lin28, c-Myc, Klf4, Nanog, Sox2 and Oct4. In parallel, HUVECs were transduced with 5MOI GFP-lentivirus to assess the transduction efficiency. HUVECs were maintained in complete endothelial cell growth medium (M199, 20% FBS, 2 mM glutamine, 100 IU/ml penicillin/streptomycin and 100 μg/ml ECGS) for 4 days, which was then replaced with mTeSR1 medium. The medium was changed daily after this point. After 2 weeks, colonies with morphologic characteristics of pluripotent stem cells (round colonies with defined edges, individual cells within the colony had a high nuclear to cytoplasmic ratio, and were tightly packed with defined junctions) started to emerge. After 3 weeks, desirable colonies were selected and passaged on Matrigel according to the protocol described by the Stem Cell Technologies mTeSR1 manual.
Matrigel hesc qualified matrix
Manufactured by BD
Sourced in Canada, United States
Matrigel hESC-qualified Matrix is a soluble basement membrane extract of the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, rich in extracellular matrix proteins. It is commonly used as a substrate for the culture of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs).
Automatically generated - may contain errors
Lab products found in correlation
Mtesr1 medium, by STEMCELL (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Mtesr1, by STEMCELL (4 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Aggrewell medium, by STEMCELL (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Ultra low attachment plate, by Corning (2 mentions)
Newborn bovine serum, by Atlanta Biologicals (2 mentions)
Rpmi b27 culture media, by Thermo Fisher Scientific (2 mentions)
Rpmi b27 culture media, by Atlanta Biologicals (2 mentions)
Detach 2 kit, by PromoCell (2 mentions)
Collagen type 1, by Merck Group (2 mentions)
Amphotericin b, by Merck Group (2 mentions)
Mtesr1 medium kit, by STEMCELL (2 mentions)
Gf144, by Merck Group (2 mentions)
Gf003, by Merck Group (2 mentions)
Tryple express, by Thermo Fisher Scientific (2 mentions)
Serum replacement, by Thermo Fisher Scientific (1 mentions)
Psc neural induction medium, by Thermo Fisher Scientific (1 mentions)
28 protocols using matrigel hesc qualified matrix
1
Generation of iPSCs from HUVECs
2
Differentiation of hESCs into MSCs
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To induce differentiation of the naïve-hESCs into mesenchymal lineage cells, we first adapted the hESCs on a feeder-free culture condition using the mTeSR medium kit (STEMCELL Technologies Inc., Canada) on Matrigel hESC-qualified Matrix (BD Biosciences) with 20% (v/v) serum replacement (Gibco) for 5 d to verify the xeno-free differentiation condition. Then, the cells were differentiated into the MSC cell population via treatment with 100 ng/ml of CTGF for an additional 5 d in same cell culture medium. Finally, the differentiated cells were maturated into the MSC population in EBM cell culture medium (Lonza, USA) treated with 25 ng/ml of CTGF. The EBM medium consists of ascorbic acid, GA-1000, hydrocortisone, and 2% SR. After the final differentiation, the CTGF-induced hESC-MSCs were cultured in EMB medium and expanded through the typical trypsinization method.
3
Directed Differentiation of iPSCs
4
Derivation of 201B7 iPSC-derived neural stem cells
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The human iPSC line 201B7 was provided by the RIKEN BioResource Center in Japan. This iPSC line was derived from human dermal fibroblasts from the facial dermis of 36-year old Caucasian female [21 (link)]. The 201B7 iPSCs were maintained in primate embryonic stem (ES) cell medium (ReproCELL, Japan) supplemented with 4 ng/mL recombinant human fibroblast growth factor (FGF)-basic (146 aa), and penicillin–streptomycin (Life Technologies, USA) on mitomycin C (Kyowa kirin, Japan)-treated mouse embryonic fibroblasts (SNL 76/7; DS Pharma Biomedical, Japan) as feeder cells. These cultures were maintained at 37°C in a humidified incubator with 5% CO2. The iPSCs were then cultured with an mTeSR1 medium kit (Stem Cell Technologies, Canada) on Matrigel hESC-qualified matrix (BD, USA) without feeder cells. Subsequently, the iPSCs were induced to form NSCs by treatment with PSC neural induction medium (Thermo Fisher Scientific, USA) for 7 days according to the manufacturer’s instructions.
5
Generation of Retinal Pigment Epithelial Cells
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iPSC colonies were cultured on Matrigel hESC-qualified matrix (354277, BD Biosciences) in Essential 8 (A1517001, Thermo Fisher). Once 80% confluent, the media was changed to Differentiation Media17 (link). From here on, cultures were fed twice weekly for at least a further 8 weeks until pigmented foci were observed. These pigmented foci were isolated manually and purified, fed twice weekly with X-VIVO 10 (LZBE 04-743Q, Lonza) with gentamicin (ThermoFisher). All experiments were performed with passage 2 RPE aged 8 to 16 weeks.
6
Pluripotent Stem Cell Culture Protocol
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M199, MEM medium, RPMI1640, AIM-V T cell culture medium, FBS, glutamine, penicillin/streptomycin, ECGS, both B27 with insulin and without insulin, essential amino acids, sodium pyruvate, and dextrose were purchased from Invitrogen, Life Technologies (Burlington, ON). Matrigel hESC-qualified Matrix was from BD Biosciences, ON. Lentiviral vectors were from viPS Vector Kit (ThermoFisher Scientific, catalogue number IPS5449). Activin A was from R&D Systems (Minneapolis, MN). mTeSR1 medium, Accutase, Aggrewell plates, Aggrewell medium, Rock inhibitor (Y-27632), and DMEM/F12 medium were from Stem Cell Technologies Inc, BC; RNeasy Plus mini kit was from Qiagen, Ontario, and qScript synthesis kit was purchased from Quanta Biosciences, VWR international.
7
Directed Differentiation of iPSCs
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iPS cells grown on Matrigel hESC-qualified matrix (BD Biosciences) in mTESR1 medium (StemCell Technologies, Inc.) were washed with phosphate buffered saline (PBS) and dissociated with collagenase for 10 minutes at 37°C. After partial dissociation, the colonies were harvested using a cell scraper and pelleted at 200×g for 5 minutes. The colonies were resuspended in Aggrewell medium (StemCell Technologies, Inc.) and transferred to ultra-low attachment plates (Corning) to form embryoid bodies. The medium was changed every 3 days until day 7, when the embryoid bodies were transferred to 0.1% gelatin-coated plates in culture medium (DMEM with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1% penicillin/streptomycin (Life Technologies)). The medium was changed every 3 days until day 7, when RNA was isolated in TRIzol. RT-PCR was performed for genes representative of the three germ layers.
8
Feeder-free culture of human pluripotent stem cells
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The hESC lines KhES-1, KhES-3, H9, and HES3 (Reubinoff et al., 2000; Suemori et al., 2006; Thomson et al., 1998 ) and the hiPSC lines 253G1 (Takahashi et al., 2007 (link)) and IMR90-1 (Yu et al., 2007 (link)) were used for this study. The hESC lines were used in accordance with the Guidelines for the Derivation and Utilization of Human Embryonic Stem Cells of the Ministry of Education, Culture, Sports, Science and Technology, Japan.
For the conventional adherent culture, hPSCs were grown on a mouse fibroblast feeder cell layer in DMEM/F12 with 20% KSR (Life Technologies) and 5 ng/ml recombinant bFGF (Wako Pure Chemical Industries) as reported previously (Suemori et al., 2006 (link)). For feeder-free adherent culture, hPSCs were grown on Matrigel (hESC-qualified Matrix, BD Biosciences) in mTeSR culture medium (STEMCELL Technologies).
For the conventional adherent culture, hPSCs were grown on a mouse fibroblast feeder cell layer in DMEM/F12 with 20% KSR (Life Technologies) and 5 ng/ml recombinant bFGF (Wako Pure Chemical Industries) as reported previously (Suemori et al., 2006 (link)). For feeder-free adherent culture, hPSCs were grown on Matrigel (hESC-qualified Matrix, BD Biosciences) in mTeSR culture medium (STEMCELL Technologies).
9
Culturing Human Cell Lines and hESCs
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The human cervical carcinoma cell lines HeLa, SiHa, C33A and CaSki were purchased from the American Type Culture Collection (ATCC). HeLa, SiHa and C33A cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, St. Louis, Mo) supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA) at 37°C in an atmosphere of 5% CO2. CaSki cells were maintained in McCoy’s 5A Medium (Sigma-Aldrich) with 10% FBS.
The human Embryonic Stem Cell (hESC) line H7 (given by the professor Huayan Wang, Department of Animal Biotechnology, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China) was obtained the approval from the Ethical Committee of the Xi’an Jiaotong University. Cells were cultured feeder-free in mTeSR medium (Stem Cell Technologies, Vancouver, Canada) on Matrigel (hESC-qualified Matrix, BD Biosciences, CA, USA) in a 5% CO2 normoxic humidified incubator and passaged 1∶6 using accutase solution (Millipore) every 3–7 days.
The human Embryonic Stem Cell (hESC) line H7 (given by the professor Huayan Wang, Department of Animal Biotechnology, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China) was obtained the approval from the Ethical Committee of the Xi’an Jiaotong University. Cells were cultured feeder-free in mTeSR medium (Stem Cell Technologies, Vancouver, Canada) on Matrigel (hESC-qualified Matrix, BD Biosciences, CA, USA) in a 5% CO2 normoxic humidified incubator and passaged 1∶6 using accutase solution (Millipore) every 3–7 days.
10
Reprogramming Fibroblasts into iPSCs Using mRNA, microRNA, and Lentiviral Approaches
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WBS1, WBS2, WBS3, WBS4, 7dupASD1, atWBS1, and CTL2 fibroblasts were reprogrammed using the mRNA Reprogramming Kit (Stemgent), while the 7dupASD2 and CTL1R lines were reprogrammed with the microRNA Booster Kit (Stemgent). The CTL3 line was reprogrammed by transfection with the STEMCCA polycistronic lentiviral vector followed by Cre-mediated excision of the integrated polycistron. 7dupASD3 and CTL4R fibroblasts were reprogrammed using the Simplicon RNA Reprogramming Kit (Millipore).
Before differentiation, iPSC lines were cultured on Matrigel hESC-qualified Matrix (BD Biosciences)–coated plates, diluted 1:40 in Dulbecco’s minimum essential medium/F-12, and grown in mTeSR 1 medium (STEMCELL Technologies). They were passaged upon treatment with Accutase (Sigma-Aldrich) and then plated in mTeSR 1 medium supplemented with 5 μM Y-27632 (Sigma-Aldrich).
Before differentiation, iPSC lines were cultured on Matrigel hESC-qualified Matrix (BD Biosciences)–coated plates, diluted 1:40 in Dulbecco’s minimum essential medium/F-12, and grown in mTeSR 1 medium (STEMCELL Technologies). They were passaged upon treatment with Accutase (Sigma-Aldrich) and then plated in mTeSR 1 medium supplemented with 5 μM Y-27632 (Sigma-Aldrich).
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