Human iPSCs were generated according to a published report [26 (link)] with some modifications. Briefly, 5 × 105 hUCMSCs were electroporated with 3 μg of each episomal plasmid, including pCXLE-hSK, pCXLE-hUL, and pCXLE-hOCT3/4-shp53-F (expressing SOX2, KLF4, L-MYC, LIN28, OCT3/4 and p53-targeting shRNA, Addgene, Cambridge, MA, USA) using the following conditions: 900 μV, 500 ms and 1 pulse. After electroporation, cells were reseeded on a Matrigel (BD Bioscience, San Jose, USA) -coated cell culture dish and cultured in DMEM/F12 containing 10% FBS. Two days later, medium was changed into mTeSRTM1 medium (STEMCELL Technologies Inc., Vancouver, Canada,) containing 0.25 mM Sodium butyrate (Sigma, St. Louis, MO, USA). The iPSC colonies were picked out by cloning ring and subcultured in mTeSRTM1 medium.
Pcxle hoct3 4 shp53 f
Manufactured by Addgene
Sourced in United States
PCXLE-hOCT3/4-shp53-F is a plasmid construct containing the human OCT3/4 gene and a short hairpin RNA (shRNA) targeting the p53 gene. The plasmid is designed for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Pcxle hsk, by Addgene (17 mentions)
Pcxle hul, by Addgene (17 mentions)
Pcxwb ebna1, by Addgene (5 mentions)
Mtesr1, by STEMCELL (4 mentions)
Matrigel, by BD (3 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Matrigel, by Corning (2 mentions)
Pcxle egfp, by Addgene (2 mentions)
Neon transfection system, by Thermo Fisher Scientific (2 mentions)
P2 primary cell 4d nucleofector x kit, by Lonza (2 mentions)
Collagenase type 4, by Thermo Fisher Scientific (2 mentions)
Versene solution, by Thermo Fisher Scientific (2 mentions)
Cultrex bme, by Bio-Techne (2 mentions)
Stemflex medium, by Thermo Fisher Scientific (2 mentions)
Matrigel, by R&D Systems (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (2 mentions)
23 protocols using pcxle hoct3 4 shp53 f
1
Generation of Human iPSCs from UCMSCs
2
Episomal Reprogramming of Equine Fibroblasts
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Equine fibroblasts were transfected with different combinations of episomal reprogramming plasmids that have demonstrated to reprogram human cells [38 (link)–40 (link)]: pCXLE-hOCT3/4-shp53-F (addgene N°27077), pCXLE-hSK (addgene N°27078), pCXLE-hUL (addgene N°27080), pEP4 E02S CK2M EN2L (addgene N°20924) and pCXWB-EBNA1 (addgene N°37624). Either the 100 μl tip NEON system or FuGENE6 transfection reagent (Roche, 1814443) was used for the transfection of the plasmids, following the manufacturer´s instructions in both cases. The equine fibroblasts were transfected with different conditions of plasmid concentrations, plasmid combinations and NEON settings, which are detailed in S3 Table . All the conditions were evaluated twice and the EGFP-N1 plasmid was used as control of transfection in each procedure. Once transfected, the cells were cultured in gelatin coated p100 in DMEM 10% SFB medium for 5 days. After that time, they were trypsinized and plated in different dilutions over iMEFs in eqHES medium.
3
Generation of Human iPSC Lines
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The iPSCs were generated from human dermal fibroblasts HDFa (Cascade Biologics) and asF5 (CellResearch Corporation Pte Ltd). Somatic reprogramming of skin fibroblasts was performed using a modification of the previously described reprogramming method30 (link). The episomal vectors pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, and pCXLE-hUL were purchased from Addgene (27077, 27078, 27080, respectively). Actively growing fibroblasts were co-transfected with the three vectors using the Neon Transfection System. The iPSC lines were maintained feeder-free in mTeSR1 (StemCell Technologies 85850). Human ESCs (H1 and H9) were cultured as described previously20 (link).
4
Construction of Polycistronic Reprogramming Vector
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A detailed construction strategy is illustrated in Supplementary Fig. 1a .
A CLEB-B4 miniGene was synthesized by Integrated DNA Technologies (IDT) and inserted into pCLEB to produce the backbone vector pCLEB-B4. The CMV expression cassette was removed from pCEP4 to make pCEP4.2, and then an oriP-EBNA1 fragment was transferred from pCEP4.2 to pCLEB-B4 to generate pCLEB-EBV.
OCT3/4-P2A-SOX2-P2A-KLF4 was transferred from pPK33237 (link) to the shuttle vector pM7.5-EF1α to produce pM7.5E-OSK and, subsequently, the woodchuck hepatitis post-transcriptional regulatory element (WPRE) was inserted upstream of the polyadenylation signal to produce pM7.5E-OSKw.
Shp53 from pCXLE-hOCT3/4-shp53-F (Addgene: plasmid #27077) and hUL from pCXLE-hUL (Addgene: plasmid #27080) were transferred sequentially to the shuttle vector pM.3-CAG to produce pM7.3Cgh-shp53-hUL, which was combined with elements of pM7.5E-OSKw to result in pM7-R6.
The six reprogramming factors were then transferred from pM7-R6 to pCLEB-EBV to obtain pCLEB-R6.
A 17 kb stuffer DNA fragment was inserted into pCLEB-R6 to generate pCLAE-R6.
A CLEB-B4 miniGene was synthesized by Integrated DNA Technologies (IDT) and inserted into pCLEB to produce the backbone vector pCLEB-B4. The CMV expression cassette was removed from pCEP4 to make pCEP4.2, and then an oriP-EBNA1 fragment was transferred from pCEP4.2 to pCLEB-B4 to generate pCLEB-EBV.
OCT3/4-P2A-SOX2-P2A-KLF4 was transferred from pPK33237 (link) to the shuttle vector pM7.5-EF1α to produce pM7.5E-OSK and, subsequently, the woodchuck hepatitis post-transcriptional regulatory element (WPRE) was inserted upstream of the polyadenylation signal to produce pM7.5E-OSKw.
Shp53 from pCXLE-hOCT3/4-shp53-F (Addgene: plasmid #27077) and hUL from pCXLE-hUL (Addgene: plasmid #27080) were transferred sequentially to the shuttle vector pM.3-CAG to produce pM7.3Cgh-shp53-hUL, which was combined with elements of pM7.5E-OSKw to result in pM7-R6.
The six reprogramming factors were then transferred from pM7-R6 to pCLEB-EBV to obtain pCLEB-R6.
A 17 kb stuffer DNA fragment was inserted into pCLEB-R6 to generate pCLAE-R6.
5
Generation of Human iPSCs from Fibroblasts
6
Establishment of iPSCs from Urine Cells
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Establishment of iPSCs was performed as reported before18 (link). 0.5 × 106 primary urine cells were reprogrammed using the Amaxa Basic Nucleofector Kit (Lonza) according to the protocol of the manufacturer. 1 μg of each plasmid pCXLE-hOCT3/4-shp53-F (Addgene #27077), pCXLE-hSK (Addgene #27078), and pCXLE-hUL (Addgene #27080) was used. After nucleofection, the cells were transferred to a Matrigel (Corning) coated 6 well. After 24 h, the UC medium was replaced by mTeSR-1 (Stemcell Technologies), supplemented with 1% PenStrep, and changed every second day. After about two weeks the emerged iPSC colonies were picked and transferred to a new Matrigel-precoated 6 well plate. The mTeSR-1 cell medium (was replaced on a daily basis thereafter and the iPSC colonies were subcultured every seven days using 1 U/ml Dispase (Stemcell Technologies). During cultivation, colonies with atypical morphology were removed by scraping. HepaRG cells served as biliary reference cell line and were maintained in RPMI/10% FCS.
7
Generation and Characterization of HCM hiPSCs
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BRAF1 dermal fibroblasts were obtained from an 18-year-old female with HCM. BRAF1 hiPSCs were generated using retroviral pMX-based vectors encoding human OCT3/4, SOX2, KLF4, and c-MYC as previously published (Carvajal-Vergara et al., 2010 (link)) with modifications. Retrovirus was added to fibroblasts 24 and 48 hr after plating. After day 4 the medium was changed every 2 days, and hESC-like colonies were isolated after 30 days. BRAF2 and BRAF3 dermal fibroblasts were obtained from a 13-year-old female with HCM and a 2-year-old male without evidence of HCM, respectively. BRAF2 and BRAF3 hiPSC lines were generated using episomal-based vectors as previously described (Okita et al., 2011 (link)), with modifications. One microgram of each plasmid pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCXLE-EGFP (Addgene) was mixed with Resuspension Buffer R (Life Technologies) and added to fibroblasts. Cells were electroporated using the Neon Transfection System (Life Technologies), re-plated, and placed at 37°C. After 5 days, fibroblasts were plated on MEFs and maintained in hiPSC medium. Clones were chosen based on morphology and growth. Pluripotency was verified by immunofluorescence, gene expression, southern blot, and teratoma formation or in vitro tri-lineage differentiation assays. Fully characterized retrovirally generated WT hiPSC lines were separately provided.
8
Reprogramming B-lymphocytes to iPSCs
9
Reprogramming Dura-Derived Fibroblasts into iPSCs
10
Optimized Fibroblast Reprogramming for iPSCs
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pCXLE-hOCT3/4-shp53-F, pCXLE-hUL, pCXLE-hSK, pCXLE-hGLIS1, and pCXWB-EBNA1 were purchased from Addgene. pCXLE-hTET-1 was constructed. iMATRIX-511 was purchased from Nippi® (Tokyo, Japan). Fibroblasts (1.0 × 106) from each tissue sample were prepared, then gene transfection for iPS cell reprogramming was performed with the electroporation device Nucleofector 2b (Lonza Japan®, Tokyo, Japan) using the U-020 protocol, VPI-1002 transfection kit (Lonza Japan®), 1.2 µg each of pCXLE-hOCT3/4-shp53-F, pCXLE-hUL, pCXLE-hSK, pCXLE-hGLIS1, and pCXLE-hTET-1, and 1.0 µg of pCXWB-EBNA1. To reduce the possibility of the induction efficiency being decreased by the entry of a nick when using the stocked vectors, we irradiated the vectors before use. After electroporation, the cells were seeded on 100-mm dishes that had previously been coated with collagen and 0.125 µg/cm2 of iMATRIX-511. In protocol 1, after day 1 post-transfection, they were cultured in an incubator set at 37 °C with 5% O2. In protocol 2, from day 1 to day 5 post-transfection, the cells were cultured in an incubator set at 37 °C with 20% O2; after day 5 post-transfection, they were cultured in an incubator set at 37 °C with 5% O2.
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