All experimentation using hPSC lines was approved by the University of Melbourne Human Ethics Committee (approval # 0605017) and conducted according to the National Health and Medical Research Council of Australia Guidelines for the Use of Human Stem Cells in Research (The National Statement, Chapter 2.1, 2007). The hPSC line H9 ([14 (link)], WA-09 (WiCell)) and human foreskin fibroblasts (CCD-1079Sk; ATCC) were used in this study.
Ccd 1079sk
Manufactured by American Type Culture Collection
Sourced in United States
The CCD-1079Sk is a charge-coupled device (CCD) camera designed for laboratory applications. It features a high-resolution CCD sensor that captures detailed images. The camera is capable of providing accurate and reliable data acquisition for various scientific and research purposes.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Sh sy5y, by American Type Culture Collection (2 mentions)
Sk n be 2, by American Type Culture Collection (2 mentions)
Human umbilical vein endothelial cells (huvec), by American Type Culture Collection (2 mentions)
Dmem high glucose media, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Mtesrtm1 medium, by STEMCELL (1 mentions)
Short tandem repeat profiling, by Eurofins (1 mentions)
Mccoy s 5a, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Y 27632, by Bio-Techne (1 mentions)
H14 wa14, by WiCell (1 mentions)
H9 wa09, by WiCell (1 mentions)
Dmi8 microscope, by Leica (1 mentions)
Sk n sh cells, by Korean Cell Line Bank (1 mentions)
T 47d cells, by Merck Group (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Mcf 10a, by American Type Culture Collection (1 mentions)
Bt474, by American Type Culture Collection (1 mentions)
14 protocols using ccd 1079sk
1
Ethical hPSC Research Protocols
2
Fibroblast Culture for Reprogramming
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Human fibroblast lines PCS201-012 (PCS-201-012) and CCD-1079Sk (CRL-2097) were obtained from the ATCC, and cultured in DMEM high-glucose media (Life Technologies 11195-073) containing 10% fetal bovine serum, penicillin/streptomycin (Life technologies 10378-016), non-essential amino acids (Life Technologies 11140-050) and L-glutamine (2 mM) until several days into reprogramming.
3
Culturing Human Stem Cells and Fibroblasts
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The NIH-registered human embryonic stem cell (hESC) lines H1 and H9 were cultured in the chemically defined media as described previously [4 (link),5 (link),8 (link),13 (link)]. Briefly, hESCs were cultured on Matrigel-coated vessels with E8 media [60 (link)], and passaged using EDTA-mediated dissociation when they reach 80% confluency. Human iPSC lines were culture in the same way as for hESCs.
Human primary fibroblasts (BJ, ATCC, Cat#, CRL-2522; CCD-1079Sk, ATCC, Cat#, CRL-2097) were culture in the fibroblast medium, Dulbecco’s modified Eagle’s medium, with high glucose, supplemented with 10% heat-inactivated fetal bovine serum, 0.1 mM 2-mercaptoethanol, 1× penicillin–streptomycin, 0.1 mM minimum essential medium non-essential amino acids, and 4 ng/mL human fibroblast growth factor 2 (FGF2).
Human primary fibroblasts (BJ, ATCC, Cat#, CRL-2522; CCD-1079Sk, ATCC, Cat#, CRL-2097) were culture in the fibroblast medium, Dulbecco’s modified Eagle’s medium, with high glucose, supplemented with 10% heat-inactivated fetal bovine serum, 0.1 mM 2-mercaptoethanol, 1× penicillin–streptomycin, 0.1 mM minimum essential medium non-essential amino acids, and 4 ng/mL human fibroblast growth factor 2 (FGF2).
4
Derivation of iPSCs from MCF-7 Breast Cancer Cells
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MCF-7 cells (Mcfs) was a human breast carcinoma line, which was useful for in vitro BC studies because the cell line had retained several ideal characteristics. It was gifted from Professor Liu Feng (Cancer Hospital of Harbin Medical University). Human iPSCs (Hips) were purchased from Innovative Cellular Therapeutics, which were generated by reprogramming human fibroblasts (CCD-1079SK, ATCC) using four transcription factors (TFs) (Oct4, Sox2, Klf4, and c-Myc) delivered by lentiviral vectors. MCFs were cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen). Human iPSCs were grown in iPSCs medium (knockout DMEM/F12 plus 20% KOSR, 1% penicillin/streptomycin, 1% NEAA, 1% Glutamax, 100μM β-mercaptoethanol and 10 ng/ml bFGF).
293T cells were transfected with Oct4, Sox2, c-Myc and Klf4 plasmids. After 48h, the viruses supernatant were collected and transduced into MCFs. Then, MCFs were cultured in 10% FBF-DMEM medium for 6 days. At Day7, the induced cells were reseeded on the irradiated mouse embryonic fibroblasts and cultured in iPSCs medium. Cells were subsequently observed for colony formation. About day 20, iPS colonies were isolated and transferred to matrigel-coated plate and maintained in mTeSRTM1 medium (Stem Cell).
293T cells were transfected with Oct4, Sox2, c-Myc and Klf4 plasmids. After 48h, the viruses supernatant were collected and transduced into MCFs. Then, MCFs were cultured in 10% FBF-DMEM medium for 6 days. At Day7, the induced cells were reseeded on the irradiated mouse embryonic fibroblasts and cultured in iPSCs medium. Cells were subsequently observed for colony formation. About day 20, iPS colonies were isolated and transferred to matrigel-coated plate and maintained in mTeSRTM1 medium (Stem Cell).
5
Human Embryonic Stem Cell Cultivation and Fibroblast Culture
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The NIH-registered human embryonic stem cell (ESC) lines H1 (WiCell, Madison, WI) and H9 (WiCell, Madison, WI) were cultured in the chemically defined media as described before2 (link),3 (link),9 (link),10 (link). Briefly, hESCs were cultured on Matrigel-coated vessels with the E8 media25 (link), and passaged using the EDTA-mediated dissociation when they reach 80% confluency.
Human primary fibroblasts (BJ, ATCC, CRL-2522, and CCD-1079Sk, ATCC, CRL-2097) with normal karyotypes were cultured in the fibroblast medium: Dulbecco’s Modified Eagle Medium (DMEM) with high glucose, supplemented with 10% heat-inactivated fetal bovine serum, 0.1 mM 2-mercaptoethanol, 1 × penicillin–streptomycin, 0.1 mM Minimum Essential Medium Non-Essential Amino Acids, and 4 ng/mL human FGF2.
Human primary fibroblasts (BJ, ATCC, CRL-2522, and CCD-1079Sk, ATCC, CRL-2097) with normal karyotypes were cultured in the fibroblast medium: Dulbecco’s Modified Eagle Medium (DMEM) with high glucose, supplemented with 10% heat-inactivated fetal bovine serum, 0.1 mM 2-mercaptoethanol, 1 × penicillin–streptomycin, 0.1 mM Minimum Essential Medium Non-Essential Amino Acids, and 4 ng/mL human FGF2.
6
Culturing Diverse Cell Lines for Research
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Karyotypically normal hTERT RPE-1 cells (ATCC CRL-4000) were derived by transfecting the retinal pigmented epithelial line RPE-340 cell line with pGRN145 hTERT-expressing plasmid (40 (link)). Cells were maintained in DMEM/F-12 Ham (1/1) medium (Sigma-Aldrich) containing 2 mM l -glutamine, 10% (vol/vol) FBS, and 0.25% (vol/vol) sodium bicarbonate. Human male newborn skin fibroblasts, CCD-1079Sk (ATCC CRL-2097), are karyotypically normal. CCD-1079Sk fibroblasts and the normal adult male fibroblast line 1BR3 were maintained in DMEM/F-12 Ham (1/1) medium containing 2 mM l -glutamine, 10% (vol/vol) FBS, and nonessential amino acids. Cancer cell lines HT1080 and HeLa were cultured in DMEM (Gibco) with 10% FBS. U2OS cells were grown in McCoys 5A (Sigma-Aldrich) supplemented with 10% FBS. GM10063, A9 cells containing an Xder21 reciprocal translocation product, were obtained from the Coriell Institute and cultured as specified. Cell line authentication was done by short tandem repeat profiling (Eurofins).
7
Cultivation of Human Fibroblasts and BHK21 Cells
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All growth media, fetal calf serum (FCS), antibiotics, and other supplements were supplied by Life Technologies/Gibco, except when stated otherwise. Human foreskin fibroblasts obtained from System Bioscience (HFF, neonatal) or ATCC (CCD-1079Sk) were cultivated in minimum essential media (MEM) containing 15% FCS, 1 IU/mL of penicillin, 1 μg/mL of streptomycin, 1% non-essential amino acids, and 1 mM of sodium pyruvate at 37°C. Cells were grown at 37°C in a humidified atmosphere equilibrated to 5% CO2. BHK21 cells (ATCC; CCL10) were grown in Eagle's MEM supplemented with 10% FCS.
8
Establishment of Induced Pluripotent Stem Cells
9
Quantifying Cell Death in hES Cells
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Human embryonic stem cell lines H9 (WA09), H14 (WA14) and WA22 were obtained from WiCell Research Institute. hES cells were maintained on hES cell qualified matrigel in mTeSR1 medium. Human normal skin fibroblasts CCD-1079Sk were acquired from ATCC and maintained in DMEM with 10% Fetal Bovine Serum and MEM non-essential amino acids. Cells were maintained at 37℃ in 5% CO2.
For cell death assays, cells were seeded at a density of 600,000 cells per well in 6-well plates.
Approximately after 24 hours, cells were treated with etoposide or tunicamycin. When MCL1 inhibitors (S63845 and AZD5991) were used, cells were treated with inhibitors in presence or absence of etoposide. Cells were stained with Nuclear Blue to label cell nuclei. Images were captured using Leica DMi8 microscope. Cell death was quantified on the basis of cellular and nuclear morphology, average of more than 800 cells were analyzed per condition. For siRNA transfections, cells were seeded at 350,000 cells per well in 6-well. Approximately after 24 hours, control siRNA (MISSION siRNA Fluorescent Universal Negative Control #1, Cyanine 3) and MCL1 siRNA (GGACUUUUAUACCUGUUAUtt) were transfected using lipofectamine 3000 reagent at 50 nM concentration. Approximately 24 hours post transfection cells, cells were treated with DMSO or etoposide and cell death was quantified as described above.
For cell death assays, cells were seeded at a density of 600,000 cells per well in 6-well plates.
Approximately after 24 hours, cells were treated with etoposide or tunicamycin. When MCL1 inhibitors (S63845 and AZD5991) were used, cells were treated with inhibitors in presence or absence of etoposide. Cells were stained with Nuclear Blue to label cell nuclei. Images were captured using Leica DMi8 microscope. Cell death was quantified on the basis of cellular and nuclear morphology, average of more than 800 cells were analyzed per condition. For siRNA transfections, cells were seeded at 350,000 cells per well in 6-well. Approximately after 24 hours, control siRNA (MISSION siRNA Fluorescent Universal Negative Control #1, Cyanine 3) and MCL1 siRNA (GGACUUUUAUACCUGUUAUtt) were transfected using lipofectamine 3000 reagent at 50 nM concentration. Approximately 24 hours post transfection cells, cells were treated with DMSO or etoposide and cell death was quantified as described above.
10
Cell Culture of H9c2 and CCD-1079Sk
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Rat cardiomyoblasts H9c2 (CRL-1446™) and human skin fibroblasts CCD-1079Sk (CRL-2097™) were purchased from American Type Culture Collection (ATCC, VA, USA), and cultured in DMEM-F12 medium supplemented with 10% fetal bovine serum and 1 % penicillin–streptomycin (Gibco, USA). Sub-culturing was done every 2–3 days.
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