Normal Control iPSC Lines
WTC iPSC Line
The WTC line is used as a normal control by research groups all over the world. This iPSC line was derived from a skin biopsy from an healthy adult Asian male donor in his early thirties, who showed normal function in a battery of tests including EKG and head MRI. The original fibroblasts were reprogrammed using episomal methods with the following factors: LIN28A, MYC (L-MYC), POU5F1 (OCT4), and SOX2(Okita el al. Nat Methods 2011, PMID: 21460823, Addgene# 27077, 27078, 27080). The iPSC line was tested for genomic integration of reprogramming factors using qPCR, but no integration was detected. It also has a normal karyotype (46, XY), as shown by cytogenetic testing using FISH methodologies. Immunostaining and RT-PCR have been used to characterize the undifferentiated iPSC line for the pluripotency state, and its pluripotency and differentiation potential have been characterized via teratoma formation, embryoid formation, and cardiomyocyte and neural crest differentiation.
WTC is the parental line for the Allen Institute Cell Collection.
Primary References:
Isolation of single-base genome-edited human iPS cells without antibiotic selection.Yuichiro Miyaoka, Amanda H. Chan, Luke M. Judge, Jennie Yoo, Miller Huang, Trieu D. Nguyen, Paweena P. Lizarraga, Po-Lin So, and Bruce R. Conklin. Nat Methods. Mar 2014; 11(3): 291–293. PMCID: PMC4063274
CRISPR interference efficiently induces specific and reversible gene silencing in human iPSCs. Mo A. Mandegar, Nathaniel Huebsch, Ekaterina B. Frolov, Edward Shin, Truong A, Michael P. Olvera, Amanda H. Chan, Yuichiro Miyaoka, Kristen Holmes, Ian C. Spencer, Luke M. Judge, Gordon DE, Tilda V. Eskildsen , Jacqueline E. Villalta, Max A. Horlbeck, Luke A. Gilbert, Nevan J. Krogan, Soren P. Sheikh, Jonathan S. Weissman, Stanley L. Qi, Po-Lin So, and Bruce R. Conklin. Cell Stem Cell. Apr 2016; 18(4):541-53. PMID: 26971820
Other References:
Stem cells: the new "model organism". Drubin David G., Hyman Anthony A. Mol Biol Cell. 2017 June1;28 (11):1409-1411. PMID:28559439. PMCID: PMC5449140
A robust method to derive functional neural crest cells from human pluripotent stem cells. Faith R. Kreitzer, Nathan Salomonis, Alice Sheehan, Miller Huang, Jason S. Park, Matthew J. Spindler, Paweena Lizarraga, William A. Weiss, Po-Lin So, and Bruce R. Conklin. Am J Stem Cell 2013;2(2):119-131. PMID:23862100, PMCID:PMC3708511
Automated Video-Based Analysis of Contractility and Calcium Flux in Human-Induced Pluripotent Stem-Derived Cardiomyocytes Cultured over Different Spatial Scales. Nathaniel Huebsch, Peter Loskill, Mohammad A. Mandegar, Natalie C. Marks, Alice S. Sheehan, Zhen Ma, Anurag Mathur, Trieu N. Nguyen, Jennie C. Yoo, Luke M. Judge, C. Ian Spencer, Anand C. Chukka, Caitlin R. Russell, Po-Lin So, Bruce R. Conklin, and Kevin E. Healy. Tissue Eng Part C Methods. 2014 Oct 21. PMID:25333967
Miniaturized iPS-cell-derived cardiac muscles for physiologically relevant drug response analyses. NathanielHuebsch, Peter Loskill, Nikhil Deveshwar, Ian C Spencer, Luke M. Judge, Mo A. Mandegar, Cade B. Fox, Tamer M. Mohamed, Zhen Ma, Alice M. Sheehan, Annie Truong, Michael Saxton, Jennie Yoo, Deepak Srivastava, Tejal A. Desai, Po-Lin So, Kevin E. Healy, Bruce R. Conklin. Sci Rep. Apr 2016; 6:24726. PMID: 27095412
A BAG3 Chaperone Complex Maintains Cardiomyocyte Function During Proteotoxic Stress. Luke M. Judge, Juan A. Perez-Bermejo, Annie Truong, Alexandre J. Ribeiro, Jennie C. Yoo, Chrisina L. Jensen, Mohammad A. Mandegar, Nathaniel Huebsch, Robyn M. Kaake, Po-Lin So, Deepak Srivastava, Beth L. Pruitt, Nevan J. Krogan, Bruce R. Conklin. JCI Insight. 2017 Jul 20;2 (14). PMID:2872793
WTB iPSC Line
This iPSC line was derived from a skin biopsy of a 42 year old Hispanic female donor, who was in good health and had a normal EKG at the time of skin biopsy. The original fibroblasts were reprogrammed using episomal methods with the following factors: LIN28A, MYC (L-MYC), POU5F1 (OCT4), and SOX2 (Okita et al. Nat Methods 2011, PMID: 21460823, Addgene# 27077, 27078, 27080). The iPSC line was tested for genomic integration of reprogramming vectors using qPCR, but no integration was detected. It also has a normal karyotype (46, XX), as shown by cytogenetic testing using FISH methodologies. Immunostaining and RT-PCR have been used to characterize the undifferentiated iPSC line for the pluripotency state, and its pluripotency and differentiation potential have been characterized via teratoma formation, embryoid formation, and cardiomyocyte and neural crest differentiation.
Publications:
Isolation of single-base genome-edited human iPS cells without antibiotic selection. Yuichiro Miyaoka, Amanda H. Chan, Luke M. Judge, Jennie Yoo, Miller Huang, Trieu D. Nguyen, Paweena P. Lizarraga, Po-Lin So, and Bruce R. Conklin. Nat Methods. Mar 2014; 11(3): 291–293. PMCID: PMC4063274
A robust method to derive functional neural crest cells from human pluripotent stem cells. Faith R. Kreitzer, Nathan Salomonis, Alice Sheehan, Miller Huang, Jason S. Park, Matthew J. Spindler, Paweena Lizarraga, William A. Weiss, Po-Lin So, and Bruce R. Conklin. Am J Stem Cell 2013;2(2):119-131. PMCID:PMC3708511
Genetically Engineered Versions of WTC and WTB Lines
WTC GCaMP
The calcium-activated version of GFP (GCaMPf6) was targeted to the AAVS1 locus of WTC cells. A strong constitutive promoter (CAG) drives the expression of the GCaMP6f ORF. Puromycin-resistant at 0.5 µg/ml (Sensitive to doses greater than 2.0 µg/ml).
Available from Gladstone Stem Cell Core.
CRISPRi Gen1C
WTC iPSC background, inducible with mCherry and GCaMP6
Mandegar et al. 2016, Cell Stem Cell, 18, 541–553
Available from Gladstone Stem Cell Core.
CRISPRi Gen2C
WTC iPSC background, inducible with no fluorophore
Mandegar et al. 2016, Cell Stem Cell, 18, 541–553
Available from Gladstone Stem Cell Core.
CRISPRi Gen1B
WTB iPSC background, inducible with mCherry and GCaMP6
Mandegar et al. 2016, Cell Stem Cell, 18, 541–553
Available from Gladstone Stem Cell Core.
CRISPRn Gen1C
WTC iPSC background, inducible Cas9 cutting with no fluorophore
Mandegar et al. 2016, Cell Stem Cell, 18, 541–553
Available from Gladstone Stem Cell Core.
CRISPRn Gen1B
WTB iPSC background, inducible Cas9 cutting with no fluorophore
Mandegar et al. 2016, Cell Stem Cell, 18, 541–553
Available from Gladstone Stem Cell Core.
Genomic Sequence Data and RNA Sequence for Normal Control Lines
WTC-public 100X genome sequence from the Allen Institute hg38
The genome analysis of the WTC line is provided by the Allen Institute for Cell Science. We thank the Institute and their founder Paul G. Allen for making this work possible.WTB RNA-Seq data of iPSC-cardiomyocyte differentiation by Wnt pathway modulation:11 time-points of the iPSC to CM.
The protocol used for cardiomyocyte differentiation is adapted from: Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Lian X, Hsiao C, Wilson G, Zhu K, Hazeltine LB, Azarin SM, Raval KK, Zhang J, Kamp TJ, Palecek SP. (2012). Proc. Natl. Acad. Sci. U S A. Jul 3;109(27). PMID: 22645348.
Patient-Derived iPSC Lines
LQT2 iPSC Line
These cells were derived from a skin biopsy from an Asian female donor with a known A422T point mutation (HERG 1264 G>A) associated with Long QT Syndrome, Type 2. An EKG reveals QT prolongation in iPSC-cardiomyocytes derived from this line. The original patient fibroblasts were reprogrammed using retroviral methods with factors: KLF4, MYC (c-MYC), POU5F1 (OCT4), and SOX2. The iPSC line also has a normal karyotype (46 XX). Immunostaining and RT-PCR were carried out to confirm reprogramming to the pluripotent state. Its pluripotency and differentiation potential were characterized using teratoma formation, embryoid formation, and cardiomyocyte differentiation.
Publications:
Calcium transients closely reflect prolonged action potentials in iPSC models of inherited cardiac arrhythmia. C. Ian Spencer, Shiro Baba, Kenta Nakamura, Ethan A. Hua, Marie A.F. Sears, Chi-cheng Fu, Jianhua Zhang, Sadguna Balijepalli, Kiichiro Tomoda, Yohei Hayashi, Paweena Lizarraga, Julianne Wojciak, Melvin M. Scheinman, Katriina Aalto-Setälä, Jonathan C. Makielski, Craig T. January, Kevin E. Healy, Timothy J. Kamp, Shinya Yamanaka, and Bruce R. Conklin. Stem Cell Reports. 2014 Aug 12;3(2):269-81. PMID: 25254341
LQT3 iPSC Line
This iPSC line was derived from a skin biopsy from a Hispanic female donor with a known N406K point mutation (SCN5A 1219 C>A) associated with Long QT Syndrome, Type 3. An EKG reveals QT prolongation in iPSC-cardiomyocytes derived from this line. The original patient fibroblasts were reprogrammed using retroviral methods with factors: KLF4, MYC (c-MYC), POU5F1 (OCT4), and SOX2. The iPSC line has a normal karyotype (46 XX). Immunostaining and RT-PCR were carried out to confirm reprogramming to the pluripotent state. Its pluripotency and differentiation potential were characterized using teratoma formation, embryoid formation, and cardiomyocyte differentiation.
Publications:
Three-dimensional filamentous human diseased cardiac tissue model. Zhen Ma, Sangmoo Koo, Micaela A. Finnegan, Peter Loskill, Nathaniel Huebsch, Natalie C. Marks, Bruce R. Conklin, Costas P. Grigoropoulos, and Kevin E. Healy. Biomaterials. 2014 Feb;35(5):1367-77. PMID: 24268663