Generation of a human induced pluripotent stem cell (iPSC) line from a 64 year old male patient with multiple schwannoma Shaokun Zhang, Zhenshan Lv, Lidi Liu, Weiquan Gong, Qiao Li, Hong Wu PII: DOI: Reference:
S1873-5061(16)30223-9 doi: 10.1016/j.scr.2016.12.023 SCR 905
To appear in:
Stem Cell Research
Received date: Accepted date:
9 December 2016 15 December 2016
Please cite this article as: Zhang, Shaokun, Lv, Zhenshan, Liu, Lidi, Gong, Weiquan, Li, Qiao, Wu, Hong, Generation of a human induced pluripotent stem cell (iPSC) line from a 64 year old male patient with multiple schwannoma, Stem Cell Research (2016), doi: 10.1016/j.scr.2016.12.023
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ACCEPTED MANUSCRIPT Stem Cell Research: Lab Resource Title: Generation of a human induced pluripotent stem cell (iPSC) line from a 64 year old male patient with multiple schwannoma
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Authors: Shaokun Zhang1, Zhenshan Lv1, Lidi Liu1, Weiquan Gong 1, Qiao Li1, Hong Wu2, *
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Affiliations:
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1. Department of Spine Surgery, the 1st Hospital of Jilin University, Changchun 130021, China 2. Department of Ophthalmology, the 2nd Hospital of Jilin University, Changchun 130041, China
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* Correspondence author Abstract:
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Resource Table:
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Peripheral blood was collected from a clinically diagnosed 64-year old male multiple schwannoma patient. Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the Yamanaka KMOS reprogramming factors using the Sendai-virus reprogramming system. The transgene-free iPSC line showed pluripotency verified by immunofluorescent staining for pluripotency markers, and the iPSC line was able to differentiate into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This in vitro cellular model will be useful for further pathological studies of multiple schwannoma.
Name of Stem Cell line
MS-BP003-iPSC
Institution
The 1st Hospital of Jilin University Hong Wu
Contact person and email
Hong Wu,
[email protected]
Date archived/stock date
June 2016
Origin
Peripheral blood mononuclear cells (PBMCs)
Type of resource
Biological reagent: human induced pluripotent stem cell (iPSC) line
Sub-type
cell line
Key transcription factors
Klf-4, c-Myc, Oct-4, Sox-2
Authentication
Identity and purity of cell line confirmed (Figure 1)
Link to related literature
Not available
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Person who created resource
ACCEPTED MANUSCRIPT Not available
Ethics
Patient informed consent obtained, Ethics Review Board authority approval obtained
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Information in public databases
Resource Details
Materials and Methods
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The study was approved by the ethics committee of 1st and 2nd Hospital of Jilin University, written informed consent was obtained from the patient. 1ml of peripheral blood sample was extracted from a 64-year-old male multiple schwannoma (MS) patient. This patient does not harbour SMARCB1 and NF2 mutations, which was often observed in familial MS cases (Jacoby et al., 1997; Smith et al., 2012). The MS-BP003 iPSC lines were derived using the CytoTune®-iPS 2.0 Reprogramming System (Thermo Fisher Scientific), including four Yamanaka factors Klf-4, c-Myc, Oct-4 and Sox-2 (Ban et al., 2011). The derived hiPSC lines displayed a typical ES cell morphology, a high nucleus/cytoplasm ratio and prominent nucleoli. The identity of derived iPSC lines was confirmed by immunofluorescence staining. After 7-9 passages, the absence of exogenous reprogramming transgenes was checked by RT-PCR. The differentiation capacity of hiPSC lines into three germ layers was investigated by in vivo teratoma formation assay. The derived hiPSC lines showed normal karyotype (46, XY).
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Expansion and reprogramming of PBMCs
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1ml of peripheral blood sample was collected, and was lysed in red blood cell (RBC) lysis buffer (eBioscience, San Diego, CA). Purified PBMCs were collected and seeded into one well of a 12-well tissue culture plate in 1ml of cell expansion medium, which contained StemSpan expansion medium (StemCell Technologies, Vancouver) supplemented with 1× penicillin/streptomycin (pen/strep) (Thermo Fisher Scientific), 1× L-glutamine (Thermo Fisher Scientific), 1× nonessential amino acids (Thermo Fisher Scientific), 50 µg/ml L-ascorbic acid (Sigma-Aldrich, St. Louis, MO), 50 ng/ml stem cell factor (Peprotech, Rocky Hill, NJ), 10 ng/ml interleukin-3 (Peprotech), 40 ng/ml insulin-like growth factor-1 (Peprotech), 2 U/ml erythropoietin (Peprotech), 1 µM dexamethasone (Sigma-Aldrich) and 10 ng/ml interleukin-6 (Peprotech). After expanding for 10-15 days, a total of 200,000 PMBCs were incubated with Klf-4, c-Myc, Oct-4 and Sox-2 Sendai virus (CytoTune-iPS Reprogramming Kit, Thermo Fisher Scientific) with a MOI of 5 following the manufacturer’s instruction (Figure 1A). 1ml of fresh PBMC expansion medium was added 24 hours later and cells were replated onto 1% Matrigel (BD Biosciences) coated dish 2 days later. The cell colonies with an ES cell like appearance were manually identified and picked between Day 16 to Day 22 post infection. The human iPSC cultures were maintained on culture plates coated with 1% Matrigel in mTesR-1 medium (Stem Cell Technologies) following the manufacturer's instruction (Figure 1B). All cells were cultured at 37 °C in humidified atmosphere containing 5% CO2. Immunofluorescence (IF) staining The expression of pluripotency markers were studies using IF staining. hiPSCs were fixed with 4% paraformaldehyde (PFA, Sigma), permeabilized with 1% TritonX-100 for 10 minutes
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Reverse transcription polymerase chain reaction (RT-PCR)
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(Sigma) and blocked with 5% goat serum for 45 minutes at room temperature. Oct-4 (Santa Cruz), SSEA-4 (Abcam) and TRA-1-81 (Millipore) primary antibodies diluted in 5% goat serum were applied and incubated at room temperature for 1 h. Alexfluo568-conjugated secondary antibodies (Thermo Fisher Scientific) were incubated for 45 minutes at room temperature. Cells were visualized under inverted fluorescence microscope equipped with AxioVision 4.8.1 software (Carl Zeiss) (Figure 1C).
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Total RNA was isolated from hiPSCs using RNeasy Kit (Qiagen) and 1µg RNA was reverse transcribed with SuperScript III Reverse Transcription Kit (Thermo Fisher Scientific). The PCR reactions were performed using Platinum PCR Master Mix (Thermo Fisher Scientific). To confirm the transgene-free status of the iPSC lines, SeV specific primers (Forward: 5’GGATCACTAGGTGATATCGAGC-3’; Reverse: 5’-ACCAGACAAGAGTTTAAGAGATATGTATC-3’; 181bp) were used, which was described in CytoTune®-iPS 2.0 Sendai Reprogramming Kit protocol (Thermo Fisher Scientific), and GAPDH (Forward: 5’-ACCACAGTCCATGCCATCAC-3’; Reverse: 5’-TCCACCACCCTGTTGCTGTA-3’; 498bp) was used as internal control (Figure 1F). Karyotype analysis
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Karyotype analysis was performed by GTG-banding analysis at Cytogenetics lab in the 1st Hospital of Jilin University. Cells were treated with KaryoMAX® Colcemid™ Solution (Thermo Fisher Scientific) and processed with standard methods. Following the International System Cytogenetics Nomenclature recommendations, the hiPSC lines displayed a normal karyotype (46, XY) (Figure 1D).
Teratoma formation assay
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Teratomas were generated by subcutaneous injection of iPSCs onto NOD SCID mice. In brief, 0.5 × 106 hiPSCs cultured on Matrigel were harvested and injected into the dorsal flanks of 8 week-old male NOD SCID mice. About 8 weeks after injection, tumors were dissected and fixed in 10% formalin (Sigma); paraffin sections were prepared and stained with hematoxylin/eosin. The presence of differentiated tissues which representate the three embryonic germ layers was identified (Figure 1E).
Figures and Tables Figure.1. Characterization of iPSC lines. (A) The patient peripheral blood mononuclear cells (PBMCs) were grown in PBMC expansion medium, cells reach confluent after 10-15 days of culture. Scale bar: 100µM (X10). (B) Derived iPSCs displayed typical ES cell round shape colony morphology with small, tightly packed cells, scale bar: 100µM (X10). (C) Immunofluorescence staining of iPSCs showed high expression of pluripotency marker Oct-4, SSEA-4, TRA-1-81. Scale bar: 100µM. (X20). (D) Karyotype analysis of patient iPSCs showed a normal karyotype of 46, XY. (E) In vivo differentiation capacity of iPSCs was confirmed by teratoma formation assay. Three germ layers were observed in teratoma tissue via hematoxylin/eosin staining (H&E). Scale bar: 100µM. (X10) (F) Silencing of Sendai reprogramming factors was confirmed by RT-PCR.
ACCEPTED MANUSCRIPT Author disclosure statement There are no competing financial interests in this study. Acknowledgments
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This study was supported by fund from National Medical Research Council (NMRC) of China to SZ (fund number: 30400447) and HW (fund number: 30500548).
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References Ban, H., Nishishita, N., Fusaki, N., Tabata, T., Saeki, K., Shikamura, M., Takada, N., Inoue, M., Hasegawa, M., Kawamata, S., et al. (2011). Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Proceedings of the National Academy of Sciences of the United States of America 108, 14234-14239. Jacoby, L.B., Jones, D., Davis, K., Kronn, D., Short, M.P., Gusella, J., and MacCollin, M. (1997). Molecular analysis of the NF2 tumor-suppressor gene in schwannomatosis. American journal of human genetics 61, 1293-1302. Smith, M.J., Wallace, A.J., Bowers, N.L., Rustad, C.F., Woods, C.G., Leschziner, G.D., Ferner, R.E., and Evans, D.G. (2012). Frequency of SMARCB1 mutations in familial and sporadic schwannomatosis. Neurogenetics 13, 141-145.
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Fig. 1
