- Email: [email protected]
Generation of a human induced pluripotent stem cell (iPSC) line carrying the Parkinson’s disease linked LRRK2 variant S1647T
Stem Cell Research 18 (2017) 54–56
Contents lists available at ScienceDirect
Stem Cell Research journal homepage: www.elsevier.com/locate/scr
Lab Resource: Stem Cell Line
Generation of a human induced pluripotent stem cell (iPSC) line carrying the Parkinson’s disease linked LRRK2 variant S1647T Dongrui Ma a, Shin Hui Ng b, Li Zeng b, Yi Zhao c, Eng King Tan a,b,d,⁎ a
Neurology, Singapore General Hospital, 169608, Singapore Neurology, National Neuroscience Institute of Singapore, 308433, Singapore Department of Clinical Research, Singapore General Hospital, 169608, Singapore d Duke NUS Graduate Medical School, 169857, Singapore b c
a r t i c l e
i n f o
Article history: Received 18 November 2016 Accepted 6 December 2016 Available online 9 December 2016
a b s t r a c t Peripheral blood mononuclear cells (PBMCs) were collected from a clinically diagnosed 64-year old male Parkinson's disease (PD) patient with S1647T variant in the LRRK2 gene. The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus reprogramming system. The transgene-free iPSC showed pluripotency confirmed by immunofluorescent staining for pluripotency markers and differentiated into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This cellular model will be useful for further function studies and therapeutic screening. © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Resource table. Name of stem cell line Institution Person who created resource Contact person and email Date archived/stock date Origin Type of resource Sub-type Key transcription factors Authentication Link to related literature Information in public databases Ethics
PD-BP44-iPSC National Neuroscience Institute of Singapore Dongrui Ma Dongrui Ma, [email protected] July 2016 Peripheral blood mononuclear cells (PBMCs) Biological reagent: human induced pluripotent stem cell (iPSC) line Cell line Oct4, Sox2, cMyc, Klf4 Identity and purity of cell line confirmed (Fig. 1) Not available Not available Patient informed consent obtained/Ethics Review Board-competent authority approval obtained
using CytoTune®-iPS 2.0 Reprogramming System (Thermo Fisher Scientific), which carry the 4 Yamanaka reprogramming factors OCT4, SOX2, cMYC and KLF4 (Ban et al., 2011). This reprogramming followed the previous published protocol (Tan et al., 2014). The derived hiPSC lines displayed a typical round shape ESC-like morphology with small and tightly packed cells, and a high nucleus/cytoplasm ratio and prominent nucleoli (Fig. 1A). The presence of the S1647T variant was confirmed in hiPSC lines by Sanger sequencing (Fig. 1B), and the expression of pluripotency markers was verified by immunofluorescence staining (Fig. 1C). Moreover, the absence of exogenous reprogramming transgenes was observed by RT-PCR after 5–8 passages (Fig. 1D). The differentiation capacity of hiPSC lines into three germ layers was demonstrated by in vivo teratoma formation assay (Fig. 1E). The derived hiPSC lines showed normal karyotype (46, XY) (Fig. 1F). 2. Materials and methods
1. Resource details
2.1. Reprogramming of peripheral blood mononuclear cells (PBMC)
The study was approved by the Singhealth ethics committee (protocol number SHSIBC-2014-031), and written informed consent was obtained from the patient. 2 ml of peripheral blood sample was obtained from a male 64-year-old Parkinson's disease patient carrying a heterozygous S1647T variant in LRRK2 gene. LRRK2 S1647T variant is associated with the risk of PD incidence among Asian population (Tan et al., 2010; Wu et al., 2013). PD-BP44-iPSC lines were derived
1 ml of peripheral blood sample was collected, and lysed in 2 ml of 1 × red blood cell (RBC) lysis buffer (eBioscience, San Diego, CA) for 10 mins; purified PBMCs were resuspended in 500 μl of cell expansion medium and seeded into one well of a 24-well tissue culture plate. The PBMC expansion medium 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
⁎ Corresponding author at: Neurology, National Neuroscience Institute, 308433, Singapore.
http://dx.doi.org/10.1016/j.scr.2016.12.010 1873-5061/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
D. Ma et al. / Stem Cell Research 18 (2017) 54–56
55
Fig. 1. Characterizaton of iPSC lines. (A) The PD patient iPSCs cells displayed a typical round shape colony morphology with small, tightly packed cells under inverted microscopy, scale bar: 100 μM (×10). (B) Sanger sequencing of the LRRK2 gene in iPSCs showed a heterozygous S1647T TNA substitution. (C) Immunofluorescence staining of iPSCs showed high expression of pluripotency marker POU5F1, NANOG and SSEA-4. Scale bar: 100 μM (×20). (D) Silencing of exogenous Sendai reprogramming factors was confirmed by RT-PCR. (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: 100 μM. (F) Karyotype analysis of patient iPSCs showed a normal karyotype of 46, XY.
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D. Ma et al. / Stem Cell Research 18 (2017) 54–56
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 7–12 days, a total of 100,000 PMBCs were infected with OCT4, SOX2, KLF4, and cMYC Sendai virus (CytoTune-iPS Reprogramming Kit, Thermo Fisher Scientific) with a MOI of 5. The infected cells were fed with 0.5 ml of fresh cell expansion medium 24 h later and plated onto Matrigel (BD Biosciences) coated dish 2 days later. The hiPSC colonies with an ES-like appearance were manually identified and isolated between Day 18 and Day 25 postinfection. The iPSC cultures were maintained on plates coated with Matrigel in mTESR-1 medium (Stem Cell Technologies) following the manufacturer's instruction (Fig. 1A). All cells were cultured at 37 °C in humidified atmosphere containing 5% CO2.
2.2. Reverse transcription polymerase chain reaction (RT-PCR) Total RNA was isolated from cultured cells with RNeasy Kit (Qiagen) and reverse transcribed with SuperScript III Reverse Transcription Kit (Thermo Fisher Scientific). The PCR reactions were performed using GoTaq Hot Start Green Master Mix (Promega). To confirm the transgene-free status of the iPSC lines, SeV specific primers (Forward: 5′GGATCACTAGGTGATATCGAGC-3′; Reverse: 5′-ACCAGACAAGAGTTT AAGAGATATGTATC-3′; 181 bp) 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′; 498 bp) was used as internal control.
Table 1 Primers used for detection of LRRK2 S1647T variant using Sanger sequencing. LRRK2
Sequence
S1647T forward primer S1647T reverse primer S1647T sequencing primer
5’-CGAATCTTTCAGATTTTGACAGTG-3’ 5’-GTCAGATGCAATGTTTTATTCCAA-3’ 5’-GTCAGATGCAATGTTTTATTCCAA-3’
2.5. In vivo teratoma formation assay Teratomas were generated by subcutaneous injection as described (Zapata-Linares et al., 2016). In brief, 0.5 × 106 hiPSCs cultured on Matrigel were harvested and injected into the dorsal flanks of 6– 8 week-old male NOD SCID mice. About 6–8 weeks after injection, tumors were dissected, fixed in 10% formalin (Sigma), paraffin-embedded, sectioned and stained with hematoxylin/eosin. The presence of differentiated tissues representative of the three embryonic germ layers was analyzed (Fig. 1E). 3. Karyotype analysis Chromosomal analysis was performed by GTG-banding analysis at Cytogenetics lab in Singapore General Hospital. Following the International System Cytogenetics Nomenclature recommendations, all hiPSC lines displayed a normal karyotype (46, XY) (Fig. 1F). Author disclosure statement There are no competing financial interests in this study. Acknowledgments
2.3. Genomic DNA extraction and genotyping Genomic DNA was extracted from PBMCs and hiPSCs using NucleoSpin tissue kit (Macherey-Nagel). For genotyping 50 ng of genomic DNA was amplified using Platinum® Taq DNA Polymerase High Fidelity (Invitrogen) using specific primers (Table 1) and the presence of the S1647T variant in LRRK2 gene was analyzed by Sanger sequencing (Fig. 1B).
2.4. Immunofluorescence (IF) IF was performed as described (Tan et al., 2014). In brief, hiPSCs were fixed with 4% paraformaldehyde (PFA, Sigma), permeabilized for 10 min with 1% TritonX-100 (Sigma) in PBS and blocked with 5% goat serum for 45 mins at room temperature. POU5F1 (Santa Cruz), NANOG (Abcam) and SSEA-4 (Chemicon) primary antibodies were diluted in 5% goat serum and incubated for 1 h at room temperature. Alexfluo568-conjugated secondary antibodies (Thermo Fisher Scientific) were incubated for 1 h at room temperature. Samples were visualized under an inverted fluorescence microscope (Nikon Eclipse Ti-S) (Fig. 1C).
This study was supported by fund from National Medical Research Council (NMRC) of Singapore, the Translational Clinical Research (TCR) award (NMRC/TCR/013-NNI/2014) and STaR award (NMRC/ STaR/014/2013). We thank Ms. Fiona Setiawan for collecting the patient blood samples. 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. Proc. Natl. Acad. Sci. U. S. A. 108, 14234–14239. Tan, E.K., Peng, R., Teo, Y.Y., Tan, L.C., Angeles, D., Ho, P., Chen, M.L., Lin, C.H., Mao, X.Y., Chang, X.L., et al., 2010. Multiple LRRK2 variants modulate risk of Parkinson disease: a Chinese multicenter study. Hum. Mutat. 31, 561–568. Tan, H.K., Toh, C.X., Ma, D., Yang, B., Liu, T.M., Lu, J., Wong, C.W., Tan, T.K., Li, H., Syn, C., et al., 2014. Human finger-prick induced pluripotent stem cells facilitate the development of stem cell banking. Stem Cells Transl. Med. 3, 586–598. Wu, Y.R., Chang, K.H., Chang, W.T., Hsiao, Y.C., Hsu, H.C., Jiang, P.R., Chen, Y.C., Chao, C.Y., Chang, Y.C., Lee, B.H., et al., 2013. Genetic variants ofLRRK2 in Taiwanese Parkinson's disease. PLoS One 8, e82001. Zapata-Linares, N., Rodriguez, S., Mazo, M., Abizanda, G., Andreu, E.J., Barajas, M., Prósper, F., Rodríguez-Madoz, J.R., 2016. Generation and characterization of human iPSC line generated from mesenchymal stem cells derived from adipose tissue. Stem Cell Res. 16:20–23. http://dx.doi.org/10.1016/j.scr.2015.12.002.
Contents lists available at ScienceDirect
Stem Cell Research journal homepage: www.elsevier.com/locate/scr
Lab Resource: Stem Cell Line
Generation of a human induced pluripotent stem cell (iPSC) line carrying the Parkinson’s disease linked LRRK2 variant S1647T Dongrui Ma a, Shin Hui Ng b, Li Zeng b, Yi Zhao c, Eng King Tan a,b,d,⁎ a
Neurology, Singapore General Hospital, 169608, Singapore Neurology, National Neuroscience Institute of Singapore, 308433, Singapore Department of Clinical Research, Singapore General Hospital, 169608, Singapore d Duke NUS Graduate Medical School, 169857, Singapore b c
a r t i c l e
i n f o
Article history: Received 18 November 2016 Accepted 6 December 2016 Available online 9 December 2016
a b s t r a c t Peripheral blood mononuclear cells (PBMCs) were collected from a clinically diagnosed 64-year old male Parkinson's disease (PD) patient with S1647T variant in the LRRK2 gene. The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus reprogramming system. The transgene-free iPSC showed pluripotency confirmed by immunofluorescent staining for pluripotency markers and differentiated into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This cellular model will be useful for further function studies and therapeutic screening. © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Resource table. Name of stem cell line Institution Person who created resource Contact person and email Date archived/stock date Origin Type of resource Sub-type Key transcription factors Authentication Link to related literature Information in public databases Ethics
PD-BP44-iPSC National Neuroscience Institute of Singapore Dongrui Ma Dongrui Ma, [email protected] July 2016 Peripheral blood mononuclear cells (PBMCs) Biological reagent: human induced pluripotent stem cell (iPSC) line Cell line Oct4, Sox2, cMyc, Klf4 Identity and purity of cell line confirmed (Fig. 1) Not available Not available Patient informed consent obtained/Ethics Review Board-competent authority approval obtained
using CytoTune®-iPS 2.0 Reprogramming System (Thermo Fisher Scientific), which carry the 4 Yamanaka reprogramming factors OCT4, SOX2, cMYC and KLF4 (Ban et al., 2011). This reprogramming followed the previous published protocol (Tan et al., 2014). The derived hiPSC lines displayed a typical round shape ESC-like morphology with small and tightly packed cells, and a high nucleus/cytoplasm ratio and prominent nucleoli (Fig. 1A). The presence of the S1647T variant was confirmed in hiPSC lines by Sanger sequencing (Fig. 1B), and the expression of pluripotency markers was verified by immunofluorescence staining (Fig. 1C). Moreover, the absence of exogenous reprogramming transgenes was observed by RT-PCR after 5–8 passages (Fig. 1D). The differentiation capacity of hiPSC lines into three germ layers was demonstrated by in vivo teratoma formation assay (Fig. 1E). The derived hiPSC lines showed normal karyotype (46, XY) (Fig. 1F). 2. Materials and methods
1. Resource details
2.1. Reprogramming of peripheral blood mononuclear cells (PBMC)
The study was approved by the Singhealth ethics committee (protocol number SHSIBC-2014-031), and written informed consent was obtained from the patient. 2 ml of peripheral blood sample was obtained from a male 64-year-old Parkinson's disease patient carrying a heterozygous S1647T variant in LRRK2 gene. LRRK2 S1647T variant is associated with the risk of PD incidence among Asian population (Tan et al., 2010; Wu et al., 2013). PD-BP44-iPSC lines were derived
1 ml of peripheral blood sample was collected, and lysed in 2 ml of 1 × red blood cell (RBC) lysis buffer (eBioscience, San Diego, CA) for 10 mins; purified PBMCs were resuspended in 500 μl of cell expansion medium and seeded into one well of a 24-well tissue culture plate. The PBMC expansion medium 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
⁎ Corresponding author at: Neurology, National Neuroscience Institute, 308433, Singapore.
http://dx.doi.org/10.1016/j.scr.2016.12.010 1873-5061/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
D. Ma et al. / Stem Cell Research 18 (2017) 54–56
55
Fig. 1. Characterizaton of iPSC lines. (A) The PD patient iPSCs cells displayed a typical round shape colony morphology with small, tightly packed cells under inverted microscopy, scale bar: 100 μM (×10). (B) Sanger sequencing of the LRRK2 gene in iPSCs showed a heterozygous S1647T TNA substitution. (C) Immunofluorescence staining of iPSCs showed high expression of pluripotency marker POU5F1, NANOG and SSEA-4. Scale bar: 100 μM (×20). (D) Silencing of exogenous Sendai reprogramming factors was confirmed by RT-PCR. (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: 100 μM. (F) Karyotype analysis of patient iPSCs showed a normal karyotype of 46, XY.
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D. Ma et al. / Stem Cell Research 18 (2017) 54–56
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 7–12 days, a total of 100,000 PMBCs were infected with OCT4, SOX2, KLF4, and cMYC Sendai virus (CytoTune-iPS Reprogramming Kit, Thermo Fisher Scientific) with a MOI of 5. The infected cells were fed with 0.5 ml of fresh cell expansion medium 24 h later and plated onto Matrigel (BD Biosciences) coated dish 2 days later. The hiPSC colonies with an ES-like appearance were manually identified and isolated between Day 18 and Day 25 postinfection. The iPSC cultures were maintained on plates coated with Matrigel in mTESR-1 medium (Stem Cell Technologies) following the manufacturer's instruction (Fig. 1A). All cells were cultured at 37 °C in humidified atmosphere containing 5% CO2.
2.2. Reverse transcription polymerase chain reaction (RT-PCR) Total RNA was isolated from cultured cells with RNeasy Kit (Qiagen) and reverse transcribed with SuperScript III Reverse Transcription Kit (Thermo Fisher Scientific). The PCR reactions were performed using GoTaq Hot Start Green Master Mix (Promega). To confirm the transgene-free status of the iPSC lines, SeV specific primers (Forward: 5′GGATCACTAGGTGATATCGAGC-3′; Reverse: 5′-ACCAGACAAGAGTTT AAGAGATATGTATC-3′; 181 bp) 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′; 498 bp) was used as internal control.
Table 1 Primers used for detection of LRRK2 S1647T variant using Sanger sequencing. LRRK2
Sequence
S1647T forward primer S1647T reverse primer S1647T sequencing primer
5’-CGAATCTTTCAGATTTTGACAGTG-3’ 5’-GTCAGATGCAATGTTTTATTCCAA-3’ 5’-GTCAGATGCAATGTTTTATTCCAA-3’
2.5. In vivo teratoma formation assay Teratomas were generated by subcutaneous injection as described (Zapata-Linares et al., 2016). In brief, 0.5 × 106 hiPSCs cultured on Matrigel were harvested and injected into the dorsal flanks of 6– 8 week-old male NOD SCID mice. About 6–8 weeks after injection, tumors were dissected, fixed in 10% formalin (Sigma), paraffin-embedded, sectioned and stained with hematoxylin/eosin. The presence of differentiated tissues representative of the three embryonic germ layers was analyzed (Fig. 1E). 3. Karyotype analysis Chromosomal analysis was performed by GTG-banding analysis at Cytogenetics lab in Singapore General Hospital. Following the International System Cytogenetics Nomenclature recommendations, all hiPSC lines displayed a normal karyotype (46, XY) (Fig. 1F). Author disclosure statement There are no competing financial interests in this study. Acknowledgments
2.3. Genomic DNA extraction and genotyping Genomic DNA was extracted from PBMCs and hiPSCs using NucleoSpin tissue kit (Macherey-Nagel). For genotyping 50 ng of genomic DNA was amplified using Platinum® Taq DNA Polymerase High Fidelity (Invitrogen) using specific primers (Table 1) and the presence of the S1647T variant in LRRK2 gene was analyzed by Sanger sequencing (Fig. 1B).
2.4. Immunofluorescence (IF) IF was performed as described (Tan et al., 2014). In brief, hiPSCs were fixed with 4% paraformaldehyde (PFA, Sigma), permeabilized for 10 min with 1% TritonX-100 (Sigma) in PBS and blocked with 5% goat serum for 45 mins at room temperature. POU5F1 (Santa Cruz), NANOG (Abcam) and SSEA-4 (Chemicon) primary antibodies were diluted in 5% goat serum and incubated for 1 h at room temperature. Alexfluo568-conjugated secondary antibodies (Thermo Fisher Scientific) were incubated for 1 h at room temperature. Samples were visualized under an inverted fluorescence microscope (Nikon Eclipse Ti-S) (Fig. 1C).
This study was supported by fund from National Medical Research Council (NMRC) of Singapore, the Translational Clinical Research (TCR) award (NMRC/TCR/013-NNI/2014) and STaR award (NMRC/ STaR/014/2013). We thank Ms. Fiona Setiawan for collecting the patient blood samples. 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. Proc. Natl. Acad. Sci. U. S. A. 108, 14234–14239. Tan, E.K., Peng, R., Teo, Y.Y., Tan, L.C., Angeles, D., Ho, P., Chen, M.L., Lin, C.H., Mao, X.Y., Chang, X.L., et al., 2010. Multiple LRRK2 variants modulate risk of Parkinson disease: a Chinese multicenter study. Hum. Mutat. 31, 561–568. Tan, H.K., Toh, C.X., Ma, D., Yang, B., Liu, T.M., Lu, J., Wong, C.W., Tan, T.K., Li, H., Syn, C., et al., 2014. Human finger-prick induced pluripotent stem cells facilitate the development of stem cell banking. Stem Cells Transl. Med. 3, 586–598. Wu, Y.R., Chang, K.H., Chang, W.T., Hsiao, Y.C., Hsu, H.C., Jiang, P.R., Chen, Y.C., Chao, C.Y., Chang, Y.C., Lee, B.H., et al., 2013. Genetic variants ofLRRK2 in Taiwanese Parkinson's disease. PLoS One 8, e82001. Zapata-Linares, N., Rodriguez, S., Mazo, M., Abizanda, G., Andreu, E.J., Barajas, M., Prósper, F., Rodríguez-Madoz, J.R., 2016. Generation and characterization of human iPSC line generated from mesenchymal stem cells derived from adipose tissue. Stem Cell Res. 16:20–23. http://dx.doi.org/10.1016/j.scr.2015.12.002.