- Email: [email protected]
Generation of human induced pluripotent stem cell lines from human dermal fibroblasts using a non-integration system
Stem Cell Research 21 (2017) 13–15
Contents lists available at ScienceDirect
Stem Cell Research journal homepage: www.elsevier.com/locate/scr
Lab resource: Stem cell research
Generation of human induced pluripotent stem cell lines from human dermal fibroblasts using a non-integration system Kyung-Ok Uhm, So-Jung Kim, Eun Hee Jo, Gue Youn Go, Hye Young Choi, Young Sam Im, Hye-Yeong Ha, Jung-Hyun Kim, Soo Kyung Koo ⁎ Division of Intractable Disease, Center for Biomedical Sciences, Korea National Institute of Health, Chungcheongbuk-do, Republic of Korea
a r t i c l e
i n f o
Article history: Received 7 March 2017 Accepted 12 March 2017 Available online 21 March 2017
a b s t r a c t We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts using a Sendai virus (SeV)based gene delivery method. The generated hiPSC line, KSCBi002-A, has a normal karyotype (46,XY). The pluripotency and differentiation capacity were characterized by comparison with those of a human embryonic stem cell line. This cell line is registered and available from the National Stem Cell Bank, Korea National Institute of Health. © 2017 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
1. Resource details
KSCBi002-A (hFSiPS1) Korea National Institute of Health Kyung-Ok Uhm Soo Kyung Koo, [email protected] August, 2013 Human dermal fibroblasts Biological reagent: Human induced pluripotent stem cell (iPSC); derived from dermal fibroblasts of a male donor (ScienCell, 2320) hiPSC line hOct3/4, hSox2,h Klf4, hc-MYC (CytoTune™-iPS 2.0-Thermo Fisher Scientific) Identity and purity of cell line confirmed: expression of pluripotency genes by immunofluorescence staining, expression of SeV specific genome by RT-PCR, teratoma formation, STR analysis, karyotyping by GTG-banding http://www.ncbi.nlm.nih.gov/pubmed/?term = 28105056 http://kscr.nih.go.kr http://hpscreg.eu Institutional Review Board approval obtained (2013-06EXP-06-R, 2014-10CON-04-1C-A).
Human dermal fibroblasts (ScienCell, 2320) were obtained and induced to hiPSCs using Sendai virus based non-integration vectors. Morphologies of donor cells, HDF, and generated hiPSCs, KSCBi002-A are shown in Fig. 1A. Pluripotency of generated hiPSCs, KSCBi002-A was characterized by immunofluorescence staining markers for Oct4, SSEA4, Tra-1-60, and Tra-1-81 (Fig. 1B). KSCBi002-A has a normal karyotype (46,XY) analysed by GTG-banding with 550 bands resolution (Fig. 1C). All of Sendai viral transgenes were silenced in KSCBi002-A, as analysed by RT-PCR using Sendai viral specification markers (Fig. 1D). All three germ layers are shown in teratoma derived from KSCBi002-A with H&E staining and there was no teratocarcinoma (Fig. 1E). All 16 allele loci of KSCBi002-A are consistent with donor cells, as confirmed by STR analysis (Fig. 1F). 2. Materials and methods 2.1. Donor cells Human dermal fibroblasts were obtained from a male donor (ScienCell, 2320). The cells were maintained in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum (Gibco) and grown at 37 °C in a 5% CO2 atmosphere. 2.2. Reprogramming and hiPSC maintenance
⁎ Corresponding author at: 202, Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do 28160, Republic of Korea. E-mail address: [email protected] (S.K. Koo).
Obtained dermal fibroblasts were transduced using CytoTune™-iPS 2.0 according to the manufacturer's suggested protocol (Thermo Fisher
http://dx.doi.org/10.1016/j.scr.2017.03.009 1873-5061/© 2017 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/).
14
K.-O. Uhm et al. / Stem Cell Research 21 (2017) 13–15
Fig. 1. Characterization of KSCBi002-A cell line. (A) Phase contrast images of donor cells, HDF, and generated hiPSCs, KSCBi002-A (Scale bar: 100 μm). (B) Immunofluorescence staining of pluripotent markers Oct4, SSEA4, Tra-1-60, and Tra-1-81 (Scale bar: 200 μm) and (C) karyotype (46,XY) of KSCBi002-A. (D) Analysis of Sendai viral transgene expression in KSCBi002-A passage no. 5. RT-PCR was performed to analyze the expression of Sendai viral transgenes, SeV-Oct3/4, SeV-Sox2, SeV-Klf4, and SeV-cMyc. Donor_HDF: human dermal fibroblasts from a donor. SeV HDF (d7): donor cells harvested 7 days after transduction. (E) All three germ layers are shown in teratoma derived from KSCBi002-A with H&E staining (Scale bar: 200 μm). (F) Result of STR and (G) mycoplasma test.
Scientific, A13780) (Fukaki et al., 2009). Clones were picked and cultivated onto feeder STO (ATCC, CRL1503) cells with DMEM/F12 media (Gibco) supplemented with 20% (v/v) Knock-out™ Serum Replacement (Gibco), 0.1 mM NEAA (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), and 1% (v/v) antibiotic-antimycotic (Gibco) with basic fibroblast growth factor (bFGF) (4 ng/ml). The culture medium was changed every once per day. 2.3. Immunofluorescence staining Cells were fixed with 4% paraformaldehyde (Wako) for 20 min, blocked with 5% (v/v) goat serum in 0.25% (v/v) Triton X, and incubated with primary antibodies for Oct4 (FITC, Santa Cruz, 1:200), SSEA4 (FITC,
Millipore, 1:200), Tra-1-60 (FITC, Millipore, 1:200), and Tra-1-81 (FITC, Millipore, 1:200). Nuclei were stained with Hoechst33342 (Invitrogen). Images were acquired using a fluorescence microscope (Scale bar: 200 μm). 2.4. Detection of reprogramming vector To test whether transgenes were silenced, the KSCBi002-A passage no. 5 was confirmed by reverse transcription PCR (RT-PCR). Total RNA was isolated using the RNeasy Mini kit (Qiagen), and cDNA was synthesized by reverse transcription using RNA to cDNA EcoDry Premix (Clontech). Cells harvested 7 d after transduction with SeV vectors and donor cells were used as the positive control and negative controls,
K.-O. Uhm et al. / Stem Cell Research 21 (2017) 13–15
respectively (Donor_HDF: human dermal fibroblasts from a donor, SeV HDF (d7): donor cells harvested 7 days after transduction. The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was amplified concurrently and used as a control for RNA integrity.). The primers specific for the SeV genome are presented as follows: Primer sequence of Sendai Virus test. Target
Primer sequence
Product size (bp)
SeV-Oct3/4
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
483
SeV-Sox2 SeV-Klf4 SeV-cMyc GAPDH
CCC GAA AGA GAA AGC GAA CCA G AAT GTA TCG AAG GTG CTC AA ATG CAC CGC TAC GAC GTG AGC GC AAT GTA TCG AAG GTG CTC AA TTC CTG CAT GCC AGA GGA GCC C AAT GTA TCG AAG GTG CTC AA TAA CTG ACT AGC AGG CTT GTC G TCC ACA TAC AGT CCT GGA TGA TGA TG CAT GTT CGT CAT GGG TGT GAA GGA CTG TGG TCA TGA GTC CTT
451 410
15
45 min with colcemid (Thermo Fisher Scientific), harvested in fixative (acetic acid:methanol, 1:3), and the metaphase slides were prepared. After Giemsa-Trypsin banding, we karyotyped KSCBi002-A. The karyotype was analysed according to the International System for Human Cytogenetic Nomenclature.
2.7. STR analysis Short Tandem Repeat (STR) analysis was performed on the original cells and generated hUSiPS2 using PowerPlex®16 System (Promega) with detection of 16 loci D3S1358, TH01, D21S11, D18S51, PentaE, D5S818, D13S317, D7S820, D16S539, CSF1PO, PentaD, vWA, D8S1179, TPOX, FGA, Amelogenin.
532 150
2.5. Teratoma formation To test the in vivo differentiation of KSCBi002-A, teratoma formation was performed using KSCBi002-A suspended at 5 × 106 cells/ml in 50% (v/v) hiPSCs culture medium and 50% (v/v) matrigel (BD). Next, 200 μl of suspended cells were injected subcutaneously into the dorsal flank of NOD-SCID mice anesthetized with diethyl ether. Twelve weeks after injection, tumors were surgically dissected from the mice, fixed in PBS containing 4% (v/v) formaldehyde, and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin (H&E) (Scale bar: 200 μm).
2.8. Mycoplasma test For checking whether hiPSC is contaminated with mycoplasma, M. hyopneumoniae, M. neurolyticum, M. fermentans, M. pulmonis, M. hyorhinis, M. orale, M. capricolum, M. arthritidis, M. salivarium, M. hominis, M. argini, U. urealyticum, nested PCR was carried out using mycoplasma detection set (TaKaRa) (Fig. 1G). The PCR products were confirmed on 2% agarose gel by electrophoresis.
Acknowledgement This work was supported by the Korea National Institute of Health (grant number 2016-NG61002-00, 2016). References
2.6. Karyotyping Generated hiPSCs were karyotyped by standard cytogenetic procedures using the GTG-band method. The cultured cells were treated for
Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., Hasegawa, M., 2009. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc. Jpn. Acad. Ser. B. Phys. Biol. Sci. 85, 348–362.
Contents lists available at ScienceDirect
Stem Cell Research journal homepage: www.elsevier.com/locate/scr
Lab resource: Stem cell research
Generation of human induced pluripotent stem cell lines from human dermal fibroblasts using a non-integration system Kyung-Ok Uhm, So-Jung Kim, Eun Hee Jo, Gue Youn Go, Hye Young Choi, Young Sam Im, Hye-Yeong Ha, Jung-Hyun Kim, Soo Kyung Koo ⁎ Division of Intractable Disease, Center for Biomedical Sciences, Korea National Institute of Health, Chungcheongbuk-do, Republic of Korea
a r t i c l e
i n f o
Article history: Received 7 March 2017 Accepted 12 March 2017 Available online 21 March 2017
a b s t r a c t We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts using a Sendai virus (SeV)based gene delivery method. The generated hiPSC line, KSCBi002-A, has a normal karyotype (46,XY). The pluripotency and differentiation capacity were characterized by comparison with those of a human embryonic stem cell line. This cell line is registered and available from the National Stem Cell Bank, Korea National Institute of Health. © 2017 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
1. Resource details
KSCBi002-A (hFSiPS1) Korea National Institute of Health Kyung-Ok Uhm Soo Kyung Koo, [email protected] August, 2013 Human dermal fibroblasts Biological reagent: Human induced pluripotent stem cell (iPSC); derived from dermal fibroblasts of a male donor (ScienCell, 2320) hiPSC line hOct3/4, hSox2,h Klf4, hc-MYC (CytoTune™-iPS 2.0-Thermo Fisher Scientific) Identity and purity of cell line confirmed: expression of pluripotency genes by immunofluorescence staining, expression of SeV specific genome by RT-PCR, teratoma formation, STR analysis, karyotyping by GTG-banding http://www.ncbi.nlm.nih.gov/pubmed/?term = 28105056 http://kscr.nih.go.kr http://hpscreg.eu Institutional Review Board approval obtained (2013-06EXP-06-R, 2014-10CON-04-1C-A).
Human dermal fibroblasts (ScienCell, 2320) were obtained and induced to hiPSCs using Sendai virus based non-integration vectors. Morphologies of donor cells, HDF, and generated hiPSCs, KSCBi002-A are shown in Fig. 1A. Pluripotency of generated hiPSCs, KSCBi002-A was characterized by immunofluorescence staining markers for Oct4, SSEA4, Tra-1-60, and Tra-1-81 (Fig. 1B). KSCBi002-A has a normal karyotype (46,XY) analysed by GTG-banding with 550 bands resolution (Fig. 1C). All of Sendai viral transgenes were silenced in KSCBi002-A, as analysed by RT-PCR using Sendai viral specification markers (Fig. 1D). All three germ layers are shown in teratoma derived from KSCBi002-A with H&E staining and there was no teratocarcinoma (Fig. 1E). All 16 allele loci of KSCBi002-A are consistent with donor cells, as confirmed by STR analysis (Fig. 1F). 2. Materials and methods 2.1. Donor cells Human dermal fibroblasts were obtained from a male donor (ScienCell, 2320). The cells were maintained in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum (Gibco) and grown at 37 °C in a 5% CO2 atmosphere. 2.2. Reprogramming and hiPSC maintenance
⁎ Corresponding author at: 202, Osongsaengmyeong 2-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do 28160, Republic of Korea. E-mail address: [email protected] (S.K. Koo).
Obtained dermal fibroblasts were transduced using CytoTune™-iPS 2.0 according to the manufacturer's suggested protocol (Thermo Fisher
http://dx.doi.org/10.1016/j.scr.2017.03.009 1873-5061/© 2017 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/).
14
K.-O. Uhm et al. / Stem Cell Research 21 (2017) 13–15
Fig. 1. Characterization of KSCBi002-A cell line. (A) Phase contrast images of donor cells, HDF, and generated hiPSCs, KSCBi002-A (Scale bar: 100 μm). (B) Immunofluorescence staining of pluripotent markers Oct4, SSEA4, Tra-1-60, and Tra-1-81 (Scale bar: 200 μm) and (C) karyotype (46,XY) of KSCBi002-A. (D) Analysis of Sendai viral transgene expression in KSCBi002-A passage no. 5. RT-PCR was performed to analyze the expression of Sendai viral transgenes, SeV-Oct3/4, SeV-Sox2, SeV-Klf4, and SeV-cMyc. Donor_HDF: human dermal fibroblasts from a donor. SeV HDF (d7): donor cells harvested 7 days after transduction. (E) All three germ layers are shown in teratoma derived from KSCBi002-A with H&E staining (Scale bar: 200 μm). (F) Result of STR and (G) mycoplasma test.
Scientific, A13780) (Fukaki et al., 2009). Clones were picked and cultivated onto feeder STO (ATCC, CRL1503) cells with DMEM/F12 media (Gibco) supplemented with 20% (v/v) Knock-out™ Serum Replacement (Gibco), 0.1 mM NEAA (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), and 1% (v/v) antibiotic-antimycotic (Gibco) with basic fibroblast growth factor (bFGF) (4 ng/ml). The culture medium was changed every once per day. 2.3. Immunofluorescence staining Cells were fixed with 4% paraformaldehyde (Wako) for 20 min, blocked with 5% (v/v) goat serum in 0.25% (v/v) Triton X, and incubated with primary antibodies for Oct4 (FITC, Santa Cruz, 1:200), SSEA4 (FITC,
Millipore, 1:200), Tra-1-60 (FITC, Millipore, 1:200), and Tra-1-81 (FITC, Millipore, 1:200). Nuclei were stained with Hoechst33342 (Invitrogen). Images were acquired using a fluorescence microscope (Scale bar: 200 μm). 2.4. Detection of reprogramming vector To test whether transgenes were silenced, the KSCBi002-A passage no. 5 was confirmed by reverse transcription PCR (RT-PCR). Total RNA was isolated using the RNeasy Mini kit (Qiagen), and cDNA was synthesized by reverse transcription using RNA to cDNA EcoDry Premix (Clontech). Cells harvested 7 d after transduction with SeV vectors and donor cells were used as the positive control and negative controls,
K.-O. Uhm et al. / Stem Cell Research 21 (2017) 13–15
respectively (Donor_HDF: human dermal fibroblasts from a donor, SeV HDF (d7): donor cells harvested 7 days after transduction. The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was amplified concurrently and used as a control for RNA integrity.). The primers specific for the SeV genome are presented as follows: Primer sequence of Sendai Virus test. Target
Primer sequence
Product size (bp)
SeV-Oct3/4
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
483
SeV-Sox2 SeV-Klf4 SeV-cMyc GAPDH
CCC GAA AGA GAA AGC GAA CCA G AAT GTA TCG AAG GTG CTC AA ATG CAC CGC TAC GAC GTG AGC GC AAT GTA TCG AAG GTG CTC AA TTC CTG CAT GCC AGA GGA GCC C AAT GTA TCG AAG GTG CTC AA TAA CTG ACT AGC AGG CTT GTC G TCC ACA TAC AGT CCT GGA TGA TGA TG CAT GTT CGT CAT GGG TGT GAA GGA CTG TGG TCA TGA GTC CTT
451 410
15
45 min with colcemid (Thermo Fisher Scientific), harvested in fixative (acetic acid:methanol, 1:3), and the metaphase slides were prepared. After Giemsa-Trypsin banding, we karyotyped KSCBi002-A. The karyotype was analysed according to the International System for Human Cytogenetic Nomenclature.
2.7. STR analysis Short Tandem Repeat (STR) analysis was performed on the original cells and generated hUSiPS2 using PowerPlex®16 System (Promega) with detection of 16 loci D3S1358, TH01, D21S11, D18S51, PentaE, D5S818, D13S317, D7S820, D16S539, CSF1PO, PentaD, vWA, D8S1179, TPOX, FGA, Amelogenin.
532 150
2.5. Teratoma formation To test the in vivo differentiation of KSCBi002-A, teratoma formation was performed using KSCBi002-A suspended at 5 × 106 cells/ml in 50% (v/v) hiPSCs culture medium and 50% (v/v) matrigel (BD). Next, 200 μl of suspended cells were injected subcutaneously into the dorsal flank of NOD-SCID mice anesthetized with diethyl ether. Twelve weeks after injection, tumors were surgically dissected from the mice, fixed in PBS containing 4% (v/v) formaldehyde, and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin (H&E) (Scale bar: 200 μm).
2.8. Mycoplasma test For checking whether hiPSC is contaminated with mycoplasma, M. hyopneumoniae, M. neurolyticum, M. fermentans, M. pulmonis, M. hyorhinis, M. orale, M. capricolum, M. arthritidis, M. salivarium, M. hominis, M. argini, U. urealyticum, nested PCR was carried out using mycoplasma detection set (TaKaRa) (Fig. 1G). The PCR products were confirmed on 2% agarose gel by electrophoresis.
Acknowledgement This work was supported by the Korea National Institute of Health (grant number 2016-NG61002-00, 2016). References
2.6. Karyotyping Generated hiPSCs were karyotyped by standard cytogenetic procedures using the GTG-band method. The cultured cells were treated for
Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., Hasegawa, M., 2009. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc. Jpn. Acad. Ser. B. Phys. Biol. Sci. 85, 348–362.