Stem Cell Research 15 (2015) 640–642
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Lab resource: Stem cell line
Human embryonic stem cells derived from abnormal blastocyst donated by Marfan syndrome patient Qingqing Yang a,1, Xiaoying Zhou b,1, Hao Zhou a, Long Li a, Juan Du a,c,d, Guangxiu Lu b,c,d, Ge Lin a,b,c,d, Qi Ouyang a,b,c,⁎ a
Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, China National Engineering and Research Center of Human Stem Cell, Changsha, China Key Laboratory of Stem Cells and Reproductive Engineering, Ministry of Health, Changsha, China d Reproductive & Genetic Hospital of CITIC-Xiangya, Changsha, China b c
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 12 October 2015 Accepted 21 October 2015 Available online 23 October 2015
Human embryonic stem cell (hESC) line was derived from abnormal blastocyst donated by Marfan syndrome patient after preimpantation genetic diagnosis (PGD) treatment. DNA sequencing analysis confirmed that the hESC line carried the heterozygous deletion mutation, c.3536delA, of FBN1 gene. Characteristic tests proved that the hESC line presented typical markers of pluripotency and had the capability to form the three germ layers both in vitro and in vivo. © 2015 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 construct 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 (direct URL links and full references) Information in public databases
chHES-419 National Engineering and Research Center of Human Stem Cell Xiaoying Zhou, Qi Ouyang Qi Ouyang:
[email protected] Oct 23, 2014 Abnormal blastocyst from Marfan syndrome patient Human embryonic stem cells derived from PGD-analyzed abnormally blastocyst Cell line NANOG, OCT4, SOX2, KLF4, TERF1, THY1 Identity and purity of cell line confirmed (Fig. 1) N/A N/A
1. Resource details Human embryonic stem cell (hESC) line chHES-419 was derived from abnormal blastocyst donated by Marfan syndrome patient who underwent preimplantation genetic diagnosis (PGD) treatment. DNA sequencing analysis confirmed a heterozygous deletion mutation, ⁎ Corresponding author at: Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, China. E-mail address:
[email protected] (Q. Ouyang). 1 These authors contribute equally to this study.
c.3536delA, of FBN1 in the cells. The deletion caused a frameshift at amino acid 1179 (glutamine) resulting in early termination after 25 abnormal amino acid residues (Q1179Rfs*25), indicating the pathogenic effect of this mutation (Fig. 1A). The result is consistent with the proband. The cells displayed pluripotent cell morphology (Fig. 1B), expressed pluripotency related genes (Fig. 1C) and were positive for a set of pluripotent markers OCT3/4, NANOG, TRA-1-60 and TRA-1-81 as well as alkaline phosphatase (Fig. 1D). The cells had the capability to differentiate into the derivatives from all the three germ layers both in vitro (Fig. 1E) and in vivo (Fig. 1F). In addition, this line maintained the normal karyotype 46, XX during long-term culture (Fig. 1G).
2. Materials and methods 2.1. hESC line derivation and cell culture The ethical committee of Reproductive and Genetic Hospital of CITIC-Xiangya approved the study. A blastocyst with disease-causing mutation was donated by a Marfan syndrome patient with informed consent. The methodology used for hESC line derivation was performed as previously described (Lin et al., 2009). The inner cell mass was mechanically isolated and plated on mouse embryonic fibroblast (MEF) feeders. The cells were cultured in ES medium containing DMEM/F12 supplemented with 15% knockout serum replacement, 2 mM nonessential amino acids, 2 mM L-glutamine, 0.1 mM βmercaptoethanol and 4 ng/mL of basic Fibroblast Growth Factor (bFGF) (all from Invitrogen). Once the embryo outgrowth was observed, it was transmitted onto fresh feeders and obtained the
http://dx.doi.org/10.1016/j.scr.2015.10.012 1873-5061/© 2015 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/).
Q. Yang et al. / Stem Cell Research 15 (2015) 640–642
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Fig. 1. Characterization of chHES-419 cells. (A) A heterozygous mutation, c.3536delA, of FBN1 was identified in chHES-419 cells; (B) cells showed typical round shape colonies on the MEF feeder layer; (C) pluripotent genes expressed in chHES-419 cells, NANOG, OCT3/4, SOX2, TERF1, THY1 and KLF4 were detected by RT-PCR; (D) undifferentiated colonies were positive for the pluripotency markers of NANOG, OCT4, TRA-1-60, TRA-1-81 and AKP. Nuclei staining with DAPI was in the lower right corner. Scale bar = 100 μm; (E) derivatives from embryoid bodies of chHES-419 cells could differentiate into ectoderm (TUJ1), mesoderm (SMA) and endoderm (AFP) in vitro. Scale bar = 50 μm; (F) histological sections of teratomas formed by the chHES-419 hESC line contain tissues derived from three germ layers. Scale bar = 50 μm; (G) chHES-419 has a normal karyotype 46, XX.
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Q. Yang et al. / Stem Cell Research 15 (2015) 640–642
Table 1 List of primers used in RT-PCR. Genes NANOG OCT3/4 SOX2 TERF1 THY1 KLF4 β-ACTIN
Primer sequence 5′-TGAACCTCAGCTACAAACAG-3′ 5′-TGGTGGTAGGAAGAGTAAAG-3′ 5′-AGCGAACCAGTATCGAGAAC-3′ 5′-TTACAGAACCACACTCGGAC-3′ 5′-AGCTACAGCATGATGCAGGA-3′ 5′-GGTCATGGAGTTGTACTGCA-3′ 5′-GCAACAGCGCAGAGGCTATTATT-3′ 5′-AGGGCTGATTCCAAGGGTGTAA-3′ 5′-AGAATACCAGCAGTTCACCCATCC-3′ 5′-CCTCACACTTGACCAGTTTGTCTCTG-3′ 5′-GCCGCTCCATTACCAAGAGC-3′ 5′-GGTGTGCCTTGAGATGGGAA-3′ 5′-TCCCTGGAGAAGAGCTACGA-3′ 5′-AGCACTGTGTTGGCGTACAG-3′
anti-NANOG (1:100, Abcam), mouse anti-TRA-1-60 (1:50, Millipore), mouse anti-TRA-1-81 (1:50, Millipore), mouse anti-β-tubulin (1:800, Sigma), mouse anti-AFP (1:500, Sigma), and mouse anti-SMA (1:100, Millipore). After rinsing to remove unbound antibodies, Localization of antigens was visualized by using Alexa Fluor® 488 donkey anti-mouse lgG (1:1000, Life Technologies) or Alexa Fluor® 488 donkey antirabbit lgG (1:1000, Life Technologies). Nuclei were counterstained with DAPI (1:1000, Sigma). In addition, alkaline phosphatase activity was detected with BCIP/NBT Kit (Invitrogen). Images were acquired in an epifluorescence microscopy (Nikon Eclipse TE2000-U).
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2.5. In vitro and in vivo differentiation assay
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For in vitro differentiation assay, hESCs were detached to grow as aggregates in suspension for 7 days in ES medium without bFGF, then transmitted onto Matrigel (BD) coated 4-well plates for another 14day culture. The derivatives of embryoid bodies were processed for immunocytochemistry analysis. For in vivo differentiation assay, 1 × 106 cells were injected into muscle of right hind limb of SCID mouse. After 10 weeks, teratoma was harvested and fixed with 4% paraformaldehyde. To identify the derivatives from three germ layers, hematoxylin and eosin (H&E) staining (Fig. 1G) was carried out for the histological analysis.
hESC-colony morphology during prolonged culture. hESC colonies were mechanically passaged every 7 days. 2.2. Genetic diagnosis. Genomic DNA of the cells was extracted using QIAmp® DNA mini kit (QIAGEN). PCR was conducted using 50 μL PCR amplification reaction mixture which contained 1.5 μL of 100 ng genomic DNA, 1 μL of 1.0 μM of each primer (the forward primer: 5′-GCCAAAGTTGGAAGCT TATG-3′ and the reverse primer: 5′-TAACATAACATAACATAAAATA AAG-3′) and 25 μL of 2 × GreenMaster Mix (Promega). Cycling conditions were as follows: 95 °C for 1.5 min followed by 35 cycles of amplification (95 °C denaturation for 40 s, 56 °C annealing for 40 s, 72 °C elongation for 40 s) and a final extension at 72 °C for 5 min. The PCR products were determined by Sanger sequencing using a BigDye Terminator cycle sequencing kit v3.1 and an ABI 3130XL Genetic Analyzer (Applied Biosystems). 2.3. RT-PCR Total RNA was extracted with TRIzol (Invitrogen) and complementary DNA (cDNA) was synthesized via using Transcriptor First Strand cDNA Synthesis Kit (Roche). The RT-PCR reaction was carried out as follows: 95 °C for 2 min; 35 cycles of amplification (95 °C for 30 s, 54– 64 °C for 30 s and 72 °C for 30 s) and a final extension at 72 °C for 5 min. PCR products were separated by electrophoresis on a 1.5% agarose gel, and images were taken on a UV transluminator. The primers were listed in Table 1.
2.6. Karyotyping analysis chHES-419 cells were cultured in mTeSR™1(STEMCELL) for 3– 5 days, then treated with 0.1 μg/mL of KaryoMAX® Colcemid™ solution (Life Technologies) for 3 h and dissociated with Accutase (Millipore) into single cells for standard G-banding karyotype analysis. 2.6.1. Verification and Authentication. Karyotyping and sequencing analysis were performed at Reproductive & Genetic Hospital of CITIC-Xiangya. Twenty metaphase cells were observed and all had a normal karyotype of 46, XX. After locus specific PCR, Sanger sequencing analysis confirmed a heterozygous deletion mutation, c.3536delA, of FBN1 in chHES-419 cells that is consistent with that of the proband. Acknowledgments This work was supported by grants from the National Basic Research Program of China (973 program 2011CB964901 and 2012CB944901), the National Natural Science Foundation of China (81222007 and 31101053) and Central South University innovation project of research (2014zzts290). We thank the genetic laboratory and IVF team of the Reproductive & Genetic Hospital of CITIC-Xiangya for their assistance.
2.4. Immunocytochemistry and alkaline phosphatase staining Reference chHES-419 cells were fixed with 4% paraformaldehyde (PFA), then blocked with 10% donkey serum and incubated with primary antibodies overnight at 4 °C. For intracellular antigen, cells were permeabilized in 0.2% Triton-X 100 before blocking. The following primary antibodies were used: mouse anti-OCT4 (1:200, Santa Cruz Biotechnology), rabbit
Lin, G., Xie, Y.B., OuYang, Q., et al., 2009. HLA matching potential of an established human embryonic stem cell bank in China. Cell Stem Cell 5 (5), 461–465.
