- Email: [email protected]
Generation of iPSC line MU011.A-hiPS from homozygous α-thalassemia fetal skin fibroblasts
Stem Cell Research 15 (2015) 506–509
Contents lists available at ScienceDirect
Stem Cell Research journal homepage: www.elsevier.com/locate/scr
Lab resource: Stem cell line
Generation of iPSC line MU011.A-hiPS from homozygous α-thalassemia fetal skin fibroblasts Amornrat Tangprasittipap a, Chonthicha Satirapod b, Bunyada Jittorntrum a, Sassawat Lertritanan a, Usanarat Anurathaphan c, Phetcharat Phanthong d,e, Suparerk Borwornpinyo f, Narisorn Kitiyanant e,⁎, Suradej Hongeng c,⁎ a
Office of Research, Academic Affairs and Innovations, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Department of Obstetrics and Gynecology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand c Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand d Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand e Stem Cell Research Group, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand f Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand b
a r t i c l e
i n f o
a b s t r a c t Human iPSC line MU011.A-hiPS was generated from homozygous α-thalassemia (−SEA/−SEA) fetal skin fibroblasts using a non-integrative reprogramming method. Reprogramming factors OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA of TP53 contained in three episomal vectors were delivered using electroporation. © 2015 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/).
Article history: Received 26 August 2015 Accepted 12 September 2015 Available online 21 September 2015
Resource table: Name of stem cell construct
MU011.A-hiPS
Institution Person who created resource Contact person and email
Mahidol University Amornrat Tangprasittipap, Narisorn Kitiyanant Narisorn Kitiyanant, [email protected] Suradej Hongeng, [email protected] July 2015 Human fetal skin fibroblasts Biological reagent: Induced pluripotent stem cell (iPS); derived from aborted fetal skin fibroblasts with homologous α-thalassemia (−SEA/−SEA) Cell line OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA of TP53 Identity and purity of cell line confirmed (Fig. 1) N/A
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
N/A
Resource details: Primary fibroblasts were obtained by skin biopsy from an aborted fetus with homozygous α-thalassemia (−SEA/−SEA), according to ⁎ Corresponding authors. E-mail addresses: [email protected] (N. Kitiyanant), [email protected] (S. Hongeng).
approved institutional procedures. The written informed consent was obtained from guardians. To generate MU011.A-hiPS, we delivered episomal expression cassettes of human OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA of TP53 (Okita et al., 2011) into fetal fibroblasts using a NEON microporator (Invitrogen). After 10 passages of iPSCs to ascertain that the cells had no exogenous reprogramming factors (LIN28, OCT4, SOX2) from episomal vector backbone that has been confirmed by quantitative PCR (data not shown). Pluripotent stem cell marker analysis: MU011.A-hiPS expressed pluripotent markers viz. NANOG, OCT3/4, SOX2, LIN28, REX1 (ZFP42), DNMT3B, TDGF1 (Fig. 1A) and surface markers; OCT3/4, NANOG, SSEA4 and TRA-1-60 (Fig. 1B). Teratoma formation demonstrated that MU011.A-hiPS was able to differentiate into derivatives of three germ layers (Fig. 1C). 1. Materials and methods Cell culture and iPS generation: Fetal skin fibroblasts were isolated and cultured in DMEM (Hyclone) supplemented with 10% FBS (Hyclone) and 1% GlutaMAX (Life Technologies). Three micrograms of episomal expression vectors were delivered into 2 × 105 human fetal skin fibroblast cells by a 100-μl kit of NEON microporator (Invitrogen) using the method previously described (Okita et al., 2011). After electroporation, cells were plated and cultured in a feeder free cultivation system on Geltrex (Life Technologies) in a chemically defined medium
http://dx.doi.org/10.1016/j.scr.2015.09.003 1873-5061/© 2015 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/).
A. Tangprasittipap et al. / Stem Cell Research 15 (2015) 506–509
(E8-equivalent) without TGF-β (Chen et al., 2011). Reprogramming efficiency was enhanced by adding 100 μM sodium butyrate for 2 weeks and the culture medium was replaced with full E8-equivalent medium when iPS-like colonies appeared. The iPS-liked colonies were manual picked up during day 14 to 21 post-transfection and subcultured by EDTA-based detachment solution. The presence of Rho-kinase inhibitor (Y-27632, Tocris Bioscience) was applied after subculture or thawing. Quantitative PCR: Two micrograms of total RNA extracted from iPSlike colonies was subjected for the first strand cDNA synthesis with SuperScript® III First-Strand Synthesis System (Life Technologies) using oligo(dT) primers according to the manufacturer's protocols. The cDNA was diluted 10 times and 2 μl of diluted cDNA was used for each qPCR reaction of FastStart SYBR Green Master (Roche). All primers of pluripotent marker genes applied and used in these studies were listed in Table 1. The relative expression level of genes was normalized with GAPDH. RNA without reverse transcriptase was used as a negative control. Relative expression of each pluripotency marker was compared to parental gene expression.
507
Immunocytochemistry: Cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 30 min at room temperature. Non-specific binding was blocked with 5% BSA in PBS for 30 min. Primary antibodies against TRA-1-60 (1:250, SC21705, Santa Cruz Biotechnology), SSEA4 (1:250, Ab16287, Abcam), NANOG (1:250, SC33759, Santa Cruz Biotechnology), and OCT4 (1:250, SC5279, Santa Cruz Biotechnology) were incubated overnight at 4 °C. After washing with PBS for three times, the cells were incubated with secondary antibodies; AlexaFluor 488 conjugated goat anti-mouse IgG (1:1000, A11001, Life Technologies) or AlexaFluor 488 conjugated goat anti-rabbit IgG (1: 1000, A11003, Life Technologies). Samples without primary antibody were used as negative controls. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) in mounting reagent (Life Technologies). Teratoma formation: Approximated 1–2 × 106 cells were subcutaneously injected into nude mice (BALB/cMlac-nu). The tumor-like formations were collected after injection for 8 to 12 weeks, fixed in 10% formalin, and underwent histological processes. The sections were
Fig. 1. Generation of MU011.A-hiPS generated from fetal fibroblast. (A) Quantitative PCR analysis of pluripotent marker ZFP42, TDGF1, SOX2, OCT4, NANOG, LIN28 and TDGF1 compared to parental fibroblasts. (B) Immunostaining of NANOG, OCT4, TRA1-61 and SSEA4 (green) and nucleus-staining with DAPI (blue). (C) Teratoma formation from MU011.A-hiPS showing tissues from all three germ layers. (D) Karyotyping of MU011.A-hiPS and its parental fibroblast showed the same 46, XY karyotype. (E) Gel electrophoresis of PCR product showing homozygous of α-thalassemia (−SEA/−SEA) deletion. (F) BFU-E derived from MU011.A-hiPS. (G) qPCR analysis to quantitate hemoglobin chain from BFU-E-derived from MU011.A-hiPS.
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A. Tangprasittipap et al. / Stem Cell Research 15 (2015) 506–509
Fig. 1 (continued).
A. Tangprasittipap et al. / Stem Cell Research 15 (2015) 506–509 Table 1 Primer sequences used in this study. Genes
Forward primer sequence (5′ to 3′)
Reverse primer sequence (5′ to 3′)
hNANOG hOCT4 hSOX2 hLIN28 hZFP42 hDNMT3B hTDGF1 hGADPH hHBZ hHBA hHBQ hHBM hHBB hHBD hHBG hHBE hACTB
CAG TCT GGA CAC TGG CTG AA TGT ACT CCT CGG TCC CTT TC GCT AGT CTC CAA GCG ACG AA TGC ACC AGA GTA AGC TGC AC TGC TCA CAG TCC AGC AGG TGT TT AAG TCG AAG GTG CGT CGT GC ACA GCA CAG TAA GGA GCT AAA C TGT TGC CAT CAA TGA CCC CTT TGA GAG GAC CAT CAT TGT GTC C TGG ACC CGG TCA ACT TCA AG GAA GAA GCT GGG CAG CAA CTC AGG CTC TTC ACG GTG TA GAA GGC TCA TGG CAA GAA AG CAA AGT GAA CGT GGA TGC AG TCA CAG AGG AGG ACA AGG CTA GAA TGT GGA AGA GGC TGG AG GGC ACC ACA CCT TCT ACA ATG
CTC GCT GAT TAG GCT CCA AC TCC AGG TTT TCT TTC CCT AGC GCA AGA AGC CTC TCC TTG AA CTC CTT TTG ATC TGC GCT TC TCT GGT GTC TTG TCT TTG CCC GTT CCC CTC GGT CTT TGC CGT TGT CGT CCG TAG AAG GAG GGA GG CTC CAC GAC GTA CTC AGC G AAG TGC GGG AAG TAG GTC TT TCA CAG AAG CCA GGA ACT TGT C GTG GGC TCT GAC TTG TGA GG ATT AGC AGC GGA AAG TTG GC CAC TGG TGG GGT GAA TTC TT CTG AGA AAA AGT GCC CTT GAG GCT TTA TGG CAT CTC CCA AG GGC TTG AGG TTG TCC ATG TT GGT CTC AAA CAT GAT CTG GGT C
509
Hematopoietic potential and globin analysis: Hematopoietic differentiation capability was performed by co-culture MU011.A-hiPS with OP9 cells for 8 days and differentiated cells were seeded into a MethoCult semisolid medium (StemCell Technologies). Pooled BFU-E colonies were subjected for globin gene expression determination by qPCR. All primers of globin genes used were listed in Table 1 and the relative expression level of genes was normalized with beta-actin. 2. Verification and authentication. MU011.A-hiPS and parental fibroblast showed the same karyotype 46, XY (Fig. 1D) and indeed homozygous for α-thalassemia and revealed the presence of homozygous South-East-Asian (−SEA/−SEA) deletion (Fig. 1E). In the Methocult medium, MU011.A-hiPS differentiated into the earliest erythroid progenitor (BFU-E, Fig. 1F) and the pattern of globin gene expression showed neither alpha-globin nor mu-globin gene (pseudogene of alpha-globin gene, Fig. 1G). Acknowledgments
stained with hematoxylin and eosin and examined under a light microscope for the presence of the tissue derived from three germ layers. All experiments performed on laboratory animals were approved by the Animal Care and Use Committee, Institute of Molecular Biosciences, Mahidol University. α-Thalassemia genotype: Genomic DNA of parental fibroblast and MU011.A-hiPS were subjected for characterization of α-thalassemia typing by Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University. Karyotyping: Chromosomal G-band analyses were performed at Human Genetic Laboratory, Ramathibodi Hospital, according to the International System for Human Cytogenetic Nomenclature. At least 25 metaphases were read for each sample.
This work was supported by Mahidol University grant agreement no. 108/2558 and the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme FP7-2012-PEOPLEIAPP under REA grant agreement no. 324451. References Chen, G., et al., 2011. Chemically defined conditions for human iPSC derivation and culture. Nat. Methods 8 (5), 424–429. Okita, K., et al., 2011. A more efficient method to generate integration-free human iPS cells. Nat. Methods 8 (5), 409–412.
Contents lists available at ScienceDirect
Stem Cell Research journal homepage: www.elsevier.com/locate/scr
Lab resource: Stem cell line
Generation of iPSC line MU011.A-hiPS from homozygous α-thalassemia fetal skin fibroblasts Amornrat Tangprasittipap a, Chonthicha Satirapod b, Bunyada Jittorntrum a, Sassawat Lertritanan a, Usanarat Anurathaphan c, Phetcharat Phanthong d,e, Suparerk Borwornpinyo f, Narisorn Kitiyanant e,⁎, Suradej Hongeng c,⁎ a
Office of Research, Academic Affairs and Innovations, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Department of Obstetrics and Gynecology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand c Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand d Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand e Stem Cell Research Group, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand f Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand b
a r t i c l e
i n f o
a b s t r a c t Human iPSC line MU011.A-hiPS was generated from homozygous α-thalassemia (−SEA/−SEA) fetal skin fibroblasts using a non-integrative reprogramming method. Reprogramming factors OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA of TP53 contained in three episomal vectors were delivered using electroporation. © 2015 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/).
Article history: Received 26 August 2015 Accepted 12 September 2015 Available online 21 September 2015
Resource table: Name of stem cell construct
MU011.A-hiPS
Institution Person who created resource Contact person and email
Mahidol University Amornrat Tangprasittipap, Narisorn Kitiyanant Narisorn Kitiyanant, [email protected] Suradej Hongeng, [email protected] July 2015 Human fetal skin fibroblasts Biological reagent: Induced pluripotent stem cell (iPS); derived from aborted fetal skin fibroblasts with homologous α-thalassemia (−SEA/−SEA) Cell line OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA of TP53 Identity and purity of cell line confirmed (Fig. 1) N/A
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
N/A
Resource details: Primary fibroblasts were obtained by skin biopsy from an aborted fetus with homozygous α-thalassemia (−SEA/−SEA), according to ⁎ Corresponding authors. E-mail addresses: [email protected] (N. Kitiyanant), [email protected] (S. Hongeng).
approved institutional procedures. The written informed consent was obtained from guardians. To generate MU011.A-hiPS, we delivered episomal expression cassettes of human OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA of TP53 (Okita et al., 2011) into fetal fibroblasts using a NEON microporator (Invitrogen). After 10 passages of iPSCs to ascertain that the cells had no exogenous reprogramming factors (LIN28, OCT4, SOX2) from episomal vector backbone that has been confirmed by quantitative PCR (data not shown). Pluripotent stem cell marker analysis: MU011.A-hiPS expressed pluripotent markers viz. NANOG, OCT3/4, SOX2, LIN28, REX1 (ZFP42), DNMT3B, TDGF1 (Fig. 1A) and surface markers; OCT3/4, NANOG, SSEA4 and TRA-1-60 (Fig. 1B). Teratoma formation demonstrated that MU011.A-hiPS was able to differentiate into derivatives of three germ layers (Fig. 1C). 1. Materials and methods Cell culture and iPS generation: Fetal skin fibroblasts were isolated and cultured in DMEM (Hyclone) supplemented with 10% FBS (Hyclone) and 1% GlutaMAX (Life Technologies). Three micrograms of episomal expression vectors were delivered into 2 × 105 human fetal skin fibroblast cells by a 100-μl kit of NEON microporator (Invitrogen) using the method previously described (Okita et al., 2011). After electroporation, cells were plated and cultured in a feeder free cultivation system on Geltrex (Life Technologies) in a chemically defined medium
http://dx.doi.org/10.1016/j.scr.2015.09.003 1873-5061/© 2015 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/).
A. Tangprasittipap et al. / Stem Cell Research 15 (2015) 506–509
(E8-equivalent) without TGF-β (Chen et al., 2011). Reprogramming efficiency was enhanced by adding 100 μM sodium butyrate for 2 weeks and the culture medium was replaced with full E8-equivalent medium when iPS-like colonies appeared. The iPS-liked colonies were manual picked up during day 14 to 21 post-transfection and subcultured by EDTA-based detachment solution. The presence of Rho-kinase inhibitor (Y-27632, Tocris Bioscience) was applied after subculture or thawing. Quantitative PCR: Two micrograms of total RNA extracted from iPSlike colonies was subjected for the first strand cDNA synthesis with SuperScript® III First-Strand Synthesis System (Life Technologies) using oligo(dT) primers according to the manufacturer's protocols. The cDNA was diluted 10 times and 2 μl of diluted cDNA was used for each qPCR reaction of FastStart SYBR Green Master (Roche). All primers of pluripotent marker genes applied and used in these studies were listed in Table 1. The relative expression level of genes was normalized with GAPDH. RNA without reverse transcriptase was used as a negative control. Relative expression of each pluripotency marker was compared to parental gene expression.
507
Immunocytochemistry: Cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 30 min at room temperature. Non-specific binding was blocked with 5% BSA in PBS for 30 min. Primary antibodies against TRA-1-60 (1:250, SC21705, Santa Cruz Biotechnology), SSEA4 (1:250, Ab16287, Abcam), NANOG (1:250, SC33759, Santa Cruz Biotechnology), and OCT4 (1:250, SC5279, Santa Cruz Biotechnology) were incubated overnight at 4 °C. After washing with PBS for three times, the cells were incubated with secondary antibodies; AlexaFluor 488 conjugated goat anti-mouse IgG (1:1000, A11001, Life Technologies) or AlexaFluor 488 conjugated goat anti-rabbit IgG (1: 1000, A11003, Life Technologies). Samples without primary antibody were used as negative controls. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) in mounting reagent (Life Technologies). Teratoma formation: Approximated 1–2 × 106 cells were subcutaneously injected into nude mice (BALB/cMlac-nu). The tumor-like formations were collected after injection for 8 to 12 weeks, fixed in 10% formalin, and underwent histological processes. The sections were
Fig. 1. Generation of MU011.A-hiPS generated from fetal fibroblast. (A) Quantitative PCR analysis of pluripotent marker ZFP42, TDGF1, SOX2, OCT4, NANOG, LIN28 and TDGF1 compared to parental fibroblasts. (B) Immunostaining of NANOG, OCT4, TRA1-61 and SSEA4 (green) and nucleus-staining with DAPI (blue). (C) Teratoma formation from MU011.A-hiPS showing tissues from all three germ layers. (D) Karyotyping of MU011.A-hiPS and its parental fibroblast showed the same 46, XY karyotype. (E) Gel electrophoresis of PCR product showing homozygous of α-thalassemia (−SEA/−SEA) deletion. (F) BFU-E derived from MU011.A-hiPS. (G) qPCR analysis to quantitate hemoglobin chain from BFU-E-derived from MU011.A-hiPS.
508
A. Tangprasittipap et al. / Stem Cell Research 15 (2015) 506–509
Fig. 1 (continued).
A. Tangprasittipap et al. / Stem Cell Research 15 (2015) 506–509 Table 1 Primer sequences used in this study. Genes
Forward primer sequence (5′ to 3′)
Reverse primer sequence (5′ to 3′)
hNANOG hOCT4 hSOX2 hLIN28 hZFP42 hDNMT3B hTDGF1 hGADPH hHBZ hHBA hHBQ hHBM hHBB hHBD hHBG hHBE hACTB
CAG TCT GGA CAC TGG CTG AA TGT ACT CCT CGG TCC CTT TC GCT AGT CTC CAA GCG ACG AA TGC ACC AGA GTA AGC TGC AC TGC TCA CAG TCC AGC AGG TGT TT AAG TCG AAG GTG CGT CGT GC ACA GCA CAG TAA GGA GCT AAA C TGT TGC CAT CAA TGA CCC CTT TGA GAG GAC CAT CAT TGT GTC C TGG ACC CGG TCA ACT TCA AG GAA GAA GCT GGG CAG CAA CTC AGG CTC TTC ACG GTG TA GAA GGC TCA TGG CAA GAA AG CAA AGT GAA CGT GGA TGC AG TCA CAG AGG AGG ACA AGG CTA GAA TGT GGA AGA GGC TGG AG GGC ACC ACA CCT TCT ACA ATG
CTC GCT GAT TAG GCT CCA AC TCC AGG TTT TCT TTC CCT AGC GCA AGA AGC CTC TCC TTG AA CTC CTT TTG ATC TGC GCT TC TCT GGT GTC TTG TCT TTG CCC GTT CCC CTC GGT CTT TGC CGT TGT CGT CCG TAG AAG GAG GGA GG CTC CAC GAC GTA CTC AGC G AAG TGC GGG AAG TAG GTC TT TCA CAG AAG CCA GGA ACT TGT C GTG GGC TCT GAC TTG TGA GG ATT AGC AGC GGA AAG TTG GC CAC TGG TGG GGT GAA TTC TT CTG AGA AAA AGT GCC CTT GAG GCT TTA TGG CAT CTC CCA AG GGC TTG AGG TTG TCC ATG TT GGT CTC AAA CAT GAT CTG GGT C
509
Hematopoietic potential and globin analysis: Hematopoietic differentiation capability was performed by co-culture MU011.A-hiPS with OP9 cells for 8 days and differentiated cells were seeded into a MethoCult semisolid medium (StemCell Technologies). Pooled BFU-E colonies were subjected for globin gene expression determination by qPCR. All primers of globin genes used were listed in Table 1 and the relative expression level of genes was normalized with beta-actin. 2. Verification and authentication. MU011.A-hiPS and parental fibroblast showed the same karyotype 46, XY (Fig. 1D) and indeed homozygous for α-thalassemia and revealed the presence of homozygous South-East-Asian (−SEA/−SEA) deletion (Fig. 1E). In the Methocult medium, MU011.A-hiPS differentiated into the earliest erythroid progenitor (BFU-E, Fig. 1F) and the pattern of globin gene expression showed neither alpha-globin nor mu-globin gene (pseudogene of alpha-globin gene, Fig. 1G). Acknowledgments
stained with hematoxylin and eosin and examined under a light microscope for the presence of the tissue derived from three germ layers. All experiments performed on laboratory animals were approved by the Animal Care and Use Committee, Institute of Molecular Biosciences, Mahidol University. α-Thalassemia genotype: Genomic DNA of parental fibroblast and MU011.A-hiPS were subjected for characterization of α-thalassemia typing by Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University. Karyotyping: Chromosomal G-band analyses were performed at Human Genetic Laboratory, Ramathibodi Hospital, according to the International System for Human Cytogenetic Nomenclature. At least 25 metaphases were read for each sample.
This work was supported by Mahidol University grant agreement no. 108/2558 and the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme FP7-2012-PEOPLEIAPP under REA grant agreement no. 324451. References Chen, G., et al., 2011. Chemically defined conditions for human iPSC derivation and culture. Nat. Methods 8 (5), 424–429. Okita, K., et al., 2011. A more efficient method to generate integration-free human iPS cells. Nat. Methods 8 (5), 409–412.