Generation of Human Induced Pluripotent Stem (iPS) Cells in Serum- and Feeder-Free Defined Culture and TGF-β1 Regulation of Pluripotency
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{"title"=>"Generation of human induced Pluripotent Stem (iPS) cells in serum- and feeder-free defined culture and TGF-β1 regulation of pluripotency", "type"=>"journal", "authors"=>[{"first_name"=>"Sachiko", "last_name"=>"Yamasaki", "scopus_author_id"=>"55556868400"}, {"first_name"=>"Yuki", "last_name"=>"Taguchi", "scopus_author_id"=>"56037587500"}, {"first_name"=>"Akira", "last_name"=>"Shimamoto", "scopus_author_id"=>"7005666330"}, {"first_name"=>"Hanae", "last_name"=>"Mukasa", "scopus_author_id"=>"56037323400"}, {"first_name"=>"Hidetoshi", "last_name"=>"Tahara", "scopus_author_id"=>"16173201200"}, {"first_name"=>"Tetsuji", "last_name"=>"Okamoto", "scopus_author_id"=>"35599884100"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-84898637537", "doi"=>"10.1371/journal.pone.0087151", "sgr"=>"84898637537", "isbn"=>"1932-6203 (Electronic) 1932-6203 (Linking)", "pmid"=>"24489856", "issn"=>"19326203", "pui"=>"373059921"}, "id"=>"5b776f79-8f4d-3be9-83b6-4969f2f8a020", "abstract"=>"Human Embryonic Stem cells (hESCs) and human induced Pluripotent Stem cells (hiPSCs) are commonly maintained on inactivated mouse embryonic fibroblast as feeder cells in medium supplemented with FBS or proprietary replacements. Use of culture medium containing undefined or unknown components has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. In addition, there is no consensus as to the optimal formulation, or the nature of the cytokine requirements of the cells to promote their self-renewal and inhibit their differentiation. In this study, we successfully generated hiPSCs from human dental pulp cells (DPCs) using Yamanaka's factors (Oct3/4, Sox2, Klf4, and c-Myc) with retroviral vectors in serum- and feeder-free defined culture conditions. These hiPSCs retained the property of self-renewal as evaluated by the expression of self-renewal marker genes and proteins, morphology, cell growth rates, and pluripotency evaluated by differentiation into derivatives of all three primary germ layers in vitro and in vivo. In this study, we found that TGF-β1 increased the expression levels of pluripotency markers in a dose-dependent manner. However, increasing doses of TGF-β1 suppressed the growth rate of hiPSCs cultured under the defined conditions. Furthermore, over short time periods the hiPSCs cultured in hESF9 or hESF9T exhibited similar morphology, but hiPSCs maintained in hESF9 could not survive beyond 30 passages. This result clearly confirmed that hiPSCs cultured in hESF9 medium absolutely required TGF-β1 to maintain pluripotency. This simple serum-free adherent monoculture system will allow us to elucidate the cell responses to growth factors under defined conditions and can eliminate the risk might be brought by undefined pathogens.", "link"=>"http://www.mendeley.com/research/generation-human-induced-pluripotent-stem-ips-cells-serum-feederfree-defined-culture-tgf%CE%B21-regulatio", "reader_count"=>57, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>3, "Researcher"=>17, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>13, "Student > Postgraduate"=>4, "Other"=>10, "Student > Master"=>6, "Student > Bachelor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>3, "Researcher"=>17, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>13, "Student > Postgraduate"=>4, "Other"=>10, "Student > Master"=>6, "Student > Bachelor"=>1}, "reader_count_by_subject_area"=>{"Engineering"=>3, "Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>8, "Agricultural and Biological Sciences"=>36, "Medicine and Dentistry"=>5, "Chemistry"=>2, "Immunology and Microbiology"=>2}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>3}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>5}, "Chemistry"=>{"Chemistry"=>2}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>36}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>8}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"United States"=>1, "Poland"=>1, "France"=>1, "Germany"=>1}, "group_count"=>2}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1365411"], "description"=>"<p>A) Phase contrast photomicrograph of DP-F-iPS-CL16 (passage 28) supplemented with various concentration of TGF-β1 (0, 0.1, 1, 2, 5, 10 ng/ml). B) Digital-PCR analysis of gene expression of Nanog, Oct3/4, PAI-I and GATA4 in DP-F-iPS-CL6 in hESF9 medium supplemented with TGF-β1 (0, 0.1, 1, 2, 5, 10 ng/ml) on fibronectin. Expression levels were all normalized against GAPDH. C) Effects of TGF-β1 on hiPS cell proliferation. hiPSCs generated under hESF9 and cultured in hESF9T (CL-4 passage 38, CL-8 passage 38, CL-16 passage 33) were seeded in a 24 well plate coated with fibronectin at 1×10<sup>4</sup> cells/well and counted at every 24 hr. Each bar shows the number of cells in each concentration of TGF-β1 after 6 days of culture. Increasing the dose of TGF-β1 up to 10 ng/ml suppressed the growth of hiPSCs. Bars represent the mean±SEM. (n  = 3). D) Expression of ES cell marker genes in iPSCs derived from DPCs. We used primers that only amplified the endogenous genes. #1: DP cell (DP-A): passage 2 = before infection. #2: DP cell (DP-F): passage 4 = before infection. #3: DP-A-iPS-CL1: passage 14 = serum-free condition (hESF9/on FN). #4: DP-F-iPS-CL4: passage 37 = serum-free condition (hESF9T/on FN). #5: DP-F-iPS-CL6: passage 35 = serum-free condition (hESF9T/on FN). #6: DP-F-iPS-CL8: passage 35 = serum-free condition (hESF9T/on FN). #7: DP-A-iPS-CL1: passage 8 = KSR-based condition (KSR/on MEF). #8: DP-F-iPS-CL12: passage 36 = KSR-based condition (KSR/on MEF). #9: Tic (hiPSC: JCRB1331): passage 103 = KSR-based condition (KSR/on MEF). E) Immunocytochemistry of Nanog, Oct3/4, SSEA-4, Tra-1-60 and Tra-1-81. DP-F-iPS-CL16 grown under hESF9T-based culture conditions for 19 passages were fixed and reacted with antibodies (Nanog, Oct3/4, SSEA-4, Tra-1-61 and Tra-1-81). Binding of these antibodies was visualized with Alexa Fluor® 488-conjugated secondary antibodies (green). Nuclei were stained with DAPI (blue). Scale bars represent 100 µm.</p>", "links"=>[], "tags"=>["Biochemistry", "cytochemistry", "Extracellular matrix", "proteins", "Extracellular matrix proteins", "developmental biology", "stem cells", "Embryonic stem cells", "Induced pluripotent stem cells", "Stem cell niche", "Molecular cell biology", "Cellular types", "Signal transduction", "Signaling in cellular processes", "Extracellular matrix signaling", "hipscs", "hesf9t", "self-renewal", "pluripotent", "cells", "defined"], "article_id"=>917246, "categories"=>["Biological Sciences"], "users"=>["Sachiko Yamasaki", "Yuki Taguchi", "Akira Shimamoto", "Hanae Mukasa", "Hidetoshi Tahara", "Tetsuji Okamoto"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0087151.g005", "stats"=>{"downloads"=>0, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Culture_of_hiPSCs_in_hESF9T_and_Self_renewal_marker_expression_of_pluripotent_stem_cells_in_hiPSCs_in_defined_culture_conditions_/917246", "title"=>"Culture of hiPSCs in hESF9T and Self-renewal marker expression of pluripotent stem cells in hiPSCs in defined culture conditions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-29 02:44:31"}
  • {"files"=>["https://ndownloader.figshare.com/files/1365393"], "description"=>"<p>A) Phase contrast images of iPSCs derived from DPCs (DP-A-iPS and DP-F-iPS). i) DP-A-iPS-CL1 at passage 2, or 21 on fibronectin-coated dish with hESF9 medium. Right panel showed the cells at passage 5 cultured on MEF with KSR-based conditions. ii) DP-F-iPS-CL4 at passage 60, CL6 at passage 59 and CL16 at passage 58 on fibronectin-coated dish with hESF9T medium. Right panel showed CL31 at passage 19 on MEF with KSR-based conditions. Bars indicate 200 µm. B) Flow cytometry analysis of Oct3/4 and SSEA-4 expression in hiPSCs generated and maintained in hESF9 medium supplemented with TGF-β1 (2 ng/ml) (hESF9T) or without TGF-β1 (hESF9) (DP-F-iPS-CL-8 at passage 33). The horizontal bar indicates the gating used to score the percentage of cells antigen positive. C) Comparison of the global gene expression analysis. Unsupervised clustering was performed using microarray data from parental cell (DPCs), DP-iPSCs cultured in hESF9 or hESF9T (DP-A-iPS, DP-F-iPS) and hiPSCs (Tic, DP-F-iPS). 1.DP cell (DP-A): passage 2 = before infection. 2.DP cell (DP-F): passage 4 = before infection. 3.DP-A-iPS-CL1: passage 14 = serum-free condition (hESF9/on FN). 4.DP-F-iPS-CL12: passage 36 = KSR-based condition (KSR/on MEF). 5.DP-F-iPS-CL6: passage 37 = serum-free condition (hESF9T/on FN). 6.DP-F-iPS-CL8: passage 35 = serum-free condition (hESF9T/on FN). 7.Tic (hiPSC: JCRB1331): passage 58 = KSR-based condition (KSR/on MEF). A genome-wide gene expression profiling analysis demonstrated that hiPSCs cultured in hESF9 or hESF9T on fibronectin showed a similar gene expression pattern to those grown in a conventional feeder-dependent culture (KSR-based condition). Hierarchical combined tree on compare. (Fold change> = 20).</p>", "links"=>[], "tags"=>["Biochemistry", "cytochemistry", "Extracellular matrix", "proteins", "Extracellular matrix proteins", "developmental biology", "stem cells", "Embryonic stem cells", "Induced pluripotent stem cells", "Stem cell niche", "Molecular cell biology", "Cellular types", "Signal transduction", "Signaling in cellular processes", "Extracellular matrix signaling", "derived", "dpcs", "serum-", "feeder-free"], "article_id"=>917228, "categories"=>["Biological Sciences"], "users"=>["Sachiko Yamasaki", "Yuki Taguchi", "Akira Shimamoto", "Hanae Mukasa", "Hidetoshi Tahara", "Tetsuji Okamoto"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0087151.g004", "stats"=>{"downloads"=>1, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_hiPSCs_derived_from_DPCs_in_completely_serum_and_feeder_free_culture_conditions_/917228", "title"=>"hiPSCs derived from DPCs in completely serum- and feeder-free culture conditions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-29 02:44:31"}
  • {"files"=>["https://ndownloader.figshare.com/files/1365420"], "description"=>"<p>A) Differentiation was performed using embryoid body formation, and the differentiated iPSCs (DP-A-iPS/hESF9 or DP-F-iPS/hESF9T) were fixed and reacted with antibodies. Shown were immunocytochemistry of Nestin, βIII-tubulin, α-smooth muscle actin (α-SMA), and α-fetoprotein (AFP). Binding of these antibodies was visualized with Alexa Fluor 488-conjugated secondary antibodies (green). Oct3/4 was also investigated. Binding of these antibodies was visualized with Alexa Fluor® 594-conjugated secondary antibodies (red). Nucleuses were stained with DAPI. (passage 25). Bar indicates 100 µm. B) Teratomas were generated in SCID mice (CB17/Icr-<i>Prkdc<sup>scid</sup></i>/CrlCrlj) from DP-A-iPS and DP-F-iPS grown under hESF9 or hESF9T-based conditions. Histological analysis with HE staining or Alcian Blue staining demonstrated that teratomas formed from iPS cells cultured in KSR-based (data not shown) or in hESF9T-based conditions contained derivatives of all three germ layers. Left panel shows teratomas from DP-A-iPS-CL1 at passage 22. Right panel shows teratomas from DP-F-iPS-CL14 at passage 6. Scale bars represent 200 µm.</p>", "links"=>[], "tags"=>["Biochemistry", "cytochemistry", "Extracellular matrix", "proteins", "Extracellular matrix proteins", "developmental biology", "stem cells", "Embryonic stem cells", "Induced pluripotent stem cells", "Stem cell niche", "Molecular cell biology", "Cellular types", "Signal transduction", "Signaling in cellular processes", "Extracellular matrix signaling", "body-mediated", "differentiation", "hipscs", "derived", "dpcs", "serum-free", "feeder-free", "defined", "teratoma"], "article_id"=>917254, "categories"=>["Biological Sciences"], "users"=>["Sachiko Yamasaki", "Yuki Taguchi", "Akira Shimamoto", "Hanae Mukasa", "Hidetoshi Tahara", "Tetsuji Okamoto"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0087151.g006", "stats"=>{"downloads"=>4, "page_views"=>30, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Embryoid_body_mediated_differentiation_of_hiPSCs_derived_from_DPCs_in_serum_free_and_feeder_free_defined_culture_conditions_and_teratoma_formation_of_hiPSCs_in_the_defined_culture_conditions_/917254", "title"=>"Embryoid body-mediated differentiation of hiPSCs derived from DPCs in serum-free and feeder-free defined culture conditions and teratoma formation of hiPSCs in the defined culture conditions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-29 02:44:31"}
  • {"files"=>["https://ndownloader.figshare.com/files/1365384"], "description"=>"<p>TIG-3 was introduced with pMXs retroviruses containing the EGFP cDNA. After 3 days, cells were photographed under a fluorescence microscope and analyzed by flow cytometry. The left panel shows the images of phase contrast, fluorescent microscope and the results of flow cytometry of the cells cultured in serum-supplemented condition (DMEM+10%FBS). The right panel shows the cells in serum-free culture conditions (hESF9). Transfection efficiency of EGFP was 62.6% in serum-supplemented condition and 46.4% in serum-free culture condition. Bars indicate 200 µm.</p>", "links"=>[], "tags"=>["Biochemistry", "cytochemistry", "Extracellular matrix", "proteins", "Extracellular matrix proteins", "developmental biology", "stem cells", "Embryonic stem cells", "Induced pluripotent stem cells", "Stem cell niche", "Molecular cell biology", "Cellular types", "Signal transduction", "Signaling in cellular processes", "Extracellular matrix signaling", "retroviruses", "serum-free", "hesf9", "serum-supplemented"], "article_id"=>917219, "categories"=>["Biological Sciences"], "users"=>["Sachiko Yamasaki", "Yuki Taguchi", "Akira Shimamoto", "Hanae Mukasa", "Hidetoshi Tahara", "Tetsuji Okamoto"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0087151.g002", "stats"=>{"downloads"=>0, "page_views"=>42, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Transduction_efficiency_of_retroviruses_in_serum_free_hESF9_medium_and_serum_supplemented_medium_in_TIG_3_/917219", "title"=>"Transduction efficiency of retroviruses in serum-free hESF9 medium and serum-supplemented medium in TIG-3.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-29 02:44:31"}
  • {"files"=>["https://ndownloader.figshare.com/files/1365381"], "description"=>"<p>A) Transduced TIG-3 cells were cultured on each ECMs with hESF9 medium or on MEF with KSR-based conditions. After 20 days, iPS colony were picked up and sub-cultured on each ECMs. B) Images of sub-cultured iPS colonies seeded after 2days on each ECMs with hESF9 medium. C) ALP staining of iPSCs on gelatin, collagen, and fibronectin (infect after 36days). Bars indicate 200 µm.</p>", "links"=>[], "tags"=>["Biochemistry", "cytochemistry", "Extracellular matrix", "proteins", "Extracellular matrix proteins", "developmental biology", "stem cells", "Embryonic stem cells", "Induced pluripotent stem cells", "Stem cell niche", "Molecular cell biology", "Cellular types", "Signal transduction", "Signaling in cellular processes", "Extracellular matrix signaling", "transduced", "tig-3", "ecms", "hesf9"], "article_id"=>917217, "categories"=>["Biological Sciences"], "users"=>["Sachiko Yamasaki", "Yuki Taguchi", "Akira Shimamoto", "Hanae Mukasa", "Hidetoshi Tahara", "Tetsuji Okamoto"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0087151.g001", "stats"=>{"downloads"=>0, "page_views"=>18, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Morphology_of_transduced_TIG_3_on_each_ECMs_in_hESF9_medium_/917217", "title"=>"Morphology of transduced TIG-3 on each ECMs in hESF9 medium.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-29 02:44:31"}
  • {"files"=>["https://ndownloader.figshare.com/files/1365426", "https://ndownloader.figshare.com/files/1365427", "https://ndownloader.figshare.com/files/1365428", "https://ndownloader.figshare.com/files/1365429", "https://ndownloader.figshare.com/files/1365430", "https://ndownloader.figshare.com/files/1365431", "https://ndownloader.figshare.com/files/1365432", "https://ndownloader.figshare.com/files/1365433", "https://ndownloader.figshare.com/files/1365434"], "description"=>"<div><p>Human Embryonic Stem cells (hESCs) and human induced Pluripotent Stem cells (hiPSCs) are commonly maintained on inactivated mouse embryonic fibroblast as feeder cells in medium supplemented with FBS or proprietary replacements. Use of culture medium containing undefined or unknown components has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. In addition, there is no consensus as to the optimal formulation, or the nature of the cytokine requirements of the cells to promote their self-renewal and inhibit their differentiation. In this study, we successfully generated hiPSCs from human dental pulp cells (DPCs) using Yamanaka's factors (<i>Oct3/4, Sox2, Klf4</i>, and <i>c-Myc</i>) with retroviral vectors in serum- and feeder-free defined culture conditions. These hiPSCs retained the property of self-renewal as evaluated by the expression of self-renewal marker genes and proteins, morphology, cell growth rates, and pluripotency evaluated by differentiation into derivatives of all three primary germ layers <i>in vitro</i> and <i>in vivo</i>. In this study, we found that TGF-β1 increased the expression levels of pluripotency markers in a dose-dependent manner. However, increasing doses of TGF-β1 suppressed the growth rate of hiPSCs cultured under the defined conditions. Furthermore, over short time periods the hiPSCs cultured in hESF9 or hESF9T exhibited similar morphology, but hiPSCs maintained in hESF9 could not survive beyond 30 passages. This result clearly confirmed that hiPSCs cultured in hESF9 medium absolutely required TGF-β1 to maintain pluripotency. This simple serum-free adherent monoculture system will allow us to elucidate the cell responses to growth factors under defined conditions and can eliminate the risk might be brought by undefined pathogens.</p></div>", "links"=>[], "tags"=>["Biochemistry", "cytochemistry", "Extracellular matrix", "proteins", "Extracellular matrix proteins", "developmental biology", "stem cells", "Embryonic stem cells", "Induced pluripotent stem cells", "Stem cell niche", "Molecular cell biology", "Cellular types", "Signal transduction", "Signaling in cellular processes", "Extracellular matrix signaling", "induced", "pluripotent", "cells", "serum-", "feeder-free", "defined"], "article_id"=>917260, "categories"=>["Biological Sciences"], "users"=>["Sachiko Yamasaki", "Yuki Taguchi", "Akira Shimamoto", "Hanae Mukasa", "Hidetoshi Tahara", "Tetsuji Okamoto"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0087151.s001", "https://dx.doi.org/10.1371/journal.pone.0087151.s002", "https://dx.doi.org/10.1371/journal.pone.0087151.s003", "https://dx.doi.org/10.1371/journal.pone.0087151.s004", "https://dx.doi.org/10.1371/journal.pone.0087151.s005", "https://dx.doi.org/10.1371/journal.pone.0087151.s006", "https://dx.doi.org/10.1371/journal.pone.0087151.s007", "https://dx.doi.org/10.1371/journal.pone.0087151.s008", "https://dx.doi.org/10.1371/journal.pone.0087151.s009"], "stats"=>{"downloads"=>4, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Generation_of_Human_Induced_Pluripotent_Stem_Ips_Cells_in_Serum_and_Feeder_Free_Defined_Culture_and_TGF_914_1_Regulation_of_Pluripotency_/917260", "title"=>"Generation of Human Induced Pluripotent Stem (Ips) Cells in Serum- and Feeder-Free Defined Culture and TGF-Β1 Regulation of Pluripotency", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2014-01-29 02:44:31"}
  • {"files"=>["https://ndownloader.figshare.com/files/1365387"], "description"=>"<p>A) Time schedule of hiPSC generation. Day-7∼0: DPCs were cultured in RD6F serum-free medium on type I collagen-coated dish. Day 0∼4: Retroviral transduction (<i>Oct3/4, Sox2, KLF-4, c-Myc</i>) with hESF9 medium. Day5: re-seeding on fibronectin-coated plate with hESF9 medium. Day6∼30: replace medium every other day. B) Images of DPCs (DP-A: passage 4) on type I collagen-coated plate with RD6F medium. C) Transduced DPCs were cultured on fibronectin with hESF9 medium or on MEF with KSR-based conditions. After 20 days, iPS colony were picked up and sub-cultured on fibronectin. The reprogramming efficiency was 0.23–0.38% with a high success rate. D) ALP staining of iPSCs on fibronectin, 43 days after infection. Bars indicate 200 µm.</p>", "links"=>[], "tags"=>["Biochemistry", "cytochemistry", "Extracellular matrix", "proteins", "Extracellular matrix proteins", "developmental biology", "stem cells", "Embryonic stem cells", "Induced pluripotent stem cells", "Stem cell niche", "Molecular cell biology", "Cellular types", "Signal transduction", "Signaling in cellular processes", "Extracellular matrix signaling", "dpcs", "serum-", "feeder-free"], "article_id"=>917222, "categories"=>["Biological Sciences"], "users"=>["Sachiko Yamasaki", "Yuki Taguchi", "Akira Shimamoto", "Hanae Mukasa", "Hidetoshi Tahara", "Tetsuji Okamoto"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0087151.g003", "stats"=>{"downloads"=>4, "page_views"=>32, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_hiPSC_generation_from_DPCs_in_serum_and_feeder_free_culture_conditions_/917222", "title"=>"hiPSC generation from DPCs in serum- and feeder-free culture conditions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-01-29 02:44:31"}

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