Improved Methods for Reprogramming Human Dermal Fibroblasts Using Fluorescence Activated Cell Sorting
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{"title"=>"Improved Methods for Reprogramming Human Dermal Fibroblasts Using Fluorescence Activated Cell Sorting", "type"=>"journal", "authors"=>[{"first_name"=>"David J.", "last_name"=>"Kahler", "scopus_author_id"=>"8935930000"}, {"first_name"=>"Faizzan S.", "last_name"=>"Ahmad", "scopus_author_id"=>"53876767800"}, {"first_name"=>"Anita", "last_name"=>"Ritz", "scopus_author_id"=>"55633844800"}, {"first_name"=>"Haiqing", "last_name"=>"Hua", "scopus_author_id"=>"55208799400"}, {"first_name"=>"Dorota N.", "last_name"=>"Moroziewicz", "scopus_author_id"=>"6508167337"}, {"first_name"=>"Andrew A.", "last_name"=>"Sproul", "scopus_author_id"=>"12775910200"}, {"first_name"=>"Carmen R.", "last_name"=>"Dusenberry", "scopus_author_id"=>"55914213700"}, {"first_name"=>"Linshan", "last_name"=>"Shang", "scopus_author_id"=>"55209026700"}, {"first_name"=>"Daniel", "last_name"=>"Paull", "scopus_author_id"=>"53981787100"}, {"first_name"=>"Matthew", "last_name"=>"Zimmer", "scopus_author_id"=>"55540069100"}, {"first_name"=>"Keren A.", "last_name"=>"Weiss", "scopus_author_id"=>"55539240400"}, {"first_name"=>"Dieter", "last_name"=>"Egli", "scopus_author_id"=>"7003399539"}, {"first_name"=>"Scott A.", "last_name"=>"Noggle", "scopus_author_id"=>"6602290504"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"368612403", "sgr"=>"84875491866", "issn"=>"19326203", "pmid"=>"23555815", "scopus"=>"2-s2.0-84875491866", "doi"=>"10.1371/journal.pone.0059867", "isbn"=>"1932-6203"}, "id"=>"1f98b9ca-fbc2-37fa-b83a-68345b389cf8", "abstract"=>"Current methods to derive induced pluripotent stem cell (iPSC) lines from human dermal fibroblasts by viral infection rely on expensive and lengthy protocols. One major factor contributing to the time required to derive lines is the ability of researchers to identify fully reprogrammed unique candidate clones from a mixed cell population containing transformed or partially reprogrammed cells and fibroblasts at an early time point post infection. Failure to select high quality colonies early in the derivation process results in cell lines that require increased maintenance and unreliable experimental outcomes. Here, we describe an improved method for the derivation of iPSC lines using fluorescence activated cell sorting (FACS) to isolate single cells expressing the cell surface marker signature CD13(NEG)SSEA4(POS)Tra-1-60(POS) on day 7-10 after infection. This technique prospectively isolates fully reprogrammed iPSCs, and depletes both parental and \"contaminating\" partially reprogrammed fibroblasts, thereby substantially reducing the time and reagents required to generate iPSC lines without the use of defined small molecule cocktails. FACS derived iPSC lines express common markers of pluripotency, and possess spontaneous differentiation potential in vitro and in vivo. To demonstrate the suitability of FACS for high-throughput iPSC generation, we derived 228 individual iPSC lines using either integrating (retroviral) or non- integrating (Sendai virus) reprogramming vectors and performed extensive characterization on a subset of those lines. The iPSC lines used in this study were derived from 76 unique samples from a variety of tissue sources, including fresh or frozen fibroblasts generated from biopsies harvested from healthy or disease patients.", "link"=>"http://www.mendeley.com/research/improved-methods-reprogramming-human-dermal-fibroblasts-using-fluorescence-activated-cell-sorting-3", "reader_count"=>37, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>4, "Researcher"=>10, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>5, "Student > Postgraduate"=>5, "Other"=>3, "Student > Master"=>3, "Student > Bachelor"=>2, "Professor"=>2}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>4, "Researcher"=>10, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>5, "Student > Postgraduate"=>5, "Other"=>3, "Student > Master"=>3, "Student > Bachelor"=>2, "Professor"=>2}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Engineering"=>2, "Biochemistry, Genetics and Molecular Biology"=>7, "Materials Science"=>1, "Agricultural and Biological Sciences"=>23, "Medicine and Dentistry"=>1, "Pharmacology, Toxicology and Pharmaceutical Science"=>1, "Chemistry"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>2}, "Materials Science"=>{"Materials Science"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Chemistry"=>{"Chemistry"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>23}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>7}, "Unspecified"=>{"Unspecified"=>1}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}}, "reader_count_by_country"=>{"Czech Republic"=>1, "United States"=>2, "France"=>1}, "group_count"=>5}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1002279"], "description"=>"<p>Immunofluroescence microscopy of the 1001.131.01 line demonstrating expression of common markers of pluripotency by FACS or Manually Derived IPSC lines. Nuclear Transcription Factors shown in Green, Surface Markers shown in Red, Nucleus stained with DAPI in Blue (<b>A</b>) Nanog/Tra-1-60 (<b>B</b>) Oct4/Tra-1-81 (<b>C</b>) Sox2/SSEA4 (<b>D</b>) Oct4/Alkaline Phosphatase. 10× Magnification (<b>E</b>) Expression of endogenous pluripotent transcription factors (<b>F</b>) Silencing of viral transcription factors occur by passage 5. (<b>G</b>) Expression levels of transcription factors common to the indicated germ layers from EB generated by the indicated IPSC lines.</p>", "links"=>[], "tags"=>["fluorescence", "activated", "sorted", "manually", "derived", "ipsc", "lines", "sendai"], "article_id"=>663486, "categories"=>["Microbiology", "Cell Biology", "Developmental Biology", "Virology"], "users"=>["David J. Kahler", "Faizzan S. Ahmad", "Anita Ritz", "Haiqing Hua", "Dorota N. Moroziewicz", "Andrew A. Sproul", "Carmen R. Dusenberry", "Linshan Shang", "Daniel Paull", "Matthew Zimmer", "Keren A. Weiss", "Dieter Egli", "Scott A. Noggle"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0059867.g006", "stats"=>{"downloads"=>0, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Characterization_of_Fluorescence_Activated_Cell_Sorted_and_Manually_Derived_IPSC_Lines_by_Sendai_virus_/663486", "title"=>"Characterization of Fluorescence Activated Cell Sorted and Manually Derived IPSC Lines by Sendai virus.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-30 07:23:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/1002272"], "description"=>"<p>Embryoid bodies were derived from FACS (<b>A</b>) or manually derived clones (<b>B</b>) and stained for expression of alpha fetoprotein, smooth muscle actin and beta III tubulin (Tuj1) to demonstrate differentiation potential <i>in vitro</i> potential. 10× Magnification (<b>C</b>) Differentiation potential of the derived lines for expression of germ layer genes present in the Lineage scorecard assay. (<b>D</b>) Teratomas from FACS (<b>D</b>) or manually derived (<b>E</b>) clones of 1023 fibroblast line indicating <i>in vitro</i> differentiation potential by formation of three germ layers.</p>", "links"=>[], "tags"=>["lines", "derived", "fluorescence", "activated", "sorting", "spontaneous", "differential"], "article_id"=>663482, "categories"=>["Microbiology", "Cell Biology", "Developmental Biology", "Virology"], "users"=>["David J. Kahler", "Faizzan S. Ahmad", "Anita Ritz", "Haiqing Hua", "Dorota N. Moroziewicz", "Andrew A. Sproul", "Carmen R. Dusenberry", "Linshan Shang", "Daniel Paull", "Matthew Zimmer", "Keren A. Weiss", "Dieter Egli", "Scott A. Noggle"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0059867.g004", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_hIPSC_lines_derived_by_Fluorescence_Activated_Cell_Sorting_possess_in_vitro_and_in_vivo_spontaneous_differential_potential_/663482", "title"=>"hIPSC lines derived by Fluorescence Activated Cell Sorting possess <i>in vitro</i> and <i>in vivo</i> spontaneous differential potential.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-30 07:21:59"}
  • {"files"=>["https://ndownloader.figshare.com/files/1002291", "https://ndownloader.figshare.com/files/1002292", "https://ndownloader.figshare.com/files/1002294", "https://ndownloader.figshare.com/files/1002295", "https://ndownloader.figshare.com/files/1002297", "https://ndownloader.figshare.com/files/1002298", "https://ndownloader.figshare.com/files/1002299", "https://ndownloader.figshare.com/files/1002302", "https://ndownloader.figshare.com/files/1002304", "https://ndownloader.figshare.com/files/1002305", "https://ndownloader.figshare.com/files/1002306", "https://ndownloader.figshare.com/files/1002307", "https://ndownloader.figshare.com/files/1002309"], "description"=>"<div><p>Current methods to derive induced pluripotent stem cell (iPSC) lines from human dermal fibroblasts by viral infection rely on expensive and lengthy protocols. One major factor contributing to the time required to derive lines is the ability of researchers to identify fully reprogrammed unique candidate clones from a mixed cell population containing transformed or partially reprogrammed cells and fibroblasts at an early time point post infection. Failure to select high quality colonies early in the derivation process results in cell lines that require increased maintenance and unreliable experimental outcomes. Here, we describe an improved method for the derivation of iPSC lines using fluorescence activated cell sorting (FACS) to isolate single cells expressing the cell surface marker signature CD13<sup>NEG</sup>SSEA4<sup>POS</sup>Tra-1-60<sup>POS</sup> on day 7–10 after infection. This technique prospectively isolates fully reprogrammed iPSCs, and depletes both parental and “contaminating” partially reprogrammed fibroblasts, thereby substantially reducing the time and reagents required to generate iPSC lines without the use of defined small molecule cocktails. FACS derived iPSC lines express common markers of pluripotency, and possess spontaneous differentiation potential <i>in vitro</i> and <i>in vivo</i>. To demonstrate the suitability of FACS for high-throughput iPSC generation, we derived 228 individual iPSC lines using either integrating (retroviral) or non- integrating (Sendai virus) reprogramming vectors and performed extensive characterization on a subset of those lines. The iPSC lines used in this study were derived from 76 unique samples from a variety of tissue sources, including fresh or frozen fibroblasts generated from biopsies harvested from healthy or disease patients.</p> </div>", "links"=>[], "tags"=>["methods", "Reprogramming", "dermal", "fibroblasts", "fluorescence", "activated"], "article_id"=>663496, "categories"=>["Microbiology", "Cell Biology", "Developmental Biology", "Virology"], "users"=>["David J. Kahler", "Faizzan S. Ahmad", "Anita Ritz", "Haiqing Hua", "Dorota N. Moroziewicz", "Andrew A. Sproul", "Carmen R. Dusenberry", "Linshan Shang", "Daniel Paull", "Matthew Zimmer", "Keren A. Weiss", "Dieter Egli", "Scott A. Noggle"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0059867.s001", "https://dx.doi.org/10.1371/journal.pone.0059867.s002", "https://dx.doi.org/10.1371/journal.pone.0059867.s003", "https://dx.doi.org/10.1371/journal.pone.0059867.s004", "https://dx.doi.org/10.1371/journal.pone.0059867.s005", "https://dx.doi.org/10.1371/journal.pone.0059867.s006", "https://dx.doi.org/10.1371/journal.pone.0059867.s007", "https://dx.doi.org/10.1371/journal.pone.0059867.s008", "https://dx.doi.org/10.1371/journal.pone.0059867.s009", "https://dx.doi.org/10.1371/journal.pone.0059867.s010", "https://dx.doi.org/10.1371/journal.pone.0059867.s011", "https://dx.doi.org/10.1371/journal.pone.0059867.s012", "https://dx.doi.org/10.1371/journal.pone.0059867.s013"], "stats"=>{"downloads"=>9, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Improved_Methods_for_Reprogramming_Human_Dermal_Fibroblasts_Using_Fluorescence_Activated_Cell_Sorting_/663496", "title"=>"Improved Methods for Reprogramming Human Dermal Fibroblasts Using Fluorescence Activated Cell Sorting", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-03-30 07:26:25"}
  • {"files"=>["https://ndownloader.figshare.com/files/1002274"], "description"=>"<p>Three individual clones were selected from foreskin (0819) fibroblasts lines which previously underwent four factor retroviral reprogramming and were derived by either FACS (<b>A, C<sub>1</sub>–C<sub>3</sub></b>) or manual (<b>B, C<sub>4</sub>–C<sub>6</sub></b>) techniques were analyzed by flow cytometry for pluripotent surface marker expression following expansion on murine embryonic fibroblasts for 12–14 passages. Clones C3 and C6 were adapted to Matrigel and mTSER media around passage11 and expanded for several passages prior to surface marker analysis by flow cytometry to demonstrate stability following changes in culture conditions. Events displayed in the 2D scatter plots are “live” cells as defined by forward and side scatter properties expressing indicated surface markers.</p>", "links"=>[], "tags"=>["fluorescence", "activated", "sorted", "manually", "derived", "ipsc"], "article_id"=>663484, "categories"=>["Microbiology", "Cell Biology", "Developmental Biology", "Virology"], "users"=>["David J. Kahler", "Faizzan S. Ahmad", "Anita Ritz", "Haiqing Hua", "Dorota N. Moroziewicz", "Andrew A. Sproul", "Carmen R. Dusenberry", "Linshan Shang", "Daniel Paull", "Matthew Zimmer", "Keren A. Weiss", "Dieter Egli", "Scott A. Noggle"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0059867.g005", "stats"=>{"downloads"=>1, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Stability_of_Fluorescence_Activated_Cell_Sorted_and_Manually_Derived_IPSC_Lines_/663484", "title"=>"Stability of Fluorescence Activated Cell Sorted and Manually Derived IPSC Lines.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-30 07:22:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/1002271"], "description"=>"<p>Modified pluripotent scorecard assay was performed on manually and FACS derived clones to demonstrate (<b>A</b>) activation of endogenous gene expression and (<b>B</b>) silencing of gene expression and presence of unreprogrammed and transformed fibroblasts CD13<sup>POS</sup> in manually derived clones. (<b>C</b>) Three sorted and three picked lines from patient 1023 were used to compare the ability of both methods to generate independent clones. 10 μ of genomic DNA were cut overnight with BglII and submitted to Southern blotting. The HUES line HES2 was used as a positive control for endogenous KLF4/OCT4, and as a negative control for transgene insertions. Samples were first blotted for KLF4, then stripped and reblotted for OCT4. Picked clones 1023 C and E are consistent with being the same clone by both KLF4 and OCT4 blotting. * indicated the predicted endogenous KLF4/OCT4 bands, and ** indicated a consistent band found in all samples blotted with OCT4.</p>", "links"=>[], "tags"=>["activated", "sorting", "generates", "higher", "clones"], "article_id"=>663481, "categories"=>["Microbiology", "Cell Biology", "Developmental Biology", "Virology"], "users"=>["David J. Kahler", "Faizzan S. Ahmad", "Anita Ritz", "Haiqing Hua", "Dorota N. Moroziewicz", "Andrew A. Sproul", "Carmen R. Dusenberry", "Linshan Shang", "Daniel Paull", "Matthew Zimmer", "Keren A. Weiss", "Dieter Egli", "Scott A. Noggle"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0059867.g003", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Fluorescence_Activated_Cell_Sorting_generates_higher_quality_independent_clones_than_manual_derivation_/663481", "title"=>"Fluorescence Activated Cell Sorting generates higher quality independent clones than manual derivation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-30 07:21:27"}
  • {"files"=>["https://ndownloader.figshare.com/files/1002269"], "description"=>"<p>(<b>A</b>) CD13<sup>NEG</sup>SSEA4<sup>POS</sup>Tra-1-60<sup>NEG</sup> and CD13<sup>NEG</sup>SSEA4<sup>POS</sup>Tra-1-60<sup>POS</sup> populations from the manually derived 1018 clone were sorted onto MEF feeder layers and expanded for 20 days prior to reanalysis by flow cytometry to assess retention of sorted surface markers. dpi = days post infection. dps = days post sort. (<b>B</b>) CD13<sup>NEG</sup>SSEA4<sup>POS</sup> and CD13<sup>NEG</sup>SSEA4<sup>POS</sup>Tra-1-60<sup>POS</sup> populations were sorted onto MEF layers at seven days post infection and imaged at 3 and 17 dps to assess colony formation. (<b>C</b>) Colony counts arising from the sorted cell populations shown in Panel B at 17 dps (25 dpi). (<b>D</b>) Gating structure used in the analysis of CD13<sup>POS</sup> cells present within the SSEA4<sup>POS</sup>Tra-1-60<sup>POS</sup> population at 7 dpi. (<b>E</b>) Fluorescence microscopy demonstrating NANOG expression in CD13<sup>POS</sup> cell at 7 dpi. 40× magnification. CD13 shown in red. Nanog in shown Green. Values designated %T indicates proportion of total cells within the culture positive for the indicated combinations of surface markers. Values without T designation indicate the proportion of CD13<sup>NEG</sup>SSEA4<sup>POS</sup> cells that are Tra-1-60<sup>POS</sup> or Tra-1-60<sup>NEG</sup> in Panel A and D.</p>", "links"=>[], "tags"=>["derivation", "virally", "reprogrammed", "fibroblasts", "fluorescence", "activated"], "article_id"=>663479, "categories"=>["Microbiology", "Cell Biology", "Developmental Biology", "Virology"], "users"=>["David J. Kahler", "Faizzan S. Ahmad", "Anita Ritz", "Haiqing Hua", "Dorota N. Moroziewicz", "Andrew A. Sproul", "Carmen R. Dusenberry", "Linshan Shang", "Daniel Paull", "Matthew Zimmer", "Keren A. Weiss", "Dieter Egli", "Scott A. Noggle"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0059867.g001", "stats"=>{"downloads"=>2, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Enhanced_derivation_and_maintenance_of_virally_reprogrammed_fibroblasts_using_Fluorescence_Activated_Cell_Sorting_/663479", "title"=>"Enhanced derivation and maintenance of virally reprogrammed fibroblasts using Fluorescence Activated Cell Sorting.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-30 07:20:25"}
  • {"files"=>["https://ndownloader.figshare.com/files/1002270"], "description"=>"<p>(<b>A</b>) Foreskin (0825) and adult dermal fibroblast (1018 and 1023) lines underwent four factor retroviral reprogramming and were analyzed by flow cytometry for the emergence of the CD13<sup>NEG</sup>SSEA4<sup>POS</sup>Tra-1-60<sup>POS</sup> population at seven day intervals post infection. Values designated %T indicates proportion of total cells within the culture positive for the indicated combinations of surface markers. Values without T designation indicate the proportion of cells positive within the parent gate. (<b>B</b>) Gating structure used to sort the CD13<sup>NEG</sup>SSEA4<sup>POS</sup>Tra-1-60<sup>POS</sup> populations for all cell lines derived in this study. Live cell are first defined using forward (FSC) and Side (SSC) light scattering properties. The CD13<sup>NEG</sup>SSEA4<sup>POS</sup> population is then selected from the live cell gate (blue cells). The highest Tra-1-60<sup>POS</sup> expressing cells are then selected from the CD13<sup>NEG</sup>SSEA4<sup>POS</sup> population (Green cells) and sorted for expansion and characterization. (<b>C</b>) Comparison of SSEA4<sup>POS</sup>Tra-1-60<sup>POS</sup> populations present in Retro (R) or Sendai (S) viral infected fibroblast cultures during first two weeks of programming. (<b>D</b>) Comparison of CD13<sup>POS</sup> cells present within the SSEA4<sup>POS</sup>Tra-1-60<sup>POS</sup> populations during first two weeks of programming following Retro (R) or Sendai (S) infection. Dpi 1–7:(R) n = 29, (S) n = 21. Dpi 8–14: (R) n = 32, (S) n = 46. Total n = 228. Statistical significance was assessed via Student’s t-Test. * p< = 0.05, ** p< = 0.001, *** p< = 0.001, ****p< = 0.0001.</p>", "links"=>[], "tags"=>["undergoing", "viral", "Reprogramming", "markers", "points"], "article_id"=>663480, "categories"=>["Microbiology", "Cell Biology", "Developmental Biology", "Virology"], "users"=>["David J. Kahler", "Faizzan S. Ahmad", "Anita Ritz", "Haiqing Hua", "Dorota N. Moroziewicz", "Andrew A. Sproul", "Carmen R. Dusenberry", "Linshan Shang", "Daniel Paull", "Matthew Zimmer", "Keren A. Weiss", "Dieter Egli", "Scott A. Noggle"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0059867.g002", "stats"=>{"downloads"=>1, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Fibroblasts_undergoing_viral_reprogramming_exhibit_characteristic_expression_levels_of_surface_markers_at_early_time_points_post_infection_/663480", "title"=>"Fibroblasts undergoing viral reprogramming exhibit characteristic expression levels of surface markers at early time points post infection.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-30 07:20:58"}

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Relative Metric

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