Microbioreactor Array Screening of Wnt Modulators and Microenvironmental Factors in Osteogenic Differentiation of Mesenchymal Progenitor Cells
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{"title"=>"Microbioreactor array screening of wnt modulators and microenvironmental factors in osteogenic differentiation of mesenchymal progenitor cells", "type"=>"journal", "authors"=>[{"first_name"=>"Jessica E.", "last_name"=>"Frith", "scopus_author_id"=>"36336778100"}, {"first_name"=>"Drew M.", "last_name"=>"Titmarsh", "scopus_author_id"=>"35485875900"}, {"first_name"=>"Harish", "last_name"=>"Padmanabhan", "scopus_author_id"=>"37081854800"}, {"first_name"=>"Justin J.", "last_name"=>"Cooper-White", "scopus_author_id"=>"56051263100"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"24376608", "sgr"=>"84893498597", "doi"=>"10.1371/journal.pone.0082931", "scopus"=>"2-s2.0-84893498597", "pui"=>"372294842", "issn"=>"19326203"}, "id"=>"1dc79594-5c92-39ec-a7a9-2b3c7624892a", "abstract"=>"Cellular microenvironmental conditions coordinate to regulate stem cell populations and their differentiation. Mesenchymal precursor cells (MPCs), which have significant potential for a wide range of therapeutic applications, can be expanded or differentiated into osteo- chondro- and adipogenic lineages. The ability to establish, screen, and control aspects of the microenvironment is paramount if we are to elucidate the complex interplay of signaling events that direct cell fate. Whilst modulation of Wnt signaling may be useful to direct osteogenesis in MPCs, there is still significant controversy over how the Wnt signaling pathway influences osteogenesis. In this study, we utilised a full-factorial microbioreactor array (MBA) to rapidly, combinatorially screen several Wnt modulatory compounds (CHIR99021, IWP-4 and IWR-1) and characterise their effects upon osteogenesis. The MBA screening system showed excellent consistency between donors and experimental runs. CHIR99021 (a Wnt agonist) had a profoundly inhibitory effect upon osteogenesis, contrary to expectations, whilst the effects of the IWP-4 and IWR-1 (Wnt antagonists) were confirmed to be inhibitory to osteogenesis, but to a lesser extent than observed for CHIR99021. Importantly, we demonstrated that these results were translatable to standard culture conditions. Using RT-qPCR of osteogenic and Wnt pathway markers, we showed that CHIR exerted its effects via inhibition of ALP and SPP1 expression, even though other osteogenic markers (RUNX2, MSX2, DLX, COL1A1) were upregulated. Lastly, this MBA platform, due to the continuous provision of medium from the first to the last of ten serially connected culture chambers, permitted new insight into the impacts of paracrine signaling on osteogenic differentiation in MPCs, with factors secreted by the MPCs in upstream chambers enhancing the differentiation of cells in downstream chambers. Insights provided by this cell-based assay system will be key to better understanding signaling mechanisms, as well as optimizing MPC growth and differentiation conditions for therapeutic applications.", "link"=>"http://www.mendeley.com/research/microbioreactor-array-screening-wnt-modulators-microenvironmental-factors-osteogenic-differentiation", "reader_count"=>19, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>3, "Researcher"=>4, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>2, "Student > Master"=>3, "Student > Bachelor"=>3, "Lecturer > Senior Lecturer"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>3, "Researcher"=>4, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>2, "Student > Master"=>3, "Student > Bachelor"=>3, "Lecturer > Senior Lecturer"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Engineering"=>2, "Biochemistry, Genetics and Molecular Biology"=>3, "Materials Science"=>3, "Medicine and Dentistry"=>2, "Agricultural and Biological Sciences"=>7, "Psychology"=>1, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>2}, "Materials Science"=>{"Materials Science"=>3}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Psychology"=>{"Psychology"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>7}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>3}}, "group_count"=>1}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1326099"], "description"=>"<p>Significance of factor effects from factorial analysis.</p>", "links"=>[], "tags"=>["factorial"], "article_id"=>884717, "categories"=>["Biological Sciences"], "users"=>["Jessica E. Frith", "Drew M. Titmarsh", "Harish Padmanabhan", "Justin J. Cooper-White"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0082931.t003", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Significance_of_factor_effects_from_factorial_analysis_/884717", "title"=>"Significance of factor effects from factorial analysis.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-12-23 02:48:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1326098"], "description"=>"<p>qPCR Primer Sequences.</p>", "links"=>[], "tags"=>["primer"], "article_id"=>884716, "categories"=>["Biological Sciences"], "users"=>["Jessica E. Frith", "Drew M. Titmarsh", "Harish Padmanabhan", "Justin J. Cooper-White"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0082931.t001", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_qPCR_Primer_Sequences_/884716", "title"=>"qPCR Primer Sequences.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-12-23 02:48:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1326094"], "description"=>"<p>A ELF97 (green) and PI (red) staining of MPCs treated with CHIR, IWP-4 and IWR-1 for 7 days. Scale bar, 100 µm. B Alizarin red staining of MPCs treated with combinations of CHIR, IWP-4 and IWR-1 for 21 days. Scale bar, 100 µm. C) RT-qPCR determination of expression of osteogenic marker genes after 7 days D) qPCR determination of expression of osteogenic markers genes after 21 days. RT-qPCR data is shown as mean±SEM. N = 3, p<0.05 (*), p<0.01 (**), p<0.001 (***).</p>", "links"=>[], "tags"=>["inhibitor", "concentrations", "osteogenesis"], "article_id"=>884712, "categories"=>["Biological Sciences"], "users"=>["Jessica E. Frith", "Drew M. Titmarsh", "Harish Padmanabhan", "Justin J. Cooper-White"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0082931.g003", "stats"=>{"downloads"=>0, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Analysis_of_selected_inhibitor_concentrations_on_osteogenesis_under_standard_conditions_/884712", "title"=>"Analysis of selected inhibitor concentrations on osteogenesis under standard conditions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-23 02:48:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1326107", "https://ndownloader.figshare.com/files/1326108", "https://ndownloader.figshare.com/files/1326109", "https://ndownloader.figshare.com/files/1326110", "https://ndownloader.figshare.com/files/1326111", "https://ndownloader.figshare.com/files/1326112", "https://ndownloader.figshare.com/files/1326113", "https://ndownloader.figshare.com/files/1326114", "https://ndownloader.figshare.com/files/1326115", "https://ndownloader.figshare.com/files/1326116", "https://ndownloader.figshare.com/files/1326117"], "description"=>"<div><p>Cellular microenvironmental conditions coordinate to regulate stem cell populations and their differentiation. Mesenchymal precursor cells (MPCs), which have significant potential for a wide range of therapeutic applications, can be expanded or differentiated into osteo- chondro- and adipogenic lineages. The ability to establish, screen, and control aspects of the microenvironment is paramount if we are to elucidate the complex interplay of signaling events that direct cell fate. Whilst modulation of Wnt signaling may be useful to direct osteogenesis in MPCs, there is still significant controversy over how the Wnt signaling pathway influences osteogenesis. In this study, we utilised a full-factorial microbioreactor array (MBA) to rapidly, combinatorially screen several Wnt modulatory compounds (CHIR99021, IWP-4 and IWR-1) and characterise their effects upon osteogenesis. The MBA screening system showed excellent consistency between donors and experimental runs. CHIR99021 (a Wnt agonist) had a profoundly inhibitory effect upon osteogenesis, contrary to expectations, whilst the effects of the IWP-4 and IWR-1 (Wnt antagonists) were confirmed to be inhibitory to osteogenesis, but to a lesser extent than observed for CHIR99021. Importantly, we demonstrated that these results were translatable to standard culture conditions. Using RT-qPCR of osteogenic and Wnt pathway markers, we showed that CHIR exerted its effects via inhibition of <i>ALP</i> and <i>SPP1</i> expression, even though other osteogenic markers (<i>RUNX2</i>, <i>MSX2</i>, <i>DLX, COL1A1)</i> were upregulated. Lastly, this MBA platform, due to the continuous provision of medium from the first to the last of ten serially connected culture chambers, permitted new insight into the impacts of paracrine signaling on osteogenic differentiation in MPCs, with factors secreted by the MPCs in upstream chambers enhancing the differentiation of cells in downstream chambers. Insights provided by this cell-based assay system will be key to better understanding signaling mechanisms, as well as optimizing MPC growth and differentiation conditions for therapeutic applications.</p></div>", "links"=>[], "tags"=>["wnt", "modulators", "microenvironmental", "osteogenic", "differentiation", "mesenchymal", "progenitor"], "article_id"=>884725, "categories"=>["Biological Sciences"], "users"=>["Jessica E. Frith", "Drew M. Titmarsh", "Harish Padmanabhan", "Justin J. Cooper-White"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0082931.s001", "https://dx.doi.org/10.1371/journal.pone.0082931.s002", "https://dx.doi.org/10.1371/journal.pone.0082931.s003", "https://dx.doi.org/10.1371/journal.pone.0082931.s004", "https://dx.doi.org/10.1371/journal.pone.0082931.s005", "https://dx.doi.org/10.1371/journal.pone.0082931.s006", "https://dx.doi.org/10.1371/journal.pone.0082931.s007", "https://dx.doi.org/10.1371/journal.pone.0082931.s008", "https://dx.doi.org/10.1371/journal.pone.0082931.s009", "https://dx.doi.org/10.1371/journal.pone.0082931.s010", "https://dx.doi.org/10.1371/journal.pone.0082931.s011"], "stats"=>{"downloads"=>7, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Microbioreactor_Array_Screening_of_Wnt_Modulators_and_Microenvironmental_Factors_in_Osteogenic_Differentiation_of_Mesenchymal_Progenitor_Cells_/884725", "title"=>"Microbioreactor Array Screening of Wnt Modulators and Microenvironmental Factors in Osteogenic Differentiation of Mesenchymal Progenitor Cells", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-12-23 02:48:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1326100"], "description"=>"<p>Pearson’s correlation coefficients for paired chambers from individual MBA experiments.</p>", "links"=>[], "tags"=>["coefficients", "paired", "chambers", "mba"], "article_id"=>884718, "categories"=>["Biological Sciences"], "users"=>["Jessica E. Frith", "Drew M. Titmarsh", "Harish Padmanabhan", "Justin J. Cooper-White"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0082931.t002", "stats"=>{"downloads"=>0, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Pearson_8217_s_correlation_coefficients_for_paired_chambers_from_individual_MBA_experiments_/884718", "title"=>"Pearson’s correlation coefficients for paired chambers from individual MBA experiments.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-12-23 02:48:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1326097"], "description"=>"<p>A Traces of ELF97/DNA expression index against row, from column 1 of all microbioreactor runs from <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082931#pone-0082931-g002\" target=\"_blank\">Figure 2</a> (pooled arrays), and the average value. B Panel of conditions formed in conditioned medium screening experiment. C Heatmaps of total expression intensities (arbitrary units) for DNA, ELF97, and ELF97/DNA ratio. The average response of 3 technical replicates from one experimental run is shown. D Main effects plot showing effect of ROW, Growth-conditioned medium and Osteo-conditioned medium on expression index for ELF97/DNA ratio. E Interaction effects plot showing effects of 2 combined factors on ELF97/DNA ratio. F Traces of ELF97/DNA expression index against row, from columns 1, 8 and 9 (average of 3 technical replicates).</p>", "links"=>[], "tags"=>["mpc", "growth-", "differentiation-conditioned"], "article_id"=>884715, "categories"=>["Biological Sciences"], "users"=>["Jessica E. Frith", "Drew M. Titmarsh", "Harish Padmanabhan", "Justin J. Cooper-White"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0082931.g005", "stats"=>{"downloads"=>0, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Screening_MPC_growth_and_differentiation_conditioned_medium_in_MBAs_/884715", "title"=>"Screening MPC growth- and differentiation-conditioned medium in MBAs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-23 02:48:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1326096"], "description"=>"<p>qPCR determination of expression of Wnt pathway genes in MPCs after 7 and 21 days treatment. Data is shown as mean±SEM. N = 3, p<0.05 (*), p<0.01 (**), p<0.001 (***).</p>", "links"=>[], "tags"=>["wnt"], "article_id"=>884714, "categories"=>["Biological Sciences"], "users"=>["Jessica E. Frith", "Drew M. Titmarsh", "Harish Padmanabhan", "Justin J. Cooper-White"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0082931.g004", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_qPCR_determination_of_the_expression_of_Wnt_related_factors_/884714", "title"=>"qPCR determination of the expression of Wnt related factors.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-23 02:48:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1326091"], "description"=>"<p>A Panel of screening conditions in MBAs. Numbers denote concentrations of the various molecules, in µM. B Confocal microscopy images of endpoint PI (DNA) and ELF97 (alkaline phosphatase activity) staining from a representative experiment. Direction of fluid flow was from top to bottom. C Heatmaps of expression indices (see Methods) for DNA, ELF97, and ELF97/DNA ratio. The average expression index of 2 runs from each of 2 MPC donors (4 in total) is shown, and units represent global standard deviations of difference relative to the global mean. For data from individual runs, see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082931#pone.0082931.s002\" target=\"_blank\">Figs. S2</a>–<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082931#pone.0082931.s005\" target=\"_blank\">S5</a>. D Higher magnification fluorescence images of representative MPCs in MBA displaying alkaline phosphatase activity (ELF97) and DNA staining (PI). Scale bar: 200 µm. E Main effects plot showing effect of DONOR, CHIR99021 (CHIR), IWP-4, IWR-1 and POSITION on expression index for ELF97/DNA ratio. F Interaction effects plot showing effects of 2 combined factors on ELF97/DNA ratio.</p>", "links"=>[], "tags"=>["wnt", "modulators", "mpc"], "article_id"=>884709, "categories"=>["Biological Sciences"], "users"=>["Jessica E. Frith", "Drew M. Titmarsh", "Harish Padmanabhan", "Justin J. Cooper-White"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0082931.g002", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_MBA_screening_of_Wnt_modulators_in_MPC_osteogenesis_/884709", "title"=>"MBA screening of Wnt modulators in MPC osteogenesis.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-23 02:48:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1326090"], "description"=>"<p>A Comparison of cell morphology in 100 µm (top) versus 250 µm-high (bottom) devices. Scale bar, 200 µm. B Comparison of medium exchange regimes varied from conditions in top panel of A –10 µL/h flowrate (top) and periodic flow-stop (bottom). Scale bar, 200 µm. C Comparison of surface coating regimes varied from conditions in top panel of A – FBS-coated substrate (top) and collagen-I-coated substrate (bottom). Scale bar, 200 µm. D Phase contrast micrographs of MPCs in static plate controls and microbioreactor arrays in suspension directly after seeding, and attached after 4–5 h, just prior to the start of fluid flow. Scale bar, 200 µm. E Heatmap showing distribution of MPCs seeded into a MBA at representative experimental densities. F Graph showing average cells per chamber as a function of row. G Graph showing average cells per chamber as a function of column. H Live/dead staining of MPCs after 7 days. Scale bar, 100 µm.</p>", "links"=>[], "tags"=>["mba", "mpc"], "article_id"=>884708, "categories"=>["Biological Sciences"], "users"=>["Jessica E. Frith", "Drew M. Titmarsh", "Harish Padmanabhan", "Justin J. Cooper-White"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0082931.g001", "stats"=>{"downloads"=>2, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Validation_of_MBA_culture_parameters_and_MPC_seeding_/884708", "title"=>"Validation of MBA culture parameters and MPC seeding.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-23 02:48:42"}

PMC Usage Stats | Further Information

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  • {"unique-ip"=>"9", "full-text"=>"11", "pdf"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"3"}
  • {"unique-ip"=>"9", "full-text"=>"4", "pdf"=>"5", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"5", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2019", "month"=>"4"}
  • {"unique-ip"=>"7", "full-text"=>"5", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"4", "cited-by"=>"0", "year"=>"2019", "month"=>"5"}

Relative Metric

{"start_date"=>"2013-01-01T00:00:00Z", "end_date"=>"2013-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences", "average_usage"=>[269, 466, 588, 697, 800, 896, 988, 1076, 1165, 1254, 1340, 1417]}, {"subject_area"=>"/Biology and life sciences/Biochemistry", "average_usage"=>[266, 468, 593, 703, 804, 903, 993, 1084, 1171, 1256, 1339, 1422, 1492]}, {"subject_area"=>"/Medicine and health sciences", "average_usage"=>[264, 460, 584, 692, 794, 887, 978, 1067, 1154, 1241, 1328, 1408, 1474]}, {"subject_area"=>"/Medicine and health sciences/Endocrinology", "average_usage"=>[255, 447, 575, 680, 781, 872, 965, 1055, 1143, 1236, 1317, 1397, 1457]}, {"subject_area"=>"/Medicine and health sciences/Physiology", "average_usage"=>[258, 449, 572, 679, 775, 866, 956, 1041, 1124, 1211, 1291, 1371, 1437]}, {"subject_area"=>"/Physical sciences/Physics", "average_usage"=>[254, 421, 527, 626, 720, 813, 900, 983, 1063, 1136, 1210, 1283, 1342]}]}
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