PDX1+ cell budding morphogenesis in a stem cell-derived islet spheroid system.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
13 Jul 2024
13 Jul 2024
Historique:
received:
06
01
2024
accepted:
01
07
2024
medline:
14
7
2024
pubmed:
14
7
2024
entrez:
13
7
2024
Statut:
epublish
Résumé
Remarkable advances in protocol development have been achieved to manufacture insulin-secreting islets from human pluripotent stem cells (hPSCs). Distinct from current approaches, we devised a tunable strategy to generate islet spheroids enriched for major islet cell types by incorporating PDX1+ cell budding morphogenesis into staged differentiation. In this process that appears to mimic normal islet morphogenesis, the differentiating islet spheroids organize with endocrine cells that are intermingled or arranged in a core-mantle architecture, accompanied with functional heterogeneity. Through in vitro modelling of human pancreas development, we illustrate the importance of PDX1 and the requirement for EphB3/4 signaling in eliciting cell budding morphogenesis. Using this new approach, we model Mitchell-Riley syndrome with RFX6 knockout hPSCs illustrating unexpected morphogenesis defects in the differentiation towards islet cells. The tunable differentiation system and stem cell-derived islet models described in this work may facilitate addressing fundamental questions in islet biology and probing human pancreas diseases.
Identifiants
pubmed: 39003281
doi: 10.1038/s41467-024-50109-2
pii: 10.1038/s41467-024-50109-2
doi:
Substances chimiques
pancreatic and duodenal homeobox 1 protein
0
Homeodomain Proteins
0
Trans-Activators
0
Receptors, Eph Family
EC 2.7.10.1
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
5894Informations de copyright
© 2024. The Author(s).
Références
Powers, A. C. Type 1 diabetes mellitus: much progress, many opportunities. J. Clin. Investig. 131, e142242 (2021).
Atkinson, M. A., Eisenbarth, G. S. & Michels, A. W. Type 1 diabetes. Lancet 383, 69–82 (2014).
pubmed: 23890997
doi: 10.1016/S0140-6736(13)60591-7
Katsarou, A. et al. Type 1 diabetes mellitus. Nat. Rev. Dis. Prim. 3, 17016 (2017).
pubmed: 28358037
doi: 10.1038/nrdp.2017.16
Shapiro, A. M. et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N. Engl. J. Med. 343, 230–238 (2000).
pubmed: 10911004
doi: 10.1056/NEJM200007273430401
Thompson, D. M. et al. Reduced progression of diabetic microvascular complications with islet cell transplantation compared with intensive medical therapy. Transplantation 91, 373–378 (2011).
pubmed: 21258272
doi: 10.1097/TP.0b013e31820437f3
Marfil-Garza, B. A. et al. Pancreatic islet transplantation in type 1 diabetes: 20-year experience from a single-centre cohort in Canada. Lancet Diabetes Endocrinol. 10, 519–532 (2022).
pubmed: 35588757
doi: 10.1016/S2213-8587(22)00114-0
Latres, E., Finan, D. A., Greenstein, J. L., Kowalski, A. & Kieffer, T. J. Navigating two roads to glucose normalization in diabetes: automated insulin delivery devices and cell therapy. Cell Metab. 29, 545–563 (2019).
pubmed: 30840911
doi: 10.1016/j.cmet.2019.02.007
Rezania, A. et al. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat. Biotechnol. 32, 1121–1133 (2014).
pubmed: 25211370
doi: 10.1038/nbt.3033
Pagliuca, F. W. et al. Generation of functional human pancreatic beta cells in vitro. Cell 159, 428–439 (2014).
pubmed: 25303535
pmcid: 4617632
doi: 10.1016/j.cell.2014.09.040
Russ, H. A. et al. Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro. EMBO J. 34, 1759–1772 (2015).
pubmed: 25908839
pmcid: 4516429
doi: 10.15252/embj.201591058
Nair, G. G. et al. Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived beta cells. Nat. Cell Biol. 21, 263–274 (2019).
pubmed: 30710150
pmcid: 6746427
doi: 10.1038/s41556-018-0271-4
Mahaddalkar, P. U. et al. Generation of pancreatic beta cells from CD177(+) anterior definitive endoderm. Nat. Biotechnol. 38, 1061–1072 (2020).
pubmed: 32341565
doi: 10.1038/s41587-020-0492-5
Hogrebe, N. J., Augsornworawat, P., Maxwell, K. G., Velazco-Cruz, L. & Millman, J. R. Targeting the cytoskeleton to direct pancreatic differentiation of human pluripotent stem cells. Nat. Biotechnol. 38, 460–470 (2020).
pubmed: 32094658
pmcid: 7274216
doi: 10.1038/s41587-020-0430-6
Balboa, D. et al. Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells. Nat. Biotechnol. 40, 1042–1055 (2022).
pubmed: 35241836
pmcid: 9287162
doi: 10.1038/s41587-022-01219-z
Yoshihara, E. et al. Immune-evasive human islet-like organoids ameliorate diabetes. Nature 586, 606–611 (2020).
pubmed: 32814902
pmcid: 7872080
doi: 10.1038/s41586-020-2631-z
Tran, R., Moraes, C. & Hoesli, C. A. Developmentally-inspired biomimetic culture models to produce functional islet-like cells from pluripotent precursors. Front Bioeng. Biotechnol. 8, 583970 (2020).
pubmed: 33117786
pmcid: 7576674
doi: 10.3389/fbioe.2020.583970
Pan, F. C. & Wright, C. Pancreas organogenesis: from bud to plexus to gland. Dev. Dyn. 240, 530–565 (2011).
pubmed: 21337462
doi: 10.1002/dvdy.22584
Flasse, L., Schewin, C. & Grapin-Botton, A. Pancreas morphogenesis: branching in and then out. Curr. Top. Dev. Biol. 143, 75–110 (2021).
pubmed: 33820626
doi: 10.1016/bs.ctdb.2020.10.006
Larsen, H. L. & Grapin-Botton, A. The molecular and morphogenetic basis of pancreas organogenesis. Semin Cell Dev. Biol. 66, 51–68 (2017).
pubmed: 28089869
doi: 10.1016/j.semcdb.2017.01.005
Jennings, R. E. et al. Development of the human pancreas from foregut to endocrine commitment. Diabetes 62, 3514–3522 (2013).
pubmed: 23630303
pmcid: 3781486
doi: 10.2337/db12-1479
Jennings, R. E., Berry, A. A., Strutt, J. P., Gerrard, D. T. & Hanley, N. A. Human pancreas development. Development 142, 3126–3137 (2015).
pubmed: 26395141
doi: 10.1242/dev.120063
Nair, G. & Hebrok, M. Islet formation in mice and men: lessons for the generation of functional insulin-producing beta-cells from human pluripotent stem cells. Curr. Opin. Genet. Dev. 32, 171–180 (2015).
pubmed: 25909383
pmcid: 4523641
doi: 10.1016/j.gde.2015.03.004
Liang, S. et al. Differentiation of stem cell-derived pancreatic progenitors into insulin-secreting islet clusters in a multiwell-based static 3D culture system. Cell Rep. Methods 3, 100466 (2023).
pubmed: 37323565
pmcid: 10261893
doi: 10.1016/j.crmeth.2023.100466
Braam, M. J. S. et al. Protocol development to further differentiate and transition stem cell-derived pancreatic progenitors from a monolayer into endocrine cells in suspension culture. Sci. Rep. 13, 8877 (2023).
pubmed: 37264038
pmcid: 10235054
doi: 10.1038/s41598-023-35716-1
Docherty, F. M. et al. ENTPD3 marks mature stem cell-derived beta-cells formed by self-aggregation in vitro. Diabetes 70, 2554–2567 (2021).
pubmed: 34380694
pmcid: 8564403
doi: 10.2337/db20-0873
Micallef, S. J. et al. INS(GFP/w) human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells. Diabetologia 55, 694–706 (2012).
pubmed: 22120512
doi: 10.1007/s00125-011-2379-y
Ameri, J. et al. Efficient generation of glucose-responsive beta cells from isolated GP2(+) human pancreatic progenitors. Cell Rep. 19, 36–49 (2017).
pubmed: 28380361
doi: 10.1016/j.celrep.2017.03.032
Ren, H. et al. Pancreatic alpha and beta cells are globally phase-locked. Nat. Commun. 13, 3721 (2022).
pubmed: 35764654
pmcid: 9240067
doi: 10.1038/s41467-022-31373-6
Goedhart, J. PlotTwist: a web app for plotting and annotating continuous data. PLoS Biol. 18, e3000581 (2020).
pubmed: 31929523
pmcid: 6980690
doi: 10.1371/journal.pbio.3000581
Offield, M. F. et al. PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development 122, 983–995 (1996).
pubmed: 8631275
doi: 10.1242/dev.122.3.983
Stoffers, D. A., Zinkin, N. T., Stanojevic, V., Clarke, W. L. & Habener, J. F. Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence. Nat. Genet 15, 106–110 (1997).
pubmed: 8988180
doi: 10.1038/ng0197-106
Zhu, Z. et al. Genome editing of lineage determinants in human pluripotent stem cells reveals mechanisms of pancreatic development and diabetes. Cell Stem. Cell 18, 755–768 (2016).
pubmed: 27133796
pmcid: 4892994
doi: 10.1016/j.stem.2016.03.015
Hashimoto, H. et al. Expression of pancreatic and duodenal homeobox1 (PDX1) protein in the interior and exterior regions of the intestine, revealed by development and analysis of Pdx1 knockout mice. Lab Anim. Res 31, 93–98, (2015).
pubmed: 26155204
pmcid: 4490151
doi: 10.5625/lar.2015.31.2.93
Mitchell, J. et al. Neonatal diabetes, with hypoplastic pancreas, intestinal atresia and gall bladder hypoplasia: search for the aetiology of a new autosomal recessive syndrome. Diabetologia 47, 2160–2167 (2004).
pubmed: 15592663
doi: 10.1007/s00125-004-1576-3
Smith, S. B. et al. Rfx6 directs islet formation and insulin production in mice and humans. Nature 463, 775–780 (2010).
pubmed: 20148032
pmcid: 2896718
doi: 10.1038/nature08748
Piccand, J. et al. Rfx6 maintains the functional identity of adult pancreatic beta cells. Cell Rep. 9, 2219–2232 (2014).
pubmed: 25497096
pmcid: 4542305
doi: 10.1016/j.celrep.2014.11.033
Coykendall, V. M. N. et al. RFX6 maintains gene expression and function of adult human islet alpha-cells. Diabetes 73, 448–460 (2024).
pubmed: 38064570
doi: 10.2337/db23-0483
Adams, M. T., Gilbert, J. M., Hinojosa Paiz, J., Bowman, F. M. & Blum, B. Endocrine cell type sorting and mature architecture in the islets of Langerhans require expression of Roundabout receptors in beta cells. Sci. Rep. 8, 10876 (2018).
pubmed: 30022126
pmcid: 6052079
doi: 10.1038/s41598-018-29118-x
Escot, S., Willnow, D., Naumann, H., Di Francescantonio, S. & Spagnoli, F. M. Robo signalling controls pancreatic progenitor identity by regulating Tead transcription factors. Nat. Commun. 9, 5082 (2018).
pubmed: 30504829
pmcid: 6269453
doi: 10.1038/s41467-018-07474-6
Cozzitorto, C. et al. A specialized niche in the pancreatic microenvironment promotes endocrine differentiation. Dev. Cell 55, 150–162 e156 (2020).
pubmed: 32857951
pmcid: 7720791
doi: 10.1016/j.devcel.2020.08.003
Miralles, F., Battelino, T., Czernichow, P. & Scharfmann, R. TGF-beta plays a key role in morphogenesis of the pancreatic islets of Langerhans by controlling the activity of the matrix metalloproteinase MMP-2. J. Cell Biol. 143, 827–836 (1998).
pubmed: 9813100
pmcid: 2148155
doi: 10.1083/jcb.143.3.827
Tran, R., Moraes, C. & Hoesli, C. A. Controlled clustering enhances PDX1 and NKX6.1 expression in pancreatic endoderm cells derived from pluripotent stem cells. Sci. Rep. 10, 1190 (2020).
pubmed: 31988329
pmcid: 6985188
doi: 10.1038/s41598-020-57787-0
Lin, S., Gordon, K., Kaplan, N. & Getsios, S. Ligand targeting of EphA2 enhances keratinocyte adhesion and differentiation via desmoglein 1. Mol. Biol. Cell 21, 3902–3914 (2010).
pubmed: 20861311
pmcid: 2982116
doi: 10.1091/mbc.e10-03-0242
Poliakov, A., Cotrina, M. & Wilkinson, D. G. Diverse roles of Eph receptors and ephrins in the regulation of cell migration and tissue assembly. Dev. Cell 7, 465–480 (2004).
pubmed: 15469835
doi: 10.1016/j.devcel.2004.09.006
Yang, Y. H., Wills, Q. F. & Johnson, J. D. A live-cell, high-content imaging survey of 206 endogenous factors across five stress conditions reveals context-dependent survival effects in mouse primary beta cells. Diabetologia 58, 1239–1249 (2015).
pubmed: 25773404
pmcid: 4415993
doi: 10.1007/s00125-015-3552-5
Sharon, N. et al. Wnt signaling separates the progenitor and endocrine compartments during pancreas development. Cell Rep. 27, 2281–2291 e2285 (2019).
pubmed: 31116975
pmcid: 6933053
doi: 10.1016/j.celrep.2019.04.083
Adams, R. H. et al. Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains, vascular morphogenesis, and sprouting angiogenesis. Genes Dev. 13, 295–306 (1999).
pubmed: 9990854
pmcid: 316426
doi: 10.1101/gad.13.3.295
Zhang, J., Jiang, Z., Liu, X. & Meng, A. Eph/ephrin signaling maintains the boundary of dorsal forerunner cell cluster during morphogenesis of the zebrafish embryonic left-right organizer. Development 143, 2603–2615 (2016).
pubmed: 27287807
pmcid: 4958335
Kindberg, A. A. et al. EPH/EPHRIN regulates cellular organization by actomyosin contractility effects on cell contacts. J. Cell. Biol. 220, https://doi.org/10.1083/jcb.202005216 (2021).
Canty, L., Zarour, E., Kashkooli, L., Francois, P. & Fagotto, F. Sorting at embryonic boundaries requires high heterotypic interfacial tension. Nat. Commun. 8, 157 (2017).
pubmed: 28761157
pmcid: 5537356
doi: 10.1038/s41467-017-00146-x
Cayuso, J. et al. EphrinB1/EphB3b coordinate bidirectional epithelial-mesenchymal interactions controlling liver morphogenesis and laterality. Dev. Cell 39, 316–328 (2016).
pubmed: 27825440
pmcid: 5107609
doi: 10.1016/j.devcel.2016.10.009
Villasenor, A., Chong, D. C., Henkemeyer, M. & Cleaver, O. Epithelial dynamics of pancreatic branching morphogenesis. Development 137, 4295–4305 (2010).
pubmed: 21098570
pmcid: 2990215
doi: 10.1242/dev.052993
Villasenor, A. et al. EphB3 marks delaminating endocrine progenitor cells in the developing pancreas. Dev. Dyn. 241, 1008–1019 (2012).
pubmed: 22434763
pmcid: 3328632
doi: 10.1002/dvdy.23781
Greggio, C. et al. Artificial three-dimensional niches deconstruct pancreas development in vitro. Development 140, 4452–4462 (2013).
pubmed: 24130330
pmcid: 4007719
doi: 10.1242/dev.096628
Goncalves, C. A. et al. A 3D system to model human pancreas development and its reference single-cell transcriptome atlas identify signaling pathways required for progenitor expansion. Nat. Commun. 12, 3144 (2021).
pubmed: 34035279
pmcid: 8149728
doi: 10.1038/s41467-021-23295-6
Beydag-Tasoz, B. S., Yennek, S. & Grapin-Botton, A. Towards a better understanding of diabetes mellitus using organoid models. Nat. Rev. Endocrinol. 19, 232–248 (2023).
pubmed: 36670309
pmcid: 9857923
Jiang, Y. et al. Generation of pancreatic progenitors from human pluripotent stem cells by small molecules. Stem Cell Rep. 16, 2395–2409 (2021).
doi: 10.1016/j.stemcr.2021.07.021
Blauwkamp, T. A., Nigam, S., Ardehali, R., Weissman, I. L. & Nusse, R. Endogenous Wnt signalling in human embryonic stem cells generates an equilibrium of distinct lineage-specified progenitors. Nat. Commun. 3, 1070 (2012).
pubmed: 22990866
doi: 10.1038/ncomms2064
Huang, H., Vogel, S. S., Liu, N., Melton, D. A. & Lin, S. Analysis of pancreatic development in living transgenic zebrafish embryos. Mol. Cell Endocrinol. 177, 117–124 (2001).
pubmed: 11377827
doi: 10.1016/S0303-7207(01)00408-7
Otsuka, T., Tsukahara, T. & Takeda, H. Development of the pancreas in medaka, Oryzias latipes, from embryo to adult. Dev. Growth Differ. 57, 557–569 (2015).
pubmed: 26435359
doi: 10.1111/dgd.12237
Grapin-Botton, A., Majithia, A. R. & Melton, D. A. Key events of pancreas formation are triggered in gut endoderm by ectopic expression of pancreatic regulatory genes. Genes Dev. 15, 444–454 (2001).
pubmed: 11230152
pmcid: 312631
doi: 10.1101/gad.846001
Mamidi, A. et al. Mechanosignalling via integrins directs fate decisions of pancreatic progenitors. Nature 564, 114–118 (2018).
pubmed: 30487608
doi: 10.1038/s41586-018-0762-2
Bonner-Weir, S., Sullivan, B. A. & Weir, G. C. Human islet morphology revisited: human and rodent islets are not so different after all. J. Histochem Cytochem 63, 604–612 (2015).
pubmed: 25604813
pmcid: 4530393
doi: 10.1369/0022155415570969
Steiner, D. J., Kim, A., Miller, K. & Hara, M. Pancreatic islet plasticity: interspecies comparison of islet architecture and composition. Islets 2, 135–145 (2010).
pubmed: 20657742
doi: 10.4161/isl.2.3.11815
Levetan, C. S. & Pierce, S. M. Distinctions between the islets of mice and men: implications for new therapies for type 1 and 2 diabetes. Endocr. Pr. 19, 301–312 (2013).
doi: 10.4158/EP12138.RA
Brissova, M. et al. Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy. J. Histochem Cytochem 53, 1087–1097 (2005).
pubmed: 15923354
doi: 10.1369/jhc.5C6684.2005
Jeon, J., Correa-Medina, M., Ricordi, C., Edlund, H. & Diez, J. A. Endocrine cell clustering during human pancreas development. J. Histochem Cytochem 57, 811–824 (2009).
pubmed: 19365093
pmcid: 2728126
doi: 10.1369/jhc.2009.953307
Riedel, M. J. et al. Immunohistochemical characterisation of cells co-producing insulin and glucagon in the developing human pancreas. Diabetologia 55, 372–381 (2012).
pubmed: 22038519
doi: 10.1007/s00125-011-2344-9
Tixi, W. et al. Coordination between ECM and cell-cell adhesion regulates the development of islet aggregation, architecture, and functional maturation. Elife 12, https://doi.org/10.7554/eLife.90006 (2023).
Krishnamurthy, M. et al. Using human induced pluripotent stem cell-derived organoids to identify new pathologies in patients with PDX1 mutations. Gastroenterology 163, 1053–1063 e1057 (2022).
pubmed: 35803312
doi: 10.1053/j.gastro.2022.06.083
Ibrahim, H. B. et al. RFX6 haploinsufficiency predisposes to diabetes through impaired beta cell functionality. Diabetologia. https://doi.org/10.1007/s00125-024-06163-y (2024). Epub ahead of print.
Rukstalis, J. M. & Habener, J. F. Snail2, a mediator of epithelial-mesenchymal transitions, expressed in progenitor cells of the developing endocrine pancreas. Gene Expr. Patterns 7, 471–479 (2007).
pubmed: 17185046
doi: 10.1016/j.modgep.2006.11.001
Sharon, N. et al. A peninsular structure coordinates asynchronous differentiation with morphogenesis to generate pancreatic islets. Cell 176, 790–804 e713 (2019).
pubmed: 30661759
pmcid: 6705176
doi: 10.1016/j.cell.2018.12.003
Miller, K. et al. Islet formation during the neonatal development in mice. PLoS ONE 4, e7739 (2009).
pubmed: 19893748
pmcid: 2770846
doi: 10.1371/journal.pone.0007739
Hard, W. L. The origin and differentiation of the alpha and beta cells in the pancreatic islets of the rat. Am. J. Anat. 75, 369–403 (1944).
doi: 10.1002/aja.1000750305
Matsuda, H. Zebrafish as a model for studying functional pancreatic beta cells development and regeneration. Dev. Growth Differ. 60, 393–399 (2018).
pubmed: 30133710
doi: 10.1111/dgd.12565
Field, H. A., Dong, P. D., Beis, D. & Stainier, D. Y. Formation of the digestive system in zebrafish. II. Pancreas morphogenesis. Dev. Biol. 261, 197–208 (2003).
pubmed: 12941629
doi: 10.1016/S0012-1606(03)00308-7
Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).
pubmed: 9804556
doi: 10.1126/science.282.5391.1145
Mandegar, M. A. et al. CRISPR interference efficiently induces specific and reversible gene silencing in human iPSCs. Cell Stem Cell 18, 541–553 (2016).
pubmed: 26971820
pmcid: 4830697
doi: 10.1016/j.stem.2016.01.022
Zhao, J. et al. Differentiation of human pluripotent stem cells into insulin-producing islet clusters. J. Vis. Exp. https://doi.org/10.3791/64840 (2023).
Babicki, S. et al. Heatmapper: web-enabled heat mapping for all. Nucleic Acids Res 44, W147–153, (2016).
pubmed: 27190236
pmcid: 4987948
doi: 10.1093/nar/gkw419
Ewald, J. D. et al. Web-based multi-omics integration using the Analyst software suite. Nat. Protoc. https://doi.org/10.1038/s41596-023-00950-4 (2024).
Liang, S. et al. Carbon monoxide enhances calcium transients and glucose-stimulated insulin secretion from pancreatic beta-cells by activating Phospholipase C signal pathway in diabetic mice. Biochem. Biophys. Res. Commun. 582, 1–7 (2021).
pubmed: 34678590
doi: 10.1016/j.bbrc.2021.10.030
Zhao, J. et al. In vivo imaging of beta-cell function reveals glucose-mediated heterogeneity of beta-cell functional development. Elife 8, https://doi.org/10.7554/eLife.41540 (2019).
Zhao, J. et al. In vivo imaging of calcium activities from pancreatic beta-cells in zebrafish embryos using spinning-disc confocal and two-photon light-sheet microscopy. Bio Protoc. 11, e4245 (2021).
pubmed: 35005090
pmcid: 8678548
doi: 10.21769/BioProtoc.4245
Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).
pubmed: 22930834
pmcid: 5554542
doi: 10.1038/nmeth.2089
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
pubmed: 25516281
pmcid: 4302049
doi: 10.1186/s13059-014-0550-8
Gu, Z., Eils, R. & Schlesner, M. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics 32, 2847–2849 (2016).
pubmed: 27207943
doi: 10.1093/bioinformatics/btw313
Yu, G., Wang, L. G., Han, Y. & He, Q. Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16, 284–287 (2012).
pubmed: 22455463
pmcid: 3339379
doi: 10.1089/omi.2011.0118