(19) TZZ ¥_Z _T (11) EP 2 310 492 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 5/07 (2010.01) 22.07.2015 Bulletin 2015/30 (86) International application number: (21) Application number: 09774297.7 PCT/US2009/049183 (22) Date of filing: 30.06.2009 (87) International publication number: WO 2010/002846 (07.01.2010 Gazette 2010/01) (54) DIFFERENTIATION OF PLURIPOTENT STEM CELLS DIFFERENZIERUNG PLURIPOTENTER STAMMZELLEN DIFFÉRENCIATION DE CELLULES SOUCHES PLURIPOTENTES (84) Designated Contracting States: • MCLEAN AMANDA B ET AL: "Activin a efficiently AT BE BG CH CY CZ DE DK EE ES FI FR GB GR specifies definitive endoderm from human HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL embryonic stem cells only when PT RO SE SI SK TR phosphatidylinositol 3-kinase signaling is suppressed" STEM CELLS, ALPHAMED PRESS, (30) Priority: 30.06.2008 US 76908 P DAYTON, OH, US, vol. 25, no. 1, 1 January 2007 30.06.2008 US 76900 P (2007-01-01), pages 29-38, XP002482637 ISSN: 30.06.2008 US 76915 P 1066-5099 • ARMSTRONG LYLE ET AL: "The role of (43) Date of publication of application: PI3K/AKT, MAPK/ERK and NF kappa beta 20.04.2011 Bulletin 2011/16 signalling in the maintenance of human embryonic stem cell pluripotency and viability (60) Divisional application: highlighted by transcriptional profiling and 15171064.7 functional analysis" HUMAN MOLECULAR GENETICS, OXFORD UNIVERSITY PRESS, (73) Proprietor: Janssen Biotech, Inc. SURREY,vol. 15, no. 11,1 June 2006 (2006-06-01), Horsham, PA 19044 (US) pages 1894-1913, XP002471543 ISSN: 0964-6906 • PALING N R D ET AL: "REGULATION OF (72) Inventors: EMBRYONIC STEM CELL SELF-RENEWAL BY • LIU, Jiajian PHOSPHOINOSITIDE 3-KINASE-DEPENDENT Skillman, NJ 08558 (US) SIGNALING" JOURNAL OF BIOLOGICAL • DAVIS, Janet CHEMISTRY, AMERICAN SOCIETY OF Skillman, NJ 08558 (US) BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM, • PARMENTER, Christine US, vol. 279, no. 46, 24 August 2004 (2004-08-24), San Diego, CA 92121-1126 (US) pages 48063-48070, XP002380573 ISSN: • BONNET, Pascal, Ghislain, André 0021-9258 B-2340 Beerse (BE) • HORI YUICHI ET AL: "Growth inhibitors promote differentiation of insulin-producing tissue from (74) Representative: Goodfellow, Hugh Robin embryonic stem cells" PROCEEDINGS OF THE Carpmaels & Ransford LLP NATIONAL ACADEMY OF SCIENCES OF USA, One Southampton Row NATIONAL ACADEMY OF SCIENCE, London WC1B 5HA (GB) WASHINGTON, DC, US, vol. 99, no. 25, 10 December2002 (2002-12-10), pages 16105-16110, (56) References cited: XP002392968 ISSN: 0027-8424 cited in the WO-A-2008/048647 application Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 310 492 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 310 492 B1 • MCKIERNAN EADAOIN ET AL: "Directed • KUBO A ET AL: "Development of definitive differentiation of mouse embryonic stem cells endoderm from embryonic stem cells in culture" into pancreatic-like or neuronal- and glial-like DEVELOPMENT, COMPANY OF BIOLOGISTS, phenotypes." TISSUE ENGINEERING OCT 2007, CAMBRIDGE, GB, vol. 131, no. 7, 1 April 2004 vol. 13, no. 10, October 2007 (2007-10), pages (2004-04-01), pages 1651-1662, XP002985523 2419-2430, XP002547893 ISSN: 1076-3279 ISSN: 0950-1991 • SHIRAKI NOBUAKI ET AL: "Guided differentiation of embryonic stem cells into Pdx1- expressing regional-specific definitive endoderm." STEM CELLS (DAYTON, OHIO) APR 2008, vol. 26, no. 4, April 2008 (2008-04), pages 874-885, XP002547894 ISSN: 1549-4918 2 EP 2 310 492 B1 Description FIELD OF THE INVENTION 5 [0001] The present invention is directed to methods to differentiate pluripotent stem cells. In particular, the present invention is directed to methods and compositions to differentiate pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage comprising culturing the pluripotent stem cells in medium comprising a sufficient amount of GDF-8 to cause the differentiation of the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage. 10 BACKGROUND [0002] Advances in cell-replacement therapy for Type I diabetes mellitus and a shortage of transplantable islets of Langerhanshave focusedinterest on developing sourcesof insulin-producingcells, or βcells, appropriate for engraftment. 15 Oneapproach is thegeneration of functional β cellsfrom pluripotent stem cells, such as, for example, embryonic stem cells. [0003] In vertebrate embryonic development, a pluripotent cell gives rise to a group of cells comprising three germ layers (ectoderm, mesoderm, and endoderm) in a process known as gastrulation. Tissues such as, for example, thyroid, thymus, pancreas, gut, and liver, will develop from the endoderm, via an intermediate stage. The intermediate stage in this process is the formation of definitive endoderm. Definitive endoderm cells express a number of markers, such as, 20 for example, HNF-3beta, GATA4, MIXL1, CXCR4 and SOX17. [0004] Formation of the pancreas arises from the differentiation of definitive endoderm into pancreatic endoderm. Cells of the pancreatic endoderm express the pancreatic-duodenal homeobox gene, PDX1. In the absence of PDX1, the pancreas fails to develop beyond the formation of ventral and dorsal buds. Thus, PDX1 expression marks a critical step in pancreatic organogenesis. The mature pancreas contains, among other cell types, exocrine tissue and endocrine 25 tissue. Exocrine and endocrine tissues arise from the differentiation of pancreatic endoderm. [0005] Cells bearing the features of islet cells have reportedly been derived from embryonic cells of the mouse. For example, Lumelsky et al. (Science 292:1389, 2001) report differentiation of mouse embryonic stem cells to insulin- secreting structures similar to pancreatic islets. Soria et al. (Diabetes 49:157, 2000) report that insulin-secreting cells derived from mouse embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. 30 [0006] In one example, Hori et al. (PNAS 99: 16105, 2002) discloses that treatment of mouse embryonic stem cells with inhibitors of phosphoinositide 3-kinase (LY294002) produced cells that resembled β cells. [0007] In another example, Blyszczuk et al. (PNAS 100:998, 2003) reports the generation of insulin-producing cells from mouse embryonic stem cells constitutively expressing Pax4. [0008] Micallef et al. reports that retinoic acid can regulate the commitment of embryonic stem cells to form Pdx1 35 positive pancreatic endoderm. Retinoic acid is most effective at inducing Pdx1 expression when added to cultures at day 4 of embryonic stem cell differentiation during a period corresponding to the end of gastrulation in the embryo (Diabetes 54:301, 2005). [0009] Miyazaki et al. reports a mouse embryonic stem cell line over-expressing Pdx1. Their results show that exog- enous Pdx1 expression clearly enhanced the expression of insulin, somatostatin, glucokinase, neurogenin3, p48, Pax6, 40 and HNF6 genes in the resulting differentiated cells (Diabetes 53: 1030, 2004). [0010] Skoudy et al. reports that activin A (a member of the TGF- β superfamily) up-regulates the expression of exocrine pancreatic genes (p48 and amylase) and endocrine genes (Pdx1, insulin, and glucagon) in mouse embryonic stem cells. [0011] The maximal effect was observed using 1 nM activin A. They also observed that the expression level of insulin and Pdx1 mRNA was not affected by retinoic acid; however, 3 nM FGF7 treatment resulted in an increased level of the 45 transcript for Pdx1 (Biochem. J. 379: 749, 2004). [0012] Shiraki et al. studied the effects of growth factors that specifically enhance differentiation of embryonic stem cells into Pdx1 positive cells. They observed that TGFβ2 reproducibly yielded a higher proportion of Pdx1 positive cells (Genes Cells. 2005 June; 10(6): 503-16). [0013] Gordon et al. demonstrated the induction of brachyury [positive]/ HNF-3beta [positive] endoderm cells from 50 mouse embryonic stem cells in the absence of serum and in the presence of activin along with an inhibitor of Wnt signaling (US 2006/0003446A 1). [0014] Gordon et al. (PNAS, Vol 103, page 16806, 2006) states: "Wnt and TGF-beta/ nodal/ activin signaling simul- taneously were required for the generation of the anterior primitive streak." [0015] However, the mouse model of embryonic stem cell development may not exactly mimic the developmental 55 program in higher mammals, such as, for example, humans. [0016] D’Amour et al. describes the production of enriched cultures of human embryonic stem cell-derived definitive endoderm in the presence of a high concentration of activin and low serum (D’Amour K Aet al. 2005). Transplanting these cells under the kidney capsule of mice resulted in differentiation into more mature cells with characteristics of 3 EP 2 310 492 B1 some endodermal organs. Human embryonic stem cell-derived definitive endoderm cells can be further differentiated into PDX1 positive cells after addition of FGF-10 (US 2005/0266554A1). [0017] D’Amour et al. (Nature Biotechnology--24, 1392-1401 (2006)) states: "We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable