(19) TZZ ¥_T (11) EP 2 669 366 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 5/0735 (2010.01) C12N 5/074 (2010.01) 31.01.2018 Bulletin 2018/05 (21) Application number: 13163321.6 (22) Date of filing: 22.04.2009 (54) Pluripotent cells Pluripotente Zellen Cellules pluripotentes (84) Designated Contracting States: • KROON EVERT ET AL: "Pancreatic endoderm AT BE BG CH CY CZ DE DK EE ES FI FR GB GR derived from human embryonic stem cells HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL generates glucose-responsive insulin-secreting PT RO SE SI SK TR cells in vivo", NATURE BIOTECHNOLOGY APR 2008,, vol. 26, no. 4, 1 April 2008 (2008-04-01), (30) Priority: 24.04.2008 US 108872 pages 443-452, XP002561975, • D’AMOUR K A ET AL: "Efficient differentiation of (43) Date of publication of application: human embryonic stem cells to definitive 04.12.2013 Bulletin 2013/49 endoderm", NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP, NEW YORK, NY, (62) Document number(s) of the earlier application(s) in US, vol. 23, no. 12, 28 October 2005 (2005-10-28), accordance with Art. 76 EPC: pages 1534-1541, XP002385437, ISSN: 1087-0156 09734026.9 / 2 283 113 • SHIRAKI N ET AL: "Guided differentiation of embryonic stem cells into Pdx1-expressing (73) Proprietor: Janssen Biotech, Inc. regional-specific definitive endoderm", STEM Horsham, PA 19044 (US) CELLS, ALPHAMED PRESS, DAYTON, OH, US, vol. 26, no. 4, 1 April 2008 (2008-04-01), pages (72) Inventor: Rezania, Alireza 874-885, XP002547894, ISSN: 1066-5099 Hillsborough, NJ 08844 (US) [retrieved on 2008-01-31] • LUDWIG T E ET AL: "Derivation of human (74) Representative: Wise, Daniel Joseph embryonic stem cells in defined conditions", Carpmaels & Ransford LLP NATURE BIOTECHNOLOGY 200602 US, vol. 24, One Southampton Row no. 2, February 2006 (2006-02), pages 185-187, London WC1B 5HA (GB) XP002564246, • MORRISON SEAN J ET AL: "Culture in reduced (56) References cited: levels of oxygen promotes clonogenic WO-A-00/29549 WO-A2-2009/012428 sympathoadrenal differentiation by isolated neural crest stem cells", JOURNAL OF • EZASHI T ET AL: "Low O2 tensions and the NEUROSCIENCE, vol. 20, no. 19, 1 October 2000 prevention of differentiation of hES cells", (2000-10-01), pages 7370-7376, XP002552625, PROCEEDINGS OF THE NATIONAL ACADEMY ISSN: 0270-6474 OFSCIENCES OF USA,NATIONAL ACADEMY OF SCIENCE, WASHINGTON, DC, US, vol. 102, no. 13, 29 March 2005 (2005-03-29), pages 4783-4788, XP002466326, ISSN: 0027-8424 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 2 669 366 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 669 366 B1 • KOLLERM R ET AL: "EFFECTS OF SYNERGISTIC • YASUDA EMIKO ET AL: "Development of cystic CYTOKINE COMBINATIONS, LOW OXYGEN, embryoid bodies with visceral yolk-sac-like ANDIRRADIATED STROMA ON THE EXPANSION structures from mouse embryonic stem cells OF HUMAN CORD BLOOD PROGENITORS", using low-adherence 96-well plate.", JOURNAL BLOOD, AMERICAN SOCIETY OF OF BIOSCIENCE AND BIOENGINEERING APR HEMATOLOGY, US, vol. 80, no. 2, 15 July 1992 2009, vol. 107, no. 4, April 2009 (2009-04), pages (1992-07-15), pages403-411, XP000604784, ISSN: 442-446, XP002564247, ISSN: 1347-4421 0006-4971 2 EP 2 669 366 B1 Description FIELD OF THE INVENTION 5 [0001] The present invention provides a method to expand cells expressing markers characteristic of the definitive endoderm lineage, comprising the steps of culturing the cells under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix. The present disclosure is directed to pluripotent stem cells that can be readily expanded in culture on tissue culture polystyrene and do not require a feeder cell line. The present disclosure also provides methods to derive the pluripotent stem cell line from human embryonic stem cells. 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 HNF-3 beta, GATA-4, Mix11, CXCR4 and SOX-17. [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, PDX-1. In the absence of PDX-1, the pancreas fails to develop beyond the formation of ventral and dorsal buds. Thus, PDX-1 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) disclose 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 PDX-1 35 positive pancreatic endoderm. Retinoic acid is most effective at inducing PDX-1 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 PDX-1. Their results show that exogenous PDX-1 expression clearly enhanced the expression of insulin, somatostatin, glucokinase, neurogenin3, P48, 40 Pax6, 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) upregulates the expression of exocrine pancreatic genes (p48 and amylase) and endocrine genes (PDX-1, insulin, and glucagon) in mouse embryonic stem cells. The maximal effect was observed using 1nM activin-A. They also observed that the expression level of insulin and PDX-1 mRNA was not affected by retinoic acid; however, 3nM FGF-7 treatment resulted in an increased level of the 45 transcript for PDX-1 (Biochem. J. 379: 749,2004). [0011] Shiraki et al. studied the effects of growth factors that specifically enhance differentiation of embryonic stem cells into PDX-1 positive cells. They observed that TGFβ2 reproducibly yielded a higher proportion of PDX-1 positive cells (Genes Cells. 2005 Jun; 10(6): 503-16.). [0012] Gordon et al. demonstrated the induction of brachyury+/HNF-3 beta+ endoderm cells from mouse embryonic 50 stem cells in the absence of serum and in the presence of activin along with an inhibitor of Wnt signaling (US2006/0003446A1). [0013] Gordon et al. (PNAS, Vol 103, page 16806, 2006) states "Wnt and TGF-beta/nodal/ activin signaling simulta- neously were required for the generation of the anterior primitive streak". [0014] 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. [0015] Thomson et al. isolated embryonic stem cells from human blastocysts (Science 282:114, 1998). Concurrently, Gearhart and coworkers derived human embryonic germ (hEG) cell lines from fetal gonadal tissue (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Unlike mouse embryonic stem cells, which can be prevented from differ- 3 EP 2 669 366 B1 entiating simply by culturing with Leukemia Inhibitory Factor (LIF), human embryonic stem cells must be maintained under very special conditions (U.S. Pat. No. 6,200,806; WO 99/20741; WO 01/51616). [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 KA et al. 2005). Transplanting 5 these cells under the kidney capsule of mice resulted in differentiation into more mature cells with characteristics of some endodermal organs. Human embryonic stem cell-derived definitive endoderm cells can be further differentiated into PDX-1 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 of synthesizing the pancreatic 10 hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin.