AU2015268664B2 - Methods for purifying cells derived from pluripotent stem cells - Google Patents

Abstract

Abstract The present invention is directed to methods to differentiate pluripotent stem cells. In particular, the present invention provides methods of characterization of cells differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage utilizing unique surface markers. The present invention also provides methods to enrich or sort cells expressing markers characteristic of the pancreatic endocrine lineage. The present invention also provides methods to deplete cells that may contaminate populations of cells expressing markers characteristic of the pancreatic endocrine lineage formed by the methods of the present invention, thereby reducing the incidence of tumor formation in vivo following transplantation.

Description

The present invention is directed to methods to differentiate pluripotent stem cells. In particular, the present invention provides methods of characterization of cells differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage utilizing unique surface markers. The present invention also provides methods to enrich or sort cells expressing markers characteristic of the pancreatic endocrine lineage. The present invention also provides methods to deplete cells that may contaminate populations of cells expressing markers characteristic of the pancreatic endocrine lineage formed by the methods of the present invention, thereby reducing the incidence of tumor formation in vivo following transplantation.

2015268664 01 Mar 2016

METHODS FOR PURIFYING CELLS DERIVED FROM PLURIPOTENT STEM CELLS [0000] The present application is a divisional application of Australian Application No. 2011223900, which is incorporated in its entirety herein by reference.

CROSS REFERENCE TO RELATED APPLICATION [0001] This application claims priority to provisional application serial number 61/309, 193, filed March 1, 2010.

FIELD OF THE INVENTION [0002] The present invention is directed to methods to differentiate pluripotent stem cells.

In particular, the present invention provides methods of characterization of cells differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage utilizing unique surface markers. The present invention also provides methods to enrich or sort cells expressing markers characteristic of the pancreatic endocrine lineage. The present invention also provides methods to deplete cells that may contaminate populations of cells expressing markers characteristic of the pancreatic endocrine lineage formed by the methods of the present invention, thereby reducing the incidence of tumor formation in vivo following transplantation.

[0002a] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

BACKGROUND [0003] Pluripotent stem cells have the potential to produce differentiated cell types comprising all somatic tissues and organs. Treatment of diabetes using cell therapy is facilitated by the production of large numbers of cells that are able to function similarly to human islets. Accordingly, there is need for producing these cells derived from pluripotent stem cells, as well as reliable methods for purifying such cells.

10004] Proteins and other cell surface markers found on pluripotent stem cell and cell populations derived from pluripotent stem cells are useful in preparing reagents for the separation and isolation of these populations. Cell surface markers are also useful in the further characterization of these cells.

2015268664 01 Mar 2016

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1005] In one example, W02009131568 discloses a method of purifying a gut endoderm cell comprising: a) exposing a population of cells derived from pluripotent stem cells comprising a gut endoderm cell to a ligand which binds to a cell surface marker expressed on the gut endoderm cell, wherein said cell surface marker is selected from the group consisting of CD49e, CD99, CD 165, and CD334; and b) separating the gut endoderm cell from cells derived from pluripotent stem cells which do not bind to the ligand, thereby purifying said gut endoderm cell.

¢006] In another example, W02010000415 discloses the use of an antibody that binds to the antigen TNAP, or functional fragments of the antibody, alone or i n combination with an antibody that binds to CD56, or functional fragments of the antibody, for the isolation of stem cells having adipocytic, chondrocytic and pancreatic differentiation potential.

1007] In another example, US7371576 discloses the discovery' of a selective cell surface marker that permits the selection of a unique subset of pancreatic stents cells having a high propensity to differentiate into insulin producing cells or info insulin producing cell aggregates.

1003] In another example, OS7585672 discloses a method to enrich a culture derived from human embryonic stem cells for cells of endoderm and pancreatic lineages, the method comprising the steps of (a) cu lturing intact colonies of human embryonic stem ceils to form whole, intact embryoid bodies surrounded by visceral yolk sac (V YS) cells, wherein the human embryonic stem cells express Oct-4, surface stage-specific embryonic antigen-3/4 (SSEA 3/4) and epithelial cell adhesion molecule (EpCAM); (b) culturing the embryoid bodies of step (a) under conditions that permit the embryoid body cells to differentiate into a cell population containing cells of the endoderm and pancreatic lineages; (c) dispersing the cell population of step (b) into single cells; (d) selecting against the expression of SSEA 3/4 positive cells to remove undifferentiated cells from the cells of step (c); (e) selecting against the expression of SSEA-1 positive cells to remove VYS cells from the remaining ceils of step (d): and (f) selecting from among the remaining cells of step (e) for the expression of EpCAM posi tive ceils to enrich for cells of endoderm and pancreatic lineages.

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2015268664 11 Dec 2015 >009] US7585672 also discloses a method to enrich a culture derived from human embryonic stem cells for cells of endoderm and pancreatic lineages, t he method comprising the steps of (a) culturing intact colonies of human embryonic stem cells to form whole, intact embryoid bodies surrounded by visceral yolk sac (VYS) cells, wherein the human embryonic stem cells express Oct-4, surface stage-specific embryonic antigen-3/4 (SSEA 3/4) and epithelial cell adhesion molecule (EpCAM); (b) culturing the embryoid bodies of step (a) under conditions that permit the embryoid body cells to differentiate into a cell population containing cells of the endoderm and pancreatic lineages; (e) treating the cell population of step (b) with an effective amount of fibroblast growth factor 1.0 (EGF10); and (d) dispersing the cell population of step (c) into single cells enriched for cells of endoderm and pancreatic lineages (e) selecting against the expression of SSEA-3/4 positive cells to remove undifferentiated stem cells front the cells of step (d); (f) selecting against the expression of SSEA-1 pos itive cells to remove VYS cells front the cells of step (e); and (g) selecting from among the remaining cells of step (f) for the expression of EpCAM posi tive ceils io enrich for cells of endoderm and pancreatic lineages, (0010] US7585672 also discloses an enrichment method for the creation of a stem ceil.

derived cell population which docs not have tumorigenic capability comprising the steps of (a) culturing intact colonies of human embryonic stem cells to form whole, intact embryoid bodies surrounded by visceral yolk sac (VYS) ceils, wherein the human embryonic stem cells express Oct-4, surface stage-specific embryonic antigen3/4 (SSEA 3/4) and epithelial cell adhesion molecule (EpCAM): (b) culturing the embryoid bodies of step (a) under conditions that permit the embryoid body cells to differentiate into a cell population containing cells of the endoderm and pancreatic lineages; (c) dispersing the cell population of s tep (b) into single cells; (d) selecting against the expression of SSEA 3/4 positive cells to remove undifferentiated cells from the cells of step (c); (e) selecting against the expression of SSEA-i positive cells to remove VYS cells from the cells of step (d);and (f) selecting from among the remaining cells of step (e) for the expression of EpCAM positive cells, the resulting cells not forming teratomas when injected in immunocompromised mice,

J0010] In another example, US20050260749 discloses a method to enrich a culture derived from stem cells for cells of endoderm and pancreatic lineages, the method comprising 3

2015268664 11 Dec 2015 the steps of culturing stem cells into the forma tion of embryotd bodies; and selecting among embry old bodies for the expression of the spec ies appropriate cel l surface stage-specific embryonic and culturing only the embryoid bodies which do not. express cell surface stage-specific antigen for differentiation into endoderm and pancreatic cells.

1011] In another example, 0820100003749 discloses an isolated pancreatic stem cell population, wherein the pancreatic stem cell population is enriched for

CD 133+CD49ft-pancreatic stem cells.

1012] US20100003749 further discloses the isolation of pancreatic stem cells from primary pancreatic tissue occurs by selecting .from a population of pancreatic cells, pancreaticderived cells, or gastrointestinal-derived cells for cells that are GDI33+, CD49f+, or CD1.33+CD49f+; removing the cells that are GDI 5+, wherein the remaining ceils are CD15-; introducing the remaining cells to a serum-free culture medium containing one or more growth factors; and proliferating the remaining cells in the culture medium.

(013] In another example, Dorrell et al s tate; “We have developed a novel panel of cellsurface markers for the isolation and study of ail major cell types of the human pancreas. Hybridomas were selected after subtractive immunization of Balb/C mice with intact or dissociated human islets and assessed for cell-type specifici ty and cellsurface reactivity by immunohistochemistry and flow cytometry¹. Antibodies were identified by specific binding of surface antigens on islet (panendocnne or a-specific) and nonislet pancreatic cell, subsets (exocrine and duct). These antibodies were used individually or in combination to isolate populations of α, β, exocrine, or duet cells from primary human pancreas by FACS and to characterize the detailed cell composi tion of human islet preparations. They were also employed to show that human islet expansion cultures originated from nonendocrine cells and that insulin expression levels could be increased to up to 1% of normal islet cel ls by snbpopula tion sorting and overexpression of the transcription factors Fdx-1 and ngn3, an improvement over previous results with this culture system. These methods permit the analysis and isolation of fonctionally distinct pancreatic cell populations with

2015268664 11 Dec 2015 potential for ceil therapy.” (Stew Cell Research, Volume 1, Issue 3 , September 2008, Pages 155-156).

(014] In another example, Sngiyama ef al state: “We eventually identified two antigens, called CD 133 and CD49f, useful for purifying NGN3+ cells from mice. CD 133 (also called prominin-1) is a transmembrane protein of unknown function and a known marker of haematopoietic progenitor and neural stem cells. CD49f is also called «6integrm, and a receptor subunit for laminin. By combining antibodies that recognize CD 133 and CD49f, we fractionated four distinct pancreatic cell populations. Ihununostaming and RT-PCR revealed that the CD49fhigh CD133+ cell population (’fraction Γ, 50% of input) comprised mainly differentiated exocrine cells that express CatbA. The CD49flow CD133- fraction ('fraction 11Γ, 10% of input) included honnonet cells expressing endocrine products like insulin and glucagon. By contrast, the CD49fiow CD133·*- fraction (called 'fraction IT, 13% of input) contained NGN3-r cells, but not hormone ·(· cells. Approximately 8% of fraction Π ceils produced immnnostainabie NGN3. In the CD49f~ CD 133- fraction (’fraction IV’, 25% of input), we did not detect cells expressing NGN3, CarbA or islet hormones,” (Diabetes, Obesity and Metabolism, Volume 10, Issue s4, Pages 179-185).

1015] In another example, Fujikawa et al state: ‘- When CD45-TER119- side-scatterlow GFPhigh cells were sorted, a-fetoprotein-positive immature endoderm-characterized, cells, having high growth potential, were present in this population. Clonal analysis and electron microscopic evaluation revealed that each single cell of this population could differentiate not only into hepatocytes, but also into biliary¹ epithelial cells, showing their biimeage differentiation activity. When surface markers were analyzed, they were positive for integrm-ab and -pi, but negative for c-Kit and Thy! ,1.” (Journal offfepatlogy, Vol 39, pages 162-170), |Θ016] In another example, Zhao et al state; “la this study, we first identified N-cadherin as a surface marker of hepatic endoderm cells for purification from hES cell-dertvates, and generated hepatic progenitor cells from purified hepatic endoderm cells by coculture with murine embryonic stromal feeders (STO) cells. These hepatic progenitor cells could expand and be passaged for more than 100 days. Interestingly, they coexpressed the early hepatic marker AFP and biliary lineage marker KRT7, suggesting

2015268664 11 Dec 2015 that they are a common ancestor of both hepatocytes and cholangioeytes, Moreover, these progenitor cells could be expanded extensively while still maintaining the bipotential of differentiation into hepatocyte-like ceils and cholangiocyte-like cells, as verified by both gene express ion and functional assays. Therefore, this work offers a new in vitro model for studying liver development, as well as a new source for cell therapy based on hepatic progenitors.” (PloS OAK 4(7): ¢6468.

doi: 10,137 1/joumai.pone.0006468), >017] In another example, Cai et al state: “To further increase the PDX1T cell purity, we sorted the activin A-induced ceils using CXCR4..., a marker for ES cell-derived endodermal cells. Sorting with CXC.R4 enriched the endodermal cell population because nearly all the cells in the CXCR4+ popula tion were positive for the endodermal cell marker SOX17, and >90% of the cells were positive for FOXA2.” (Journal of Molecular Cell Biology Advance Access originally published online on November 12, 2009. .Journal of Molecular Ceil Biology 2010 2(1):50-60; doi: 10.1093/jmcb/mjp037),

1018] In another example, Koblas et al state: “We found that population of human CD133positive pancreatic cells contains endocrine progenitors expressing neurogenin~3 and cells expressing human telomerase, ABCG2, Oet-3/4, Nanog, and Rex-1, markers of pluripotent stem cells. These cells were able to differentiate into insulin-producing cells in vitro and secreted C-peptide in a glucose-dependent manner. Based on our results, we suppose that the CD 133 molecule represents another cell surface marker suitable for identification and isolation of pancreatic endocrine progenitors”, (TransplantProc. 2008 Mar;40(2):415-8)

10019] hi another example, Sugiyarna et al slate: “we found CD 133 was expressed by

NGN3+ cells. CD 133 appeared to be localized to the apical membrane of pancreatic ductal epithelial ceils.” (ΡΛΟίΝ 2007 104:175-180; published online before print December 26, 2006, doi: 10.1073/pnas.0609490104).

[0020] In another example, Kobayashi ez al state: ‘The embryonic pancreatic epithelium, and later the ductal epithelium, is known to give rise to the endocrine and exocrine cells of the developing pancreas, but no specific surface marker for these cells has

2015268664 20 Dec 2017

-7been identified. Here, we utilized Dolichos Biflorus Agglutinin (DBA) as a specific marker of these epithelial cells in developing mouse pancreas. From the results of an immunofluorescence study using fluorescein-DBA and pancreatic specific cell markers, we found that DBA detects specifically epithelial, but neither differentiating endocrine cells nor acinar cells. We further applied this marker in an immunomagnetic separation system (Dynabead system) to purify these putative multipotential cells from a mixed developing pancreatic cell population. This procedure could be applied to study differentiation and cell lineage selections in the developing pancreas, and also may be applicable to selecting pancreatic precursor cells for potential cellular engineering.” (Biochemical and Biophysical Research Communications, Volume 293, Issue 2, 3 May 2002, Pages 691-697).

[0021] Identification of markers expressed by cells derived from pluripotent stem cells would expand the understanding of these cells, aid in their identification in vivo and in vitro, and would enable their positive enrichment in vitro for study and use. Thus, there remains a need for tools that are useful in isolating and characterizing cells derived from pluripotent stem cells, in particular, cells expressing markers characteristic of the pancreatic endocrine lineage.

SUMMARY [0021a] According to a first aspect of the present invention there is provided a method to enhance the generation of insulin-producing cells from a population of cells expressing markers characteristic of the pancreatic endocrine lineage comprising:

a. differentiating a population of pluripotent stem cells into a population of cells expressing markers characteristic of the definitive endoderm lineage;

b. differentiating the population of cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the primitive gut tube lineage;

c. differentiating the population of cells expressing markers characteristic of the primitive gut tube lineage into a population of cells expressing markers characteristic of the pancreatic endoderm lineage;

d. differentiating the population of cells expressing markers characteristic of the pancreatic endoderm lineage into a population cells expressing markers characteristic of the pancreatic endocrine lineage; and

e. enriching the population of cells expressing markers characteristic of the pancreatic endocrine lineage for expression of NEUROD, NGN3, PDX1 and NKX6.1 by isolating a population of cells expressing markers characteristic of the pancreatic endocrine lineage that is positive for CD56 and negative for CD13.

2015268664 20 Dec 2017

-7a[0021b] According to a second aspect of the present invention there is provided a method of enriching a population of cells expressing markers characteristic of the pancreatic endocrine lineage for expression of NEUROD, NGN3, PDX1 and NKX6.1, comprising providing a population of cells expressing markers characteristic of the pancreatic endocrine lineage and isolating a population of cells expressing markers characteristic of the pancreatic endocrine lineage that is positive for CD56 and negative for CD13.

[0021c] According to a third aspect of the present invention there is provided a method of enhancing the generation of human insulin-producing cells from a population of cells expressing markers characteristic of the pancreatic endocrine lineage, comprising enriching a population of cells expressing markers characteristic of the pancreatic endocrine lineage for NEUROD, NGN3, PDX1 and NKX6.1 by isolating a population of cells expressing markers characteristic of the pancreatic endocrine lineage that is positive for CD56 and negative for CD13.

[0021 d] According to a fourth aspect of the present invention there is provided a method of enriching a population of cells expressing markers characteristic of the pancreatic endocrine lineage for NeuroD, NGN3, PDX-1, and NKX6.1 by screening the population of cells for positive for CD56 and negative for CD13 to thereby produce an enriched population of cells. [0021 e] According to a fifth aspect of the present invention there is provided an isolated population of insulin-producing cells produced by performing the method of any one of the first to fourth aspects.

[0021 f] According to a sixth aspect of the present invention there is provided use of the isolated population of cells of the fifth aspect in medicine.

[0021 g] According to a seventh aspect the present invention provides a method of treating or preventing an insulin-related disease or disorder comprising transplanting a population of insulin producing cells of the fifth aspect to a patient in need thereof [0021 h] According to an eighth aspect of the present invention there is provided an isolated enriched population of cells expressing markers characteristic of the pancreatic endocrine lineage produced by performing the method of the second aspect.

[0021 i] According to a ninth aspect of the present invention there is provided use of the isolated enriched population of cells of the eighth aspect in medicine.

[0021j] According to a tenth aspect of the present invention there is provided a method of treating or preventing an insulin-related disease or disorder comprising transplanting a population cells of the eighth aspect to a patient in need thereof.

[0021k] According to an eleventh aspect of the present invention there is provided use of a population of insulin producing cells of the fifth aspect for the manufacture of a medicament for treating or preventing an insulin-related disease or disorder.

2015268664 12 Dec 2017

-7b[00211] According to a twelfth aspect of the present invention there is provided use of a population cells of the eighth aspect for the manufacture of a medicament for treating or preventing an insulin-related disease or disorder.

[0021m] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0022] In one embodiment, the present invention provides a method to differentiate a population of pluripotent stem cells into a population of cells expressing markers characteristic of the pancreatic endocrine lineage, comprising the steps of:

a. Culturing a population of pluripotent stem cells,

b. Differentiating the population of pluripotent stem cells into a population of cells expressing markers characteristic of the definitive endoderm lineage,

c. Differentiating the population of cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the primitive gut tube lineage,

2015268664 11 Dec 2015

d. Differentiating the population of cells expressing markers characteristic of the primitive gut tube lineage into a population of cells expressing markers characteristic of the pancreatic endoderm lineage, and

e. Differentiating the population of cells expressing markers characteristic of the pancreatic endoderm lineage into a population cells expressing markers characteristic of the pancreatic endocrine lineage.

(023] In one embodiment, the population of cells expressing markers characteristic of the pancreatic endocrine lineage Is transplanted into an animal, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage form insulin producing cells. In one embodiment, the efficiency of the formation of insulin producing cells is enhanced by enriching the population for ceils expressing markers characteristic of the pancreatic endocrine lineage prior to transplantation.

(024] In one embodiment, the efficiency of the formation of msulin producing cells is determined by measuring the time taken for the expression of C-peptide to reach detectable levels following transplantation.

1025] In an alternate embodiment, the enrichment decreases the ability of the transplanted cells to form teratomas following transplantation,

BRIEF DESCRIPTION OF THE DRAWINGS

1020] Figure 1 shows the expression of N EU ROD (panel a), NGN3 (panel b), PDXl (panel c), NKX6,1 (panel d), NKX2.2 (panel ¢), and PAX4 (panel f) in populations of CD56’CD13', CD56CD13 andCDSbCDB' cells, as detected via real-time PCR. Fold expression is shown relative to undifferentiated H1 embryonic stem cells.

[0027] Figure 2 shows the expression of NEUROD (panel a), NGN3 (panel b), PDXl (panel c), NKX6J (panel d), NKX2.2 (panel ¢), and PAX4 (panel f), as detected via realtime PCR, in popula tions of cells sorted using an antibody to CD 133, Fold expression is shown relative to undifferentiated Hl embryonic stem cells.

[0028] Figure 3 shows the expression of NEUROD (panel a), N0N3 (panel b), PDX i (panel c), andNKX6J (panel d), as detected via real-time PCR, in populations of cells

2015268664 11 Dec 2015 sorted using an antibody to CD49c. Fold expression is shown relative to undifferentiated II1 embryonic stem cells, ►029] Figure 4 shows the expression of NEUROD (panel a), NGN3 (panel b), PDXI (panel c), NKX6.1 (panel d), insulin (panel e), and glucagon (panel f), as detected via realtime PCR, in populations of cells sorted using antibodies to CD56 and C.D15, Fold expression is shown relative to undifferentiated Hl embryonic stem cells.

(030] Figure 5 shows fee expression of NEUROD (panel a), NGN3 (panel b), PDXi (panel c), NKX6.1 (panel d), NKX2.2 (panel ¢), PAX-4 (panel f), glucagon (panel g) and insulin (panel h) as detected via real-time PCR, in populations of cells sorted using an antibody to GDIS, Fold expression is shown relative to undifferentiated Hl embryonic stem cells. .

1031] Figure 6 shows fee expression of NEUROD (panel a), NGN3 (panel b), PDX1 (panel c), NKX6..1 (panel d), NKX2.2 (panel e), insulin (panel f), and glucagon (panel g) as detected via real-time PCR, in populations of cells sorted using antibodies to CD56 and CD57. Fold expression is shown relative to undifferentiated Hl embryonic stem cells.

(032] Figure 7 shows fee expression of ZIC1 (panel a), albumin (panel b), CDX2 (panel e), NGN3 (panel d), PAX4 (panel e), NEUROD (panel f)_s NKX6.1 (panel g), PTF1 alpha (panel h), and PDXI. (panel 1), as detected via real-time PCR, in populations of cells sorted using antibodies to CD56 and CD 184, Fold expression is shown relative to undifferentiated Hl embryonic stem cells.

|0033] Figure 8 shows fee expression of NEUROD (panel a), NGN3 (panel b), insulin (panel

c), and glucagon (panel d), as detected via real-time PCR., in populations of cells sorted using an antibody to CD98. Fold expression is shown relative to undifferentiated Hl embryonic stem cells.

[0034] Figure 9 shows the expression of NEUROD (panel a), NGN3 (panel b), PDX 1 (panel c), NK.X6.1 (panel, d), NKX2.2 (panel e), and PAX4 (panel f), as detected via realtime PCR, in populations of ceils sorted using an antibody to CD47. Fold expression is shown relative to undifferentiated Hl embryonic stem cells.

2015268664 11 Dec 2015

1035] Figure 10 shows the expression ofPDX-i (panel a), NKX6.1 (panel b), NKX2.2 (panel c), PAX-4 (panel d), PTFla (panel e), NGN3 (panel f), insulin (panel g) and glucagon (panel h) as detected via real-time PCR, in populations of cells sorted using an antibody to CD47. Fold expression is shown relative to undifferentiated HI embryonic stem cells,

1036] Figure 11 shows the expression of HNF4 alpha (panel a), and LIF receptor (panel h), as detected via real-time PCR, in populations of cells sorted using an antibody to the LIP receptor. Fold expression is shown relative to unsorted cells at DAY 2 of Stage II of the differentiation protocol outlined in Example L

1037] Figure 12 shows the expression of OCT4 (panel a), NANOG (panel b), SOX2 (panel ¢), and goosecoid (panel d), as detected via real-time PCR, in populations of cells depleted of cells expressing SSEA4 using magnetic beads. Fold expression is shown relative to undifferentiated Pi t embryonic stem cells.

1038] Figure 13 shows the expression of OCT4 (panel a), NANOG (panel h), SOX2 (panel c), and goosecoid (panel d), as detected, via real-time PCR, in populati ons of cells depleted of cells expressing SSEA4 using FACS. Fold expression is shown relative to undifferentiated PH embryonic stem cells.

DETAILED P) FSCR i P P1OY

S039| For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the following subsections that describe or illustrate certain features, embodiments or applications of the present invention.

Definitions [0040] β-cell lineage refers to cells with positive gene expression for the transcription factor PDX-1 and at least one of the following transcription factors: NGN3, NKX2.2, NKX6.1, NEUROD, ISL1, PtNF-3 beta, MAFA, PAX4, and PAX6, Cells expressing markers characteristic of the β cell lineage include β cells, [0041] “Cells expressing markers characteristic of the definitive endoderm lineage” as used herein refers to cells expressing at least one of the following markers: SOX 17,

2015268664 01 Mar 2016 [0042] [0043] [0044] [0045]

- 11 GATA4, HNF-3 beta, GSC, CER1, Nodal, FGF8, Brachyury, Mix-like homeobox protein, FGF4, CD48, eomesodermin (EOMES), DKK4, FGF17, GATA6, CD184, CKit, CD99, or OTX2. Cells expressing markers characteristic of the definitive endoderm lineage include primitive streak precursor cells, primitive streak cells, mesendoderm cells and definitive endoderm cells.

Cells expressing markers characteristic of the primitive gut tube lineage refers to cells expressing at least one of the following markers: HNF-1 beta, or HNF-4 alpha.

Cells expressing markers characteristic of the pancreatic endoderm lineage as used herein refers to cells expressing at least one of the following markers: PDX1, HNF-1 beta, PTF-1 alpha, HNF6, or HB9. Cells expressing markers characteristic of the pancreatic endoderm lineage include pancreatic endoderm cells.

Cells expressing markers characteristic of the pancreatic endocrine lineage as used herein refers to cells expressing at least one of the following markers: NGN3, NEUROD, ISL1, PDX1, NKX6.1, PAX4, or PTF-1 alpha. Cells expressing markers characteristic of the pancreatic endocrine lineage include pancreatic endocrine cells, pancreatic hormone expressing cells, and pancreatic hormone secreting cells, and cells of the β-cell lineage.

Definitive endoderm as used herein refers to cells which bear the 'characteristics of cells arising from the epiblast during gastrulation and which form the gastrointestinal tract and its derivatives. Definitive endoderm cells express the following markers: CD184, HNF-3 beta, GATA4, SOX17, Cerberus, OTX2, goosecoid, c-Kit, CD99, and Mixll.

Markers as used herein, are nucleic acid or polypeptide molecules that are differentially expressed in a cell of interest. In this context, differential expression means an increased level for a positive marker and a decreased level for a negative marker. The detectable level of the marker nucleic acid or polypeptide is sufficiently higher or lower in the cells of interest compared to other cells, such that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.

[0046]

2015268664 11 Dec 2015 >047| “Pancreatic endocrine cell” or “pancreatic hormone expressing cell” as used herein refers to a cell capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide.

(0481 “Pancreatic hormone secreting ceil*' as used herein refers to a cell capable of secreting at least one of the-following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide.

»G49| “Pre-primitive streak cell” as used herein refers to a cell expressing at least one of the following markers: Nodal, or FGF8.

10501 “Primitive streak cell” as used herein refers to a cell expressing at least one of the following markers; Brachyury, Mix-like homeobox protein, or FGF4, (051 j Stem cells are undifferentiated cells defined by their ability at the single cell level to both self-renew and differentiate to produce progeny cells, including self-renewiug progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm and ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation and to contribute substantially to most, if not all, tissues following injection into blastocysts, (0521 Stem cells are classified by their developmental potential as: (i) totipotent, meaning able to give rise to all embryonic and extraembryonic cell types; (ii) pluripotent, meaning able to give rise to all embryonic cell types; (iii) multipotent, meaning able to give rise to a subset of cell lineages, but all within a parrienZe/· tissue, organ, or physiological system, (for example, hematopoietic stem cells (HSC) can produce progeny that include HSC (self- renewal), blood cell restricted oligopotent progenitors and all cell types and elements (e.g., platelets) that are norma l components of the blood); (iv) ol igopotent, meaning able to gi ve rise to a .more restricted subset of cell lineages than multipotent stem cells; and (v) unipotent, meaning able to give rise to a single cell lineage (e.g,, sperraatogenic stem cells), {00531 Differentiation is the process by which an unspecialized (’toncoinmitted’') or less specialized ceil acquires the features of a specialized cell such as, for example, a nerve

2015268664 11 Dec 2015 cell or a muscle cell. A differentiated or diflerentiation-hiduced cell is one that has taken on a more specialized (committed”) position within foe /foenge of a ceil. The term committed”, when applied to the process of differentiation., refers to a cell that has proceeded in the differentiation pat/mw zo a point where, under normal circumstances, it will continue to differentiate into a specific cell type· or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. Dedifierentiation refers to the process by which a cell reverts to a less specialized (or committed) position within the lineage of a cell. As used, herein, the lineage of a cell defines the heredi ty of the cell, that is, which cells it came from and what cells it can give rise to. The lineage of a cell places the ceil within a hereditary scheme of development and d ifferentiation, A lineage-specific marker refers to a characteristic specifically associated w ith the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted cell to the lineage of interest »054] Various terms are used to describe cells in culture. “Maintenance” refers generally to cells placed in a growth medium under conditions that facilitate ceil growth and/or division that may or may not result in a larger population of the cells, “Passaging” refers to the process of removing the cells from one culture vexveZ and plac ing them in a second culture vessel under conditions that facilitate cell growth and/or division,

1055] A specific population of cells, or a cell fine, is sometimes referred to or characterized by the number of times it has been passaged. For example, a cultured cell population that has been passaged ten times may be referred to as a Pl 0 culture. The primary culture, that is, the first culture following the isolation of cells from tissue, is designated P0. Follow ing the first subculture, the cells are described as a secondary culture (P l or passage I). After the second subculture, the cells become a tertiary·· culture (P2 or passage 2), and so on. It will he understood by those of skill in the art that there may be many population doublings during the period of passaging; therefore the number of population doublings of a culture is greater than the passage number. The expansion of cells (that is, the number of population doublings) during the period between passaging depends on many factors, including but not limited to the seeding density, substrate, medium, growth conditions, and time between passaging.

2015268664 11 Dec 2015

Enrichment of Cells Expressing Markers Characteristic of the Pancreatic Endocrine Lineage ►056] In one embodiment the present invention provides a method to differentiate a population of pluripotent stem cells Into a population of cells expressing markers characteristic of the pancreatic endocrine lineage, comprising the steps of

a. Culturing a population of pluripotent stem cells,

b. Differentiating the population of pluripotent stent cells into a population of celis expressing markers characteristic of the definitive endoderm lineage,

c. Differentiating the population of cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the primitive gut tube lineage,

d. Differentiating the population of cells expressing markers characteristic of the primitive gut tube lineage into a population of cells expressing markers characteristic of the pancreatic endoderm lineage, and

e. Differentiating the population of cells expressing markers characteristic of the pancreatic endoderm lineage into a population cells expressing markers characterist ic of the pancreatic endocrine lineage,

1057] In one embodiment, the population of cells expressing markers characteristic of the pancreatic endocrine lineage is transplanted into an animal, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage form insulin producing cells. In one embodiment, the efficiency of the formation of insulin producing cells Is enhanced by enriching the population for ceils expressing markers characteristic of the pancreatic endocrine lineage prior to transplantation, [0058] In one embodiment, the efficiency of the formation of insulin producing cells is determined by measuring the time taken for the expression of C-peptide to reach detectable levels following transplantation, [0059] In an alternate embodiment, the enrichment decreases the ability of the transplanted cells to form teratomas following transplantation,

2015268664 11 Dec 2015 (060] Cells expressing markers of the pancreatic endocrine lineage are identified or selected through the binding of antigens, found on the surfaces of the cells, to reagents that specifically bind the cell surface antigen.

(0611 In an alternate embodiment, cells expressing markers characteristic of the pancreatic endocrine lineage are further differentiated into insulin producing ceils, prior to transplantation into an animal. Insulin producing cells are identified or selected through the binding of antigens, found on the surfaces of the cells, to reagents that speci fically bind the cell surface antigen.

1062] In an alternate embodiment, the present invention provides a method to differentiate a population of pluripotent stem cells into a population of cells expressing markers characteristic of the pancreatic endocrine lineage, comprising the steps of:

a. Culturing a population of pluripotent stem ceils,

b. Differentiating the population of plu ripoten t stem cells into a population of cells expressing markers characteristic of the definitive endoderm lineage,

c. Differentiating the population of cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the primiti ve gu t tube lineage,

d. Enriching the population of cells that express markers characteristic of the primitive gut tube lineage,

e. Differentiating the population of cells expressing markers characteristic of the primitive gut tube lineage into a population of cells expressing markers characteristic of the pancreatic endoderm lineage, and

f. Differentiating the population of cells expressing markers characteristic of the pancreatic endoderm lineage into a population cells expressing markers characteristic of the pancreatic endocrine lineage.

[0063] In one embodiment, the population of cells expressing markers characteristic of the pancreatic endocrine lineage is transplanted into an animal wherein the cells expressing markers characteristic of the pancreatic endocrine lineage form insulin 15

2015268664 11 Dec 2015 producing ceils. in one embodiment, the efficiency of the formation of insulin producing cells is enhanced by enriching the population of cells that express markers characteristic of the primitive gut tube lineage prior to transplantation.

1064] Cells expressing markers of the primitive gut tube lineage are identified or selected through the binding of antigens, found on the surfaces of the cells, to reagents that specifically bind the cell surface antigen.

Surface Antigens that Facilitate Enrichment of Cells Expressing Markers Characteristic of the Pancreatic Endocrine Lineage

1065] In one embodiment, prior to transplantation into an animal, the population of cells expressing markers characteristic of the pancreatic endocrine lineage is treated with at least one reagent that is capable of binding to a marker selected from the group consisting of CD9, CD13, CD 15, CD47, CD56, CD73, CD! 17, CD! 33, CD 184, CD200, CD3I8, CD326 and SSEA4.

(066] In one embodiment, treatment with the at least one reagent results in a population of cells expressing markers characteristic of the pancreatic endocrine lineage that are positive for the expression of the marker CD56 and negative for the expression of the marker CD 13.

1067] in one embodiment, treatment with the at least one reagent results in a population of cells expressing markers characteristic of the pancreatic endocrine lineage that are positive for the expression of the marker CD56 and negative for the expression of the marker CD 15.

10068] In one embodiment, treatment with the at least one reagent results in a population of cells expressing markers characteristic of the pancreatic endocrine lineage that are nega ti ve for the expression of the marker CD 133.

[0069] In one embodiment, treatment with the at least one reagent results in a population of cells expressing markers characteristic of the pancreatic endocrine lineage that are negative for the expression of the marker CD 15,

2015268664 11 Dec 2015 (070] In one embodiment, treatment with the at least one reagent results in a population of cells expressing markers characteristic of the pancreatic endocrine lineage that are positi ve for the expression of the marker CD 184.

►07'tj In one embodiment, treatment with the at least one reagent results in a population of cells expressing markers characteristic of the pancreatic endocrine lineage that are negative for the expression of the marker SSEA4.

S«r/otx' Antigens that Facilitate Enrichment of Insulin Producing Ceifo

1072] In one embodiment, prior to transplantation into an animal, the population of cells expressing markers characteristic of the pancreatic endocrine lineage is further differentiated into a population of insulin producing cells. The population of insulin producing cells is treated with at least one reagent that is capable of binding to a marker selected from the group consisting of CD47, CD56, CDS7 CD98 and SSEA4.

(073] In one embodiment, treatment with the at least one reagent results in a population of insulin producing cells that are positive for the expression of the marker CD56 and CD57, Alternatively, the population of insulin producing cells may be positive for the expression of CD98. Alternatively, the population of insulin producing cells may be negative for the expression of CD47.

(074] In one embodiment, treatment with the at least one reagent results in a population of insulin producing cells that are negative for the expression of the marker SSEA4.

tdOTSj CD13 is expressed on the majority of peripheral blood monocytes and granulocytes.

It is also expressed by the majority of acute myeloid leukemias, chronic myeloid leukemias in myeloid blast crisis, a smaller percentage of lymphoid leukemias and myeloid cell lines. CD13 is also found in several types of non hematopoietic cells such as fibroblasts and endothelial cells and in a soluble form in blood plasma. GDI 3 is not expressed on B cells, T cells, platelets or erythrocytes. CD 13 plays a rote in biologically active peptide metabolism, in the control of growth and differentiation, in phagocytosis and in bacterieidaVtumorieidal activities, CD1.3 also serves as a receptor for human coronaviruses (HCV),

2015268664 11 Dec 2015

1076] CD 15 is a carbohydrate adhesion molecule that can be expressed on glycoproteins, glycolipids and proteoglycans, CD 15 mediates phagocytosis and chemotaxis, found on neutrophils; expressed in patients with Hodgkin disease, some B-cell chronic lymphocytic leukemias, acute lymphoblastic leukemias, and most acute nonlymphocytic leukemias. It is also called Lewis x and SSEA-i (stage specific embryonic antigen I) and represents a marker for murine pluripotent stem cells, in which it plays an important role in adhesion and migration of the cells in the preimplantation embryo,

1077] CO47 is a membrane protein, which is involved in the increase in intracellular calcium concentration that occurs upon cell adhesion to extracellular matrix. The protein is also a receptor for the C-terminal cell binding domain of thrombospondin, and it may play a role in membrane transport and signal transduction.

1078] CD56, also known as Neural Cell Adhesion Molecule (NCAM) is a homophilie binding glycoprotein expressed on the surface of neurons, glia, skeletal muscle and natural kilter cells. NCAM has been implicated as having a role in cell-cell adhesion, neurite outgrowth, synaptic plasticity, and learning and memory,

1079] CDS? also known as HNK-1 or Leu-7, is an antigenic oligosaccharide moiety detected on extracellular proteins of certain cell types, in blood, CD57 is found on 15-20% of mononuclear cells, including subsets of NK andT cells, though not on erythrocytes, monocy tes, granulocytes, or platelets. Also, CDS7 expression can be found on a variety of neural cell types.

[0080] CD98 is a. glycoprotein that comprises the light subunit of the Large neutral Amino acid Transporter (LATI). I,ATI is a heterodimeric membrane transport protein that preferentially transports neutral branched (valine, leucine, isoleucine) and aromatic (tryptophan, tyrosine) amino acids, [0081] CD133 is a glycoprotein also known in humans and rodents as Prominin 1 (PROMI).

It is a member of pentaspan transmembrane glycoproteins (5-traasmerabrane, 5-TM), which specifically localizes to cellular protrusions. CD 133 is expressed in hematopoietic stem cells, endothelial progenitor cells, glioblastomas, neuronal and

2015268664 11 Dec 2015 glial stem cells. See Corbeil et al_t Biochem Biophys Res Cmnmun 285 (4): 939-44, 2001. doi:10,1006/bbrc,200.1.5271. PMID 11467842.

Surface Antigens that Facilitate .Enrichment of Cells .gapmssing Afarfers Characteristic of the Primitive Gut Tube Lineage >082j In an alternate embodiment, the present invention provides a method to differentiate a population of pluripotent stem cells into a population of cells expressing markers characteristic of the pancreatic endocrine lineage, comprising the steps of:

a. Culturing a population of pluripotent stem cells,

b. Differentiating the population of pluripotent stem cells into a population of cells expressing markers characteristic of the definitive endoderm lineage,

c. Differentiating the population of cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the primitive gut tube lineage,

d. En riching the popul ation of cells that express markers characteri stic of the primitive gut tube lineage,

e. Differentiating the population of cells expressing markers characteristic of the primitive gut tube lineage into a population of cells expressing markers characterist ic of the pancreatic endoderm lineage, and

f. Differentiating the population of cells expressing markers characteristic of the pancreatic endoderm lineage into a population cells expressing markers characteristic of the pancreatic endocrine lineage.

J 0983 j In one embodiment, the population, of cells expressing markers characteri stic of the pancreatic endocrine lineage is transplanted into an animal, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage form insulin producing cells. In one embodiment, the efficiency of the formation of insulin producing cells is enhanced by enriching the population of cells that express markers characteristic of the primitive gut tube lineage prior to transplantation.

2015268664 11 Dec 2015

1084] The population of cells that express markers characteristic of the primitive gut tube lineage is treated with at least one reagen t that is capable of binding to the LIF receptor,

1085] The ceils expressing markers characteristic of the pancreatic endocrine lineage, cells expressing markers characteristic of the primitive gut tube lineage, or insulin producing cells may be enriched, depleted, isolated, separated, sorted and/or purified as further described in the examples. As used herein, the terms “enriched or ’’purified or enriched or purified due to depletion of other known cell populations, indicate that the cells has been subject to some selection process so that the population is enriched and/or purified. Also, the subject cells are also considered relatively enriched and/or purified, i.e, there is significantly more of a particular differentiated cell population as compared to another cell population, or as compared to pluripotent stem cells before enrichment or ’’purification’’, or as compared to the original or initial cell culture.

1086] Enriching or purifying for a given differentiated cell type may involve depleting or ’’separating or sorting one or more known cell, types from another cell type, in one embodiment, a populati on of cells may be- purified by depleting an un wanted differentiated cell type. It may be advantageous to enrich and purify a cell expressing markers characteristic of the pancreatic endocrine lineage by depleting the culture of known or unknown cell types, in this way, the enriched or purified cell population would not have the bound or attached antibody. Because there is no need to remove the antibody from the purified population, the use of the enriched or purified cells for cell therapies may be improved.

[0087] Methods for enriching, depleting, isolating, separating, sorting and/or purifying may include, for example, selective culture conditions, wherein the culture conditions are detrimental to any undesirable ceil types.

[0088] Methods for enriching, depleting, isolating, separating, sorting and/or purifying may also include, for example, antibody-coated magnetic beads, affinity chromatography and panning with antibody attached to a solid matrix or solid phase capture medium, e.g, plate, column or other convenient and available technique. Techniques

2015268664 11 Dec 2015 providing accurate separation include flow cytometry methods which are useful for measuring cell surface and intracellular parameters, as well as shape change and granularity and for analyses of beads used as antibody- or probe-linked reagents. Readouts from flow cytometry assays include, but are not limited to, the mean fluorescence associated with individual fluorescent antibody-detected cell surface molecules or cytokines, or the average fluorescence intensity, the median fluorescence intensity, the variance in fluorescence intensity, or some relationship among these.

(089] In some aspects of embodiments with analytical steps involving flow cytometry, minimal parameters or characteristics of the beads are scatter (FS and/or SS) and at least one fluorescent wavelengths. Flow cytometry can be used to quantitate parameters such as the presence of ceil surface proteins or conformational or posttranslational modification thereof; intracellular or secreted protein, where permeabiiization allows antibody (or probe) access, and the like. Flow' cytometry methods are known in the art, and described in the following: Flow Cytometry and Cell Storing (Springer t ab .Manual), Radbruch, Ed., Springer V'erlag, 2000; Ormerod, Flow' Cytometry, Springer Verlag, 1999; Flow Cytometry Protocols (Methods in Molecular Biology , No 91), Jaroszeski and Heller, Eds,, Humana Press, 1998;

Current Protocols in Cytomehy, Robinson et ak, eds, John Wiley & Sons, New York, RY., 2000.

(0901 The staining intensity of cells may be monitored by flow cy tometry , where lasers detect the quantitative levels of fluorochrome (which is proportional to the amount of cell surface marker bound by specific reagents, e.g, antibodies). Flow' cytometry, or FACS, may also be used to separate cell populations based on the intensity of binding to a specific reagent, as well as other parameters such as cell size and light scatter. Although the absolute level of staining can differ wi th a particular fluorochrome and reagent preparation, the data can be normalized to a control. In order to normalize the distribution to a control, each cell is recorded as a data point having a particular intensify of staining, (00911 In order to normalize the distribution to a control, each cell is recorded as a data point having a particular intensity of staining. These data points may be displayed according to a log scale, where the unit of measure· is arbitrary staining intensity. In

2015268664 11 Dec 2015 one example, the brightest cells in a population are designated as 4 logs more intense than the cells having the lowest level of staining. When displayed in this manner, it is clear that the ceils felling in the highest log of staining intensity are bright, while those in the lowest intensity are negative. The “low” staining cells, which fall in the 2-3 log of staining intensity, may have properties that are unique from the negative and positive cells. An alternative control may utilize a substrate having a defined density of marker on its surface, for example a fabricated bead or cell line, which provides the positive control for intensity. The “low” designation indicates that the level of staining is above the brightness of an isotype· matched control, but is not as intense as the most brightly staining cells normally found in the population, >092] The readouts of selected parameters are capable of being read simultaneously, or in sequence during a single analysts, as for example through the use of fluorescent antibodies to cell surface molecules. As an example, these can be tagged with different ffuoroehromes, fluorescent bead, tags, e.g. quantum dots, etc., allowing analysis of up to 4 or more fluorescent colors simultaneously by flow cytometry. For example, a negative designation indicates that the level of staining is at or below the bri ghtness of an isolype matched negative control; whereas a di m designation indicates that the level of staining can be near the level of a. negative stain, but can also be brighter than an isotype .matched control, >093] Identifiers of individual cells, for example different cell types or cell type variants, may be fluorescent, as for example labeling of different unit cell types with different levels of a fluorescent compound, and the like as described herein above. In some aspects of embodiments where two cell types are to be mixed, one is labeled and the other not. in some aspects of embodiments where three or more cell types are to be included, each cell type may labeled to different levels of fluorescence by incubation with different concentrations of a labeling compound, or for different times. As identifiers of large number's of cells, a matrix of fluorescence labeling in tensities of two or more different fluorescent colors may be used, such that the number of distinct uni t ceil types that are identifi ed is a number of fluorescent l evels of one color, e.g., carboxyfluorescein succinitnidyl ester (CFSE), times the number of fluorescence levels employed of the second color, e.g, tetramethylrhodamine isothiocyanate (TRJTC), or the like, times the number of levels of a third color, etc. Alternatively,

2015268664 11 Dec 2015 intrinsic light scattering properties of the different cell types, or characteristics of the BioMAPs of the test parameters included in the analysis, may be used in addition to or in place of fluorescent labels as unit cell type identifiers.

1094] In another aspect, cells may be enriched, depleted, separated, sorted and/or purified, using conventional affinity or antibody techniques. For example, the ligand and/or an tibody may be conjugated with labels to allow for ease of separation of the particular cell type, e.g, magnetic beads; biotin, which binds with high affinity to avidin or streptavidin; tluoroehromes, which can be used with a fluorescence activated ceO sorter; haptens; and the like.

1095] In one embodiment, the ligand, agent, and/or antibodies described herein may be directly or indirectly conjugated to a magnetic reagent, such as a super-paramagnetic microparticle (microparticle). Direct conjugation to a magnetic particle .may be achieved by use of various chemical linking groups, as known in the art. in some embodiments,the antibody is coupled to the microparticles through side chain amino or su.fhydryl groups and heierofunctional cross-linking reagents.

1090] A large number of hetemfunctional compounds are available for linking to entities. For example, at least, 3-(2.-pyridyid.ithio)propionic aeidN-hydroxysuecimmide ester (SPDP) or 4-(N~maleimidomethyi)-cycfohexane-l~carboxylic acid Nhydroxysuccinimide ester (SMCC) with a reactive suifltydfyl group on the antibody and a reactive amino group on the magnetic particle can be used. An example of a magnetic separation device is described in WO 90/07380, PCT/US96/00953, and EP 438,520, incorporated herein by reference in its entirety.

{0097] The purified cell population may be collected in any appropriate medium. Suitable media may include, for example, Dulbecco’s Modified Eagle Medium (d'MEM), Hank's Basic Salt Solution (BBSS), Dulbecco's phosphate buffered saline (dPBS), RPMI, 'lscove's modified Dalbecco's medium (IMDM), phosphate buffered saline (PBS) with. 5 mM EDTA, etc,, frequently supplemented with fetal calf serum (PCS), bovine serum albumin (BSA), human serum albumin (HSA), and StemPro®hESC SFM.

2015268664 11 Dec 2015

1098] In one embodiment, the celts expressing markers characteristic of the pancreatic endocrine lineage are enriched by treatment with at least one agent that selects celts that do not express markers characteristic of the pancreatic endocrine lineage. In an alternate embodiment, the cells expressing markers characteristic of the pancreatic endocrine lineage are enriched by treatment with at least one agent that selects for insu lin-producing cells,

1099] Using the methods described herein, cell populations or cell cultures may be enriched in cell content by at least about 2- to about 1000-fold as compared to untreated cell populations or cell cultures. In some embodiments, cells expressing markers characteristic of the pancreatic endocrine lineage may be enriched by at least abou t 5to about 500-fold as compared to untreated cd! populations or cell cultures. In other embodiments, cells expressing markers characteristic of the pancreatic endocrine lineage may be enriched from at. least about 10- to about 200-fold as compared to untreated cell populations or ceil cultures. In still other embodiments, cells expressing markers characteristic of the pancreatic endocri ne lineage may be enriched from at least about 20- to about 100-fold as compared to untreated cell populations or ceil cultures. In yet other embodiments, cells expressing markers characteristic of the pancreatic endocrine lineage may be enriched from at least about 40- to about 80-fold as compared to untreated cell populations or cell cultures. In certain embodiments, cells expressing markers characteristic of the pancreatic endocrine lineage may be enriched from at least about 2- to about 20-fold as compared to untreated cell populations or cell cultures.

Characterization of Ceils Derived from Pluripotent Stem Cells [01001 The formation of differentiated cells from pluripotent stem ceils may be determined bv determining the expression of markers characteristic of a given differentiated cel! type. In some embodiments, the identification and characterization of a differentiated cell is by expression of a certain marker or different expression levels and patterns of more than one marker.

[0101 ] Specifically, the presence or absence, the high or low expression, of one or more the marker(s) can typify and identify a cell-type. Also, certain markers may have

2015268664 11 Dec 2015 transient expression, whereby the marker is highly expressed during one stage of development and poorly expressed in another stage of development. The expression of certain markers can be determined by measuring the level at which the marker is present in the cells of the cell culture or cell population as compared to a standardized or normalized control marker. In. such processes, the measurement of marker expression can be qualitative or quantitative. One method of quantitating the expression of markers that are produced by marker genes is through the use of quantitative PCR (Q-PCR), Methods of performing Q-PCR are well known in the art. Other methods which are known in the art can also be used to quantitate marker gene expression. For example, the expression of a marker gene product can be detected by u sing antibodies specific for the marker gene product of interest (e.g. Western blot, flow cytometry analysis, and the like). In certain embodiments, the expression of marker genes characteristic of differentiated cells as well as the lack of significant expression of marker genes characteristic of differentiated cells may be determined.

U02] The expression of tissue-specific gene products can also be detected at the mRNA level by Northern blot analysis, dot-blot hybridization analysis, or by reverse transcriptase initiated polymerase chain reaction (RT-PCR) using sequence-specific primers in standard amplification methods. See U.S. Pat, No. 5,843,780 for further details. Sequence data for particular markers listed in this disclosure can be obtained from public databases such as GeuRank.

U03] Pluripotent stem cells may express one or more of the stage-specific embryonic antigens (SSEA) 3 and 4, and markers detectable using antibodies designated Tra-I 60 and Tra-l-8t (Thomson et al., Science 282:1145, 1998). Differentiation of pluripotent stern cells in vitro results in the loss of SSEA-4, Tra 1-60, and Tra 1-81 expression (if present) and increased expression of SSEA-1. Undifferentiated pluripotent stem cells typically have alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde, and then developing with Vector Red as a substrate, as described, by the manufacturer (Vector Laboratories, Burlingame Calif ). Undifferentiated pluripotent stem cells also typically express OCT4 and TERT, as detected by RT-PCR.

2015268664 11 Dec 2015

U04] Markers characteristic of the pancreatic endoderm lineage are selected from the group consisting of PDX1, HNF1 beta, PTF1 alpha, HNF6_S F1B9 and PROXL Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endoderm lineage, i n one aspec t of the present invention, a ceil expressing markers characteristic of the pancreatic endoderm lineage is a pancreatic endoderm cell.

(105] Markers characteristic of the definitive endoderm lineage are selected from the group consisting of SOX 17, GATA4, 'HNF3 beta, GSC, CER1, Nodal, FGF8, Brachyury, Mix-like homeobox protein, FGF4, CD48, eomesodermin (EOMES), DKK4, FGF 17, GATA6, CD1.84, C-Kit, CD99, and OTX2. Suitable for use in the present invention is a ceil tha t expresses at least one of the markers characteristic of the definitive endoderm lineage. In one aspect of the present invention, a cell expressing markers characteristic of the definitive endoderm lineage is a primitive streak precursor cell.

In an alternate aspect, a cell expressing markers characteristic of the definitive endoderm lineage is a mesendoderm cell In an alternate aspect, a cell expressing markets characteristic of the definitive endoderm lineage is a definitive endoderm cell

1106] Markers characteristic of the pancreatic endocrine lineage are selected from the group consisting of NGN3, NEGROD, ISLI, PDX1, NKX6.1, FAX4, NGN3, and FFF-1 alpha, in one embodiment, a pancreatic endocrine cell is capable of expressing at least one of the following hormones: insulin, glucagon, soma tostatin, and pancreatic polypeptide. Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endocrine lineage, in one aspect of the present invention, a cell expressing markers characteristic of the pancreatic endocrine lineage is a pancreatic endocrine cell. The pancreatic endocrine cell may be a pancreatic hormone-expressing cell. Alternatively, the pancreatic endocrine cell may be a pancreatic hormone-secreting cell (0107] In one aspect of the present invention, the pancreatic endocrine cell is a cell expressing markers characteristic of the β cell lineage. A cell expressing markers characteristic of the β cell lineage expresses PDX1 and at least one of the following transcription factors: NGN3, NKX2.2, NKX6.1, NEGROD, ISLI, HNF3 beta.

2015268664 11 Dec 2015

MAFA, PAX4, and PAX6. in one aspect of the present invention, a cell expressing markers characteristic of the β cell lineage is a β cell.

Pluripotent Stem Cells

Characterization of Pluripotent Stem Ceils

1108] Pluripotent stem cells may express one or more of the stage-specific embryonic antigens (SSEA) 3 and 4, and markers detectable using antibodies designated Tra-i60 and Tra-1-81 (Thomson et al, Science 282; 1145 1998), Differentiation of pluripotent stem cells in vitro results in the loss of SSEA-4, Tra- 1-60, and Tra-1-81 expression (if present) and increased expression of SSEA-1, Undifferentiated pluripotent stem cells typically have alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde and then developing with Vector Red as a substrate, as described by the manufacturer (Vector Laboratories, Burlingame Calif.). Undifferentiated pluripotent stem cells also typically express Oct-4 and TERT, as defected by RT-PCR,

1109] Another desirable phenotype of propagated pluripotent stem cells is a potential to differentiate into cells of all three germinal layers: endoderm, mesoderm, and ectoderm tissues. Pluripotency of stem cells can be confirmed, for example, by injecting cells into severe combined immnnodefieieni (SCID) mice, fixing the teratomas that form using 4% paraformaldehyde, and then examining them histologically for evidence of cell types from the three germ lay era. Alternatively, pluripotency may be determined by the creation of embry oid bodies and assessing the embryoid bodies for the presence of markers associated with the three germinal lay era.

|0110] Propagated pluripotent stem cell lines may be karyotyped using a standard G-banding technique and compared to published, karyotypes of the corresponding primate species. It Is desirable to obtain cells that have a ’’normal karyotype,” which means that the cells are euploid, wherein all human chromosomes arc present and not noticeably altered.

Sources of Pluripoten t Stem Ceils

2015268664 11 Dec 2015

HU] The types of pluripotent stem ceils that may be used include established lines of pluripotent ceils derived from tissue formed after gestation, including pre-embryonie tissue (such as, for example, a blastocyst), embryonic tissue, or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation. Non-limiting examples are established lines of human embryonic stem cells or human embryonic germ cells, such as, for example the human embryonic stem cell lines Hl, H7, and H9 (WiCell). Also contemplated is use of the compositions of this disclosure during the initial establishment or stabilization of such cells, in which case the source cells would be primary pluripotent cells taken directly from the source tissues. Also suitable are cells taken from a pluripotent stem cell population already cultured in the absence of feeder cells, as well as a pluripotent stem cell population already cultured in the presence of feeder cells. Also suitable are mutant human embryonic stem cell lines, such as, for example, BGOlv (BresaGen, Athens, GA). Also suitable are cells derived from adult human somatic cells, such as, for examples, cells disclosed in Takahashi et al, Cell 131: 1-12 (2007), (112] In one embodiment, human embryonic stem cells are prepared as described by

Thomson et al. (U.S. Pat. No, 5,843,780; Science 282:1145, 1998; Curr, Top, Dev; Biol, 38:133 ff„ 1998; Proc, Natl, Acad. Sci. U.S.A. 92:7844, 1995),

I'll31 Also contemplated, are pluripotent stem cells that are derived from somatic cells, in one embodiment, pluripotent stem cells suitable for use In the present invention may be derived according to the methods described in Takahashi er al (Cell 126: 663-676, 2006).

(0.114] In an alternate embodiment, pluripotent stem cells suitable for use in the present invention may be derived according to the methods described in Li etal (Ceil Stem Cell 4: 16-19,2009).

|Θ11$] In an alternate embodiment, pluripotent stem celts suitable for use in the present invention may be derived according to the methods described in Maherali et at (Cell Stem Cell I: 55-70,2007),

2015268664 11 Dec 2015

1116} In an alternate embodiment, pluripotent stem cells suitable for use in the present invention may be derived according to the methods described in Stadtfeld et «/(Ceil Stem Cell 2; 230-240), (117) In an alternate embodiment, pluripotent stem cells suitable for use in the present invention may be derived according to the methods described in Nakagawa el al {Nature Biotechnology 26: 101 -106,2008).

(118] In an alternate embodiment, pluripotent stem cells suitable for use in the present invention may be derived according to the methods described in Takahashi et al (Cell 131: 861-872,2007), (1.19] In an alternate embodiment, pluripotent stem cells suitable for use in the present invention may be derived according to the methods described in US patent application Ser, No. 61/256,149, assigned to Centocor R&D, Inc.

Culture of Pluripotent Stem Cells (120] In one embodiment, pluripotent stem cells are cultured on a layer of feeder cells or extracellular matrix protein that support the pluripotent stem cells in various ways, prior to cultu ring according to the methods of the present invention. For example, pluripotent stem cells are cultured on a feeder cell layer that supports proliferation of pluripotent stem cells without undergoing substantial differentiation. The growth of pluripotent stem cells on a feeder cell layer without differentiation is supported using (i) Obtaining a culture vessel containing a feeder cell layer; and (ii) a medium conditioned by culturing previously with another eel! type, or a non-conditioned medium, for example, free of serum or even chemically defined.

(0121] In another example, pluripotent stem cells are cultured in a culture system that is essentially free of feeder cells, but nonetheless supports proliferation of plan potent stem ceils without undergoing substantial differentiation. The growth of pluripotent stem cells in feeder-cell .free culture without differentiation is supported using (i) an adlayer on a solid substrate surface with one or more extracellular matrix proteins; and (ii) a medium conditioned by culturing previously with, another cell type, or a non-conditioned medium, for example, free of serum or even chemically defined.

2015268664 11 Dec 2015

112-21 In an alternate embodiment, pluripotent stem cells are cultured on a surface modified plate containing from at least about 0.5% N, a sum of O and N of greater than or equal to 17.2% and a contact angle of at least about 13.9 degrees in a medium conditioned by culturing previously with another ceil type, or a non-conditioned medium, for example, free of serum or even chemically defined.

11.33] Culture medium: An example of cell, culture medium suitable for use in the present invention may be found in US20020072117, Another example of cell culture medi um suitable for use in the present invention may be found in US6642048. Another example of cell culture medium suitable for use in the present invention may be found in WO2005014799. Another example of cell culture medium suitable for use in the present invention may be found in Xu et al (Stem Cells 22: 972-980, 2004). Another example of cell culture medium suitable lor use in the present invention may be found in 13820070010011, Another example of cell culture medium suitable for use in the present Invention may be found in Cheon et al (BioReprod

DOR 10.1095/biolrep.rod, 105,046870; 19 Oct 2005), Another example of cell culture medium suitable for use in the present invention may be found in Levenslein et al (Stem Cells 24; 568-574, 2006), Another example of cell culture medium suitable for use in the present invention may be found in US20050148070. Another example of cell culture medium suitable for use in the present invention may be found in {JS20050233446. Another example of cell culture medium suitable for use in the present invention may be found in US6800480. Another example of cell culture medium suitable for use in the present invention may be found in US20050244962. Another example of ceil culture medium suitable for use in the present invention may be found in WO2005065354. Another example of cell culture medium suitable for use in the present invention may be found in WO2005086845.

f 0124] Suitable culture media may also be made from the following components, such as, for example, Dulbecco's modified Eagle’s medium (DMEM), Gibco # 11965-092; Knockout Dulbecco’s modified Eagle’s medium (KO DMEM), Gibco # 10829-018; Ham's F12/50% DMEM. basal medium; 200 mM L-glutamine, Gibco # 15039-027; non-essential amino acid solution, Gibco 11140-050; β-mercaptoethanol, Sigma # M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco # 13256029.

2015268664 11 Dec 2015

Differentiation of Pluripotent Stem Cells

H25] In one embodiment, pluripotent stem cells are propagated in culture and then treated in a manner that promotes their differentiation into another cell type. For example, pluripotent stem cells formed using the methods of the present inven tion may be differentiated into neural progenitors or cardiomyocytes according to the methods disclosed in WO2O07030870.

(126] In another example, pluripotent stem cells formed using the methods of the presen t invention may be differentiated into hepatocytes according to the methods disclosed in US patent 6,458,589.

Differentiation of Pluripotent Stem Cells Formed Using the Methods of the Present Invention into Cells Expressing Markers Characteristic of the Definitive Endoderm Lineage

1127] Pluripotent stem cells formed using the methods of the present invention may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by any method in the art,

1.128] For example, pluripotent stein cells formed using the methods of the present invention may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in D’Amour et til, Nature Biotechnology 23, 1534- 1541 (2005).

^129] For example, pluripotent stem cells formed using the methods of the present inven tion may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in Shinozaki et al, Development 131, 1651 ~ 1662(2004), [0130] For example, pluripotent stem cells formed using the methods of the present inven tion may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in McLean et id, Stem Cells 25, 29 - 38 (2007).

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H31] Fo r exampl e, pluripo tent stem cells formed using the me thods of the present invention may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in D’Amour et«/, Nature Biotechnology 24,1392 - 1401 (2006), tl32f In another example, pluripotent stem cells formed using the methods of the present invention may be differentiated into cells expressing markers charac teristic of the definiti ve endoderm lineage according to the methods disclosed in US patent application Ser, No. 11/736,908, assigned to LifeScan, Inc.

1133] tit another example, pluripotent stem cells formed using the methods of the present invention may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in US patent application Ser, No. 11/779,311, assigned to LifeScan, Inc,

H34j In another example, pluripotent stem cells formed using the methods of the present invention may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in US patent application Ser, No. 12/493,741, assigned to LifeScan, Inc,

H35] In another example, pluripo tent stem cells formed using the methods of the presen t invention may be differentiated into ceils expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in US patent application Ser. No, 12/494,789, assigned to LifeScan, Inc.

(03.36] Formation of cells expressing markers characteristic of the definitive endoderm lineage may be determined by testing for the presence of the markers before and after following a particular protocol . Pluripotent stem cells typically do not express such markers. Thus, differentiation of pluripotent cells i s detected when cells begin to express them,

Differentiation of Pluripotent Stem Cells Formed Using the Methods of the Present Invention into Cells Expressing Markers Charaeierisfie of the Pancreatic Endoderm

Lineage

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H37] Pluripotent -stem cells formed using the methods of the present invention may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage by any method in the art.

1138] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage according to the methods disclosed in D’Amour et at, Nature Biotechnology 24,1392 - 1401 ¢2006).

(139] For example, cells expressi ng markers characteristic of the definitive endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with a fibroblast growth factor and the hedgehog signaling pathway inhibitor KAAD-cyclopamine, then removing the medium containing the fibroblast growth factor and KAAD-eyelopamine and subsequently culturing the cells in. medium containing retinoic acid, a fibroblast growth factor and KAAD-eyelopamine, An example of this method is disclosed in Nature Biotechnology 24, 1392 - 1401 (2006).

1140] For example, cells expressing markers characteristic of the definitive endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid one fibroblast growth factor for a period of time, according to the methods disclosed in US patent application Ser. No, 1.1/736,908, assigned to LifeScan, Inc.

[01.41 ] For example, ceils expressing markers characteristic of the definitive endoderm lineage obtained according to the methods of the present in vention are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the ceils expressing markers characteristic of the definitive endoderm lineage with retinoic acid (Sigma-Aidrteh, MO) and exendin 4, then removing the medium containing DAPT (Sigma-Aldrieh, MO) and exendin 4 and subsequently culturing the cells in medium containing exendin 1, IGF-1 and HGF.

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An example of ibis method is disclosed in Nature Biotechnology 24, 1392 - 1401 (2006)..

1142] For example, cells expressing markers characteristic of the pancreatic endoderm lineage obtained seconding to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characterist ic of the pancreatic endoderm lineage in medium containing exendin 4, then removing the medium containing exendin 4 and subsequently culturing the cells in medium containing exendin 1,IGF-1 and HGF, An example of th is method is disclosed in D’ Amour et al, Nature Biotechnology, 2006,

1143] For example, cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the me thods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing DAFT (Sigma-Aldrich, MO) and exendin 4, An example of this method is disclosed in D’ Amour et a/, Nature Biotechnology , 2006.

1144] For ex ample, cells expressin g markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present inven tion are further differen tiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by cul turing the cells expressing markers characterist ic of the pancreatic endoderm lineage in medium containing exendin 4. An example of this method is disclosed in D’ Amour et al, Nature Biotechnology, 2006, {01.45] For example, cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present in vention are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent application Ser. No, 11/736,908» assigned to LifeScan, Inc.

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H46] For example, cells expressing markers characteristic ofthe pancreatic endoderm lineage obtained according to the methods of the present invention are farther differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by trea ting the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent application Ser, No. 11/779,31 b assigned to LifeSean, Inc.

»147| For example, ceils expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods ofthe present invention are farther differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent application Ser. No. 60/953,178, assigned to LifeSean, inc.

1148] For example, ceils expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present invention are farther differentiated into cells expressing markers characteristic of (he pancreatic endocrine lineage, by trea ting the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent application Ser. No. 60/990,529, assigned to LifeSean, Inc.

Differentiation ofPluripotent Stem Celt's Formed iAing the Methods ofthe Present invention into Cells Expressing Markers Characteristic ofthe Panereatie Endocrine

Lineage (0149] Pluripotent stem cells formed using the methods of the present invention, may be differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage bv any method in the art.

[Θ150] For example, cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods ofthe present invention ate farther differentiated into cells expressing markers characteristic of the pancreatic endocrine •35

2015268664 11 Dec 2015 lineage, by culturing the cells expressing markers characterist ic of the pancreatic endoderm lineage in medium containing exendin 4, then removing the medium containing exendin 4 and subsequently culturing the cells in medium containing exendin 1, IGF-1 and HGF, An example of this method is disclosed in D’ Amour et cti, Nature Biotechnology, 2()06.

H51] For example, cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing DAPT (Sigma-Aldrich, MO) and excndin 4. An example of this method is disclosed in D’ Amour et cii. Nature Biotechnology, 2006.

1152] For example, cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by cul turing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing exendin 4, An example of this method is disclosed in D’ Amour etaf Nature Biotechnology, 2006, (153] For example, ceils expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent application Ser. No, 11/736,908» assigned to LifeSean, Inc, |0154] For example, cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present invention are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according

2015268664 11 Dec 2015 to the methods disclosed in US patent application Ser. No. 1 1/779,311, assigned to LifeScan, Inc.

1155] For example, cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of'the present invention are further differentiated into cells expressing markers characteristic of Ac pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent applica tion Ser. No. 60/953,178, assigned to LifeScan, Inc.

(156] For example, cells expressing markers characteristic of the pancreatic endoderm lineage obtained according to the methods of the present in vention are farther differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent application Ser. No, 60/990,529, assigned to LifeScan, Inc,

1157] The present invention is further illustrated, but not limited by, the following examples.

EXAMPLES

Example 1

Differentiation of Human Embryonic Stem Cells of the Cell Line Hl to

Pancreatic Endocrine Cells in the Absence of Fetal Bovine Serum {(1158] Cells of the human embryonic stem cells line Hl at various passages ( p40 to p52) were cultured on MATRIGEL (1:30 dilution) coated dishes and differentiated into pancreatic lineages using a multi-step protocol as follows:

a. Stage I (Definitive Endoderm): Human embryonic stem cells were cultured in RPM'l medium supplemented with 2% fatty acid-free BSA (Catalog# 68700, Proliant, 1A), and 100 ng/rnl activin A (R&D Systems, MN) plus 20 ng/ml WNT

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3a (Catalog# 1324-WN-002, R&D Systems, MN) plus 8 ng/ml of bFGF (Catalog# 100-18B, PeproTech, NJ), for one day. Ceils were then treated with RPMI medium supplemented with 2% BSA and 100 ng/ml activin A plus 8 ng/ml of bFGF for an additional two days, then

b. Stage II (Primitive gut tube): Cells were treated with RPMI -t 2% fatty acid-free BSA and 50 ng/ml FGF7 and. 0,25 μΜ SANT-1 (#84572, Sigma, MO), for two to three days, then

c. Stage HI (Posterior foregut); Cells were treated with DMEM/High-Glucose supplemented with 1:200 dilution of ITS-X (invitrogen, CA) and 0,1 % BSA (Lipid Rich) (invitrogen, Ca No, 11021-045), 50 ng/ml FGF7, 0.25 μΜ SΑΝΤΙ, 2 μΜ Retinoic acid(RA) (Sigma, MO), 100 ng/ml of Noggin (R & D Systems, MN), and Activin A at 20 ng/ml for four day s; In certain variations, Noggin was replaced with the A.MPK inhibitor 6-(4-(2-Piperidin-I -ylethoxy)phenyl]-3pyridin-4-ylpyTazolo[I,5-a]pyriinidine (Sigma, No. P5499) at a concentration of 2 μΜ. In yet other variations, a P38 inhibitor (4-[4-(4-Fluorophenyl)-l-(3phenylpropyl)-5-pyridin-4-yl-lH-imidazol-2-yi3but-3-yn-l-oi) (disclosed in US Patent 6,521655) was added at 2,5 μΜ, then

d. Stage IV (Pancreatic endocrine precursor): Cells were treated with DMEM/HighGlucose supplemented with 1:200 dilution of ITS-X (Invitrogen, CA) and 0,1% BSA (invitrogen, Ca), 100 ng/ml Noggin, 1 μΜ ALK5 inhibitor (SB-208, disclosed in Molecular Pharmacology 2007 72; 152-161) for three days, then

e. Stage V (Pancreatic endocrine cells): Cell were treated with DMEM/HighGlucose supplemented with 1:200 dilution of ITS-X (Invitrogen, CA), 0,1% BSA (Invitrogen, Ca), 1 μΜ ALK5 inhibitor II (Catalog# 616452, Calhiochem, Ca) for seven days., then

f. Stage VI (Mature Pancreatic endocrine cells): Cells were treated with DMEM/High-Glucose supplemented with 1:200 dilution of ITS-X (Invitrogen, CA), 0,1% BSA (Invitrogen, Ca) for seven days, with media changes every other day.

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Example 2

Flow Cytometric Characterization and Sorting of Enriched Various Pancreatic Ceil Lineage»

1159] To facilitate the isolation and characterization of novel cell populations form various stages of the differentiation process outlined in Example 1, a detailed characterization of the cells obtained from the various stages was done by flow cytometry'. A complete list of antibodies used and the expression levels of surface markers at various stages of differentiation is shown in Table I.

H60] Cells of the human embryonic stem cell line Η1 at various passages (p40 to p52) were cultured on MATKIGEL-coated plates, and differentiated into pancreatic endocrine cells using the protocol described in Example I,

U61] Cells at different stages of maturation (posterior foregut (Stage HI), endocrine precursor cells (Stage IV), pancreatic endocrine cells (Stage V) or mature pancreatic endocrine cells (Stage VI) were gently released by incubation in TrypLE Express (Invitrogen # 12604, CA) for 2-3 minutes at 37'’C and washed twice in BD FACS staining buffer containing 2% BSA (BD # 554657, CA). Approximately 0,5-1 χίΟ” cells were re-suspended in 100-200 μΐ blocking buffer (0.5% human gamma-globulin diluted 1:4 in staining buffer (BD, CA) for staining. For staining with directly conjugated primary antibodies, the appropriate antibody was added to the cells at a final dilution of 1:20, and ceils and incubated for 30 min at 4° C. For unconjugated antibodies, primary antibodies were added to cells at 1:50-1:100 dilution and cells incubated for 30 min at 4*C followed two washes in staining buffer. Cells were then incubated in the appropriate secondary antibodies at 1:500 dilution. Stained cells were re-suspended in 300 μΐ staining buffer and 5-10 μϊ of 7 A AD added for live/dead cell discrimination prior to analysis on the BD FACS Canto 11, [0162] For cell sorting, approximately 30-40 million cells were similarly processed as for flow cytometric analysis. Cells were stained with the appropriate antibodies as shown in Table 11, Cells were sorted either into two or three sub-populations as summarized in Table 11. Cell sorting gates were established based on the isotype matched controls. An aliquot of sorted cells were analyzed for purity following the sorting followed by

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PCR analysis for expression of key pancreatic markers. RNA was collected using the Rneasy Mini Kit» Qiagen, CA) was collected from presort sample, and the various fractions.

1163] Cell surface markers used for sorting were selected based on the expression of various markers in populations of cells analyzed at different stages of the differentiation protocol outlined in Example 1. The markers employed in this study are disclosed in Table Π. Briefly, the surface markers disclosed in Table Π were- used either singly or in combination to sort various populations of cell s. Samples of the sorted cells were taken to analyze the expression of markers characteristic of the pancreatic endocrine lineage by real-time PCR,

Stinting of Cells Expressing Markers Charaetefisiie of th e Pancreatic .£«dbcrz»e Lineage

H64] Antibodies to CD56 and CD 13 were· used to sort a population of cells obtained from Stage I V of the differentiation protocol outlined in Example 1. Three populations of cells were identified: a) CD56 ’CD l 3”, b) CD56CD13' and c) CD56 CD13 *' populations of cells. The CD56⁺CD13' popu lation was enriched approximately 1,3 fold following sorting, and the sorted cells were highly enriched for the expression of markers characteristic of the pancreatic endocrine lineage» including NEUROD, NGN3, PDX1, NKX6.1, NKX2.2 and PAX-4,when compared, to unsorted cells at stage IV, or populations of CD56 CD13 cells, or populations of CD56 CD13* cells. See Figure 1, panels a~f.

{0165] In a second series of experiments, antibodies to CD.133 were used to sort a population of cells obtained from Stage IV of the differentiation protocol outlined in Example 1« Two populations of cells were identified: a) CD 133 y and b) GDI.33 popula tions of cells. The CD! 33' population was enriched approximately 1,9 fold following .sorting, and the sorted cells were highly enriched for the expression of markers characteristic of the pancreatic endocrine lineage, including NEUROD, NGN3, PDXl, NR.X6.1, N.KX2.2 and PAX-4,when compared to unsorted cells at stage IV, or populations of CD 133’ cells. See Figure 2, panels a-f.

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U66] In a third series of experiments, antibodies to CD49c were used to sort a population of cells obtained from Stage IV of the differentiation protocol outlined in Example 1, Two populations of cells were identified' a) CD49c^H\ and b) CD49e^!t) populations of ceils. CD49c^u> ceils were enriched approximately 3.1 fold following sorting, and the sorted cells were highly enriched for the expression of markers characteristic of the pancreatic endocrine lineage, including NEUROD, NGN3, PDXI, andNKXtiJ when compared to nnsorted cells or CD49e^fii cells. See Figure 3, panels a~d,

H671 In a fourth series of experiments, antibodies to CD56 and CD 15 were used to sort a population of cells obtained from Stage IV of the differentiation protocol outlined in Example 1. The following populations of cells were identified: a) CD56*CD15^U\ b) CD56 XD15^}a, c) CD 15^: and d) CD 15’ populations of cells. Populations of CD 15* cells were enriched approximately 1.1 fold following sorting. Populations of CD56 'CD15^!ii cells were highly enriched for the expression of markers characteristic of the pancreatic endocrine lineage i ncluding NEUROD, NGN3 , PDXI, NKX6.1, Insulin and glucagon compared to unsorted cells, or populations of CD56^rCD15^h‘ cells. See Figure 4, panels a-fi Similarly, populations of CD 15' cells sorted using a s ingle marker were hi ghly enriched for the expression o f markers characteristic of the pancreatic endocrine lineage including NEUROD, NGN3, ΡΌΧ1, NKX6.1, NK.X2.2, PAX-4, glucagon and insulin, when compared to nnsorted cells or populations of CD! 5 ^s cells. See Figure 5, panels a-h.

1168] In a fifth series of experiments, antibodies io CD56 and CDS? were used to sort a population of cells obtained from Stage IV of the differentiation protocol outlined in Example 1, Two populations of cells were identified: a) CD56^!CD57, and b)

CD56 CD57’populations of cells. Populations of CD56’‘CD57^r cells were enriched approximately 1.9 fold following sorting. CD56XD57’ cells were highly enriched for the expressi on of markers characteristic of the pancreatic endoderm lineage, including NEUROD, NGN3, PDXI, NKX6.1, NKX2.2, as wells as insulin and glucagon, when compared to unsorted cells or populations of CD56 CD57' cells. See Figure 6, panel a~g. Similar results were observed when populations of cells at Stage V of the differentiation protocol outlined in Example 1 were sorted using antibodies to CD56 and CD57.

2015268664 11 Dec 2015 >169] In a sixth series of experiments, antibodies to CD56 and CD 184 were used to sort a population of cells obtained from Stage IV of the differentiation protocol outlined in Example 1, Three populations of cells were identified' a) CD 184’, b) CD184‘, and ¢) CD56'CD1S4' populations of ceils. Table IV summarizes the expression of CD 184 in cells before and after the enrichment. Populations of CD! 84’ cells were enriched for the expression of markers characteristic of the pancreatic endocrine lineage, including PAX4, NEUROD, NKX6.1, PDXI and PTFI alpha. The expression of Z.TC1, Albumin and CDX2 was decreased. See Figure 7, panels a-i.

Sorting qf'fnsitlm Producing CWZs

H70J Antibodies to CD98 were used to sort a population of cells obtained from Stage VI of the differentiation protocol outlined in Example 1. . Two populations of cells were identified: a) €098^-^, and b) 0098^ populations of cells. Populations of CD98^+<i*‘^! cells were enriched approximately 1. .6 fold following sorting. CD98’^1::ili cells were enriched for the expression of NEUROD, NGN3, .insulin, and glucagon.

See Figure 8, panels a-d.

>171] In another series of experiments, antibodies to CD47 were used to sort a population of cells obtained from Stage V of the differentiation protocol outlined in Example 1.

Two populations of cells were iden tified: a) CD47^iftH, and b) CDd?⁵'*'' populations of cells. CDd?¹**'¹ cells were enriched approximately 3.3 fold following sorting, CD47^Ui) ceils were enriched for the expression, of NEUROD, NGN3, PDXI, NKX6.1, NKX2.2 and PAX4. See Figure 9, panels a-f.

[0172] In another series of experiments, antibodies to CD47 were used to sort a population of cells obtained from Stage VI of the differentiation, protocol outlined in Example 1. Two populations of cells were identified: a) CD47^sb‘”, and b) CD47^{i iX}' ^! populations of cells. CD47^Ux'* cells were enriched for the expression of PDX-1, NKX6.1, NKX2.2, PAX-4, RTF! a, NGN3, Insulin and Glucagon. See Figure 10, panels a.~h,

Example 3

Sorting of Inf receptor Positive Cells at Primitive Gut Tube stage (Stage 2)

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H73] Ceils of the human embryonic stem cell tine Hl at passage 44 were cultured on

MATRlGEE-eoated plates, and differentiated into insulin producing cells using the following protocol;

a. RPMI medium supplemented, with 2% fatty acid-free BSA (Catalog# 68700, Proliant, IA), and 100 ng/ml activin A (R&D Systems, MN) plus 20 ng/ml WNT-3a (Catalog# 1324-WN-002, R&D Systems, MN) plus 8 ng/ml of bFGF (Catalog# I00-18B, PeproTech, NJ), for one day followed by treatment with RPMI media supplemented with 2% BSA and 100 ng/ml activin A phis 8 ng/ml of bFGF for an additional two days (Stage I), then

b. RPMI + 2% BSA t- SO ng/ml FGF7 + 0.25 μΜ SANT-l (WS4572, Sigma, MO), for three days (Stage 2), then

c. DMEM/High-Glucose + I '200 dilution of ITS-X (Invitrogen, CA) + 0.1% BSA (invitrogen, Ca) 50 ng/ml FGF7 (Peprotech, NJ) + 0.25 p.M SANT-1 + 2 μΜ Retinoic acid (RA) (Sigma, MO) + 1.00 ng/ml. of Noggin (R & D Systems, MN) and 20 ng/ml. of activin A for four days (Stage 3)., then

d. DMEMZH'igh-Glucosc + I ;200 dilution of ITS-X (Invitrogen, CA) + 0.1% BSA (Invitrogen, Ca) + 100 ng/ml Noggin T 1 ttM AEK5 inhibitor (SCIO 120) + for three days (Stage 4) ►174] Stage 2 cells were dispersed into single cells using TrypLE Express (invitrogen, Carlsbad, CA) and washed in stage 4 basal media (DM~Flg + ITS-X + BSA).

Released ceils were spun and the resulting cell pellet suspended, in a staining butter consisting of 2% BSA, 0.05% sodium azide in PBS (Sigma, MO). As appropriate, the cells were Fc-receptor blocked for 15 minutes using a 0.1% γ-globulin (Sigma) solution. Aliquots (approximately 10* cells) were incubated with Lif receptorPhycoeryfhrin (PE) (R & D Systems, MN) conjugated monoclonal antibodies (5 μΐ antibody per 10⁽ⁱ ceils). Controls included appropriate isotype matched antibodies and unstained cells. All incubations with antibodies were performed for 30 mins at 4”C after which the cells were washed with the staining buffer. Stained cells were sorted, on a FACS Aria (BD, Ca). RN A (Rneasy Mini Kit, Qiagen, CA) was collected from

2015268664 11 Dec 2015 presort sample, Lif receptor*· traction and Lif receptor negative fraction. The Lif receptor expression level and pattern is summarized in Table 111.

>175} Table HI summarizes die expression of Lif receptor at days 2 and 3 of stage 2. By day 3 of stage 2. approximately?0% of the cells expressed Lif receptor. As summarized in Table HI, high expression of Lif receptor was unique to stage 2 cells, as stage 3 and 4 cells showed minimal expression of Li f receptor. As shown in Figure 11, panels a-b, stage 2 cells enriched for the Lif receptor showed a signi ficant increase in expressi on of HNF4 alpha as compared to unsorted cells or Lif receptor negative cells. Expression of Lif receptor mRNA as measured by real-time PCR was also enhanced .in cell fraction containing Lif-recqptor positive celts.

Example 4

Magnetic Bead Sorting for Cells for the Depletion of SSEA-4+ Cells to Reduce Tumor Formation in Vivo >176] Expression of the SSEA4antigen is a key indicator of pluripotency in human embryonic stem cells, and expression of this marker Is greatly down regulated during the differentiation process. However, residual SSEA-4 positive cells may be responsible for tumors and/or teratomas that are observed following transplantation of partially differentiated cells. To reduce teratoma formation, methods were developed to deplete contaminating SSEA4' cells from differentiated cells prior to transplantation.

10177] Cells of the human embryonic stem cell line Hl (passage 40-52) were differentiated to various stages of the differen tiation protocol outlined in Example 1. In order to test proof of concept and efficacy of SSEA-4 deple tion, this study was first done with cells differentiated only to the primitive gut tube stage (Stage 2 in the differentiation protocol outlined in Example I) in order to ensure cells still retained higher levels of SSEA-4 expression. In subsequent experiments, cells expressing SSEA-4 were depleted in populations of cells differentiated at Stage 4 of the differentiation protocol outline in Example 1, See Table V for the results observed. Cells were gently released into single cells by incubation in TrypLE Express (Invitrogen # 12604, CA) for 2-3 minutes at 37^,;iC, To enhance cell survival and viability during depletion, anti44

2015268664 11 Dec 2015 apoptotic agents including 10μΜ Y-27632 (Cat # Y 0503, Sigma, St Louis MO) or 0.5pM Thiazovivin (Cat # 04-0017, Stemgent, San Diego, C A) were added to the cells prior to collection and in ail isolation buffers,

Il 78] Cells were washed in Isolation Buffer containing Ca~ and Mg“⁺ free phosphate buffered saline (PBS) supplemented with 0,1 % BSA and 2mM EDTA. Between 10100 x 10^fi cells were re-suspended in isolation buffer a final cell density of 5 x 10⁶ cells per 500 μΐ. Twenty five μΐ SSEA-4 antibody was added per 500 pi of cells and cells incubated tor 15-20 minutes at room temperature on a gentle rocker to ensure continuous mixing. Cells were washed in Isolation buffer by spinning at 300xg for 8 min. Supernatant was removed and cells re-suspended in original buffer volume and 50 pi of prewashed. SSEA-4 Depletion beads (DynaBeads® SSEA-4, Invitrogen, #11160D) added for every 500 μΐ of cell suspension. Cells and beads were mixed and incubated tor 15-20 minutes at room temperature with continuous gentle tilting and rotation. Cells were mixed by gentle pipetting and placed on a magnet tor 5 min. The supernatant containing SSEA-4 negative cells was transferred to a new tube and the process repeated 2-3 times to remove residual beads. Bead-bound SSEA4 cells were released from magnetic field and both cells populations counted and processed for FACS and PCR analysis, Ihe expression levels of SSEA4 in undifferentiated Hl cells, primitive gut cells and Stage IV cells, in both pre-sorted and sorted cell fractions is summarized in Table V.

*179] In populations of cells isolated at stage II of the differentiation protocol outlined in Example 1, 20.5 % of the cells expressed SSEA4 markers in prior to sorting. In contrast, only 1.8% of the cells expressed the SSEA4 post sort (Table V). The depletion resulted in removal of 91.2 % of the SSEA-4 positive cells, in another experiment using endocrine precursor cells, 25,3% of cells expressed SSEA-4 prior to depletion, but only 0,9% expressed SSEA-4 after depletion, resulting in 95.5 % removal of SSEA-4 positive cells (Table V). In contrast to differentiated cells, 91,2% of the population of undifferentiated embryonic stem cells expressed SSEA4.

(0180] The sorted SSEA4’ cells were highly enriched for the expression of pluripotency markers, including OCT4, NANOG, SOX2 and goosecoid (Figure 12 panels a-d).

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Sorting of SSEA4^<ifii’ and SSEA4^iL<'’’ Cells by FACS

1181] In order to investigate and confirm the depletion of pluripotent-marker (SSEA-4+) enriched cells from differentiated cells by flow cytometry, cells were differentiated to Stage VI as described in Example 1., Cells were released from culture using 'TrypleB Express cell dissociation buffer and cells prepared for sorting as described in Example 2, The SSEA-4 antibody (R&D Systems, Minneapolis, MN, Cat # FAB1435P) was used to isolate two cell fractions identified as SSEA-4(+)Hi and SSEA~4(~)Lo cells. Isolated cell fractions were analyzed for expression of pluripotency markers by RTPCR as described in Example 4. Similar to SSEA-4 depleted and enriched fractions obtained using magnetic beads separation, as described in Example 5, the sorted SSEA-4(T)Hi cells were highly enriched for the expression of pluripotency markers OCT4, NANOG, SOX2 and goosecoid, unlike the SSEA-4(-)Lo cells. See Figure 13 panels a-d.

Example 6

Transplantation of SSEA-4 Depleted Populations of Cells m 17vo

1182] In pilot experiments, SSEA-4 depleted cells weredtffereniiated to Stage IV of the differentiation protocol outlined in Example 1., and then transplanted into the kidney capsule of mice to test cell survival and engraftment. The data from the transplanted mice is summarized in Table VI, (0183] Five to six-week-old male scid-beige mice (C.B-lgh- s b/CsbmsTac-Prkdc^^-Lysi⁸ N7) were purchased from Taconie Farms. Mice were housed in mieroisolator cages with free access to sterilized food and water. In preparation for surgery, mice were identified by ear tagging and their body weight measured and their blood glucose determine by a hand held glucometer (One Touch, LifeScan), Mice were anesthetized with a mixture of isolflnrane and oxygen and the surgical site was shaved with small animal clippers. Mice were dosed with 0,1 mg/kg Buprenex subcutaneously preoperatfvely. The surgical site was prepared with successive washes of 70% isopropyl alcohol, 10% povidone-iodide, and 7(1% isopropyl alcohol and a left lateral incision

2015268664 11 Dec 2015 was made through the skin and muscle layers. The left kidney was externalized and kept moist with 0.9% sodium chloride, A 24G x. %” TV, catheter was used to penetrate the kidney capsule and the needle was removed. The catheter was then advanced under the kidney capsule to the distal pole of the kidney,

11841 During the preoperati ve preparation of the mice, the cells were centrifuged in a 1.5 ml. microftige tube and most of the supernatant removed, leaving just enough to collect the pellet of cells. The ceils were collected into a Ramin Pos-D positive displacement pipette and the pipette was inverted to allow for the cells to settle by gravity. The excess media, was dispensed leaving a packed ceil preparation for transplant.

1185] For transplanta tion, the Pos-D pipette tip was placed firmly in the hub of the catheter and the cells dispensed from the pipette through the catheter under the kidney capsule and delivered to the distal pole of the kidney. The lumen of the catheter was flushed with a small volume of culture media to deliver the remaining cells and the catheter withdrawn. The kidney capsule was sealed with a low temperature cautery and the kidney was returned its original anatomical position. The muscle was closed with continuous sutures using 5-0 vicryl and the skin closed with wound clips. Mice were dosed with LG mg/kg Metacam subcutaneously post-operatively. The mouse was removed from the anesthesia and allowed to fully recover,

1186] Following transplantation, mice were weighed once per week and blood glucose measured twice a week. At various intervals following transplantation, mice were dosed with 3 g/kg glucose IP and blood drawn via the retro-orbital sinus 60 minutes following glucose injection into micro&ge tubes containing a small amount of heparin. The blood was centrifuged and the plasma placed into a second nhcrofnge tube and frozen on dry ice and then stored at -80°C until human c-peptide assay was performed. Human e-peptide levels were determined using the Mereodia/ALPCO Diagnoses Ultrasensitive C-peptide ELISA according' to the manufacturer’s instructions.

2015268664 11 Dec 2015

H87] At the time of sacrifice, blood was collected as described above and mice euthanized.

The grafts were harvested from the kidney capsule and analyzed by real-time qPCR, immunohistochemistry, and pathology,

1188] Three groups of mice were transplanted with about 3,3 million cells each comprising of i) ceil clusters it) single cells (undepleted) and iii) SSEA4 depleted single cells. Cells differentiated to Stage IV were either released, with, gentle scarping to make small cell clusters, or released with TrypleE into single cells for SSEA-4 depletion. Following SSEA-4 depletion as outlined in Example 5 , both cell clusters and single cell prepatlons were replated in low attachment plates (Costar, Coming Incorporated, NY Cat# 347.1) overnight in precursor (Stage IV) cell differentiation medium prior to transplantation. The rock inhibitor Y-27632 dihydrochrolide monohydrate (Sigma, Cat # Y0503) was added to the culture overnight at a concentration of 10 μΜ. Following transplants, mice were monitored as described, above for up to 12 weeks post transplants. Graft survival was not visibly demonstrated in the single cells recipients (depleted or undepleted.) but was shown in 2 out of 5 mice receiving cell clusters. One out of 5 mice receiving cell clusters had detectable e-peptide levels at 12 weeks post transplantation. Poor graft survival was attributed to diminished cell quality and low numbers of ceils transplanted in the pilot experiment.

1189] The multi-step differentiation of human embryonic cells into mature, pancreatic endocrine cells through several intermediate steps including definitive endoderm (DE), pancreatic endoderm (PE) and pancreatic precursors is associated with dynamic changes in expression of surface markers. Although the differentiation protocol may produce as yet undefined, heterogeneous cell popula tions of multiple lineages including ectodermal and mesodermal cell types, tracking the changes in expression of surface markers in pancreatic di fferentiation medium could identify markers potentially useful in cell enrichment and purification. Table VII shows a summary of surface markers that either demonstrated an increase or decrease in expression, that may be useful for negative of positive selection of pancreatic endoderm cells.

Markers that decreased in expression during the differentiation process include

CD117, CD133, CD181, CD 184, CD200, CD221, CD326, CD55, CD57, CD9, and

CD98. Markers that increased in expression during the differentiation process include 48

2015268664 11 Dec 2015

CD13, CD141, GDIS, CD318, CD46, CD47, CD49c, CD49e, CD56, andCD73. These markers could singly or in various combinations be used to purify cell populations enriched for pancreatic endoderm and precursors.

Example 7

Flow Cytometric Sorting Procedures

1190] Ceils at different stages of maturation were gently released by incubation in TrypLE Express (Invitrogen # 12604, CA) for 2-3 minutes at 37°C and washed twice in BD FACS staining buffer containing 2% BSA (BD # 554657, CA), Based on cell yields, 20-50 xl O⁶ single cells were re-suspended in 2-3 ml of blocking buffer (0,5% human gamma-globulin diluted 1:4 in staining buffer (BD, CA) for staining. Fluorophore conjugated primary antibodies were added to the cells at a final dilation of 1:20 and cells and incubated for 30 min at 4° C, Following washes, stained cells were resuspended in 2-3 ml staining buffer and 50-60 μ I of 7AAD added for live/dead cell discrimination prior to analysis and cell sorting. Isotype matched control IgG antibodies were used for negative control staining. For calculating fluorophore compensation values prior to sorting, cell were either left unstained or stained with single fluorphore of Fluorescein isothiocyanate (FITC), Phyeoerythrin (PE) or Atlophycocyamn (AFC) the nuclear dye 7-Amjnoactinomuein D (7-AAD).

H91] Cell sorting was done using the BD FACSAria cell sorter and the BD FACSDiva software. Isotype matched control cells were used to establish negative gates for each cell sorting. For each cell sorting experiment, the photomultiplier (PMT) voltage settings were adjusted using the appropriate fluorophore compensation values to produce a bright population (positive (+) or Hi) and dim population or cell subset (Negative (~) or Lo), Typically, positive cells populations (+ or Hi) were of the order of third decade or higher (10⁴⁾ while negative population were in the first to second decade (10²-10^?'). Using established gates, cells were sorted using a 100 μΜ nozzle and a flow' rate of 1.0, Following sorting, a small aliquot of cells were analyzed to assess the purity of the sorted cell subsets, RNA was collected from the presort and sorted cells using the Rneasy Mini Kit, Qiagen, CA) for RT-PCR analysis.

2015268664 11 Dec 2015

U92] Publications cited throughout this document arc hereby incorporated by referen.ee in their entirety. Although the various aspects of the invention have been illustrated above by reference to examples and preferred embodiments, it will be appreciated that the scope of the invention is defined not by the foregoing description but by the following claims properly construed under principles of patent law.

2015268664 11 Dec 2015

Table I, Flow Cytometric Characterization of Surface Marker Expression at

Different Stages of Endodermal/Pancreatie Differentiation

Key: ND = Not Determined; +/- = 0-10%; + = 10-50%; ++ = 50-85%; +++ = 85-100%

.te/teit-	^we«y8»	ί ΪΗΙ&ί'/Λϊί.	ttC.S	tn /5ίί#ί? //	Primitive Cut Tube (&uge2)	Posteriaf (Singe 3)	/.»>/>-«.? i«<· •ftWiBWf·) (Singe	Ο+ί/ΐκν+ϊ e CfeBs f.Sft-!§E 5)	+/(-«««’ Hiiitiii'iine Cette (Stage 6)
BL.F-R		BD6552S36	ND	4,4	+A	+/-	4/4	+/.	+/-
corns	Endogiin	MililporeffC BI.418F	+/-	77'-	+/-	-4/4	+/-	774	+/-
GDI 12	PRR2	81)6551057	-J·/ ”	.......+F........	4/4.	+/-	7*/«-	.......+/-........	..........+/-...........
cm I?	c-kit	81)834)096	4-	+-4	++	+	77 »	+:-	+/.
GDI 18	LiFR, gp!90	S&D8FA82 w	+/-	+7-	7-	+/-	--1--,4	+7-	4-,.4
CD 126	IL-6R	81)8551850	4/4	7s/ -	+/-	4/4	4/4	4/ -	+/-
CD 13	Aaiinopeptida ae N	BD8555364	4/-	77-	4/4	4/-	4/4	4/-	4/4
GDI 30	IL-0R{1, gp!30	81)6555757	4/	-47-	4-/4	+/-	+/-	+/-	4,4
GDI 32		81)6555900	+/-	+/-	4-/4-	+/-	+/-	+/-	+/-
CD133	AC 133, prominifi-likel	ΜΗΑΒΝΎ1 4 DO-090854	7-	4'	++	+	4	4	4
CD J 34	OX-40	81)8554846	4/--	7*4	+/-.	+/-	4/4	4/4	+/-
CDI3S	Hi:3/Flk2	81)8558966	47 ~	74-	+/-	+/-	474	4/-	-4/4
0)13?		81)8550866	7*4	.......+/-........	4·4	7-,-4	7*4	.......+/-........	..........+/-...........
0)13? Ligand		81)6556446	•I7 ”	+/-	47»	+/-	77--	+7-	+-,,4
CD! 40a	PDGFRa	BD6556002	47-	7-.4	+/-	-+/-	,.-..	+/.
CDS 40b	PDGFRg	806558821	+/-	+/-	77“	+/-	4-4	+/-	77 -
CD 142		81)6550312	+/,	74-	+/-	+/-	4	4-	4
CD 146	MCCiS	81)65503)5	+	+	+	+/-	+	+	ND
GDIS		81)4551376	4,.4-	4	4-	+	+	4	4
CDli/i		81)4340534	47~	74-	+/-	+/-	4-/4	7'/“	-4/4
0)164		81)8551268	77 *	+/-	4/4	+/-	7'4	4/4	+4
¢3)178	Fast, CD95L	81)::555223	7-4	+/-	47™	+/-	74	+7-	ND
<3)180		81)6551953	77-	47-	4,.4	4/’	4/»	4/-	4/-
CD181	CXCR.l, 1L- 8RA	81)6555939	+/-	4	4,.4	+/-	4/»	+7-	ND
CD 183	GXCS3	BD655096?	+/-	774	+/-	4/4	+/.	77+	+.:--
CD 184	CX.CR4, ftssin	81)6555976	+/-	++	+	+/-	+/-	4/4-	4
CD 185	CXCR5	81)6551956	+/-	4/4	+/-	+/-	4/4	4	ND
CD193	GCR3	81)4558165	47-	4	+/-.	+/-	4/4-	4/4	4/4.
CDI95	GCR5	81)8555962	+/-	7-/-	+/-	+/-	4/~	77 -	ND
CD lb		81)6555069	4-/-	7*/*	4/4	+/-	4/-.	4/4	4/4
0)20		81)85 55622	77 ~	........+/-........	+/-	+/-	4/4	........+/-........	..........+7...........
¢3)200	OX-2	81)4552475	+	4-+	++	-4	+	++	++
0)205		81)6558069	Τ’/	+/-	+/-	+/-	+/·	4/-	4,4
0)220	insubn-R	BS6559955	+/-	+/-	+/-	+/.	+/-	+7-	ND
CD22I	IGF-1 Ra	81):--555999	+	++	4-4	4	4/-	4	4/~
CD24		81):--5 55428	7*7*4-	47*4	+++	444	444	444	,f . A . >. tt ;
CD243	MDR-i; P-£P	8865 57602	+/-	+/-	+/-	+:’-	+ /4	+/--	-:-7-
CO252	OX-40 Ligand	81)6558164	+/-	7*4	-:-/-	+/-	.,./.	-:-/-
CD26		81)6555456	+/-	474	+/-	+/-	4/4	74	+/-
CD27I	NGFS.	81)4557168	4-/-	ND	4/4	4/4	+/-	4/ --	ND
CD27S		81)8552502	7-,.4	7-/4	+/-.	+/-	4/4	-4/4	+/-
CD28		81)8555728	47»	+/-	-4/4	+/-	4/ -	77»	4-/4

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CD29	fee grin β!	BO95S9883	+-4-4	4-(-4	4-4-+	-i-4-4-	444	4-4-(-	4-4-4
CD305	LAIR3	80955080	+/-	+/-	+/-	+/-	47-	+/-	NO
CD309	VBGFR2, K-Dii.	809560494	+/-	+/-	4/-	+/-	47,	+/-	+/-
CD318	cdcdpi	siA 1)4 AHI nt.CDf’	+/-	^/4	+/-	+/-	4	4	4
CD326	Ep-CAM	Β0-ΑΌΌ-,	4-+4-	444	444-	..).+	++	4-4-	++
CD33		807555450	47,	+/-	-1-/4	-4/-	+/-	+/-	7-/-
CD332	FGFR2, KGFR2	RAIMABft S4A	47,	4/.	4/4	+/-	47,	+/-	+/-
CD340	LrbB-2, HBR2/»eu	BO9340553	4/4	+./-	4/.	4/4	4/4	4/-	4/.
CD36		809559956	4/-	47-	4/4	........N-........	4/-	4/~	+
CD39		BB9555464	47-	+/-	4/-	+/-	47.	+/-	NO
CB42b		BO95S5472	+/-	+/-	4/-	+/-	+A	+/-	+/-
CD43		809555475	+/.	4/4	+/-	+/-	4/.	4/4	ND
CD44		BO9559942	+/-	4/4-	+/-	4./-	+/-	+/-	+/-
Ci>46		809555949	+	4..-4	+/-	+/-	4	4	ND
CD47		81.)4556046	+/-	4/4	4	a j i. Tit	++	4+	++
<?D49b	(?2 feegrin^ VI-/V2	808555669	4-	+/-	-¼	4	4-	4	47-
CD49c	«3 Imtegrin, VLA-3	Abeatn#»b3 0489	4	+	4	4	4	4	+
CD49c	«5 Integrin. VLA-5	809555657	4	4-4-4	444	4-4	4	4	4
CD49f	«6 Integrin, VLA-6	BD85S5735	+	4/-	4	4	+	4	4/~
CQS5		BBS555696	4	.)..1.	4-	+/-	4	4	4-
CD56	NCAM	809555513	4	4	4	4	i ,t A V	44	..+ .3..:. T
CD57		809555619	4+-+	4-44	444	4-4	+	4	4
CD5S	LFA-3	809555920	4-	4,-4	+/-	4/-	4/4	4/4	+/-
CO6)	LIMP. LAMP3	804557288	NO	+/-	+/-	4-/.	4/4	4/-.	+/-
CD66		BO955M80	4,4	+/-	+/-	+/-	4/-	+/-	4/~
CD71		BD8S5B74	4-	+	-4	4	4/-	+	+
CD73		809550257	47-	+/-	4/4	4	-¼		NO
CD74		8O95S5540	47-	+/-	4/-	+/-	+./-	+/-	NO
CD88	CSaR	BO9550494	+/.	4	+/-	44	+/.	4/4	4/4
CD9	P24, MRP-1	BO9555S72	4	4	+/-	+/-	+/-	+/-	+/-
CD91		809550496	47.	-4,-4.	+/-	4/-	4-/.	4/4	+/-
CD95	Apo-!, Pas	804555674	4/4	4,-4.	+/-	4/4	4/4	4/4.	ND
CD98		804556076	i-4-4-	444	.+.)..1.	. X 1 τ r’r	+.).	4-+	4
CO99	MIC2, 1:2		4./-	444	+-44-	4-+-4	4-4-4	444	4-+
CDw210	IL-10 R	80455695 3	4/-	4	+/-	+/-	4,4	4A	4/~
DLL!		R&D8FAB1 HiSA	ND	ND	NO	4/4	44	4/~	NO
BGFR	ErbB-L HER I	809555997	+/-	4/4	+/-	4./-	+/-	+/-	+/-
fMLP		804556056	4/-	+/-	47-	4/4-	4/4	4,,-4	4/-
MICA/B		809558352	-4/-	+	+/-	+/-	-4/-	4/4	ND
No-chi		8O95S2768	4/-	4	+/-	4/-	4/-	4/~	ND
SSbA-4		R&O9FAB1 4359	4-4-4-	4-44-	+4-	4	+	4	+
TGFBR3		LliespaiAS -C76502	4-/-	N!3	4	4/-	4-/.	4	ND
TRAI-60		BD4560193	+++	+)+	4-	4	4	+	4-
TRA1-81		BO9560S6.;	4-44-	4-4-4	4	4	4	4	4-
TWEAK		8O»552S9B	+/-	+/-	+/-	+/-	+/-	+/-	+A

2015268664 11 Dec 2015

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2015268664 11 Dec 2015

Table 11' Surface Markers used to Eurieh for Panereatie Cell Precursors

Sw&cc MwJcei* Oswd (Single CttrtiHnaktKO	Swge of Cctis Sorted	Vendor/ No,	Phenotype of Enriched Popefstions	% Starting Population	% Soncff P«f)fji;jiio;s	Void Ensieinnesn
eusftons	Κγκ&χπ««· Pmnrsnrs (S4)	.853955555,8/9 5555)3	< !!5·ΥCIS·: 5	64. i	S25	~i.3
CO! j j	Endocrine Eraccxsors (543	Mflien?«G· 95)3-5554	CP 5.33'	48,6	5)2,0
CD49c(«-3 hsicgfini	Endocrine Pt«etsr$«r$ (84)	Atotssas&sWM 89	emsc**'	Si.7	95.9	-3,5
CD5&W15	Endocrine Precessors (84)	BD955551SL/9 553376	CDSdtoOiS^''	26-80	ND	ND
CD! 5	Endocrine Precentors (84)	BUSSSS376	cm 3-	89.6	97.5	—53
CO5&WS7	'Εϊϊ<Κκ·Η jje Frwiwws ($4) Endocrine· CdSs (S5>	iSlXSJSJiS/i 555/8!)	cD&rcDsr	35.3	593	-5.9
OD9S	Endocrine CcH-s { S6j	8i39556o“6	CiW	6Ϊ.3		-5.6
CW7	Endocrine CUls (SS, SS)	8539556046	twr	22,.$	7? 4	-3 J

Table III. Expression levels of LI F Receptor

Stage of Differentiation	Stage 2, Day2	Stage 2, Day3	Stage 3, Day4	Stage 4, Day3
Expression Level (%)	47%	70%	<oz. J 2 0

2015268664 11 Dec 2015

Table IV. Expression Levels of CD 184 Before and After Enrichment

j i Ere-sort	Enriched
i	O):S4· I'Kieiffsn	CDl&l· FTaCikw
Descript«>.« : CD; ^4+CD5i> CDi5s4-M.'.'D5fri·	answ-CDss-	Ο· λΛ· CO5S·?	CDi84·;·	C05S4-
1 Expression i g% <%) i ' ' j	20%	70%	79%	0,6%

Table V. Expression Levels of SSEA-4 Antigen

Cells Stage of	SSEA-4 Exf	»ression {%)	% Fold
Differentiation	|		Depletion
	Pre-Depletion	Post-Depletion
Hl Undifferentiated	I 91.2	ND	ND
Hl Primitive Gut i ίίίΊ/,Λ fT\	| 20.5	1,8	91,2
..................................j..............ν2·Λ?§·Τ····Λ*........... Endocrine Precursors (Stage 1 IV} ’	“1................20J

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2015268664 11 Dec 2015

Table VI. Summary Data of Mice Transplanted with SSEA-4 Depleted Cells

| Group	Cell Type	Total Cell No.	No, ofMiee	Grafts' at 12 Week	C‘Peptuie at / 2 Week s
1	Cell Clusters	3.3 million	5	3/5 mice visible grafts	1/5 mice detectable e~ peptide
7	Single Cells Undepleted	3,3 million	5	0/5 mice with visible grafts	0/5 mice detectable cpeptide
3	SSEA-4 Depleted Single Cells	3.3 million		0/2 mice with visible strafts	0/2 mice detectable c- peptide

Table VII. Surface Markers Associated with Difierentiation of Human Embryonic Stem cells into Pancreatic and Endodermal Lineages,

Surface Markers Changes During Differentiation I DE~^PE~^Endocrme	Changes Associated with Surface Markers *	Surface Markers Used To Enrich Pancreatic EndoderntfEndocrine	Cell Fractions Enriched
CP117	Decrease	ND	-
CD13	Increase	Yes	CD13-
[ CP133	Decrease increase	Yes ____	CPI 33-
| CD15	increase	Yes	CD 15-
CD 181	Decrease	ND
CP184	Decrease	Yes	CD! 84+
| CD20I)	Decrease	ND	-
CP221	Decrease	ND
| CD318	Increase	ND	-
|_CP326_ | CD46	_Decrease_ Increase	ND ND	_-_
| CD47	increase	Yes	CD47-
CD49c	Increase	Yes	CD49c-
CB49e	increase	ND	-
| CP55	Decrease	ND
| CD56	increase	Yes	CD56+
| CD57	Decrease	Yes	CD57+
CD73 (TD	increase Decrease	ND ND
1 CD98	Decrease	Yes	CD98+

• Changes associated with Surface Markers Denotes if Expression level of the particular Surface Marker Increased or Decreased as cell were differentiated from Definitive Endoderm (DE, Stage I) to Pancreatic Endoderm (PE, Stage 111) and finally to Endocrine Cells (Stage V/VI),

2015268664 20 Dec 2017

Claims (21)

What is claimed is:

1/21

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1. A method to enhance the generation of insulin-producing cells from a population of cells expressing markers characteristic of the pancreatic endocrine lineage comprising:

a. differentiating a population of pluripotent stem cells into a population of cells expressing markers characteristic of the definitive endoderm lineage;

b. differentiating the population of cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the primitive gut tube lineage;

c. differentiating the population of cells expressing markers characteristic of the primitive gut tube lineage into a population of cells expressing markers characteristic of the pancreatic endoderm lineage;

d. differentiating the population of cells expressing markers characteristic of the pancreatic endoderm lineage into a population cells expressing markers characteristic of the pancreatic endocrine lineage; and

e. enriching the population of cells expressing markers characteristic of the pancreatic endocrine lineage for expression of NEUROD, NGN3, PDX1 and NKX6.1 by isolating a population of cells expressing markers characteristic of the pancreatic endocrine lineage that is positive for CD56 and negative for CD13.

2/21

2015268664 11 Dec 2015

2. A method of enriching a population of cells expressing markers characteristic of the pancreatic endocrine lineage for expression of NEUROD, NGN3, PDX1 and NKX6.1, comprising providing a population of cells expressing markers characteristic of the pancreatic endocrine lineage and isolating a population of cells expressing markers characteristic of the pancreatic endocrine lineage that is positive for CD56 and negative for CD13.

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3. A method of enhancing the generation of human insulin-producing cells from a population of cells expressing markers characteristic of the pancreatic endocrine lineage, comprising enriching a population of cells expressing markers characteristic of the pancreatic endocrine lineage for NEUROD, NGN3, PDX1 and NKX6.1 by isolating a population of cells expressing markers characteristic of the pancreatic endocrine lineage that is positive for CD56 and negative for CD13.

2015268664 12 Dec 2017

-584. The method of any of claims 1 to 3, wherein the enriched population of cells has increased expression for glucagon and insulin.

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5. A method of enriching a population of cells expressing markers characteristic of the pancreatic endocrine lineage for NeuroD, NGN3, PDX-1, and NKX6.1 by screening the population of cells for positive for CD56 and negative for CD13 to thereby produce an enriched population of cells.

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6. The method of any one of claims 1 to 5, wherein the cells are enriched using flow cytometry.

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7. The method of any one of claims 1 to 5, wherein the cells are enriched using FACS.

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8. The method of any one of claims 1 to 7, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are pancreatic endocrine precursor cells.

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9. An isolated population of insulin-producing cells produced by performing the method of any one of claims 1 to 8.

10/21

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10. Use of the isolated population of insulin producing cells of claim 9 in medicine.

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11. A method of treating or preventing an insulin-related disease or disorder comprising transplanting a population of insulin producing cells of claim 9 to a patient in need thereof.

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12. An isolated enriched population of cells expressing markers characteristic of the pancreatic endocrine lineage produced by performing the method of any one of claims 2, or 4 to 8.

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13. Use of the isolated enriched population of cells of claim 12 in medicine.

14. A method of treating or preventing an insulin-related disease or disorder comprising transplanting a population cells of claim 12 to a patient in need thereof.

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15. Use of a population of insulin producing cells of claim 9 for the manufacture of a medicament for treating or preventing an insulin-related disease or disorder.

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-5916. Use of a population cells of claim 12 for the manufacture of a medicament for treating or preventing an insulin-related disease or disorder.

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