WO2011084747A2

WO2011084747A2 - Compositions and methods for somatic tissue induced pluripotent stem cells having an endoderm origin

Info

Publication number: WO2011084747A2
Application number: PCT/US2010/061334
Authority: WO
Inventors: Yoon-Young Jang; Saul J. Sharkis
Original assignee: The Johns Hopkins University
Priority date: 2009-12-21
Filing date: 2010-12-20
Publication date: 2011-07-14
Also published as: WO2011084747A3

Abstract

The present invention provides compositions and methods for reprogramming human cells of endoderm origin (e.g., primary hepatocytes) to pluripotency. Hepatocyte-derived iPS cells appear indistinguishable from human embryonic stem cells in colony morphology, growth properties, expression of pluripotency-associated transcription factors and surface markers, and differentiation potential in embryoid body (EB) formation as well as teratoma assays. In addition, these iPS cells are able to directly differentiate into definitive endoderm, hepatic progenitors, and mature hepatocytes.

Description

COMPOSITIONS AND METHODS FOR SOMATIC TISSUE INDUCED PLURIPOTENT STEM CELLS HAVING AN ENDODERM ORIGIN

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

[0001] The present invention relates generally to the field of induced pluripotent stem (iPS) cells and more specifically to compositions comprising such cells and methods of generating such cells from somatic cells of endoderm origin.

BACKGROUND INFORMATION

[0002] Recent advances in induced pluripotent stem (iPS) cell research significantly changed the perspective on regenerative medicine. Patient specific iPS cells have been derived not only for disease modeling but also as sources for cell replacement therapy.

However, there have been insufficient data to prove that iPS cells are functionally equivalent to hES cells or safer than hES cells. There are several important issues which need to be addressed and foremost are the safety and efficacy of human iPS cells from different origins. Human iPS cells have been derived mostly from cells originated from mesoderm, with a few cases from ectoderm. So far there is no report of endoderm derived human iPS cells, preventing the comprehensive comparative investigations on the quality of human iPS cells from different origins.

[0003] Human iPS cells have been shown to be pluripotent in in vitro differentiation and in vivo teratoma assays, similar to human embryonic stem (hES) cells. Disease-specific iPS cell lines have been generated from fibroblasts and blood cells and some of the disease features have been recapitulated in tissue culture after directed differentiation of the iPS cells, demonstrating the power of this technology in disease modeling. However, several key issues have to be addressed in order for the iPS cells to be used for clinical purposes.

[0004] First, although pluripotency has been demonstrated, it is premature to claim that iPS ceils are functionally equivalent to hES cells. In fact, one study has suggested that iPS cells have distinct protein-coding and microRNA gene expression signatures from ES cells. These differences can not be completely explained by the reactivation of transgenes used in

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331323-000880 the reprogramming process since human iPS cells generated without viral or transgene integration also displayed a different transcriptional signature compared to hES cells.

[0005] Secondly it was demonstrated that human iPS cells retained certain gene expression of the parent cells, suggesting that iPS cells from different origins may possess different capacity to differentiate. This issue is important not only for the purposes of generating functional cell types for therapy but also for safety implications. A

comprehensive study using various mouse iPS cells has demonstrated that the origin of the iPS cells had a profound influence on the teratoma forming propensity in a cell

transplantation therapy model. It is therefore extremely important to establish human iPS cell lines from multiple origins and thoroughly examine the source impact on both the safety issues and their differentiation potentials.

[0006] In the mouse, iPS cells have been generated from derivatives of all three embryonic germ layers, including mesodermal fibroblasts, epithelial cells of endodermal origin and ectodermal keratinocytes, whereas human iPS cells have been produced mostly from mesoderm (fibroblasts and blood cells) or from ectoderm (keratinocytes and neural stem cells).

[0007] Recent advances in induced pluripotent stem cell (iPSC) research have provided new tools for regenerative medicine and for disease modeling. iPSCs resemble embryonic stem cells (ESCs) in their pluripotency, and offer potential solutions for histo-incompatibility issues that ESC-based therapies are likely to encounter. Patient specific iPSCs that retain the precise self genetic information also provide unique advantages for disease modeling.

However, questions still remain as whether or to what degree the iPSCs resemble the ESCs in their functionality and safety. It has been shown that iPSCs and ESCs can be distinguished by their gene expression signatures, even in the absence of transgene expression of the reprogramming factors. From a separate study of hemangioblastic lineage differentiation from pluripotent stem cell lines, it has been suggested that human iPSC derivatives exhibit limited expansion and early senescence compared to the ESC derivatives.

[0008] Another related issue is how the tissue origin of the cells may affect the quality of the iPSCs. In the mouse system, it has been reported that secondary neurospheres from iPSCs derived from different adult tissues varied substantially in their tumor-forming

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331323-000880 propensity. The molecular mechanism behind this phenomenon is not fully understood, but evidence from comprehensive studies in mouse iPSCs has suggested that there is epigenetic memory retained in the iPSCs from their somatic cell origins, and that the memory may influence their directed differentiation potential into blood cells. Although these studies were originated from mouse cells, it is reasonable to speculate that human iPSCs from different origins may exhibit similar characteristics. However, a majority of the reported human iPSC lines have been generated from mesoderm-derived cells (fibroblasts and blood cells) and a few ectoderm derivatives (keratinocytes and neural progenitor cells).

SUMMARY OF THE INVENTION

[0009] The present invention is partly based on reprogramming of somatic cells and specifically, human endoderm derived cells (e.g., primary hepatocytes), to pluripotency. Hepatocyte-derived iPS cells appear indistinguishable from human embryonic stem cells in colony morphology, growth properties, expression of pluripotency-associated transcription factors and surface markers, and differentiation potential in embryoid body formation and teratoma assays. In addition, these cells are able to directly differentiate into definitive endoderm, hepatic progenitors, and mature hepatocytes. The technology to develop endoderm derived human iPS cell lines, together with other established cell lines, provides a foundation to elucidate the mechanisms of cellular reprogramming and to study the safety and efficacy of differentially originated human iPS cells for cell therapy. For studying liver disease pathogenesis, the invention methodology provides a more amenable system to generate liver disease specific iPS cells. In addition, the methodology provides for the development of patient specific stem cell lines for treatment of liver diseases.

[0010] In one embodiment, the present invention provides a method of generating induced pluripotent stem (iPS) cells from cells of endoderm origin. The method includes (a) introducing at least one pluripotent factor into the cells of endoderm origin under a first culture condition for a duration of less than about two weeks, and (b) maintaining the cells of (a) under a second culture condition different from the first. In another embodiment, the present invention provides an isolated population of human endoderm origin iPS cells according to the method provided herein.

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331323-000880 [0011] In one aspect, the first culture condition includes Williams' Medium E, HGF, and EGF. In another aspect, the first culture condition has a duration of less than about four days. In one aspect, the second culture condition includes feeder cells and/or feeder-free matrigel- coated support. In an additional aspect, the feeder-free matrigel-coated support includes a feeder-free matrigel-coated plate. In another aspect, the second culture condition includes hESC medium.

[0012] In another embodiment, the present invention provides a somatic tissue derived induced plunpotent stem (iPS) cell having an equal or greater in vivo engraftment potential as compared to an embryonic stem (ES) cell or an iPS cell derived from an embryonic tissue. In one aspect, the somatic tissue has an endoderm origin.

[0013] In one aspect, the somatic tissue comprises hepatocytes. In an additional aspect, the somatic tissue comprises human hepatocytes. In another aspect, the somatic tissue comprises human primary hepatocytes. In another aspect, the in vivo engraftment potential is examined in liver. In one aspect, Sodium Butyrate (NaB) is used to improve reprogramming efficiency. In an additional aspect, Sodium Butyrate (NaB) used is 0.5 mM. In another aspect, the reprogramming efficiency is improved ten folds. In an additional aspect, the reprogramming efficiency is improved from about 0.001 to about 0.01 (i.e., from about 0.1% to 1%).

[0014] In one aspect, the somatic tissue derived iPS cell has a differentiation potential to differentiate into cells of endoderm origin.. In an additional aspect, the cells of endoderm origin express an endoderm marker CXCR4. In another aspect, the cells of endoderm origin include hepatic cells, cells of pancreas, cells of gastrointestinal tract, cells of respiratory tract, cells of endocrine glands, cells of urinary system, or a combination thereof. In another aspect, the cells of endoderm origin are selected from the group consisting of hepatic cells, cells of gastrointestinal tract, cells of respiratory tract, cells of endocrine glands, cells of urinary system, and a combination thereof.

[0015] In one aspect, the somatic tissue derived iPS cell has a differentiation potential to differentiate into different hematopoietic cell types. In another aspect, the somatic tissue derived iPS cell has a differentiation potential to differentiate into secondary neurospheres

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331323-000880 (SNS). In another aspect, the somatic tissue derived iPS cell has a differentiation potential to differentiate into neurons, astrocytes, or oligodendrocytes.

[0016] In one aspect, the somatic tissue derived iPS cell has a distinct epigenetic pattern as compared to an ES cell or an iPS cell derived from an embryonic tissue. In another aspect, the somatic tissue derived iPS cell has an equal or lower epigenetic pattern as compared to an ES cell or an iPS cell derived from an embryonic tissue. In an additional aspect, the epigenetic pattern comprises genomic DNA methylation. In one aspect, the somatic tissue derived iPS cell is a low-passage iPS cell. In an additional aspect, the low-passage iPS cell has a passage between seven and twelve passages, or less than ten passages. In another aspect, the low-passage iPS cell has a passage less than four passages. In another aspect, no chromatin-modifying agents are used during passages for the low-passage iPS cell. In another aspect, the somatic tissue derived iPS cell has an equal or lower tumor-forming propensity as compared to an embryonic stem (ES) cell or an iPS cell derived from an embryonic tissue.

[0017] In another embodiment, the present invention provides an intermediate stage hepatic progenitor cell derived from the somatic tissue derived iPS cell provided herein. In one aspect, a step wise differentiation protocol is used. In another aspect, the intermediate stage hepatic progenitor cell displays CYP450 enzyme activity when further differentiated into mature hepatocytes.

[0018] In one aspect, the step wise differentiation protocol includes: (a) a definitive endoderm induction step; (b) a hepatic progenitor induction and expansion step; and (c) a hepatic maturation step. In another aspect, the step wise differentiation protocol consists of: (a) a definitive endoderm induction step; (b) a hepatic progenitor induction and expansion step; and (c) a hepatic maturation step. In another aspect, the step wise differentiation protocol for further maturation uses a hepatocyte culture medium (HCM) comprising FGF-4, HGF, oncostatin M, and dexamethasone.

[0019] In another embodiment, the present invention provides a system for studying drug metabolism or liver disease modeling. The system includes the intermediate stage hepatic progenitor cell provided herein. In another embodiment, the present invention provides a method for studying drug metabolism or liver disease modeling. The method includes: (a)

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331323-000880 obtaining a somatic tissue of endoderm origin; (b) generating an iPS cell from the somatic tissue of endoderm origin of (a); (c) generate an intermediate stage hepatic progenitor cell as provided herein; (d) contacting a test agent with the intermediate stage hepatic progenitor cell of (c) or a hepatocyte derived from the intermediate stage hepatic progenitor cell; and (e) determining the effect of the test agent on expression of a gene or activity of a gene product in the cell. In one aspect, the method is for a subject having a condition or disease. In another aspect, the somatic tissue of endoderm origin is obtained from the subject having a condition or disease.

[0020] In another embodiment, the present invention provides a method for treatment of liver diseases to a subject. The method includes administering an effective amount of the somatic tissue derived iPS cells provided herein to the subject, thereby treating the disease. In another embodiment, the present invention provides a method for treatment of liver diseases to a subject. The method includes administering an effective amount of the intermediate stage hepatic progenitor cells provided herein or hepatocytes derived from the intermediate stage hepatic progenitor cell to the subject, thereby treating the disease. In various aspects, the administering step is local or systemic.

[0021] In another embodiment, the present invention provides a method for in vivo engraftment to a subject. The method includes administering an effective amount of the somatic tissue derived iPS cells provided herein to the subject. In another embodiment, the present invention provides a method for in vivo engraftment to a subject. The method includes administering an effective amount of the intermediate stage hepatic progenitor cells provided herein or hepatocytes derived from the intermediate stage hepatic progenitor cell to the subject. In another embodiment, the present invention provides a method for in vivo engraftment to a subject. The method includes administering an effective amount of definitive endoderm stage cells derived from the somatic tissue derived iPS cell provided herein to the subject. In various aspects, the hepatocytes or definitive endoderm stage cells express an endoderm marker CXCR4. In various aspects, the administering step is local or systemic.

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331323-000880 BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 shows exemplary steps for generating Human hepatocyte derived iPS (hHiPS) cell colony formation and characterization. Figure 1 A shows albumin expression of primary human hepatocytes before iPS reprogramming. Figure IB shows an exemplary diagram of hHiPS generation protocol.

[0023] Figure 2 shows an exemplary diagram of human MSG derived iPS generation protocol.

[0024] Figure 3 shows differentiation of hHiPS cells into DE and hepatic progenitors. Efficient endoderm induction of human iPS cells and ES cells in the presence of 100 ng/ml Activin A. The FACS analysis showed that at least 90% of the induced cells expressed the definitive endoderm marker CXCR4. Figure 3A shows H9 cells; Figure 3B shows HI cells; Figure 3C shows hHiPS6 cells; Figure 3D shows hHiPSlO cells; and Figure 3E shows hHiPSl l cells.

[0025] Figure 4 shows AFP expression of primary human hepatocytes before iPS reprogramming.

[0026] Figure 5 shows genome-scale DNA methylation analysis of human iPSCs and their parental cells, and hESCs. Figure 5 A shows multidimensional scaling plot illustrating the relationships among all individual cell lines analyzed in our study. Euclidian distance and the top 5% most varying autosomal loci across all samples analyzed were used. iKl, iK2, and iK3 are from keratinocytes (Kera). iM2, iM3 and iM7 are derived from bone marrow mesenchymal stem cells (MSC). iLCl and iLC2 are from fibroblasts (PLCF). iHl 1, iH14 and iHIO are from primary hepatocytes (Hep). Both HI and H9 are hESC lines. Figure 5B shows hierarchical clustering using Euclidian distance and top 5% most varying autosomal loci across all analyzed samples. Color bars on the side identify the different sample groups. The log2 methylation ratios are shown in different gray scales. Figure 5C shows Venn diagrams illustrating the number of autosomal loci differentially methylated between each iPSC group and the corresponding parental cells (MES=mesoderm, ECT=Ectoderm, END=endoderm), as obtained from the linear model analysis (Adjusted p-value < 0.001). Some of the representative genes associated with the differentially methylated features, based upon functional gene set enrichment analysis, are listed for both common and distinct

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331323-000880 changes occurring during reprogramming among different source-derived iPSCs (The upper numbers, 1513, 1370, 923, 871, 141, 219, 111, and 8742, indicate hypermethylation; the lower numbers 530, 114, 249, 96, 160, 61, 270, and 12410, indicate hypomethylation).

[0027] Figure 6 shows comparison of directed hepatic differentiation potentials in vitro among distinct developmental origin human iPSCs. Figure 6A shows a schematic

representation of differentiation steps of human iPSCs into hepatocytes. The key factors that are responsible for each stage of differentiation and the time course of hepatic commitment is shown in this diagram. Stage specific markers including genes and surface phenotypes are indicated under each stage cell types. Using this protocol, all human iPSCs of different origin are able to differentiate into definitive endoderm (Figure 6B; DE, SSEA3-CXCR4+), hepatic progenitors (AFP+), and mature hepatocytes (ALB+ and AAT+) with a comparable efficiency to one another. Figures 6C and 6D show that all these human iPSC derived mature hepatocytes display cytochrome P450 metabolism. Day-20 mature hepatocytes are incubated with CYP3A4/CYP1A2 pGlo substrates. At 4 hours after treatment, 50 μΐ, of culture medium was removed and read on a luminometer. CYP1A2 and CYP3A4 activity is expressed as relative light units (RLU)/ml of culture medium (n=6). All iPSCs exhibit similar levels of both CYP450 enzyme activities. Representative results of distinct origin iPSCs are shown.

[0028] Figure 7 shows in vivo engraftment potentials of human iPSC derived hepatic cells. The engraftment percentage of multistage hepatic cells derived from human iPSCs in mouse liver is calculated based upon human-specific ALB positivity and the large area scan images of multiple different liver lobes/lobules obtained from the recipient NSG mice 8 weeks posttransplantation of 2x10⁶ cells. DE: definitive endoderm, HP; hepatic progenitors, MH;

mature hepatocytes derived from human iPSCs.

[0029] Figure 8 shows a diagram of keratinocyte derived iPSC generation protocol.

Human keratinocyte derived iPSC (iKera) colonies can be formed and characterized with expression of the ESC surface antigens SSEA4 and TRA-1-60, and the nuclear transcription factors OCT4 and NANOG.

[0030] Figure 9 shows multidimensional scaling plot based upon genome-scale DNA methylation analysis of human iPSCs and their parental cells, and hESCs. Euclidian distance

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331323-000880 and 25% most varying autosomal loci across all samples analyzed are used. iM2, iM3 and iM7 are derived from bone marrow mesenchymal stem cells (MSC). iHl 1, iH14 and iHIO are from primary hepatocytes (Hep). iKl, iK2, and iK3 are from keratinocytes (Kera). iLCl and iLC2 are from fibroblasts (PLCF). Both HI and H9 are hESC lines.

[0031] Figure 10 shows effect of passaging on pluripotency gene OCT4 expression.

Quantification of the expression level of OCT4 in different origin human iPSC lines harvested at early (p7-12) and later passages (p34-40) by quantitative PCR. HI and H9 ESCs were used as control as shown in the figure. No significant differences were observed between early and later passage iPSCs of different origin and the values were similar to hESCs. The values were normalized by 18S expression and expressed as relative quantitation (RQ), the error bars depict the SD (n=3).

[0032] Figure 11 shows CYP450 metabolism in hHiPS cell-derived mature hepatocytes. Mature hHiPS cell-derived hepatocytes displayed CYP450 metabolism. iPS cell-derived hepatocytes are incubated with hepatocyte culture medium supplemented with CYP3 A4 or CYP1 A2 pGlo substrates (Promega, Madison, WI) according to the manufacturer's instructions. After 4 hours of treatment, 50 μΐ, of the culture medium is removed and read on a GloMax luminometer. The CYP1A2 and CYP3A4 activity is expressed as relative light units per milliliter of culture medium (n-6). All three hHiPS cell lines (ihH6, ihHlO, and ihHl 1) exhibit both CYP450 enzyme activities.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention is based on the seminal discovery of methods for reprogramming human endoderm derived cells (e.g., primary hepatocytes) to pluripotency. Hepatocyte-derived iPS cells appear indistinguishable from human embryonic stem cells in colony morphology, growth properties, expression of pluripotency-associated transcription factors and surface markers, and differentiation potential in embryoid body formation and teratoma assays. In addition, these cells were able to directly differentiate into definitive endoderm, hepatic progenitors, and mature hepatocytes. The technology to develop endoderm derived human iPS cell lines, together with other established cell lines, will provide a foundation to elucidate the mechanisms of cellular reprogramming and to study the safety and efficacy of differentially originated human iPS cells for cell therapy. For studying

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331323-000880 liver disease pathogenesis, this technology also provides a potentially more amenable system to generate liver disease specific iPS cells. In addition, the technology provides for the development of patient specific stem cell lines for treatment of liver diseases.

[0034] The present invention provides compositions and methods for reprogramming of human primary hepatocytes (endoderm) to pluripotency. Hepatocyte-derived iPS cells appear indistinguishable from human embryonic stem cells in colony morphology, growth properties, expression of pluripotency-associated transcription factors and surface markers, and differentiation potential in embryoid body (EB) formation as well as teratoma assays. In addition these cells were able to directly differentiate into definitive endoderm, hepatic progenitors, and mature hepatocytes.

[0035] The present invention provides the ground work necessary to elucidate the mechanisms of cellular reprogramming and to study the safety and efficacy of differentially originated human iPS cells in cell therapy.

[0036] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0037] This invention provides a method of generating human induced pluripotent stem cells (iPS) from cells of endoderm origin. The method comprises a first endoderm cell culturing step and a second culturing step. In a preferred embodiment the media for the first cell culture step comprises Williams' Medium E, HGF and EGF, and the media for the second step is not Williams' Medium E. Preferably the media for the second step is hES media.

[0038] Preferably, the duration of the first culturing step is no more than about 7 days, more preferably no more than about 5 days, and most preferably no more than about 3 days. It is preferred that cells are not replated until TRA-1-60 positive colonies are selected following the second culturing step.

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331323-000880 [0039] The method of the present invention may be applied to any cell of endoderm origin. In a preferred embodiment the endoderm cells are hepatocytes. This invention further provides an isolated population of human endoderm origin iPS cells,

[0040] Human induced pluripotent stem cells (hiPSCs) have been derived from various cell types. It remains to be determined how much the hiPSCs originated from different cell types are molecularly and functionally different. The present invention provides that hiPSCs of various developmental origins exhibit highly similar hepatic differentiation and liver regeneration potentials. The global methylation patterns of a panel of hiPSCs of three distinct developmental origins are compared and their hepatic differentiation propensity are studied. Epigenetic memory of origin does exist in hiPSCs, although all hiPSCs are highly similar to human embryonic stem cells (hESCs). However, there are no significant differences in hepatic differentiation and liver engraftment potentials among these diverse origin hiPSCs. All the iPSCs are able to differentiate into multi-stage hepatic cells with a similar efficiency to hESCs. These cells can successfully repopulate livers of

immunodeficient mice and secreted human liver proteins into mouse blood. The present invention provides that cellular origin does not significantly influence hepatic differentiation potentials of hiPSCs and that retained epigenetic memory does not manifest as altered hepatic differentiation capacity.

[0041] The present invention provides methods for generating multi-stage hepatic cells from human iPSCs using a step wise differentiation protocol. The present invention also provides methods for in vitro hepatic differentiation of iPSCs to model liver diseases. The present invention provides comparisons for the epigenetic patterns (as a molecular assessment), and in vitro and in vivo hepatic differentiation potentials (as a functional assessment) of highly diverse human iPSCs. The present invention provides that a certain level of epigenetic memory exists among all three germ layer origin human iPSCs.

[0042] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

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331323-000880 [0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described.

[0044] In various embodiments, one or more pluripotent factors can be used to induce reprogramming of a differentiated cell without using eggs, embryos, or ES cells. The method may be used to conveniently and highly reproducibly establish an induced pluripotent stem cell having pluripotency and growth ability similar to those of ES cells.

[0045] As used herein, pluripotent cells include cells that have the potential to divide in vitro for an extended period of time (greater than one year) and have the unique ability to differentiate into cells derived from all three embryonic germ layers, including the endoderm, mesoderm and ectoderm.

[0046] Somatic cells for use with the present invention may be primary cells or immortalized cells. Such cells may be primary cells (non-immortalized cells), such as those freshly isolated from an animal, or may be derived from a cell line (immortalized cells). In an exemplary aspect, the somatic cells are mammalian cells, such as, for example, human cells or mouse cells. They may be obtained by well-known methods, from different organs, such as, but not limited to skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra and other urinary organs, or generally from any organ or tissue containing living somatic cells. Mammalian somatic cells include, by way of example, adult stem cells, Sertoli cells, endothelial cells, granulosa epithelial cells, neurons, pancreatic islet cells, epidermal cells, epithelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells, fibroblasts, cardiac muscle cells, other known muscle cells, and generally any live somatic cells.

[0047] As used herein, reprogramming is intended to refer to a process that alters or reverses the differentiation status of a somatic cell that is either partially or terminally differentiated. Reprogramming of a somatic cell may be a partial or complete reversion of the differentiation status of the somatic cell. In an exemplary aspect, reprogramming is

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331323-000880 complete wherein a somatic cell is reprogrammed into an induced pluripotent stem cell. However, reprogramming may be partial, such as reversion into any less differentiated state. For example, reverting a terminally differentiated cell into a cell of a less differentiated state, such as a multipotent cell.

[0048] As used herein, a gene that induces pluripotency is intended to refer to a gene that is associated with pluripotency and capable of generating a less differentiated cell, such as a pluripotent stem cell from a somatic cell upon integration and expression of the gene. The expression of a pluripotency gene is typically restricted to pluripotent stem cells, and is crucial for the functional identity of pluripotent stem cells.

[0049] In various aspects, the pluripotent factor is encoded by a gene contained in a recombinant vector introduced into the cell. In various aspects, the pluripotent factor is encoded by one or more of a SO family gene, a KLF family gene, a MYC family gene, SALL4, OCT4, NANOG, LIN28, or a combination thereof. In an exemplary aspect, the pluripotent factor is one or more of OCT4, SOX2, KLF4, C-MYC. In another aspect, the at least one pluripotent factor comprises c-Myc.

[0050] Several genes have been found to be associated with pluripotency and suitable for use with the present invention as pluripotent factors. Such genes are known in the art and include, by way of example, SOX family genes (SOX1, SOX2, SOX3, SOX15, SOX18), KLF family genes (KLFl, KLF2, KLF4, KLF5), MYC family genes (C-MYC, L-MYC, N- MYC), SALL4, OCT4, NANOG, LIN28, STELLA, NOBOX or a STAT family gene. STAT family members may include for example STAT1, STAT2, STAT3, STAT4, STAT5

(STAT5A and STAT5B), and STAT6. While in some instances, use of only one gene to induce pluripotency may be possible, in general, expression of more than one gene is required to induce pluripotency. For example, two, three, four or more genes may be simultaneously integrated into the somatic cell genome as a polycistronic construct to allow simultaneous expression of such genes. In an exemplary aspect, four genes are utilized to induce pluripotency including OCT4, SOX2, KLF4 and C-MYC. Additional genes known as reprogramming factors suitable for use with the present invention are disclosed in U.S. Patent Application No. 10/997,146 and U.S. Patent Application No. 12/289,873, incorporated herein by reference.

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331323-000880 [0051] All of these genes commonly exist in mammals, including human, and thus homologues from any mammals may be used in the present invention, such as genes derived from mammals including, but not limited to mouse, rat, bovine, ovine, horse, and ape.

Further, in addition to wild-type gene products, mutant gene products including substitution, insertion, and/or deletion of several (e.g., 1 to 10, 1 to 6, 1 to 4, 1 to 3, and 1 or 2) amino acids and having similar function to that of the wild-type gene products can also be used. Furthermore, the combinations of factors are not limited to the use of wild-type genes or gene products. For example, Myc chimeras or other Myc variants can be used instead of wild-type Myc.

[0052] The present invention is not limited to any particular combination of pluripotent factors. As discussed herein a pluripotent factor may comprise one or more gene products. The pluripotent factor may also comprise a combination of gene products as discussed herein. Each pluripotent factor may be used alone or in combination with other pluripotent factors as disclosed herein. Further, pluripotent factors of the present invention can be identified by screening methods, for example, as discussed in U.S. Patent Application No. 10/997,146, incorporated herein by reference. Additionally, the pluripotent factor of the present invention may contain one or more factors relating to differentiation, development, proliferation or the like and factors having other physiological activities, as well as other gene products which can function as a pluripotent factor.

[0053] Further analysis may be performed to assess the pluripotency characteristics of a reprogrammed cell. The cells may be analyzed for different growth characteristics and embryonic stem cell like morphology. For example, cells may be differentiated in vitro by adding certain growth factors known to drive differentiation into specific cell types.

Reprogrammed cells capable of forming only a few cell types of the body are multipotent, while reprogrammed cells capable of forming any cell type of the body are pluripotent.

[0054] Expression profiling of reprogrammed somatic cells to assess their pluripotency characteristics may also be conducted. Expression of individual genes associated with pluripotency may also be examined. Additionally, expression of embryonic stem cell surface markers may be analyzed.

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331323-000880 [0055] As used herein, "expression" refers to the production of a material or substance as well as the level or amount of production of a material or substance. Thus, determining the expression of a specific marker refers to detecting either the relative or absolute amount of the marker that is expressed or simply detecting the presence or absence of the marker. As used herein, "marker" refers to any molecule that can be observed or detected. For example, a marker can include, but is not limited to, a nucleic acid, such as a transcript of a specific gene, a polypeptide product of a gene, a non-gene product polypeptide, a glycoprotein, a carbohydrate, a glycolipd, a lipid, a lipoprotein or a small molecule.

[0056] Detection and analysis of a variety of genes known in the art to be associated with pluripotent stem cells may include analysis of genes such as, but not limited to OCT4, NANOG, SALL4, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, or a combination thereof. iPS cells may express any number of pluripotent cell markers, including: alkaline phosphatase (AP); ABCG2; stage specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60; TRA-1-81; Tra-2-49/6E; ERas/ECAT5, E-cadherin; β-tubulin ΠΙ; a-smooth muscle actin (a-SMA); fibroblast growth factor 4 (FGF4), Cripto, Daxl; zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Natl); ES cell associated transcript 1 (ECAT1);

ESG1/DPPA5/ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECATIO; ECAT15-1; ECAT15-2; Fthll7; Sall4; undifferentiated embryonic cell transcription factor (Utfl); Rexl; p53; G3PDH; telomerase, including TERT; silent X chromosome genes; Dnmt3a; Dnmt3b; TRIM28; F-box containing protein 15 (Fbxl5); Nanog/ECAT4; Oct3/4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3; CYP25A1; developmental pluripotency- associated 2 (DPPA2); T-cell lymphoma breakpoint 1 (Tell); DPPA3/Stella; DPPA4; as well as other general markers for Pluripotency, for example any genes used during induction to reprogram the cell. IPS cells can also be characterized by the down-regulation of markers characteristic of the differentiated cell from which the iPS cell is induced.

[0057] As used herein, "differentiation" refers to a change that occurs in cells to cause those cells to assume certain specialized functions and to lose the ability to change into certain other specialized functional units. Cells capable of differentiation may be any of totipotent, pluripotent or multipotent cells. Differentiation may be partial or complete with respect to mature adult cells.

WESTV222869914.1

331323-000880 [0058] "Differentiated cell" refers to a non-embryonic, non-parthenogenetic or non- pluripotent cell that possesses a particular differentiated, i.e., non-embryonic, state. The three earliest differentiated cell types are endoderm, mesoderm, and ectoderm.

[0059] Pluripotency can also be confirmed by injecting the cells into a suitable animal, e.g., a SCID mouse, and observing the production of differentiated cells and tissues. Still another method of confirming pluripotency is using the subject pluripotent cells to generate chimeric animals and observing the contribution of the introduced cells to different cell types. Methods for producing chimeric animals are well known in the art and are described in U.S. Pat. No. 6,642,433, incorporated by reference herein.

[0060] Yet another method of confirming pluripotency is to observe cell differentiation into embryoid bodies and other differentiated cell types when cultured under conditions that favor differentiation (e.g., removal of fibroblast feeder layers).

[0061] The invention further provides iPS cells produced using the methods described herein, as well as populations of such cells. The reprogrammed cells of the present invention, capable of differentiation into a variety of cell types, have a variety of applications and therapeutic uses. The basic properties of stem cells, the capability to infinitely self-renew and the ability to differentiate into every cell type in the body make them ideal for therapeutic uses.

[0062] The pluripotent factor or nuclear reprogramming factor may comprise a protein or peptide. The protein may be produced from a gene as discussed herein, or alternatively, in the form of a fusion gene product of the protein with another protein, peptide or the like. The protein or peptide may be a fluorescent protein and/or a fusion protein. For example, a fusion protein with green fluorescence protein (GFP) or a fusion gene product with a peptide such as a histidine tag can also be used. Further, by preparing and using a fusion protein with the TAT peptide derived from the virus HIV, intracellular uptake of the pluripotent factor through cell membranes can be promoted, thereby enabling induction of reprogramming only by adding the fusion protein to a medium thus avoiding complicated operations such as gene transduction. Since preparation methods of such fusion gene products are well known to those skilled in the art, skilled artisans can easily design and prepare an appropriate fusion gene product depending on the purpose.

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331323-000880 [0063] The nucleic acid construct of the present invention, such as recombinant vectors may be introduced into a cell using a variety of well known techniques, such as non-viral based transfection of the cell. In an exemplary aspect the construct is incorporated into a vector and introduced into the cell to allow expression of the construct. Introduction into the cell may be performed by any viral or non- viral based transfection known in the art, such as, but not limited to electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion. Other methods of transfection include proprietary transfection reagents such as

Lipofectamine™, Dojindo Hilymax™, Fugene™, jetPEI™, Effectene™ and DreamFect™

[0064] Accordingly, in one aspect the present invention further provides a method of treatment or prevention of a disorder and/or condition in a subject using induced pluripotent stem cells generated using the methods described herein. The method includes obtaining a somatic cell from a subject and reprogramming the somatic cell into an induced pluripotent stem (iPS) cell using the methods described herein. The cell is then cultured under suitable conditions to differentiate the cell into a desired cell type suitable for treating the condition. The differentiated cell may then be introducing into the subject to treat or prevent the condition.

[0065] One advantage of the present invention is that it provides an essentially limitless supply of isogenic or synegenic human cells suitable for transplantation. The iPS cells are tailored specifically to the patient, avoiding immune rejection. Therefore, it will obviate the significant problem associated with current transplantation methods, such as, rejection of the transplanted tissue which may occur because of host versus graft or graft versus host rejection. Several kinds of iPS cells or fully differentiated somatic cells prepared from iPS cells from somatic cells derived from healthy humans can be stored in an iPS cell bank as a library of cells, and one kind or more kinds of the iPS cells in the library can be used for preparation of somatic cells, tissues, or organs that are free of rejection by a patient to be subjected to stem cell therapy.

[0066] The iPS cells of the present invention may be differentiated into a number of different cell types to treat a variety of disorders. For example, iPS cells may be induced to

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331323-000880 differentiate into hematopoetic stem cells, muscle cells, cardiac muscle cells, liver cells, cartilage cells, epithelial cells, urinary tract cells, neuronal cells, and the like. The differentiated cells may then be transplanted back into the patient's body to prevent or treat a condition. Thus, the methods of the present invention may be used to treat a subject having a myocardial infarction, congestive heart failure, stroke, ischemia, peripheral vascular disease, alcoholic liver disease, cirrhosis, Parkinson's disease, Alzheimer's disease, diabetes, cancer, arthritis, wound healing, immunodeficiency, aplastic anemia, anemia, Huntington's disease, amyotrophic lateral sclerosis (ALS), lysosomal storage diseases, multiple sclerosis, spinal cord injuries, genetic disorders, and similar diseases, where an increase or replacement of a particular cell type/ tissue or cellular de-differentiation is desirable.

[0067] In various embodiments, the method increases the number of cells of the tissue or organ by at least about 5%, 10%, 25%, 50%, 75% or more compared to a corresponding untreated control tissue or organ. In yet another embodiment, the method increases the biological activity of the tissue or organ by at least about 5%, 10%, 25%, 50%, 75% or more compared to a corresponding untreated control tissue or organ. In yet another embodiment, the method increases blood vessel formation in the tissue or organ by at least about 5%, 10%, 25%, 50%, 75% or more compared to a corresponding untreated control tissue or organ. In yet another embodiment, the cell is administered directly to a subject at a site where an increase in cell number is desired.

[0068] The term "polynucleotide" or "nucleotide sequence" or "nucleic acid molecule" is used broadly herein to mean a sequence of two or more deoxyribonucleotides or

ribonucleotides that are linked together by a phosphodiester bond. As such, the terms include R A and DNA, which can be a gene or a portion thereof, a cDNA, a synthetic

polydeoxyribonucleic acid sequence, or the like, and can be single stranded or double stranded, as well as a DNA RNA hybrid. Furthermore, the terms as used herein include naturally occurring nucleic acid molecules, which can be isolated from a cell, as well as synthetic polynucleotides, which can be prepared, for example, by methods of chemical synthesis or by enzymatic methods such as by the polymerase chain reaction (PCR). It should be recognized that the different terms are used only for convenience of discussion so as to distinguish, for example, different components of a composition.

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331323-000880 [0069] In general, the nucleotides comprising a polynucleotide are naturally occurring deoxyribonucleotides, such as adenine, cytosine, guanine or thymine linked to 2'- deoxyribose, or ribonucleotides such as adenine, cytosine, guanine or uracil linked to ribose. Depending on the use, however, a polynucleotide also can contain nucleotide analogs, including non-naturally occurring synthetic nucleotides or modified naturally occurring nucleotides. Nucleotide analogs are well known in the art and commercially available, as are polynucleotides containing such nucleotide analogs. The covalent bond linking the nucleotides of a polynucleotide generally is a phosphodiester bond. However, depending on the purpose for which the polynucleotide is to be used, the covalent bond also can be any of numerous other bonds, including a thiodiester bond, a phosphorothioate bond, a peptide-like bond or any other bond known to those in the art as useful for linking nucleotides to produce synthetic polynucleotides.

[0070] A polynucleotide or oligonucleotide comprising naturally occurring nucleotides and phosphodiester bonds can be chemically synthesized or can be produced using recombinant DNA methods, using an appropriate polynucleotide as a template. In comparison, a polynucleotide comprising nucleotide analogs or covalent bonds other than phosphodiester bonds generally will be chemically synthesized, although an enzyme such as T7 polymerase can incorporate certain types of nucleotide analogs into a polynucleotide and, therefore, can be used to produce such a polynucleotide recombinantly from an appropriate template.

[0071] In various embodiments antisense oligonucleotides or RNA molecules include oligonucleotides containing modifications. A variety of modification are known in the art and contemplated for use in the present invention. For example oligonucleotides containing modified backbones or non-natural internucleoside linkages are contemplated. As used herein, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified

oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0072] In various aspects modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters,

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331323-000880 aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates,

phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters,

selenophosphates and borano-phosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more intemucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Certain oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most intemucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0073] In various aspects modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and C¾ component parts.

[0074] In various aspects, oligonucleotide mimetics, both the sugar and the

intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. In various aspects, oligonucleotides may include phosphorothioate backbones and oligonucleosides with heteroatom backbones. Modified oligonucleotides may also contain one or more substituted sugar moieties. In some embodiments oligonucleotides

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331323-000880 comprise one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N- alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C\ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are 0[(CH₂)_nO]_mCH₃, 0(CH₂)_nOCH₃, 0(CH₂)_nNH₂, 0(CH₂)„CH₃, 0(CH₂)„ONH₂ and 0(CH₂)_nON[(CH₂)_nCH₃)]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: Cj to C_\Q lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, CI, Br, CN, CF₃, OCF₃, SOCH₃, S0₂CH₃, ON0₂, N0₂, N3, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Another modification includes 2'-methoxyethoxy(2'OCH₂CH₂OCH₃, also known as 2'-0-(2-methoxyethyl) or 2'- MOE).

[0075] In related aspects, the present invention includes use of Locked Nucleic Acids (LNAs) to generate antisense nucleic acids having enhanced affinity and specificity for the target polynucleotide. LNAs are nucleic acid in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (-CH₂-)_n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2.

[0076] Other modifications include 2'-methoxy(2'-0-CH₃), 2'-aminopropoxy(2'- OCH₂CH₂CH₂NH₂), 2'-allyl (2'-CH-CH-CH₂), 2'-0-allyl (2'-0-CH₂-CH-CH₂), 2'-fluoro (2'- F), 2'-amino, 2'-thio, 2'-Omethyl, 2'-methoxymethyl, 2'-propyl, and the like. The 2'- modification may be in the arabino (up) position or ribo (down) position. A preferred 2'- arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'- 5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0077] Oligonucleotides may also include nucleobase modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified

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331323-000880 nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine, 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5- propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (lH-pyrimido[5,4- b][l,4]benzoxazi-n-2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4- b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9- (2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), carbazole cytidine (2H- pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrimido[3',2':4,5]pyrrolo[2,3- d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7- deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases are known in the art. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds described herein. These include 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 C and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications.

[0078] Another modification of the antisense oligonucleotides described herein involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The antisense oligonucleotides can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic

WEST\2228₆9914.1

331323-000880 properties of oligomers. Typical conjugates groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine,

fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific

hybridization with R A. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., dihexadecyl-rac- glycerol or triethylammonium l,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylaminocarbonyloxycholesterol moiety.

[0079] In one aspect of the invention, methylation density is determined for a region of nucleic acid. Density may be used as an indication of production of an iPS cell, for example. A density of about 0.2 to 0.7, about 0.3 to 0.7 , 0.3 to 0.6 or 0.3 to 0.4, or 0.3, may be indicative of generation of an iPS cell (the calculated DNA methylation density is the number of methylated CpGs divided by the total number of CpGs sequenced for each sample).

Methods for determining methylation density are well known in the art. For example, a method for determining methylation density of target CpG islands has been established by Luo et al. Analytical Biochemistry, Vol. 387:2 2009, pp. 143-149. In the method, DNA microarray was prepared by spotting a set of PCR products amplified from bisulfite- converted sample DNAs. This method not only allows the quantitative analysis of regional methylation density of a set of given genes but also could provide information of methylation density for a large amount of clinical samples as well as use in the methods of the invention regarding iPS cell generation and detection. Other methods are well known in the art (e.g., Holemon et al, BioTechniques, 43 :5, 2007, pp. 683-693).

[0080] The following examples are provided to further illustrate the embodiments of the present invention, but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

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331323-000880 EXAMPLE 1

Retroviral Production and Reprogramming of Hepatocytes

[0081] Cell culture: Primary human hepatocytes are obtained from Lonza plated on collagen 1 and matrigel coated dishes, and cultured in serum containing WEM (Williams' Medium E; commercially available from Lonza Walkersville Inc., Walkersville, MD, USA), Gentamicin, Dexamethasone 10 mM, FBS 5%, L-Glutamine, Hepes 15 mM, Insulin 4 rag/ml with 50 ng/ml of HGF and EGF. Medium for culturing hES cells and iPS cells is Knockout DMEM supplemented with 20% KOSR, NEAA, 2-ME, GlutaMAX, 6 ng/ml basic fibroblast growth factor (all Invitrogen). hESC lines WA09 (H9) and WAOl (HI) (WiCell) are cultured on irradiated MEF feeder layers in ES medium.

[0082] Retroviral production and Reprogramming of hepatocytes: Retroviruses for the four factors are independently produced after co-transfecting the 293T cell line with pMX retroviral vectors expressing Oct4, Sox2, Klf4 or c-Myc (Addgene) and helper plasmids. A 1:1:1 :1 mix of retroviruses containing Oct4, Sox2, Klf4 and c-Myc is added to hepatocytes (passages 1) in the presence of 6 ng/ml polybrene. After incubating for 3 days in the WEM, media is replaced with hES medium. Cells are cultured until TRA-1-60 positive colonies appear (about 6 to 9 days after retroviral transduction), and individual TRA-1-60 positive colonies are picked onto MEF coated plates.

[0083] Immunofluorescence and FACS analysis: Cells are fixed with 4%

paraformaldehyde. The following antibodies are used: TRA-1-60 (Millipore, 1 :100); SSEA- 4 (Cell Signaling, 1 :200), SSEA-3 (Millipore, 1 :200); Tujl (Covance, 1 :500), oc-fetoprotein (Dako, 1:200), SMA (DAKO, 1 :100), OCT-4 (Millipore, 1:100), NANOG (BD, 1:200), anti- SSEA-3 488 from eBiosciences; CXCR4 (BioLegend, 1:100), albumin (DAKO, 1 :200), AAT (Thermo, 1:200), CyP3A4 (Enzo, 1:200). Secondary antibodies used are all of the Alexa Fluor Series from Invitrogen.

[0084] Periodic acid-schiff assay for glycogen: Cells are fixed with 4% PFA and stained using a periodic acid-Schiff (PAS) staining system (DAKO). Cells are counterstained using Hematoxylin-QS and mounted with Vectamount AQ (all Vector Laboratories).

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331323-000880 [0085] Embryoid body formation and differentiation: Embryoid body formation is induced in ultra low attachment plates in the presence of differentiation medium (DMEM

supplemented with 20% FBS, L-glutamine, 2-ME, and NEAA). After 7 days the embryoid bodies are transferred to 0.1% gelatin-coated culture dishes and cultured for additional 2-3 days before fixation and staining.

[0086] Teratoma formation: Ten-week-old male NOD/SCID/II2yC-/- mice (Jackson Laboratories) are anesthetized and ~1 million hHiPS cells, resuspended in 20-40 μΐ of 50% matrigel, are injected subcutaneously. Mice are euthanized 12 weeks after cell injection and tumors are analyzed following H&E protocol.

EXAMPLE 2

Generation of Human Endoderm Origin iPS cells

[0087] For generation of human endoderm origin iPS cells, human primary hepatocytes are seeded in Williams' Medium E (WEM) with human HGF and EGF. These hepatocytes are over 99% albumin positive before reprogramming (Figure 1 A). Under these conditions, primary human hepatocytes only survive for a short term (about 7 to 10 days) and do not proliferate even with HGF and EGF. After hepatocyte retroviral transduction with Oct4, Sox2, Klf4 and c-Myc (Figure IB), several hundred fast-growing small colonies are observed. By days 7-10 post-infection, these colonies displayed typical human ES cell-like morphology. Expression of the ES cell surface antigens SSEA4 and TRA-1-60, the nuclear transcription factors OGT4 and NANOG can be observed. TRA-1-60 antibody live staining is performed and TRA-1-60 positive colonies are picked and seeded onto a layer of irradiated mouse embryonic fibroblasts (MEFs) in hES cell medium.

[0088] Some of these colonies show a mosaic pattern of TRA-l-60⁺ and TRA- 1 -60^" cells. These TRA-1-60 negative colonies are usually darker, thicker and proliferating faster than TRA-1-60 positive colonies during initial days (up to the first 14 days). However the majority of these non-iPS clones are not able to survive for a long time (disappeared within a few passages) and in most of cases these non-iPS colonies are easily distinguishable by morphology compared to hES cell-like iPS colonies.

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331323-000880 [0089] Seventeen (17) TRA-1-60 positive single colonies are picked and passaged onto fresh feeder cells. Most of these subcultures (16 out of 17) expanded and gave rise to stable cell lines with hES cell-like morphology. All the lines can be maintained by standard hES cell culture procedures on both feeder cells and feeder-free matrigel-coated plates. When fingerprinting is performed to amplify the microsatellite sequences by PCR, all these iPS cells display identical pattern to that of original primary hepatocytes. DNA fingerprinting analysis show that the hepatocyte-derived iPS cell lines are originated from primary hepatocytes (PH) and have a distinct signature from 293T cells and human ES (HI) cells. 100 μg of genomic DNA can be used for fingerprinting with MapPairs primers (D21S2055, Invitrogen). The PCR products can then be separated and viewed in 2.5% agarose gel.

[0090] Four human hepatocyte derived iPS (hHiPS) cell lines (hffiPS6, hHiPS 10, hHiPSl 1 , and hHiPS14) are selected for further analyses. These lines have been maintained in continuous culture for over 6 months without signs of replicative crisis. All hHiPS cells expressed transcriptional regulators and cell-surface markers characteristic of hES cells, including NANOG, OCT4, SSEA4, and TRA-1-60. These cell lines also show complete loss of hepatocyte-specific markers such as albumin. Overall, the expression of stem cell markers in hHiPS cells is indistinguishable from hES cell lines and maintained under the same conditions.

EXAMPLE 3

Embryoid Body Formation

[0091] The pluripotency of hHiPS cell lines in assays of embryoid body (EB) formation in vitro and teratoma induction in vivo are tested. All cell lines tested readily differentiate in vitro into endoderm, mesoderm and ectoderm derivatives that stained positive for a- fetoprotein (AFP), smooth muscle actin (SMA) and TuJl immunoreactivity, respectively. After generation of embryoid bodies hHiPS cells spontaneously differentiate into endoderm ( -fetoprotein-positive), mesoderm (smooth muscle actin-positive) and ectoderm (TuJl- positive neuronal cells).

[0092] About three months after injection into immunocompromised mice, independent hHiPS cell lines generate complex teratomas. Spontaneous differentiation into all three germ layers is also evident in teratomas. Structures resembling all three germ layer tissues such as

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331323-000880 glandular epithelia (endoderm), immature cartilages (mesoderm) and neural rosettes

(ectoderm) can be observed, indicating the pluripotency of these hHiPS cell lines.

[0093] Taken together, these results demonstrate that human endoderm cells (i. e., hepatocytes), like other germ layer derived cells (i.e., fibroblasts or blood cells), can be reprogrammed to pluripotency. Morphologically, nascent hHiPS cell colonies can be identified as early as 6 day post-infection. In contrast, colonies of iPS cells generated by retroviral transduction of human mesenchymal stem cells (MSCs) under the similar experimental condition only appear after day 10 post-infection (Figure 2) similar to that of blood cells.

[0094] About 50-100 hHiPS cell colonies from ~50,000 viable infected hepatocytes can be observed, based on morphological criteria and TRA-1-60 staining, representing an overall reprogramming efficiency close to 0.1-0.2% (n— 3). No significant differences of overall efficiency are observed in the generation of iPS cells from multiple independent human primary hepatocyte cultures.

EXAMPLE 4

Differentiation of HES/iPS Cells to Definitive Endoderm

[0095] Sixty percent confluent cultures are placed in RPMI medium (supplemented with GlutaMAX and 0.5% defined FBS or 1% KOSR, and 100 ng/ml Activin A (R&D Systems) for 5 days. The medium was replaced every other day.

[0096] Differentiation of definitive endoderm to hepatic progenitor cells: Day 5 endoderm cultures are passaged with 0.05% trypsin-0.53 mM EDTA and plated on collagen I-coated dishes, in RPMI medium supplemented with GlutaMAX and 2% KOSR, 10 ng/ml FGF-4, and 10 ng/ml HGF. Three days later the cells were switched to minimal MDBK-MM medium (Sigma) supplemented with GlutaMAX and 0.5 mg/ml BSA, 10 ng/ml FGF4, and 10 ng/ml HGF. Day 10 hepatic progenitors are utilized for experiments.

[0097] Differentiation of hepatic progenitor cells to mature hepatocytes: Hepatic progenitor cells are switched to complete hepatocyte culture medium (HCM) supplemented with SingleQuots (Lonza) and containing 10 ng/ml FGF-4, 10 ng/ml HGF, 10 ng/ml

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331323-000880 oncostatin M (R&D Systems), and 10^"7 M dexamethasone (Sigma- Aldrich). Differentiation is continued for another 10 days.

[0098] The present invention provides that human fibroblast (mesoderm origin) derived iPS cells could be directed to hepatocytes just like ES cells. The present invention also provides that the endodermal origin iPS cells can be more efficiently directed into endoderm cells as well as hepatocytes. The inventors utilized a hepatic differentiation protocol established for hES cells with a slight modification. The hepatic differentiation protocol of the invention is composed of three stages: definitive endoderm induction for 5 days (day 5), hepatic progenitor induction and expansion for another 5 days (day 10) and hepatic maturation for another 10 days (day 20). Cells from these three stages (day 5, day 10, and day 20) are chosen because the inventors observed these time frames are most distinct in terms of their marker expression profile i.e., CXCR4, AFP, or ALB positivity based previously for ES cells. With this protocol, hES cells, hHiPS cells, and mesenchymal stem cell-derived induced pluripotent stem (iMSC) cells are able to differentiate into hepatic cells of all three stages (Figures 3 and 11).

[0099] In the first stage, Activin A efficiently induces the endoderm differentiation of hHiPS cell lines. After 5 days of Activin A treatment, approximately 90% of the cells in culture expressed the endoderm marker CXCR4 and lost the ES/iPS cell marker SSEA3 (Figures 3A, 3B, 3C, 3D, and 3E). Similar results are obtained from multiple independent hHiPS cells.

[0100] To induce early hepatic cells or hepatic progenitor cells, hepatocyte growth factor (HGF) and fibroblast growth factor 4 (FGF4) are used. The expression of AFP, a marker of early hepatic cells, is tested in the differentiated hES cells and hHiPS cells. Approximately 90% of cells are positively stained for AFP in both ES- and hHiPS derived cells at day 10.

[0101] At the end of the final differentiation stage, the expression of hepatic markers in the differentiated hHiPS cells are tested. A majority of the day-20 cells derived from hHiPS cells express mature hepatic markers including albumin, alpha 1 -antitrypsin, and CYP3A4. The efficiency and pattern are similar if not better to hES cell derivatives.

[0102] The ability of the differentiating cell populations to store glycogen, another characteristic of functional hepatocytes, is also tested. Cells at day 20 are stained for

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331323-000880 cytoplasmic glycogen using the Periodic Acid-Schiff staining procedure. Periodic acid- Schiff assay can be performed on differentiating cells at 20 days post-initial Activin A treatment. Nuclei can be counterstained with hematoxylin. Similar to hES cell-derived cells, the majority of the differentiated hHiPS cells (up to 90%) are stained by PAS, indicating that they have the capacity to store glycogen. Hence, consistent with the gain of expression of mature hepatic markers, the differentiating cells from hHiPS cells also exhibit a gain of hepatic functionality.

EXAMPLE 5

Differentiation of IPS Cells Using a Stepwise Differentiation Method

[0103] The present invention provides that human endoderm cells (i.e., hepatocytes) can be rapidly reprogrammed to pluripotency. Although human hepatocyte reprogramming shares the main features reported for iPS cell generation from other cell types such as fibroblasts, including acquisition of self-renewal ability and pluripotency (Figure 1), the overall pace appears to be faster than that of fibroblast, MSC, or blood cell reprogramming.

[0104] Mouse hepatocytes also appear to be more easily reprogrammed than fibroblasts. It is conceivable that hepatocytes per se are more amenable to reprogramming, perhaps because, unlike fibroblasts or MSCs, they would not be required to undergo a mesenchymal- to-epithelial transition to give rise to iPS cells. However, there is a possibility that iPS colonies appear faster because in the case of hepatocytes no cell replating during the early stage of reprogramming is necessary as result of their non-proliferative nature in culture, whereas proliferating MSCs or fibroblasts require culture splitting during the early reprogramming (Figure 2) and thus it takes longer for colonies to emerge.

[0105] The cells that undergo reprogramming can be either early or mature hepatocytes. Although the hepatocyte source is homogenously albumin positive (see Figure 1 A), the inventors also observe that 20-30% of these cells are also AFP positive (Figure 4). Therefore it is possible that the reprogrammed cells are from only the AFP+ALB+ early hepatocytes. To determine the target cell type would require viable cell sorting of the primary hepatocytes but AFP and ALB are the cytoplasmic markers (not surface proteins for viable cell sorting). Moreover, primary hepatocytes would unlikely be healthy enough for reprogramming after

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331323-000880 the cell sorting and replating processes, considering the fact that primary hepatocytes even without sorting stress can only survive a week in culture.

[0106] The present invention provides that human hepatocyte derived iPS (hHiPS) cells can be directly induced to differentiate into endoderm, hepatic progenitors, and mature hepatocytes using a stepwise differentiation method (Figure 3). The hepatic differentiation efficiency of hHiPS cells is comparable if not better to that of the human ES cell lines. The inventors do not detect a significant enhancement in efficiency of hHiPS cells in either spontaneous or directed differentiation processes. This is not surprising because the differentiation efficiency is already very high in hES cells (more than 90%). A more stringent test of the differentiation ability would be in vivo functionality of the differentiated cells. The present invention provides examination whether the hepatocytes from hHiPS cells offer any advantages in the in vivo assays over differentiated cells from hES cells or from iPS cells of other origins. Comprehensive studies comparing human iPS cells from all three developmentally distinct germ layers are needed to determine whether the cell origin for reprogramming has a critical influence on functionality or safety of differentiated cells.

[0107] An important potential use of human iPS cell-derived hepatic cells is for drug development. Most drugs rely on liver cytochrome P450 activity for detoxification, which cannot be tested in animal liver cells due to species differences. The present invention provides the expression of CYP in the differentiated cells and found that the hHiPS cell derived hepatic cells expressed Cyp3 A4, as detected by immune staining in addition to fully differentiated hepatic markers, ALB and AAT expression. The functionality is also confirmed by glycogen storage activity. These data suggest that human iPS cell-derived hepatic cells may be used as a potential cell source for the generation of hepatocytes for drug metabolism analysis.

[0108] The present invention provides methods for reprograming human endoderm derived cells into iPS cells and also provides that they can be directly differentiated into hepatic cells. Reprogramming of primary hepatocytes to pluripotency should provide a valuable experimental model for investigating the bases of cellular reprogramming of other human endoderm cells. Equally important is the generation of liver disease specific iPS cells for studying liver disease pathogenesis, including hepatocellular carcinoma and liver cirrhosis, utilizing available liver tissues which can be obtained after partial hepatectomy or

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331323-000880 diagnostic liver biopsy. The generation of endoderm derived human iPS cells also facilitates the comparative studies to determine the most suitable iPS cells in terms of safety and efficacy for treating particular diseases.

EXAMPLE 6

Differentiation of Human IPSCs to Multistage Hepatic cells

[0109] Cell culture: ESC and iPSCs are cultured in standard ESC maintenance media; Knockout DMEM supplemented with 20% KOSR, NEAA, 2-ME, GlutaMAX, 8 ng/ml basic fibroblast growth factor (all Invitrogen). hESC lines WA09 (H9) and WA01 (HI) (WiCell) are cultured on irradiated MEF feeder layers in the ESC medium. Alternatively cells are cultured on matrigel using MEF conditioned media or mTeSR (Stem Cell Technology).

[0110] Differentiation of human iPSCs to multistage hepatic cells : Fifty to sixty percent confluent cultures are placed in RPMI medium (supplemented with GlutaMAX and 0.5% defined FBS, and 100 ng/ml Activin A (R&D Systems) for 5-6 days to induce DE stage cells. The medium is replaced every other day. DE cells are passaged with 0.05% trypsin-0.53 mM EDTA and plated on collagen I-coated dishes in minimal MDBK-MM medium (Sigma) supplemented with GlutaMAX and 0.5 mg/ml BSA, 10 ng ml FGF4, and 10 ng/ml HGF. Day 10 hepatic progenitors are utilized for experiments. Hepatic progenitor cells are switched to complete hepatocyte culture medium (HCM) containing 5% defined FBS, 10 ng/ml FGF-4, 10 ng/ml HGF, 10 ng/ml oncostatin M (R&D Systems), and 10^"7 M

dexamethasone (Sigma). Differentiation is continued for another 10 days to generate mature hepatocytes.

[0111] Transplantation of iPSC derived hepatic cells: The NOD/Lt-SCID/IL-2Rf'^' (NSG, Jackson Laboratories) mice is used as recipients of the human cells because the NSG mouse is a new generation of severely immunodeficient mouse which accepts human cells much more easily compared with any other type of immunodeficient rodent models. NSG mice receive a hepatotoxin dimethylnitrosamine (5 mg/kg DMN, 1.0% dissolved in saline) intraperitoneally 3 consecutive days per week for 4 weeks to induce chronic liver injury. One day after the final DMN injection, 2 xlO⁶ human iPSC/ESC derived hepatic cells are intravenously transplanted to seven to ten week old male mice. Due to a high mortality rate associated with surgery for intraspenic/portal injection in the highly immunodeficient NSG

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331323-000880 mice especially after hepatotoxin treatment, tail vein injections are used for all the experiments. 8 weeks after human cell transplantation, recipient mouse liver and blood is harvested. Human hepatocytes that are producing the albumin protein are identified in mouse liver by an antibody specifically recognizing human but not mouse albumin. Liver images are taken using the Nikon Ti-E microscope with Coolsnap HQ2 camera. Serum and plasma are separated from mouse blood and stored at -80 °C for liver function tests.

[0112] FACS analysis and Immunofluorescence: Anti-SSEA-3 488 from eBiosciences (1:100), CXCR4 (BioLegend, 1 :100), albumin (ALB, DAKO, 1 :200), alpha- 1 -antitrypsin (AAT, Thermo, 1:200), and CyP2El (Thermo, 1 :100) are used. Secondary antibodies used are all of the Alexa Fluor Series from Invitrogen. For immunofluorescence, cells are fixed with 4% paraformaldehyde and permeabilized/blocked with 0.1% Triton X-100 and 0.3% bovine serum albumin in PBS.

[0113] Cytochrome P450 Assay: CYP 1 A2 and CYP3A4 activity is assessed using the pGlo kit (Promega) according to manufacturer's instruction for non-lytic CYP450 activity estimation. iPSC-derived mature hepatocytes are incubated with hepatocyte culture medium supplemented with CYP3A4 or CYP1A2 pGlo substrates. At 4 hours after exposure, 50 μΐ of culture medium was removed and read on a luminometer (GLOMAX, model 9101-002). CYP activities are expressed as relative light units (RLU) per mL of media, normalized against percentage of albumin expressing hepatocyte-like cells.

[0114] Enzyme-linked immunoassay: Serum and plasma obtained from mice that have been transplanted with each stage of hepatic cells differentiated from iPSCs ESCs are used for detecting human specific liver protein secretion. Human ALB and Transferrin (alpha diagnostic), and AAT and Fibrinogen (GenWay Biotech, Inc.) are measured using enzyme- linked immunoassay (ELISA) kits according to the manufacturer's instruction.

EXAMPLE 7

DNA Methylation Measurement

[0115] Methylation status across the genome is measured using the Illumina® Infinium® comprehensive DNA Methylation assays, according to the manufacturer protocols, which enables the investigation of a large number of genomic loci (27,578 CpG measurements

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331323-000880 spanning 14,495 genes). Methylation estimates, as obtained from the Illumina BeadStudio Software, are processed and analyzed using functions and methods available through the R- Bioconductor biostatistical computing environment. At each genomic locus the log2 ratio between probe intensities for methylated and un-methylated probes is obtained and further used in all subsequent analyses. For the generalized linear model and gene sets enrichment analyses all genomic features located within 1500 bp from a transcription starting site of a known gene, as obtained from the NCBI ENTREZ gene data base, are averaged. For clustering analyses both original and averaged methylation estimates are used, with comparable results. All analyses are performed using all measured autosomal genomic loci, excluding the non autosomal ones, in order to avoid confounding with gender. All results described are obtained using the log2 ratio between un-methylated and methylated probes, however similar results are also obtained using the beta statistics (methylated/un- methylated+methylated) .

[0116] Multidimensional scaling plot and hierarchical clustering: Classical

multidimensional scaling (MDS) is used (principal coordinates or components analysis) to display in a multidimensional space the distance between the individual samples analyzed, based on their methylation status, using both averaged and unaveraged methylation estimates. The Euclidian distance between all analyzed samples is computed, using methylation status estimates at all measured autosomal loci on the Infinium Illumina platform. For this analysis the variance across all samples analyzed is used to filter genomic loci showing no variation across samples. Similarly hierarchical clustering is performed to further show the

relationships among the analyzed cell lines, using Euclidian distance and average linkage clustering method. All analyses are also performed using 1 -squared correlation as the distance, using both averaged and unaveraged methylation estimates with similar results.

[0117] Differential methylation analysis: Differential methylation across the analyzed sample groups is investigated using functions and methods implemented in the

R/Bioconductor package limma. At each genomic locus the averaged log2 ratio between probe intensities for methylated and un-methylated probes is used, and non autosomal loci are excluded from the analysis to avoid confounding with gender. A fixed effects linear model is fit for each individual locus to estimate methylation differences between the compared groups of samples. An empirical Bayes approach is applied to moderate standard errors of

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331323-000880 normalized logarithmic fold-change (M- values). Finally, for each feature, moderated t- statistics, log-odds ratios of differential methylation (B- statistics), raw and adjusted p-values (FDR control by the Benjamini and Hochberg method (S6) are obtained.

[0118] Analysis of Functional Annotation: Gene set enrichment analyses are performed using statistical packages from the R/Bioconductor project. Briefly, to capture biological processes and signaling pathways associated with differential methylation, Analysis of Functional Annotation (AFA) is performed, where the AFA is conceptually similar to Gene Set Enrichment Analysis (GSEA). The gene lists used are obtained from the Gene Ontology database, the KEGG pathway database, and from the Molecular Signature Database

(MsigDb). Functional themes enrichment are first evaluated for each group comparison performed by one-sided Wilcoxon test, using the moderated t-statistics obtained from the linear model analyses to rank the features/loci. Similarly, the inventors also test for gene sets over- and under-represented in overlapping and non-overlapping lists of differentially methylated features across contrasts between iPSCs and their parental counterpart using cross-tabulation and the chi-squared test. Multiple testing correction is performed using the Benjamini and Hochberg method.

EXAMPLE 8

Retroviral production and reprogramming of keratinocvtes

[0119] Retroviruses for the four factors are independently produced after co-transfecting the 293T cell line with pMX retroviral vectors expressing Oct4, Sox2, Klf4 or c-Myc (Addgene) and helper plasmids. A 1 : 1 : 1 : 1 mix of retroviruses containing Oct4, Sox2, Klf4 and c-Myc is added to keratinocytes (passages 1) in the presence of 8 μξ/ηύ polybrene. After incubating for 3 days in the serum free gold media (Lonza), media is replaced with hESC medium. After transformed colonies are observed in the reprogramming plates, a pluripotent stem cell marker, TRA-1 -60 antibody (1 :200 dilution, Millipore) and Alexa555 conjugated anti-mouse IgM antibody (1 :500 dilution, Invitrogen) are added into live cell culture (without fixation) and incubated for 1 hour at 37 °C, to distinguish the iPSC from non-iPSC colonies. TRA-1-60 positive colonies appear in about 6 days after retroviral transduction, and individual TRA-1-60 positive colonies are picked onto MEF coated plates.

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331323-000880 [0120] Embryoid body formation and spontaneous differentiation into three germ layer cells: Human iPSCs (iKl, iK2, and iK3) are dissociated by collagenase IV digestion and plated in ultra low attachment plates (Corning) at the density of ~ lxlO⁶ cells/well in the presence of differentiation medium (DMEM supplemented with 20% FBS, L-glutamine, β- mercaptoethanol, and Non-essential amino acids). Half of the medium is replaced with fresh medium every 2 days. After 7 days the embryoid bodies are transferred to 0.1% gelatin- coated culture dishes and cultured for additional 3 days before fixation and staining.

Antibodies against Tujl (Covance, 1 :500), a-fetoprotein (Dako, 1 :200), or SMA (DAKO, 1 :100) are used to detect the spontaneously differentiated cells from EBs.

[0121] Teratoma formation: Ten-week-old male NSG mice (Jackson Laboratories) are anesthetized and ~1 million iPSCs, resuspended in 20-40 μΐ of 50% matrigel, are injected subcutaneously. Mice are euthanized 8 to 12 weeks after cell injection and tumors are analyzed following H&E protocol.

[0122] Immunofluorescence: Cells are fixed with 4% paraformaldehyde. The following antibodies are used: TRA-1-60 (Millipore, 1 :100); SSEA-4 (Cell Signaling, 1 :200); Tuj l (Covance, 1 :500), a-fetoprotein (Dako, 1 :200), SMA (DAKO, 1 :100), OCT-4 (Millipore, 1 :100), NANOG (BD, 1 :200). Secondary antibodies used were all of the Alexa Fluor Series from Invitrogen.

[0123] RNA isolation and Quantitative Real-Time Polymerase Chain Reaction: Total RNA is isolated from undifferentiated ESC/iPSCs cultured on matrigel and genomic DNA is removed using RNAqueous® -PCR kit (Applied Biosystems). Two μg total RNA from each sample is subjected to cDNA using High Capacity RNA-to-cDNA kit (Applied Biosystems). cDNA products are diluted 1 :100 for Oct4 and 1 : 1000 for 18s rRNA in water, and 4 μΐ of each cDNA with TaqMan® Universal PCR Master Mix and TaqMan® Gene Expression Assays for each gene (Applied Biosystems) are used real time PCR. Reactions are carried out in triplicate and analyzed on StepOnePlus Real-Time PCR System (Applied Biosystems).

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331323-000880 EXAMPLE 9

Human iPSC Generated from Ectoderm

[0124] Human hepatocyte (endoderm), bone marrow mesenchymal stem cell (MSC, mesoderm), and liver fibroblast (mesoderm) derived iPSC lines can be generated using retroviruses expressing Oct4, Sox2, Klf4 and c-Myc. In this example, iPSCs from human keratinocytes (ectoderm) are generated using the same retroviruses but slightly different conditions (Figure 8). The pluripotency of these cell lines is provided. All cell lines (15 iPSC lines derived from three germ layer origin tissues) proliferated at similar rates, express the pluripotency markers, and give rise to teratomas, demonstrating their identities as bona fide iPSC lines (Table 1).

[0125] Karyotyping of iKera cells: After 30 passages, these iPSCs (including iKl, iK2, and iK3 lines) show normal karyotypes. Embryoid bodies can be derived from iKl line and In vitro differentiation can be performed into all three primary germ cell layers. After generation of embryoid bodies, iKl cells spontaneously differentiated into endoderm (AFP+), mesoderm (SMA+) and ectoderm (TuJl+). Spontaneous differentiation into all three germ layers including glandular epithelium, cartilages, pigmented epithelium can also be observed in teratomas.

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331323-000880 [0126] To gain insight about the epigenetic patterns of the pluripotent stem cells from different origins, the present invention provides a genome- wide methylation analysis on all three germ layer-derived human iPSCs, their parental cells and ESCs. The overall methylation patterns of all human iPSCs including endoderm tissue origin-iPSCs (iHIO, iHl 1, iH14), mesoderm-iPSCs (iM2, iM3, iM7, iLCl, iLC2) and ectoderm-iPSCs (iKl, iK2, iK3) are highly similar to those of ESCs (HI and H9) and are distinct from the parental cells (Figures 5A and 9). In hierarchical clustering and heat map of these lines base upon top 5% most varying features (Figure 5B), the inventors are able to detect one cluster of CpG loci that are hypermethylated only in ESCs but not in iPSCs or parental cells (Figure 5B, indicated with *), indicating a certain level of common epigenetic memory in iPSCs retained from adult somatic cells. Methylation patterns that are common to the same germ layer origin iPSCs and their parental cells but distinct from all the rest cells (i.e., different origin iPSCs and their parental cells, and ESCs) are also found, indicating certain level of tissue specific epigenetic memory preserved in all these distinct iPSCs (Table 3). Nonetheless the overall methylation patterns of human iPSCs are highly similar to one another and to ESCs (but lower than ESC) rather than to their parental cells even in the top 5% most varying feature based hierarchical clustering/heat map (Figure 5B).

[0127] The differences and similarities among distinct origin human iPSCs including hepatocyte derived iPSCs are evaluated. The present invention provides that epigenetic memory of the parental/donor cells exists in human iPSCs (Figures 5 A, 5B and Table 3). As shown in Table 3, significant numbers of donor specific epigenetic features are still retained each germ layer origin human iPSCs indicating tissue origin associated signatures across the multiple iPSCs. This is important because these retained epigenetic features might influence differentiation propensity (including differentiation into blood; see Kim et al. (2010) Nature. 467:285-290 and Polo et al. (2010) Nat Biotechnol. 28:848-855) and importantly the Safety of iPS cells (inclduing tumor-forming propensity; see Miura et al. (2009) Nat Biotechnol. 27:743-745).

[0128] The list of loci differentially methylated between each of iPSC lines and their parental cells are also analyzed, and the lists among the distinct developmental groups is compared (Figure 5C; differentially methylated features between iPSCs versus parental cells, P-value < 0.001). In addition to common sets of loci that either gained or lost DNA

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331323-000880 methylation during reprogramming, tissue specific methylation changes are also evident in the reprogramming processes of somatic cells from each germ layer (Figure 5C). This is further confirmed by functional gene set enrichment analyses. Genes involved in tissue specific biological pathways (e.g., liver/metabolism related genes in hepatocytes) are among the ones that gained methylation during reprogramming (Figure 5C). These results suggest that human iPSCs undergo both common and distinct changes associated with their cell of origin to become pluripotent (Figure 5C). Together these results provide that human iPSCs of various sources are strongly similar to one another and to (a lesser degree) ESCs, regardless of the distinct epigenetic changes that occurred during the reprogramming process.

EXAMPLE 10

Hepatic Differentiation Potential of IPSCs

[0129] To determine if the observed molecular similarity/differences among distinct source iPSCs could influence hepatic differentiation potential in vitro, the invention provides evaluation of the directed hepatic differentiation propensities for 15 iPSC lines and 2 ESC lines (Table 2) using the multi-stage differentiation protocol (Figure 6A). All these iPSCs, regardless of their origin, generated each stage of hepatic cells [definitive endoderm (DE), hepatic progenitors, and mature hepatocytes] with comparable efficiencies (Figures 6B, 6C, and 6D). At the end of stage 1, all iPSCs give rise to DE cells with -90% efficiency as measured by SSEA3 negativity and CXCR4 positivity (Figure 6B). Virtually all the DE cells are differentiated into AFP expressing hepatic progenitors at day 10 (Figure 6C), and these cells again differentiated into albumin (ALB)/cc-l -antitrypsin (A AT) expressing mature hepatocytes at day 20 (Figure 6D). In addition, no significant differences are observed in functional capabilities of mature hepatic cells derived from the distinct origin iPSCs (i.e., CYP450 activities, Figures 6E and 6F).

[0130] Recent reports showed tendency of early-passage mouse iPSCs to differentiate preferentially into the cells of origin (i.e., blood) and increased expression levels of Oct4 during the passaging process of the mouse iPSCs. However, such passage/origin effect is not observed in the human iPSC-based hepatic differentiation. The present invention provides that this is mainly due to a high differentiation efficiency of human iPSCs into the hepatic lineage (~90%), making a small difference not easily distinguishable. Since passage related

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331323-000880 Oct4 levels have been shown to be inversely correlated to differentiation efficiencies, real time PCR for OCT4 among early and late passages of human iPSCs are performed (Figure 10). The expression levels of OCT4 of all three germ layer-derived human iPSC lines are similar to one another and to ESCs regardless of passage numbers, which correlates with the hepatic differentiation results across all iPSCs of different origin/passages.

[0131] To investigate the potentials of the different origin iPSCs in liver regeneration in vivo, the present invention provides evaluation of the in vivo engraftment capabilities of human iPSC derived multi-stage hepatic cells using highly immunodeficient NOD/Lt- SCID/IL-2Ry^~A (NSG) mice. Under conditions of chronic liver injury (4 week

dimethylnitrosamine treatment mimicking liver cirrhosis) for the purpose of increasing donor cell engraftment, human ESC/iPSC derived hepatic cells migrated to and engraft mouse liver (Figure 7). Eight weeks after human cell transplantation, mouse liver and blood are analyzed for liver engraftment and in vivo functions of the iPSCs and their hepatic derivatives (Figure 7 and Table 2). Human liver protein secretion is measured using enzyme-linked

immunoassays for each protein in serum/plasma obtained from mice that had been transplanted with each stage of hepatic cells differentiated from human iPSCs. Both undifferentiated (dO) iPSCs and human primary hepatocytes are used as controls. DE;

definitive endoderm, ND; not detected. All ESC/iPSC derived DE cells differentiate into mature and functional human hepatocytes as evidenced by the production of human ALB and CYP2E1. The inventors further evaluate hepatic progenitors and mature hepatocytes derived from human ESC/iPSCs for the level of mouse liver repopulation. Cells of every stage show a certain level of engraftment evidenced by positivity for human specific ALB and CYP2E1 in mouse liver. Mice which do not receive human cells or received human primary hepatocytes (PH) are used as a negative control and a positive control, respectively. An increased percentage of human ALB+ cells is detected in a mouse liver when a higher 7xl0⁶ dose human iPSC derived DE cells are transplanted.

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331323-000880 Table 2. Human liver protein secretion from human iPSC derived hepatic cells in mouse serum/plasma (n=6-15, Mean± SEM)

DO D10 (Hepatic D20 (Mature Primary

Unit D5 (DE)

(iPSCs) Progenitors) hepatocytes) Hepatocytes

ALB ng/ml 1.8 ± 0.5 45 ± 17 32 ± 13 39 ± 15 40 ± 11

Transferrin ng/ml 9 ± 0.8 101 ± 21 . 62 ± 13 86 ± 18 58 ± 17

AAT ng/ml 1.1 ± 0.5 8.1 ± 1.6 7.8 ± 1.2 7.9 ± 1.5 8.2 ± 1.4

Fibrinogen μ©¾^η1 ND 1.1 ± 0.2 0.9 ± 0.1 0.9 ± 0.1 0.9 ± 0.1

[0132] In order to overcome the limitation of a conventional imaging approach {i.e., taking snap shots of a small area of a tissue section) for more accurate determination of hepatic engraftment efficiency, the present invention provides the automated "Scan Large Image" method which provides scanning the entire area of a tissue section (Figure 7). By this approach the inventors are able to calculate more precise engraftment percentages of human ALB⁺ cells by scanning multiple different lobules/lobes reflecting the entire mouse livers. Regardless of tissue origin, human iPSCs/ESCs display equivalent levels of liver engraftment potentials (Figure 7). The level of engraftment is significantly higher in mice receiving differentiated hepatic cells (i.e., DE, hepatic progenitors, and mature hepatocytes) compared to those receiving undifferentiated iPSCs (Figure 7).

[0133] Unexpectedly, hepatic progenitors show a little less hepatic engraftment than those transplanted with DE or mature hepatocytes although the difference is not statistically significant (Figure 7). Distribution of human cells relative to liver histology/structures are easily detected by the scan method; many of the human cells are detected as focal patterns associated with vessels (2 million DE cells are transplanted) and some are evenly distributed in hepatic parenchyme especially in a mouse receiving a higher cell dose (7 million DE cells are transplanted). Increased engraftment with a higher cell dose is observed (>30% with 7 million) compared with lower cell dose transplantation (-13% with 2 million). The level of engraftment is sustained for a long-term period (> 7 months).

[0134] The present invention also provides in vivo secretion of human liver specific proteins in serum/plasma obtained from mice that had been transplanted with each stage of hepatic cells differentiated from human iPSCs (Table 2). Measurable levels of human liver proteins including ALB, Transferrin, AAT and Fibrinogen can be detected. The levels of

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331323-000880 these secreted proteins are comparable to those obtained from mice that have been transplanted with human primary hepatocytes (Table 2). Importantly, detected protein levels are correlated with the engraftment efficiency.

[0135] Together, these data demonstrates that human iPSC derived multi-stage hepatic cells can engraft mouse liver, and functionally produce/secrete human liver proteins regardless of their origin (Figure 7 and Table 2). The levels of engraftment and functionality of these cells are comparable to primary hepatocytes. This engraftment potential of human iPSC derived hepatic cells will be critical to create humanized liver mice with patient iPSCs for in vivo disease modeling.

[0136] The present invention provides that diverse developmental origin of human iPSCs does not significantly influence their potentials to differentiate into hepatic cells both in vitro and in vivo, despite certain levels of existing epigenetic memory of their origins. These results might suggest that non-hepatic source iPSCs can be sufficient for regeneration therapy and drug screening for many liver disorders. However disease modeling/drug discovery of many acquired liver diseases which accumulate genetic/epigenetic changes only within the affected hepatocytes, not in other cell types, will still require hepatic-sourced iPSCs. There is a possibility that for certain human cell types (e.g., blood), epigenetic memory may have a more significant impact on the efficiency of directed differentiation as observed in the mouse studies.

[0137] From the foregoing description, it will be apparent that variations and

modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0138] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or

subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. Terms listed in single tense also include multiple unless the context indicates otherwise. The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary

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331323-000880 skill in the art and are encompassed by the appended claims. All publications, databases, and patents cited herein are hereby incorporated by reference for all purposes.

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331323-O00880 27350 APOBEC3C 12 104 ADARB1 11.5 9021 SOCS3 13.1

9289 GPR56 11.7 6910 TBX5 11.5 10485 Clorf61 13.1

10578 GNLY 11.7 90050 C14orfl52 11.4 50507 NOX4 13.1

57111 RAB25 11.6 3159 HMGA1 11.4 148738 HFE2 13.1

115761 ARL11 11.6 11259 D0C1 11.3 1903 EDG3 13

90678 LRSAM1 11.5 55103 RALGPS2 11.2 221 ALDH3B1 13

3171 F0XA3 11.4 83597 RTP3 11.2 63924 □ DEC 12.7

284837 LOC284837 11.3 4773 NFATC2 11.2 5867 RAB4A 12.6

10801 9-Sep 11.3 26007 DAK 11 1280 COL2A1 12.6

128876 C20orfl28 11.2 3914 LA B3 11 113220 KIF12 12.6

1128 CHRM1 11.2 4487 MSX1 10.9 90050 C14orfl52 12.5

23413 FREQ 11.1 3856 KRT8 10.9 6770 STAR 12.4

53822 FXYD7 11.1 2706 GJB2 10.9 4313 MMP2 12.4

9051 PSTPIP1 11 5777 PTPN6 10.8 283600 C14orf68 12.2

91663 MYADM 11 1 A1BG 10.8 3386 ICA 4 12.1

54106 TLR9 11 221833 SP8 10.8 11117 EMILINl 12

343035 Clorf36 11 203100 HTRA4 10.8 22981 KIAA0980 12

27134 TJP3 11 128602 C20orf85 10.7 10864 SLC22A7 12

6614 SN 10.9 1776 DNASE1L3 10.7 3855 KRT7 11.9

65094 JMJD4 10.9 122416 ANKRD9 10.6 55237 C14orfll5 11.9

339665 SLC35E4 10.9 2041 EPHA1 10.6 6236 RRAD 11.9

4793 NFKBIB 10.8 55615 PRR5 10.5 1 A1BG 11.6

83742 ARVELD1 10.8 3898 LAD1 10.5 2030 SLC29A1 11.6

6776 STAT5A 10.7 9750 C6orf32 10.5 5575 PRKAR1B 11.6

1638 DCT 10.7 151011 10-Sep 10.4 6927 TCF1 11.6

3866 KRT15 10.6 9249 DHRS3 10.4 25999 CLIPR-59 11.5

90199 WFDC8 10.6 4320 MMP11 10.3 60436 TGIF2 11.5

6385 SDC4 10.5 780 DDR1 10.3 3083 HGFAC 11.4

56302 TRPV5 10.4 5265 SERPINA1 10.3 219928 RGPRF 11.4

81875 ISG20L2 10.4 9289 GPR56 10.1 4762 NEUROG1 11.3

23061 KIAA0676 10.4 5083 PAX9 10 718 C3 11.1

7462 LAT2 10.3 8424 BB0X1 10 3697 ITIH1 11

338773 TMEM119 10.3 8710 SERPINB7 10 23231 KIAA0746 10.9

6398 SECT 1 10.3 55620 STAP2 10 335 APOA1 10.9

80781 C0L18A1 10.2 85004 RERG 9.91 4625 MYH7 10.8

84948 TIGD5 10.2 345 APOC3 9.88 9253 NUMBL 10.7

390212 GPR152 10.1 338442 GPR109A 9.71 81493 SYNC1 10.7

26 ABP1 10.1 1030 CDKN2B 9.59 866 SERPINA6 10.7

2626 GATA4 10.1 10672 GNA13 9.46 80723 TMEM22 10.7

162514 TRPV3 10 9028 RHBDL1 9.4 222487 GPR97 10.6

22924 MAPRE3 9.98 54970 TTC12 9.37 2662 GDF10 10.6

284114 T EM102 9.97 7253 TSHR 9.32 3929 LBP 10.5

9002 F2RL3 9.95 5306 PITPNA 9.26 761 CA3 10.5

1454 CSNK1E 9.81 81029 WNT5B 9.25 29993 PACSIN1 10.5

4261 CIITA 9.81 1044 CDX1 9.22 23037 PDZK3 10.5

1760 DMPK 9.81 8673 VA P8 9.21 29881 NPC1L1 10.4

WEST\2228₆99I4.1

331323-000880 2246 FGF1 9.77 843 CASP10 9.18 51156 SERPINA10 10.4

11322 TMC6 9.74 9048 ARTN 9.17 7490 WT1 10.4

146802 FU31196 9.74 2155 F7 9.14 1749 DLX5 10.4

10614 HEXIM! 9.71 64866 CDCP1 9.11 54810 GIPC2 10.3

8435 S0AT2 9.7 146456 TMED6 9.06 6484 ST3GAL4 10.3

79748 LMAN1L 9.69 1305 C0L13A1 9.05 5648 MASP1 10.3

340205 TREML1 9.66 115572 FAM46B 8.96 4490 T1B 10.3

55205 ZNF532 9.64 7022 TFAP2C 8.95 57168 ASPHD2 10.3

55287 TMEM40 9.64 10110 SGK2 8.92 54751 FBLIM1 10.2

84985 FAM83A 9.63 5443 PO C 8.92 7416 VDAC1 10.2

2694 GIF 9.58 65012 SLC26A10 8.91 79574 EPS8L3 10.1

9362 CPNE6 9.49 8614 STC2 8.87 64757 MOSC1 10.1

79690 GAL3ST4 9.37 131669 UROC1 8.86 6506 SLC1A2 10

340061 LOC340061 9.33 114038 C21orf84 8.84 43847 KLK14 10

148423 Clorf52 9.31 57533 TBC1D14 8.82 7802 DNALI1 9.96

359710 C20orfl85 9.3 421 ARVCF 8.8 108 ADCY2 9.95

2950 GSTP1 9.26 9020 MAP3K14 8.8 9970 NR1I3 9.94

6275 S100A4 9.24 4081 MAB21L1 8.78 3857 KRT9 9.92

83401 EL0VL3 9.23 6615 SNAI1 8.74 23526 HMHA1 9.91

2709 GJB5 9.22 222894 FERD3L 8.71 8525 DGKZ 9.85

2214 FCGR3A 9.22 5493 PPL 8.7 1812 DRD1 9.84

2125 EVPL 9.2 2300 FOXL1 8.68 25956 SEC31L2 9.84

116840 CNTROB 9.2 54436 SH3TC1 8.68 9099 USP2 9.78

6273 S100A2 9.09 90990 KIFC2 8.6 11317 RBPSUHL 9.74

5159 PDGFRB 9.08 8416 ANXA9 8.6 27198 GPR81 9.65

3036 HAS1 9.04 83541 C20orf55 8.58 10335 MRVI1 9.61

80097 FU 14346 9 54682 MANSC1 8.57 7015 TERT 9.61

54578 UGT1A6 9 151278 FU32447 8.54 353134 LCE1D 9.6

6696 SPP1 8.99 5077 PAX3 8.53 353144 LCE3C 9.57

6358 CCL14 8.97 27111 SDCBP2 8.39 53841 MUCDHL 9.56

5753 PTK6 8.95 79574 EPS8L3 8.38 3889 KRTHB3 9.55

81607 PVRL4 8.91 2027 EN03 8.28 57538 ALPK3 9.5

388533 UNQ467 8.9 58473 PLEKHB1 8.23 9543 PUNC 9.48

80736 SLC44A4 8.86 1775 DNASE1L2 8.2 5118 PCOLCE 9.45

6348 CCL3 8.82 3909 LAMA3 8.2 9811 KIAA0427 9.41

137797 LYPD2 8.79 1244 ABCC2 8.19 81621 KAZALD1 9.41

56833 SLAMF8 8.77 3294 HSD17B2 8.19 4239 MFAP4 9.38

10544 PROCR 8.76 400830 DEFB32 8.12 325 APCS 9.36

11117 EMILINl 8.65 9022 CLIC3 8.12 2147 F2 9.32

136371 ASB10 8.64 2672 GFI1 8.07 10873 ME3 9.32

85456 TNKS1BP1 8.63 2147 F2 8.02 124404 FU25410 9.32

23500 DAAM2 8.63 2877 GPX2 8.02 2028 ENPEP 9.26

126353 C19orf21 8.62 8431 NR0B2 7.98 8431 NR0B2 9.21

3590 IL11RA 8.59 26232 FBX02 7.97 23542 MAPK8IP2 9.19

79413 ZBED2 8.57 3960 LGALS4 7.97 2260 FGFR1 9.18

147495 APCDD1 8.56 7850 IL1R2 7.96 8382 NME5 9.17

WEST\222869914.1

331323-000880 23650 TRIM29 8.55 50619 DEF6 7.93 8692 HYAL2 9.16

9450 LY86 8.52 2741 GLRA1 7.81 8993 PGLYRP1 9.1

348174 LOC348174 8.5 3866 KRT15 7.77 90332 XTP7 9.08

374739 TEPP 8.5 4232 MEST 7.76 60312 AFAP 9.08

253935 ANGPTL5 8.48 5345 SERPINF2 7.75 763 CA5A 9.06

8436 SDPR 8.48 10410 IFITM3 7.73 5348 FXYD1 9.06

3689 ITGB2 8.47 3200 HOXA3 7.71 30008 EFEMP2 9

80380 PDCD1LG2 8.42 108 ADCY2 7.7 116835 HSPA12B 9

8572 PDLIM4 8.38 5697 PYY 7.68 133121 ENPP6 9

26191 PTPN22 8.32 10848 PPP1R13L 7.66 5624 PROC 8.97

148170 CDC42EP5 8.29 55287 TMEM40 7.65 4489 MTIA 8.97

201294 UNC13D 8.22 130367 SGPP2 7.6 85480 TSLP 8.94

2170 FABP3 8.2 5753 PTK6 7.59 51160 VPS28 8.93

22874 PLEKHA6 8.18 3299 HSF4 7.59 1293 COL6A3 8.91

2012 EMP1 8.18 858 CAV2 7.58 3172 HNF4A 8.86

8857 FCGBP 8.17 3699 ITIH3 7.54 7849 PAX8 8.83

22941 SHANK2 8.15 149708 WFDC5 7.51 60385 TSKS 8.78

4245 MGAT1 8.13 3232 HOXD3 7.49 716 CIS 8.74

84766 MGC4266 8.12 79152 FA2H 7.49 10110 SGK2 8.74

23263 MCF2L 8.08 5970 RELA 7.47 147746 HIPK4 8.72

4720 NDUFS2 8.07 335 APOA1 7.43 119587 CPXM2 8.71

55898 UNC45A 8.07 272 AMPD3 7.39 338321 NALP9 8.68

6820 SULT2B1 8 5763 PT S 7.37 7804 LRP8 8.59

2215 FCGR3B 7.99 144568 A2ML1 7.37 131669 UROC1 8.59

84033 OBSCN 7.93 64921 NBLA04196 7.37 349136 LOC349136 8.57

55643 BTBD2 7.88 80818 ZNF436 7.36 54544 Clorf42 8.56

1606 DGKA 7.87 201780 SLC10A4 7.35 10449 ACAA2 8.56

1101 CHAD 7.86 5364 PLXNB1 7.34 3483 IGFALS 8.51

1535 CYBA 7.85 2769 GNA15 7.34 388218 UNQ5810 8.51

23429 RYBP 7.83 6899 TBX1 7.34 84101 USP44 8.5

2810 SFN 7.77 283078 IRXL1 7.31 51733 UPB1 8.49

51161 C3orfl8 7.76 89796 NAV1 7.28 4776 NFATC4 8.47

83638 Bles03 7.76 9333 TGM5 7.28 8614 STC2 8.46

2022 ENG 7.75 4485 MST1 7.26 4922 NTS 8.46

246176 GAS2L2 7.74 646 BNC1 7.26 2822 GPLD1 8.43

56944 0LFML3 7.73 2308 FOX01A 7.25 161247 LOC161247 8.42

221472 FGD2 7.7 389434 C6orf71 7.22 5368 PNOC 8.41

4222 ME0X1 7.7 1748 DLX4 7.19 344838 PAQR9 8.32

93643 TJAP1 7.69 9235 IL32 7.13 5345 SERPINF2 8.31

1742 DLG4 7.69 8692 HYAL2 7.06 3707 ITPKB 8.3

1728 NQOl 7.59 4199 MEl 7.06 5173 PDYN 8.3

51629 CGI-69 7.59 116844 LRG1 7.06 9590 AKAP12 8.29

11078 HRIHFB2122 7.54 712 C1QA 7.01 255043 TMEM86B 8.26

3846 KRTAP5-9 7.54 169166 GC39715 7 6707 SPRR3 8.23

90019 SYT8 7.54 635 BHMT 7 5376 PMP22 8.22

WESTV222869914.1

331323-000880 1212 CLTB 7.53 53832 IL20RA 6.98 2527 FUT5 8.22

25959 ANKRD25 7.52 3990 UPC 6.96 284340 UNQ473 8.22

118738 ZNF488 7.52 6717 SRI 6.95 283375 SLC39A5 8.22

113622 ADPRHL1 7.52 347733 TUBB2B 6.95 3242 HPD 8.14

8476 CDC42BPA 7.5 23562 CLDN14 6.95 29953 TRHDE 8.14

7113 TMPRSS2 7.49 4009 LMX1A 6.93 9576 SPAG6 8.13

3914 LAMB3 7.48 26281 FGF20 6.92 345 AP0C3 8.13

126308 MQBKL2A 7.45 90019 SYT8 6.91 401040 FU45964 8.12

3888 RTHB2 7.45 4250 SCGB2A2 6.86 1160 CKMT2 8.12

64333 ARHGAP9 7.45 822 CAPG 6.85 2161 F12 8.11

55273 TMEM100 7.43 79755 FU13841 6.84 4188 MDFI 8.11

220388 FU38159 7.4 202915 GC9712 6.84 60681 FKBP10 8.08

414062 CCL3L3 7.37 3651 IPF1 6.8 183 AGT 8.07

9253 NUMBL 7.36 2625 GATA3 6.76 1582 CYP8B1 8.07

374875 SCDR10 7.34 10747 MASP2 6.75 1564 CYP2D7P1 8.05

286256 LCN12 7.32 64218 SE A4A 6.74 2949 GSTM5 8.04

6538 SLC6A11 7.31 404217 CTXN1 6.73 79170 ATAD4 8.03

3265 HRAS 7.3 57104 PNPLA2 6.71 5104 SERPINA5 8.01

1639 DCTN1 7.28 977 CD151 6.71 116987 CENTG2 7.98

170575 GIMAP1 7.28 9154 SLC28A1 6.69 10255 HCG9 7.98

1066 CES1 7.26 51296 SLC15A3 6.68 7123 CLEC3B 7.98

79228 WDR58 7.25 57580 PREX1 6.66 2569 GABRR1 7.94

944 TNFSF8 7.23 54923 LIMEl 6.65 246 AL0X15 7.93

83933 HDAC10 7.2 2114 ETS2 6.64 1565 CYP2D6 7.92

114131 UCN3 7.17 7498 XDH 6.62 54674 LRRN3 7.9

3770 KC J14 7.15 10406 WFDC2 6.59 10750 GRAP 7.9

84734 Clorf90 7.12 84985 FAM83A 6.58 142683 ITLN2 7.89

2134 EXTL1 7.09 10529 NEBL 6.56 65012 SLC26A10 7.88

8915 BCL10 7.08 1271 CNTFR 6.56 710 SERPING1 7.83

5623 PSPN 7.07 57016 AKR1B10 6.55 9496 TBX4 7.83

55567 DNAH3 7.07 152189 CMTM8 6.54 7169 TPM2 7.83

5438 P0LR2I 7.07 7036 TFR2 6.5 2746 GLUD1 7.83

57010 CABP4 7.05 151473 SLC16A14 6.49 10623 P0LR3C 7.81

790 CAD 7.05 344838 PAQR9 6.49 3485 IGFBP2 7.8

84283 TMEM79 7.05 55867 SLC22A11 6.49 64240 ABCG5 7.8

53828 FXYD4 7.03 143662 MUC15 6.46 51267 CLEC1A 7.79

84292 M0RG1 7.02 55651 N0LA2 6.45 8630 HSD17B6 7.79

6678 SPARC 7.02 118856 MMP21 6.45 10395 DLC1 7.79

124975 GGT6 7 25816 TNFAIP8 6.38 388591 FU46380 7.76

23351 KIAA0323 6.98 54101 RIPK4 6.37 487 ATP2A1 7.74

84286 MGC4618 6.98 2070 EYA4 6.34 50486 G0S2 7.7

29951 PDZRN4 6.97 387990 UNQ9438 6.34 26508 HEYL 7.69

283897 C16orf54 6.96 57190 SEPN1 6.32 23589 CARHSP1 7.66

257203 DSCR9 6.96 4155 MBP 6.32 344 AP0C2 7.65

55630 SLC39A4 6.94 54546 RNF186 6.32 925 CD8A 7.61

7046 TGFBR1 6.93 5029 P2RY2 6.32 9376 SLC22A8 7.61

51475 CABP2 6.92 10417 SP0N2 6.32 1179 CLCA1 7.56

WEST\222869914.1

331323-000880 9290 GP 55 6.92 2295 FOXF2 6.31 971 CD72 7.55

6362 CCL18 6.92 25953 PNKD 6.29 83959 SLC4A11 7.53

22796 COG2 6.89 4258 MGST2 6.28 7054 TH 7.52

1325 CORT 6.86 5213 PFKM 6.27 8858 PROZ 7.52

10850 CCL27 6.86 3026 HABP2 6.23 4921 DDR2 7.49

126432 FU45909 6.84 5947 RBP1 6.22 752 FMNL1 7.47

9536 PTGES 6.83 2063 NR2F6 6.22 4485 MST1 7.47

347853 TBX10 6.83 26257 NKX2-8 6.21 5174 PDZK1 7.46

9358 ITGBL1 6.82 348094 ANKDD1A 6.18 434 ASIP 7.46

71 ACTG1 6.81 135932 FU90586 6.16 5444 PON1 7.45

79932 KIAA0319L 6.8 84931 FU14816 6.15 160622 GRASP 7.44

122622 ADSSL1 6.8 83758 RBP5 6.08 4815 NINJ2 7.44

1145 CHRNE 6.8 317749 DHRS4L2 6.07 10783 NEK6 7.4

5119 PC0LN3 6.78 2065 ERBB3 6.07 113730 KLHDC7B 7.37

57165 GJA12 6.78 124152 MGC35048 6.06 57716 PRX 7.36

4636 MYL5 6.75 4926 NUMA1 6.06 64403 CDH24 7.33

26526 TSPAN16 6.75 56241 SUSD2 6.04 56944 OLFML3 7.33

7043 TGFB3 6.75 3872 KRT17 6.04 203074 UNQ9391 7.32

1823 DSC1 6.74 2624 GATA2 6.03 5730 PTGDS 7.32

85007 MGC15875 6.74 23191 CYFIP1 6.03 131377 KBTBD5 7.31

8839 WISP2 6.73 6097 RORC 6.02 51268 PIPOX 7.3

8808 IL1RL2 6.72 64240 ABCG5 6.01 64763 ZNF574 7.3

84951 TNS4 6.72 26270 FBX06 5.96 27233 SULT1C2 7.29

6274 S100A3 6.72 83999 KREMEN1 5.96 92591 ASB16 7.28

26476 OR10J1 6.71 9032 T 4SF5 5.95 2944 GSTM1 7.27

55850 MDS032 6.71 56925 LXN 5.92 29956 LASS2 7.27

124222 PAQR4 6.71 341883 FU46156 5.92 6582 SLC22A2 7.27

400696 LOC400696 6.67 1718 DHCR24 5.91 51066 C3orf32 7.26

338785 KRT6L 6.66 3700 ITIH4 5.91 92162 T EM88 7.26

91156 DKFZp434B1231 6.65 54799 BTD1 5.9 8528 DDO 7.24

9047 SH2D2A 6.65 3772 KCNJ15 5.9 9027 NAT8 7.24

56257 BCDIN3 6.64 51066 C3orf32 5.89 389084 UNQ830 7.23

7124 TNF 6.64 274 BIN1 5.88 23550 PSD4 7.23

147949 ZNF583 6.63 94241 TP53INP1 5.87 22901 ARSG 7.22

10636 RGS14 6.6 7448 VTN 5.86 3004 GZMM 7.22

973 CD79A 6.59 3398 ID2 5.86 25792 CIZ1 7.21

4939 OAS2 6.59 94234 FOXQ1 5.85 84303 CHCHD6 7.2

84467 FBN3 6.58 374969 CCDC23 5.85 92305 TMEM129 7.17

79713 U2AF1L4 6.57 10659 CUGBP2 5.84 10344 CCL26 7.15

23616 SH3BP1 6.56 5075 PAX1 5.83 146456 TMED6 7.14

324 APC 6.55 8111 GPR68 5.83 27439 CECR6 7.12

29947 DNMT3L 6.54 4880 NPPC 5.83 22918 C1QR1 7.12

57037 ANKMY2 6.53 50604 IL20 5.83 401508 FU45202 7.08

81553 FA 49A 6.52 9963 SLC23A1 5.83 79981 FRMD1 7.08

5202 PFDN2 6.52 25791 NGEF 5.82 3717 JAK2 7.08

23704 KCNE4 6.51 115584 SLC5A11 5.82 6036 RNASE2 7.08

WEST\222869914.1

331323-000880 140446 KRT6C 6.5 9170 EDG4 5.82 337960 KRTAP13-3 7.07

29937 NENF 6.5 84733 CBX2 5.82 79812 MMRN2 7.07

29933 GPR132 6.49 64288 ZNF323 5.81 140691 RNF36 7.07

2865 FFAR3 6.48 92369 SPSB4 5.8 283298 OLFML1 7.04

9028 RHBDL1 6.48 3158 HMGCS2 5.8 6356 CCL11 7.04

1553 CYP2A13 6.48 1116 CHI3L1 5.8 43849 KLK12 7.04

1468 SLC25A10 6.47 149461 CLDN19 5.8 5602 MAPK10 7.03

6663 SOX10 6.47 81552 ECOP 5.79 2209 FCGR1A 7.03

116092 DNTTIP1 6.4 3880 KRT19 5.79 5959 RDH5 7.03

3681 ITGAD 6.4 1525 CXADR 5.78 7471 WNT1 7.02

7044 LEFTY2 6.4 3239 HOXD13 5.78 252995 FNDC5 7.01

728 C5AR1 6.38 1124 CHN2 5.78 10279^' PRSS16 7

5539 PPY 6.38 3053 SERPIND1 5.78 51296 SLC15A3 6.99

6764 ST5 6.37 112817 C10orf65 5.76 6688 SPI1 6.95

3852 KRT5 6.37 200150 PLD5 5.75 112755 STX1B2 6.93

407977 TNFSF12- 6.36 10160 FARP1 5.75 127534 GJB4 6.9

TNFSF13

9080 CLDN9 6.34 201799 FU32028 5.73 283807 FBXL22 6.9

9973 CCS 6.34 23555 TSPAN15 5.73 23187 PHLDB1 6.89

9119 6HF 6.33 338 APOB 5.71 6703 SPRR2D 6.88

3671 ISLR 6.33 64902 AGXT2 5.71 5066 PAM 6.88

89910 UBE3B 6.33 283375 SLC39A5 5.7 467 ATF3 6.87

6504 SLAMF1 6.32 64816 CYP3A43 5.7 5972 REN 6.86

54739 BIRC4BP 6.29 1001 CDH3 5.7 11095 ADAMTS8 6.86

8668 EIF3S2 6.28 79170 ATAD4 5.69 7704 ZBTB16 6.85

375061 FAM89A 6.28 1952 CELSR2 5.69 23436 ELA3B 6.84

517 ATP5G2 6.26 7851 MALL 5.67 55891 LENEP 6.84

4653 MYOC 6.26 6943 TCF21 5.65 284359 IZUMOl 6.84

126375 FU38451 6.26 115290 FBX017 5.64 115584 SLC5A11 6.83

5133 PDCD1 6.25 79852 ABHD9 5.63 8482 SEMA7A 6.82

64089 SNX16 6.24 6692 SPINT1 5.62 400841 FU45832 6.82

7054 TH 6.23 79413 ZBED2 5.62 2038 EPB42 6.82

2867 FFAR2 6.23 5045 FURIN 5.62 3956 LGALS1 6.8

3164 NR4A1 6.22 10040 TOM1L1 5.61 4063 LY9 6.78

1137 CHRNA4 6.21 338785 KRT6L 5.6 51435 SCARA3 6.75

166336 PRICKLE2 6.2 83547 RILP 5.58 254910 LCE5A 6.72

132014 IL17RE 6.15 58985 IL22RA1 5.58 83992 CTTNBP2 6.7

11170 FA 107A 6.14 25891 DKFZP586H2123 5.58 51663 ZFR 6.69

6693 SPN 6.12 170692 ADAMTS18 5.54 92749 MGC16372 6.69

114898 C1QTNF2 6.12 4688 NCF2 5.54 5820 PVT1 6.68

338323 NALP14 6.11 2593 GAMT 5.53 57139 RGL3 6.68

956 ENTPD3 6.1 8764 TNFRSF14 5.51 4332 MNDA 6.68

10964 IFI44L 6.09 1825 DSC3 5.51 4130 MAP1A 6.68

57876 MUC3B 6.08 1364 CLDN4 5.5 50649 ARHGEF4 6.67

WEST\222869914.1

331323-000880 23166 STAB1 6.08 5744 PTHLH 5.5 84561 SLC12A8 6.66

116447 TOP1MT 6.06 83850 FAM62C 5.48 5081 PAX7 6.66

5510 PPP1R7 6.05 9052 GPRC5A 5.48 3211 HOXB1 6.65

57449 PLEKHG5 6.05 3425 IDUA 5.46 430 ASCL2 6.65

64423 C14orfl73 6.02 81889 FAHD1 5.46 2547 XRCC6 6.64

1621 DBH 6.02 80145 NIF3L1BP1 5.46 149345 TMEM58 6.63

4351 MPI 6.02 148398 SAMD11 5.45 7287 TULP1 6.63

1943 EFNA2 6.02 130497 OSR1 5.44 283422 C12orf36 6.63

623 BDKRB1 6.02 3761 KCNJ4 5.43 4725 NDUFS5 6.61

3663 IRF5 6.01 63970 P53AIP1 5.43 347741 OTOP3 6.6

10335 MRVI1 6.01 23213 SULF1 5.43 55024 BANK1 6.59

339403 RLN3R2 6 23231 KIAA0746 5.41 285 ANGPT2 6.57

1847 DUSP5 5.96 163702 IL28RA 5.4 1138 CHRNA5 6.57

1805 DPT 5.96 10874 NMU 5.4 94274 PPP1R14A 6.56

128272 ARHGEF19 5.96 55937 APOM 5.37 85481 PSKH2 6.55

128178 EDARADD 5.95 94235 GNG8 5.36 114990 SLITL2 6.55

5652 PRSS8 5.94 7108 TM7SF2 5.36 255352 C10orf93 6.54

51744 CD244 5.92 776 CACNA1D 5.34 1586 CYP17A1 6.53

5552 PRG1 5.92 166012 CHST13 5.32 275 A T 6.53

126259 MGC23244 5.91 7049 TGFBR3 5.32 57699 CPNE5 6.51

2192 FBLN1 5.9 3852 KRT5 5.31 25791 NGEF 6.51

153090 DAB2IP 5.89 3694 ITGB6 5.3 84275 MGC4399 6.5

9796 PHYHIP 5.87 93611 FBX044 5.3 11167 FSTL1 6.5

3910 LAMA4 5.86 6492 SIM1 5.28 3213 HOXB3 6.49

283487 LOC283487 5.86 1041 CDSN 5.28 64843 ISL2 6.47

7106 TSPAN4 5.84 259 AMBP 5.28 8483 CI LP 6.47

51110 LACTB2 5.84 116496 Clorf24 5.27 3892 KRTHB6 6.47

342666 FU43826 5.83 30812 SOX8 5.27 7480 WNT10B 6.45

6941 TCF19 5.83 27022 FOXD3 5.27 144423 GLT1D1 6.42

1991 ELA2 5.83 3822 KLRC2 5.26 343035 Clorf36 6.41

4284 MIP 5.82 8973 CHRNA6 5.26 197407 ZNF553 6.41

29800 ZDHHC1 5.8 25946 ZNF385 5.23 1805 DPT 6.4

8567 MADD 5.8 57111 RAB25 5.19 1025 CDK9 6.39

10158 PDZK1IP1 5.8 163183 FU36445 5.19 8718 TNFRSF25 6.37

79188 TMEM43 5.79 25780 RASGRP3 5.18 146722 CD300LF 6.36

113828 FAM83F 5.78 6493 SI 2 5.17 6278 S100A7 6.34

828 CAPS 5.77 23362 PSD3 5.16 9965 FGF19 6.33

8581 LY6D 5.76 23516 SLC39A14 5.14 359 AQP2 6.32

5368 PNOC 5.75 9743 RICS 5.14 84929 FIBCDl 6.31

5745 PTHR1 5.75 189 AGXT 5.13 51274 KLF3 6.31

84690 SPATA22 5.73 79962 FU13236 5.12 9942 XYLB 6.3

150248 C22orfl5 5.71 8840 WISP1 5.12 2745 GLRX 6.3

3868 KRT16 5.7 136541 TRY1 -5.1 83596 BCL2L12 6.29

22921 MSRB2 5.7 91746 YTHDC1 -5.1 290 ANPEP 6.29

9123 SLC16A3 5.69 7106 TSPAN4 -5.1 3270 HRC 6.29

5913 RAPSN 5.69 26160 IFT172 -5.1 962 CD48 6.29

54020 SLC37A1 5.68 6484 ST3GAL4 -5.1 6663 SOX10 6.29

WEST\222869914.1

331323-000880 149699 FAM112A 5.67 81696 0R5V1 -5.1 2837 UTS2R 6.29

8789 FBP2 5.67 83992 CTTNBP2 -5.1 9020 MAP3K14 6.28

11269 DDX19B 5.67 5136 PDE1A -5.1 83597 RTP3 6.27

2980 GUCA2A 5.65 11086 ADAM29 -5.1 7448 VTN 6.27

57333 RCN3 5.65 3575 IL7R -5.1 7486 WRN 6.26

10669 CGREF1 5.64 6361 CCL17 -5.1 56981 PRDM11 6.26

6788 STK3 5.64 4166 CHST6 -5.1 400165 FU26443 6.26

6610 SMPD2 5.64 2848 GPR25 -5.1 2620 GAS2 6.25

1549 CYP2A7 5.62 3305 HSPA1L -5.1 10990 LILRB5 6.25

2779 GNAT1 5.62 317716 RP11-49G10.8 -5.1 128414 C20orf58 6.23

5738 PTGFRN 5.61 9987 HNRPDL -5.2 496 ATP4B 6.22

2525 FUT3 5.6 5452 POU2F2 -5.2 1442 CSH1 6.21

5871 MAP4K2 5.6 10648 SCGB1D1 -5.2 162333 RNF190 6.21

122060 FU30046 5.59 5156 PDGFRA -5.2 55228 FU10781 6.21

64174 DPEP2 5.59 6327 SCN2B -5.2 2898 GRIK2 6.21

9404 LPXN 5.57 153579 BTNL9 -5.2 57447 NDRG2 6.18

4486 MST1R 5.57 7594 ZNF43 -5.2 1135 CHRNA2 6.16

26232 FBX02 5.56 130560 SPATA3 -5.2 84258 SYT3 6.16

79086 MGC2747 5.56 1186 CLCN7 -5.2 6037 RNASE3 6.15

79758 FU13639 5.55 8832 CD84 -5.2 65108 MARCKSL1 6.15

50626 CYHR1 5.54 3674 ITGA2B -5.2 55908 LOC55908 6.14

1357 CPA1 5.53 64284 RAB17 -5.2 13 AADAC 6.13

54 ACP5 5.5 7015 TERT -5.2 433 ASGR2 6.12

83552 MFRP 5.5 284618 Clorfl04 -5.2 204219 LASS3 6.1

8906 AP1G2 5.5 10887 PR0KR1 -5.2 6480 ST6GAL1 6.1

170589 GPHA2 5.5 128859 BPIL3 -5.2 221188 GPR114 6.1

339789 LOC339789 5.49 2003 -5.2 23500 DAAM2 6.1

1056 CEL 5.49 146433 MGC34647 -5.2 3050 HBZ 6.09

4878 NPPA 5.48 1188 CLCNKB -5.2 56923 N UR2 6.08

55340 GIMAP5 5.47 3982 LIM2 -5.2 84310 MGC11257 6.08

5304 PIP 5.47 80739 C6orf25 -5.2 5005 ORM2 6.08

921 CD5 5.47 145946 SPATA8 -5.2 23321 TRIM2 6.08

27004 TCL6 5.46 10603 APS -5.2 112817 C10orf65 6.08

8930 MBD4 5.44 57381 RHOJ -5.2 219539 YPEL4 6.08

3460 IFNGR2 5.44 80347 COASY -5.2 5122 PCSK1 6.07

10462 CLECIOA 5.43 491 ATP2B2 -5.2 93377 TME 10 6.07

6444 SGCD 5.43 358 AQP1 -5.2 3002 GZMB 6.07

65095 FU12949 5.42 5341 PLEK -5.2 6320 CLEC11A 6.06

4689 NCF4 5.41 5731 PTGER1 -5.2 283234 FU37970 6.06

706 BZRP 5.39 10544 PROCR -5.3 80342 TRAF3IP3 6.03

150244 FU31568 5.39 6696 SPP1 -5.3 115362 GBP5 6.03

55231 FU10786 5.39 23385 NCSTN -5.3 4586 MUC5AC 6.03

917 CD3G 5.39 623 BDKRB1 -5.3 150209 AIFL 6.03

6355 CCL8 5.39 4359 MPZ -5.3 336 AP0A2 6.02

2217 FCGRT 5.37 338872 MGC48915 -5.3 266629 SEC14L3 6

WEST\222869914.1

331323-000880 91227 GGTL4 5.36 5122 PCSK1 -5.3 51208 CLDN18 6

59 ACTA2 5.36 29127 RACGAP1 -5.3 3912 LAMB1 5.99

2036 EPB41L1 5.34 914 CD2 -5.3 6699 SPRR1B 5.98

56920 SEMA3G 5.33 1991 ELA2 -5.3 2862 MLNR 5.94

54715 A2BP1 5.32 57715 SE A4G -5.3 337880 KRTAPll-1 5.93

50507 NOX4 5.32 1014 CDH16 -5.3 3902 LAG3 5.92

79414 LRFN3 5.32 126248 LOC126248 -5.3 9002 F2RL3 5.91

54777 C10orf92 5.31 2191 FAP -5.3 10044 SH2D3C 5.91

1811 SLC26A3 5.3 5920 RARRES3 -5.3 5140 PDE3B 5.9

7809 BSND 5.3 64761 PARP12 -5.3 9783 RIMS3 5.89

7169 TPM2 5.3 4477 MSMB -5.3 128864 C20orfl44 5.89

6284 S100A13 5.28 79758 FU13639 -5.3 114899 C1QTNF3 5.88

161247 LOC161247 -5.3 115560 ZNF501 -5.3 4340 MOG 5.88

2488 FSHB -5.3 2918 GR 8 -5.3 26469 PTPN18 5.87

27283 TINAG -5.3 374403 TBC1D10C -5.3 30814 PLA2G2E 5.86

7869 SEMA3B -5.3 283849 LOC283849 -5.3 5778 PTPN7 5.86

6909 TBX2 -5.3 10423 CDIPT -5.3 9060 PAPSS2 5.85

64577 ALDH8A1 -5.3 126792 B3GALT6 -5.3 3000 GUCY2D 5.85

307 ANXA4 -5.3 84218 TBC1D3 -5.3 388341 C17orf76 5.84

6528 SLC5A5 -5.3 27233 SULT1C2 -5.3 5995 RGR 5.83

132720 FU39370 -5.3 79605 PGBD5 -5.3 283171 Cllorf44 5.83

8346 HIST1H2BI -5.3 2837 UTS2R -5.3 5795 PTPRJ 5.83

.8785 MATN4 -5.3 89858 SIGLEC12 -5.3 10538 BATF 5.82

63934 ZNF667 -5.3 9706 ULK2 -5.3 4166 CHST6 5.82

6942 TCF20 -5.4 6876 TAGLN -5.3 284756 FU33860 5.82

3061 HCRTR1 -5.4 6710 SPTB -5.3 8875 VNN2 5.82

53841 MUCDHL -5.4 6538 SLC6A11 -5.3 5313 PKLR 5.82

142680 SLC34A3 -5.4 89857 KLHL6 -5.3 57473 GM632 5.81

91662 NALP12 -5.4 3663 IRF5 -5.3 121391 K6IRS4 5.8

25799 ZNF324 -5.4 4316 MMP7 -5.3 3034 HAL 5.8

283310 FU90579 -5.4 5730 PTGDS -5.3 25814 ATXN10 5.79

229 ALDOB -5.4 4350 MPG -5.3 189 AGXT 5.79

9543 PUNC -5.4 5575 PRKAR1B -5.4 85291 KRTAP4-2 5.79

3203 HOXA6 -5.4 200523 FU25369 -5.4 9358 ITGBL1 5.79

259232 VGCNL1 -5.4 4340 MOG -5.4 57381 RHOJ 5.77

57473 G 632 -5.4 22954 TRIM32 -5.4 9961 MVP 5.77

9557 CHD1L -5.4 342510 CD300E -5.4 80740 LY6G6C 5.76

9496 TBX4 -5.4 84645 C22orf23 -5.4 56270 WDR45L 5.76

84733 CBX2 -5.4 83902 KRTAP17-1 -5.4 7033 TFF3 5.75

79152 FA2H -5.4 89795 NAV3 -5.4 83888 KSP37 5.75

6514 SLC2A2 -5.4 390212 GPR152 -5.4 1510 CTSE 5.74

285367 RPUSD3 -5.4 341567 H1T2 -5.4 128488 WFDC12 5.74

WEST\222869914.1

331323-000880 3643 INS -5.4 27180 SIGLEC9 -5.4 134285 PRP2 5.73

54915 YTHDF1 -5.5 4991 0R1D2 -5.4 90102 PHLDB2 5.73

3855 KRT7 -5.5 3373 HYAL1 -5.4 54861 SNRK 5.73

85329 LGALS12 -5.5 90102 PHLDB2 -5.4 55679 LIMS2 5.73

122970 AC0T4 -5.5 9918 CNAP1 -5.4 5970 RELA 5.72

26112 CCDC69 -5.5 9153 SLC28A2 -5.4 57804 P0LD4 5.72

55350 VNN3 -5.5 6037 RNASE3 -5.4 6360 CCL16 5.71

6943 TCF21 -5.5 83742 MARVELD1 -5.4 6253 RTN2 5.69

716 CIS -5.5 81569 ACTL8 -5.4 63827 BCAN 5.68

252995 FNDC5 -5.5 3265 HRAS -5.4 283849 LOC283849 5.67

158056 AMDC4 -5.5 6749 SSRP1 -5.4 2784 GNB3 5.67

1589 CYP21A2 -5.5 54866 PPP1R14D -5.5 161198 CLEC14A 5.65

6425 SFRP5 -5.5 53831 GPR84 -5.5 4043 LRPAP1 5.65

164091 PAQR7 -5.5 84701 COX4I2 -5.5 54345 S0X18 5.64

51513 ETV7 -5.6 9811 KIAA0427 -5.5 1088 CEACAM8 5.64

84856 GC16291 -5.6 5545 PRB4 -5.5 1788 DNMT3A 5.64

339488 TFAP2E -5.6 2784 GNB3 -5.5 147429 C18orfl6 5.63

3822 KLRC2 -5.6 83443 SF3B5 -5.5 51341 ZBTB7A 5.62

1271 CNTFR -5.6 90480 GADD45GIP1 -5.5 144193 AMDHD1 5.62

10290 APEG1 -5.6 339669 MGC35206 -5.5 117194 MRGPRX2 5.62

27232 GNMT -5.6 11250 GPR45 -5.5 387332 TBPL2 5.62

5493 PPL -5.6 6366 CCL21 -5.5 83482 SCRT1 5.61

399717 FU45983 -5.6 6017 RLBP1 -5.5 10933 M0RF4L1 5.61

727 C5 -5.6 10507 SEMA4D -5.5 7052 TGM2 5.6

3131 HLF -5.6 4607 YBPC3 -5.5 56301 SLC7A10 5.59

1406 CRX -5.6 245909 DEFB106A -5.5 84465 MEGF11 5.59

399980 Cllorf39 -5.6 375686 SPATC1 -5.5 9701 SAPS2 5.58

202151 FU25422 -5.6 10538 BATF -5.5 55640 C14orf58 5.58

3752 KCND3 -5.6 126353 C19orf21 -5.5 23569 PADI4 5.58

490 ATP2B1 -5.6 3452 IFNA21 -5.5 2335 FN1 5.58

1788 DNMT3A -5.6 11105 PRD 7 -5.5 27180 SIGLEC9 5.57

64405 CDH22 -5.6 5146 PDE6C -5.5 11185 INMT 5.56

10406 WFDC2 -5.6 151254 ALS2CR11 -5.5 3273 HRG 5.55

2326 FM01 -5.6 140883 SUHW2 -5.6 1589 CYP21A2 5.55

23471 TRAM1 -5.6 5967 REG1A -5.6 84767 TRIM51 5.54

353500 BMP8A -5.6 389458 LOC389458 -5.6 54596 ECAT11 5.54

151278 FU32447 -5.7 51350 KRT2B -5.6 150350 FU25421 5.54

51659 Pfs2 -5.7 353140 LCE2C -5.6 81492 RSHL1 5.53

2905 GRIN2C -5.7 5968 REG1B -5.6 629 CFB 5.53

2444 FRK -5.7 23351 KIAA0323 -5.6 57088 PLSCR4 5.53

5476 PPGB -5.7 759 CA1 -5.6 23649 P0LA2 5.53

1401 CRP -5.7 4261 CIITA -5.6 3249 HPN 5.52

WEST\222869914.1

331323-000880 11337 GABARAP -5.7 828 CAPS -5.6 84648 LCE3D 5.52

130497 OSR1 -5.7 10225 CD96 -5.6 18 ABAT 5.52

8736 MYOM1 -5.7 4133 MAP2 -5.6 84931 FU14816 5.51

3241 HPCAL1 -5.7 85480 TSLP -5.6 9744 CENTB1 5.51

6045 RNF2 -5.7 128861 C20orf71 -5.6 622 BDH 5.49

6694 SPP2 -5.8 364 AQP7 -5.6 586 BCAT1 5.48

27439 CECR6 -5.8 148479 PHF13 -5.6 90853 SPOCDl 5.48

9214 FAI 3 -5.8 9582 AP0BEC3B -5.6 2694 GIF 5.48

6035 RNASE1 -5.8 6203 RPS9 -5.6 7036 TFR2 5.48

2686 GGTL3 -5.8 2866 GPR42 -5.6 1339 COX6A2 5.47

3902 LAG 3 -5.8 3535 -5.6 766 CA7 5.47

2981 GUCA2B -5.8 3437 IFIT3 -5.6 37 ACADVL 5.47

388759 FU45717 -5.8 10803 CCR9 -5.7 118425 GDEP 5.46

1441 CSF3R -5.8 10277 UBE4B -5.7 187 AGTRL1 5.46

4055 LTBR -5.8 284369 FU40235 -5.7 2900 GRIK4 5.45

8871 SYNJ2 -5.8 57573 ZNF471 -5.7 1773 DNASE1 5.45

5137 PDE1C -5.8 132141 IQCF1 -5.7 134 ADORA1 5.44

118471 PRAP1 -5.8 199786 BCNP1 -5.7 8572 PDLIM4 5.44

1442 CSH1 -5.8 4687 NCF1 -5.7 5452 POU2F2 5.44

1118 CHIT1 -5.8 6699 SPRR1B -5.7 90070 LACRT 5.43

54546 RNF186 -5.9 1943 EFNA2 -5.7 29949 IL19 5.43

8764 TNFRSF14 -5.9 10738 RFPL3 -5.7 8841 HDAC3 5.42

164284 FU90166 -5.9 8352 HIST1H3C -5.7 80201 HKDC1 5.4

80350 LPAL2 -5.9 23542 MAPK8IP2 -5.7 3851 KRT4 5.39

220001 VWCE -5.9 284415 UNQ3033 -5.7 121512 FGD4 5.39

6523 SLC5A1 -5.9 113802 Clorf59 -5.7 5968 REG1B 5.38

57580 PREX1 -5.9 9021 S0CS3 -5.7 140856 C20orf79 5.37

3426 CFI -5.9 64066 MMP27 -5.7 4638 MYLK 5.37

5909 RAP1GA1 -5.9 3434 IFIT1 -5.7 117156 SCGB3A2 5.37

5711 PSMD5 -5.9 90423 ATP6V1E2 -5.7 5311 PKD2 5.36

5947 RBP1 -6 5988 RFPL1 -5.7 4855 NOTCH4 5.36

3755 KCNG1 -6 79854 FU22639 -5.7 23562 CLDN14 5.36

5346 PUN -6 4807 NHLH1 -5.7 4133 MAP2 5.36

28965 SLC27A6 -6 266553 0FCC1 -5.7 59341 TRPV4 5.35

7179 TPTE -6 6404 SELPLG -5.7 164781 WDR69 5.35

5313 PKLR -6 284680 Clorflll -5.7 92579 G6PC3 5.35

6539 SLC6A12 -6 124961 ZFP3 -5.8 2178 FANCE 5.35

6690 SPINK1 -6 9172 MYO 2 -5.8 6531 SLC6A3 5.34

6392 SDHD -6 6367 CCL22 -5.8 157769 FAM91A1 5.34

283383 GPR133 -6 126123 SCRL -5.8 85397 RGS8 5.33

5140 PDE3B -6.1 220074 LRRC51 -5.8 163933 FA 43B 5.32

2099 ESR1 -6.1 57094 CPA6 -5.8 79058 ASPSCR1 5.32

7137 TNNI3 -6.1 254950 KRTAP15-1 -5.8 2068 ERCC2 5.32

350 APOH -6.1 9592 IER2 -5.8 10882 C1QL1 5.32

2027 EN03 -6.1 7681 KRN3 -5.8 7425 VGF 5.31

WESTY222869914.1

331323-000880 401105 FU42393 -6.1 5959 RDH5 -5.8 60489 AP0BEC3G 5.3

79870 BAALC -6.1 5510 PPP1R7 -5.8 55303 GIMAP4 5.3

25900 HOM-TES-103 -6.1 168391 GALNTL5 -5.8 127845 G0LT1A 5.29

5077 PAX3 -6.1 22874 PLEKHA6 -5.8 79672 FN3KRP 5.29

83595 S0X7 -6.1 841 CASP8 -5.8 51706 CYB5R1 5.29

3299 HSF4 -6.1 60676 PAPPA2 -5.8 7305 TYROBP 5.29

162333 RNF190 -6.1 8309 ACOX2 -5.8 341116 MS4A10 5.29

83999 KREMEN1 -6.2 7136 TNNI2 -5.8 8871 SYNJ2 5.28

129807 NEU4 -6.2 55423 SIRPB2 -5.8 80774 LIMD2 5.27

64757 M0SC1 -6.2 55898 UNC45A -5.8 266553 OFCC1 5.27

200634 KRTCAP3 -6.2 165100 MGC35154 -5.8 80228 C7orfl9 5.26

3200 HOXA3 -6.2 8482 SEMA7A -5.8 2912 GRM2 5.26

29993 PACSIN1 -6.2 339403 RLN3R2 -5.8 23558 WBP2 5.26

11346 SYNPO -6.2 374977 Clorfl75 -5.8 140876 C20orfl75 5.25

3354 HTR1E -6.2 3108 HLA-DMA -5.8 23601 CLEC5A 5.25

197257 LDHD -6.2 55303 GIMAP4 -5.8 84695 LOXL3 5.25

91703 ACY3 -6.2 3744 KCNA10 -5.9 527 ATP6V0C 5.24

130013 ACMSD -6.2 6275 S100A4 -5.9 10326 SIRPB1 5.24

3889 KRTHB3 -6.3 116985 CENTD2 -5.9 3949 LDLR 5.24

6935 TCF8 -6.3 55329 MNS1 -5.9 10637 LEFTY1 5.24

6799 SULT1A2 -6.3 126402 FU40365 -5.9 10382 TUBB4 5.23

7498 XDH -6.3 54453 RIN2 -5.9 170850 KCNG3 5.22

135932 FU90586 -6.3 1535 CYBA -5.9 389114 ZNF662 5.22

80833 APOL3 -6.3 5337 PLD1 -5.9 4295 MLN 5.21

84267 C9orf64 -6.3 337879 KRTAP8-1 -5.9 126393 HSPB6 5.21

4357 MPST -6.3 121214 SDR-0 -5.9 10093 ARPC4 5.2

25780 RASGRP3 -6.3 5232 PGK2 -5.9 11094 C9orf7 5.2

56241 SUSD2 -6.4 282991 BLOC1S2 -5.9 26580 BSCL2 5.2

2153 F5 -6.4 5149 PDE6H -5.9 90861 C16orf34 5.2

3987 LIMS1 -6.4 23769 FLRT1 -5.9 11093 ADAMTS13 5.19

84699 CREB3L3 -6.4 1544 CYP1A2 -5.9 1757 SARDH 5.18

3669 ISG20 -6.4 1259 CNGA1 -5.9 341 APOC1 5.18

4185 ADAM11 -6.4 353131 LCE1A -5.9 353142 LCE3A 5.18

3386 ICAM4 -6.4 79541 OR2A4 -5.9 10136 ELA3A 5.18

8614 STC2 -6.4 23434 C3orf27 -5.9 121599 SPIC -5.2

55620 STAP2 -6.4 79086 MGC2747 -5.9 10529 NEBL -5.2

8942 KYNU -6.5 339779 MGC44505 -5.9 117155 CATSPER2 -5.2

54799 MBTD1 -6.5 56547 MMP26 -5.9 8470 SORBS2 -5.2

863 CBFA2T3 -6.5 169270 ZNF596 -5.9 57003 CCDC47 -5.2

284186 T EM105 -6.5 3630 INS -5.9 79849 PDZD3 -5.2

60436 TGIF2 -6.5 64897 C12orf43 -5.9 8741 TNFSF13 -5.2

9099 USP2 -6.5 3821 KLRC1 -5.9 9049 AIP -5.2

54436 SH3TC1 -6.5 160897 ITR -5.9 6242 RTKN -5.2

388428 FU44861 -6.5 85413 SLC22A16 -5.9 55630 SLC39A4 -5.2

WEST\222869914.1

331323-000880 84931 FU 14816 -6.5 467 ATF3 -6 5510 PPP1R7 -5.2

115701 HAK -6.5 84648 LCE3D -6 5213 PFK -5.2

9675 KIAA0406 -6.6 23601 CLEC5A -6 64777 FU22318 -5.2

259 AMBP -6.6 5596 MAPK4 -6 55314 FU11155 -5.2

7139 TNNT2 -6.6 345275 HSD17B13 -6 1133 CHRM5 -5.2

5630 PRPH -6.6 388289 C16orf47 -6 1212 CLTB -5.2

9971 NR1H4 -6.6 93643 TJAP1 -6 93650 ACPT -5.2

171019 ADAMTS19 -6.6 1138 CHRNA5 -6 2013 EMP2 -5.2

83850 FAM62C -6.7 79690 GAL3ST4 -6 64866 CDCP1 -5.2

2706 GJB2 -6.7 43847 KLK14 -6 56832 IFNK -5.2

81552 ECOP -6.7 79825 CCDC48 -6 127833 SYT2 -5.2

5959 RDH5 -6.7 375061 FAM89A -6 375061 FAM89A -5.2

13 AADAC -6.7 2220 FCN2 -6 64063 PRSS22 -5.3

339327 ZNF546 -6.7 140689 CBLN4 -6 5260 PHKG1 -5.3

115290 FBX017 -6.7 1066 CES1 -6 11035 RIPK3 -5.3

1891 ECH1 -6.8 3080 CFHR2 -6 3856 KRT8 -5.3

29 ABR -6.8 51161 C3orfl8 -6 5325 P LAG LI -5.3

5744 PTHLH -6.8 270 AMPD1 -6 1917 EEF1A2 -5.3

29775 CARD10 -6.8 161497 STRC -6 7881 KCNAB1 -5.3

4829 NMBR -6.8 3903 LAIR1 -6 2863 GPR39 -5.3

24146 CLDN15 -6.8 10255 HCG9 -6 89790 SIGLEC10 -5.4

6822 SULT2A1 -6.8 53632 PRKAG3 -6 1305 C0L13A1 -5.4

421 ARVCF -6.8 7419 VDAC3 -6 22921 MSRB2 -5.4

8630 HSD17B6 -6.8 779 CACNA1S -6 2872 MKNK2 -5.4

84310 MGC11257 -6.9 2999 GZ H -6 4879 NPPB -5.4

25864 ABHD14A -6.9 57605 PITPNM2 -6 347733 TUBB2B -5.4

8673 VAMP8 -6.9 23209 MLC1 -6 93611 FBX044 -5.4

9826 ARHGEF11 -6.9 399717 FU45983 -6 4258 MGST2 -5.4

635 BHMT -6.9 2679 GGT2 -6 152816 FU23657 -5.4

4320 MMP11 -6.9 417 ART1 -6.1 140 AD0RA3 -5.4

5555 PRH2 -6.9 4351 MPI -6.1 64073 C19orf33 -5.4

8876 VNN1 -7 10750 GRAP -6.1 7253 TSHR -5.4

51171 DHRS10 -7 54658 UGT1A1 -6.1 268 AMH -5.4

127845 GOLT1A -7 921 CD5 -6.1 6910 TBX5 -5.4

54810 GIPC2 -7 57611 ISLR2 -6.1 25823 TPSG1 -5.5

4069 LYZ -7 9965 FGF19 -6.1 1672 DEFB1 -5.5

11136 SLC7A9 -7 149345 TMEM58 -6.1 145264 SERPINA12 -5.5

83482 SCRT1 -7 343 AQP8 -6.1 66005 CHID1 -5.5

149461 CLDN19 -7 8048 CSRP3 -6.1 7293 TNFRSF4 -5.5

5554 PRH1 -7 124404 FU25410 -6.1 10551 AG 2 -5.5

7104 TM4SF4 -7 79679 VTCN1 -6.1 623 BDKRB1 -5.5

92840 REEP6 -7 1668 DEFA3 -6.1 6521 SLC4A1 -5.5

255352 C10orf93 -7 114771 PGLYRP3 -6.1 55529 TMEM55A -5.5

2063 NR2F6 -7 2980 GUCA2A -6.1 3898 LAD1 -5.5

2646 GCKR -7.1 80740 LY6G6C -6.1 57817 HA P -5.5

8718 TNFRSF25 -7.1 283297 OR10A4 -6.1 170691 ADAMTS17 -5.5

WEST\222869914.1

331323-000880 79187 FSD1 -7.1 144423 GLT1D1 -6.1 26353 HSPB8 -5.5

6242 TKN -7.1 147409 DSG4 -6.1 22937 SCAP -5.5

79814 AG MAT -7.1 23495 TNFRSF13B -6.1 1999 ELF3 -5.5

29948 OKL38 -7.1 4159 MC3R -6.1 55738 ARFGAP1 -5.5

51268 PIPOX -7.2 3036 HAS1 -6.1 29766 TMOD3 -5.6

6770 STAR -7.2 84814 PPAPDC3 -6.1 51127 TRIM17 -5.6

29881 NPC1L1 -7.2 5408 PNLIPRP2 -6.1 124975 GGT6 -5.6

4224 MEP1A -7.2 80117 ARL14 -6.1 6123 RPL3L -5.6

148252 DIRAS1 -7.3 337977 KRTAP21-1 -6.1 9788 MTSS1 -5.6

7490 WT1 -7.3 165186 LOC165186 -6.2 126868 Clorfl61 -5.6

113675 SDSL -7.3 4313 MMP2 -6.2 55702 FU 10374 -5.6

2065 ERBB3 -7.3 284525 SLC9A11 -6.2 11014 KDELR2 -5.6

283078 IRXL1 -7.3 9783 RIMS3 -6.2 397 ARHGDIB -5.6

5104 SERPINA5 -7.4 8789 FBP2 -6.2 728 C5AR1 -5.6

5720 PSME1 -7.4 1258 CNGB1 -6.2 5003 SLC22A18AS -5.6

55615 PRR5 -7.4 338567 KCNK18 -6.2 147495 APCDD1 -5.6

4804 NGFR -7.4 29953 TRHDE -6.2 7517 XRCC3 -5.6

116085 SLC22A12 -7.4 820 CAMP -6.2 1525 CXADR -5.6

8424 BBOX1 -7.4 84227 RGSL2 -6.2 26281 FGF20 -5.6

1368 CPM -7.5 126014 OSCAR -6.2 1387 CREBBP -5.7

321 APBA2 -7.5 1198 CLK3 -6.2 57165 GJA12 -5.7

5648 MASP1 -7.5 131377 KBTBD5 -6.2 10801 9-Sep -5.7

3700 ITIH4 -7.5 3952 LEP -6.2 166 AES -5.7

284340 UNQ473 -7.5 4239 MFAP4 -6.2 7022 TFAP2C -5.7

6714 SRC -7.5 7465 WEE1 -6.2 2950 GSTP1 -5.7

3250 HPR -7.6 389012 FU42986 -6.3 1524 CX3CR1 -5.7

5306 PITPNA -7.6 5602 MAPK10 -6.3 345275 HSD17B13 -5.7

166012 CHST13 -7.6 283422 C12orf36 -6.3 51304 ZDHHC3 -5.7

51435 SCARA3 -7.6 7226 TRPM2 -6.3 50964 SOST -5.7

3931 LCAT -7.6 53947 A4GALT -6.3 7874 USP7 -5.7

57125 PLXDC1 -7.7 8620 NPFF -6.3 64077 LHPP -5.7

6554 SLC10A1 -7.7 220070 LOC220070 -6.3 11100 HNRPUL1 -5.7

3918 LA C2 -7.7 5032 P2RY11 -6.3 1952 CELSR2 -5.8

389434 C6orf71 -7.7 220004 FU32771 -6.3 83715 ESPN -5.8

79935 FU13265 -7.7 8896 G10 -6.3 254528 MGC35212 -5.8

6506 SLC1A2 -7.7 85360 SYDE1 -6.3 8638 OASL -5.8

496 ATP4B -7.7 495 ATP4A -6.3 4051 CYP4F3 -5.8

5004 ORM1 -7.7 4846 NOS3 -6.3 2192 FBLN1 -5.8

5174 PDZK1 -7.8 4256 MGP -6.3 10573 MRPL28 -5.8

58985 IL22RA1 -7.8 2206 MS4A2 -6.3 345274 SLC10A6 -5.8

4490 MT1B -7.8 91663 MYADM -6.3 81553 FAM49A -5.8

80004 RBM35B -7.9 3451 IFNA17 -6.3 10103 TSPAN1 -5.8

WEST\222869914.1

331323-000880 80168 MOGAT2 -7.9 5118 PCOLCE -6.3 4199 ME1 -5.8

122416 ANKRD9 -7.9 1215 CMA1 -6.3 6749 SSRP1 -5.8

7052 TG 2 -7.9 55273 TMEM100 -6.3 148022 TICAM1 -5.8

10921 RNPS1 -8 374739 TEPP -6.3 50619 DEF6 -5.8

1820 ARID3A -8 84690 SPATA22 -6.3 7991 TUSC3 -5.8

1565 CYP2D6 -8 9612 NC0R2 -6.4 26290 GALNT8 -5.9

128602 C20orf85 -8 164656 TMPRSS6 -6.4 9123 SLC16A3 -5.9

4773 NFATC2 -8.1 25770 HS747E2A -6.4 2844 GPR21 -5.9

85006 C21orfl24 -8.1 79906 M0RN1 -6.4 340061 LOC340061 -5.9

54979 HRASLS2 -8.1 4725 NDUFS5 -6.4 23166 STAB1 -5.9

325 APCS -8.1 11318 ADMR -6.4 11184 MAP4K1 -5.9

26230 TIAM2 -8.1 389197 FU46481 -6.4 140689 CBLN4 -5.9

622 BDH -8.1 1669 DEFA4 -6.4 9476 NAPSA -5.9

10659 CUGBP2 -8.1 4656 MYOG -6.4 8514 KCNAB2 -5.9

2242 FES -8.2 242 AL0X12B -6.4 79581 GPR172A -5.9

114990 SLITL2 -8.2 81027 TUBB1 -6.4 6935 TCF8 -5.9

11251 GPR44 -8.2 885 CCK -6.4 3695 ITGB7 -5.9

1749 DLX5 -8.2 551 AVP -6.4 131408 C3orf40 -6

283171 Cllorf44 -8.2 5322 PLA2G5 -6.4 2916 GRM6 -6

433 ASGR2 -8.2 219928 MRGPRF -6.4 254295 PHYHD1 -6

1244 ABCC2 -8.3 22982 DIP2C -6.4 90990 KIFC2 -6

8528 DDO -8.3 971 CD72 -6.4 115572 FA 46B -6.1

3856 KRT8 -8.4 43849 KLK12 -6.4 29079 MED4 -6.1

57168 ASPHD2 -8.5 920 CD4 -6.4 3984 LIMK1 -6.1

3176 HNMT -8.6 3479 IGF1 -6.4 64101 LRRC4 -6.1

201780 SLC10A4 -8.6 5858 PZP -6.4 51214 IGF2AS -6.1

114770 PGLYRP2 -8.6 930 CD19 -6.4 118472 ZNF511 -6.1

116496 Clorf24 -8.6 8838 WISP3 -6.4 114038 C21orf84 -6.1

1179 CLCA1 -8.7 23643 LY96 -6.4 2877 GPX2 -6.1

64902 AGXT2 -8.7 4161 MC5R -6.4 22924 MAPRE3 -6.2

2155 F7 -8.7 37 ACADVL -6.5 10256 CNKSR1 -6.2

338094 Clorfl79 -8.7 54544 Clorf42 -6.5 124857 WFIKKN2 -6.2

3158 HMGCS2 -8.7 3001 GZ A -6.5 8436 SDPR -6.2

9032 TM4SF5 -8.8 2213 FCGR2B -6.5 6005 RHAG -6.2

1903 EDG3 -8.8 9376 SLC22A8 -6.5 9625 AATK -6.2

91373 UAP1L1 -8.8 91156 DKFZp434B1231 -6.5 54680 Clorfl81 -6.2

114571 SLC22A9 -8.8 57102 C12orf4 -6.5 4071 TM4SF1 -6.3

5342 PLGLB2 -8.9 10326 SIRPB1 -6.5 8844 KSR1 -6.3

2741 GLRAl -8.9 8747 ADA 21 -6.5 9743 RICS -6.3

1018 CDK3 -9 353144 LCE3C -6.5 2701 GJA4 -6.3

8858 PROZ -9 7043 TGFB3 -6.5 632 BGLAP -6.3

712 C1QA -9 1417 CRYBB3 -6.5 3384 ICAM2 -6.3

3263 HPX -9.1 84203 TXNDC2 -6.5 192666 KRT24 -6.3

8416 ANXA9 -9.1 2022 ENG -6.5 6279 S100A8 -6.3

83729 INHBE -9.1 158062 LCN6 -6.5 55567 DNAH3 -6.4

WEST\222869914.1

331323-000880 55103 RALGPS2 -9.1 63968 PWCR1 -6.5 400830 DEFB32 -6.4

7066 THPO -9.1 255027 FU39599 -6.5 54210 TREM1 -6.4

197 AHSG -9.1 2949 GSTM5 -6.6 164091 PAQR7 -6.4

55937 APOM -9.1 10050 SLC17A4 -6.6 83742 MARVELD1 -6.4

83547 RILP -9.1 50615 IL21R -6.6 5148 PDE6G -6.4

114038 C21orf84 -9.2 7267 TTC3 -6.6 147700 KLC3 -6.4

3340 NDST1 -9.2 1667 DEFA1 -6.6 28951 TRIB2 -6.4

150135 C21orfl29 -9.2 113730 KLHDC7B -6.6 151011 10-Sep -6.5

54923 LIME1 -9.2 10382 TUBB4 -6.6 161725 OTUD7 -6.5

400830 DEFB32 -9.2 84514 LGP1 -6.6 284739 LOC284739 -6.5

1733 DIOl -9.2 54965 PIGX -6.6 3159 HMGA1 -6.5

64816 CYP3A43 -9.2 909 CD1A -6.6 199699 DAND5 -6.5

2812 GP1BB -9.3 219793 C10orf27 -6.6 57152 SLURP1 -6.5

1369 CPN1 -9.3 23533 PIK3R5 -6.6 10501 SEMA6B -6.5

55811 SAC -9.3 5184 PEPD -6.6 23263 MCF2L -6.5

3626 INHBC -9.3 83874 TBC1D10A -6.6 150244 FU31568 -6.5

84275 MGC4399 -9.3 9033 PKD2L1 -6.6 79228 WDR58 -6.5

2052 EPHX1 -9.3 112476 PRRT2 -6.6 3779 KCNMB1 -6.5

80201 HKDC1 -9.4 23371 TENC1 -6.6 29941 PKN3 -6.6

3199 HOXA2 -9.5 26279 PLA2G2D -6.7 513 ATP5D -6.6

4762 NEUROG1 -9.5 6280 S100A9 -6.7 686 BTD -6.6

717 C2 -9.5 1811 SLC26A3 -6.7 9056 SLC7A7 -6.6

133121 ENPP6 -9.5 6009 RHEB -6.7 4147 MATN2 -6.7

374969 CCDC23 -9.6 23761 PISD -6.7 6355 CCL8 -6.7

79981 FRMD1 -9.8 10536 LEPREL2 -6.7 79885 HDAC11 -6.7

8537 BCAS1 -9.8 10136 ELA3A -6.7 58985 IL22RA1 -6.7

152189 CMTM8 -9.8 286077 FU46072 -6.7 23620 NTSR2 -6.7

85004 RERG -9.9 117532 TMC2 -6.7 55612 C20orf42 -6.7

8692 HYAL2 -10 1908 EDN3 -6.7 10089 KCNK7 -6.8

83541 C20orf55 -10 90011 FU00060 -6.7 83650 AMAC1L2 -6.8

51042 ZNF593 -10 83959 SLC4A11 -6.7 116285 ACS 1 -6.8

5267 SERPINA4 -10 85453 TSPYL5 -6.7 23413 FREQ -6.8

130 ADH6 -10 128821 CST9L -6.7 390212 GPR152 -6.8

51733 UPB1 -10 79005 SCN 1 -6.7 83541 C20orf55 -6.8

2527 FUT5 -10 83693 HSDL1 -6.7 780 DDR1 -6.8

317749 DHRS4L2 -10 26291 FGF21 -6.8 54777 C10orf92 -6.8

23231 KIAA0746 -10 973 CD79A -6.8 6614 SN -6.8

1582 CYP8B1 -10 2880 GPX5 -6.8 1577 CYP3A5 -6.8

388007 SERPINA13 -10 282617 IL28B -6.8 6387 CXCL12 -6.8

5444 PON1 -10 60385 TSKS -6.8 56971 CEACAM19 -6.8

10637 LEFTY1 -10 8435 SOAT2 -6.8 85378 TUBGCP6 -6.9

6097 RORC -10 153201 SLC36A2 -6.8 5777 PTPN6 -6.9

5265 SERPINA1 -10 112755 STX1B2 -6.8 83871 RAB34 -6.9

3960 LGALS4 -10 2882 GPX7 -6.8 146802 FU31196 -6.9

WEST\222869914.1

331323-000880 116844 LRG1 -10 2515 ADA 2 -6.8 84821 MGC13168 -6.9

57402 S100A14 -10 93190 Clorfl58 -6.8 8808 IL1RL2 -6.9

2328 FM03 -10 6947 TCN1 -6.8 2853 GPR31 -6.9

57104 PNPLA2 -10 10753 CAPN9 -6.8 2041 EPHA1 -7

5867 RAB4A -10 5008 OSM -6.8 1847 DUSP5 -7

462 SERPINC1 -10 115861 TXNL6 -6.8 51759 C9orf78 -7

84258 SYT3 -10 92747 C20orfll4 -6.8 8764 TNFRSF14 -7

6339 SCNN1D -10 22902 RUFY3 -6.8 11025 LILRB3 -7

51296 SLC15A3 -10 54900 LAX1 -6.8 4081 MAB21L1 -7

5308 PITX2 -11 2208 FCER2 -6.8 286257 C9orfl42 -7

4803 NGFB -11 201305 MGC29671 -6.9 2581 GALC -7

1579 CYP4A11 -11 26998 FETUB -6.9 27071 DAPP1 -7.1

6717 SRI -11 378708 APITD1 -6.9 4487 MSX1 -7.1

5364 PLXNB1 -11 1159 CKMT1B -6.9 81889 FAHD1 -7.1

9208 LRRFIP1 -11 8021 NUP214 -6.9 80004 RBM35B -7.1

5105 PCK1 -11 7730 ZNF177 -6.9 116123 LOC116123 -7.1

57167 SALL4 -11 1208 CLPS -6.9 9796 PHYHIP -7.1

9963 SLC23A1 -11 27178 IL1F7 -6.9 132014 IL17RE -7.2

2822 GPLD1 -11 79054 TRPM8 -6.9 29108 PYCARD -7.2

3034 HAL -11 10666 CD226 -6.9 122622 ADSSL1 -7.2

55651 N0LA2 -11 10964 IFI44L -6.9 5275 SERPINB13 -7.2

725 C4BPB -11 125206 SLC5A10 -6.9 1307 C0L16A1 -7.2

432 ASGR1 -11 200909 HTR3D -6.9 360132 FKBP9L -7.2

113220 KIF12 -11 7133 TNFRSF1B -6.9 9324 HMGN3 -7.2

65012 SLC26A10 -11 147741 ZNF560 -6.9 50509 COL5A3 -7.2

83758 RBP5 -11 59351 PB0V1 -6.9 7051 TGM1 -7.2

5176 SERPINF1 -12 3577 IL8RA -6.9 85395 C21orf70 -7.3

843 CASP10 -12 4337 M0CS1 -6.9 57678 GPAM -7.3

4153 MBL2 -12 5553 PRG2 -6.9 55620 STAP2 -7.3

1564 CYP2D7 1 -12 11170 FA M 107 A -6.9 6540 SLC6A13 -7.3

3026 HABP2 -12 29949 IL19 -6.9 164284 FU90166 -7.3

3240 HP -12 57010 CABP4 -6.9 26012 NELF -7.3

221 ALDH3B1 -12 1325 CORT -7 283860 FU36701 -7.3

629 CFB -12 23615 PYY2 -7 89941 RH0T2 -7.3

336 AP0A2 -12 124989 C17orf57 -7 9073 CLDN8 -7.3

57533 TBC1D14 -12 400359 FU35695 -7 3881 KRTHA1 -7.3

3697 ITIH1 -12 8483 CI LP -7 284252 KCTD1 -7.4

9235 IL32 -13 80342 TRAF3IP3 -7 23418 CRB1 -7.4

94086 HSPB9 -13 140446 KRT6C -7 84708 LNX1 -7.4

51208 CLDN18 -13 7429 VI LI -7 5443 POMC -7.4

866 SERPINA6 -13 401508 FU45202 -7 64220 STRA6 -7.4

6360 CCL16 -13 338339 CLEC4D -7 1437 CSF2 -7.5

344 AP0C2 -13 132112 RTP1 -7 8915 BCL10 -7.5

WEST\222869914.1

331323-000880 9154 SLC28A1 -13 27237 ARHGEF16 -7 3163 HM0X2 -7.5

108 ADCY2 -13 761 CA3 -7 6398 SECTM1 -7.5

3990 LIPC -13 64579 NDST4 -7 26298 EHF -7.5

9970 NR1I3 -13 134 AD0RA1 -7 53822 FXYD7 -7.6

144193 A DHD1 -13 4640 MY01A -7 8424 BB0X1 -7.6

5005 0R 2 -13 6480 ST6GAL1 -7.1 10672 GNA13 -7.6

55908 LOC55908 -13 10517 FBXW10 -7.1 126353 C19orf21 -7.6

3233 H0XD4 -13 83698 CALN1 -7.1 11187 PKP3 -7.6

266629 SEC14L3 -13 348932 SLC6A18 -7.1 4814 NINJ1 -7.6

1757 SARDH -13 10320 ZNFN1A1 -7.1 202915 MGC9712 -7.7

60529 ALX4 -13 1473 CST5 -7.1 10045 SH2D3A -7.7

23589 CARHSP1 -13 83871 RAB34 -7.1 6692 SPINT1 -7.7

3929 LBP -13 6677 SPAM1 -7.1 5652 PRSS8 -7.7

3294 HSD17B2 -13 942 CD86 -7.1 2314 FLU -7.7

255043 TMEM86B -14 5831 PYCR1 -7.1 246176 GAS2L2 -7.7

8309 AC0X2 -14 5539 PPY -7.1 6549 SLC9A2 -7.7

290 ANPEP -14 92591 ASB16 -7.1 56925 LXN -7.7

80341 BPIL1 -14 5741 PTH -7.1 2875 GPT -7.8

3053 SERPIND1 -14 126410 FU39501 -7.1 84141 FU13391 -7.8

55867 SLC22A11 -14 283487 LOC283487 -7.1 9144 SYNGR2 -7.8

112817 C10orf65 -14 4342 MOS -7.1 53832 IL20RA -7.8

3483 IGFALS -14 79183 C20orfl21 -7.1 149428 BIMIPL -7.8

3242 HPD -14 6368 CCL23 -7.1 83549 UCK1 -7.8

183 AGT -14 324 APC -7.1 222389 C10orf30 -7.8

23562 CLDN14 -15 27290 SPINK4 -7.1 3854 KRT6B -7.8

115584 SLC5A11 -15 3717 JAK2 -7.2 353174 LGICZ1 -7.8

2886 GRB7 -15 389558 UNQ1940 -7.2 1308 C0L17A1 -7.8

3172 HNF4A -15 8749 ADAM18 -7.2 23429 RYBP -7.9

5348 FXYD1 -15 51378 ANGPT4 -7.2 1145 CHRNE -7.9

26007 DAK -15 84767 TRIM51 -7.2 26504 CNNM4 -7.9

148738 HFE2 -15 127534 GJB4 -7.2 3140 MR1 -7.9

79170 ATAD4 -15 29800 ZDHHC1 -7.2 257000 PLAC2 -7.9

51066 C3orf32 -15 57459 GATAD2B -7.2 7481 WNT11 -7.9

25791 NGEF -16 79883 FU23447 -7.2 56654 NPDC1 -8

5624 PROC -16 337960 KRTAP13-3 -7.2 401459 FU46365 -8

189 AGXT -16 2134 EXTL1 -7.2 79098 Clorfll6 -8

5524 PPP2R4 -16 347688 TUBB8 -7.2 1364 CLDN4 -8

2161 F12 -16 3270 HRC -7.2 4055 LTBR -8

344838 PAQR9 -16 3164 NR4A1 -7.2 79626 TNFAIP8L2 -8

51156 SERPINA10 -16 80039 FAM106A -7.2 116092 DNTTIP1 -8

5970 RELA -16 6703 SPRR2D -7.2 5265 SERPINA1 -8.1

7448 VTN -16 3910 LAMA4 -7.2 64132 XYLT2 -8.1

63924 CIDEC -17 3002 GZMB -7.3 57121 GPR92 -8.1

6927 TCF1 -17 4160 MC4R -7.3 285097 FU38379 -8.1

WEST\222869914.1

331323-000880 64240 ABCG5 -17 10462 CLECIOA -7.3 2651 GCNT2 -8.2

7036 TFR2 -17 944 TNFSF8 -7.3 10410 IFITM3 -8.2

5083 PAX9 -17 114131 UCN3 -7.3 54436 SH3TC1 -8.2

9020 MAP3K14 -17 389114 ZNF662 -7.3 50636 TMEM16G -8.2

335 APOA1 -17 1160 CKMT2 -7.3 5881 RAC3 -8.2

283375 SLC39A5 -17 128488 WFDC12 -7.3 9547 CXCL14 -8.2

718 C3 -17 54 ACP5 -7.3 4261 CIITA -8.2

763 CA5A -18 407977 TNFSF12- -7.3 9048 ARTN -8.3

TNFSF13

4485 ST1 -18 486 FXYD2 -7.3 55808 ST6GALNAC1 -8.3

283600 C14orf68 -18 123970 GC33367 -7.3 6363 CCL19 -8.3

222487 GPR97 -18 79932 KIAA0319L -7.3 7851 MALL -8.3

84647 PLA2G12B -19 55231 FU 10786 -7.3 55691 FRMD4A -8.3

146456 TMED6 -19 1280 COL2A1 -7.3 54546 RNF186 -8.3

90050 C14orfl52 -19 10279 PRSS16 -7.4 27350 AP0BEC3C -8.3

5345 SERPINF2 -20 4696 NDUFA3 -7.4 115761 ARL11 -8.3

2147 F2 -20 10234 LRRC17 -7.4 2593 GAMT -8.3

10110 SGK2 -21 8685 MARCO -7.4 55 ACPP -8.3

83597 RTP3 -21 6698 SPRR1A -7.4 10636 RGS14 -8.4

10864 SLC22A7 -21 54751 FBLIM1 -7.4 9537 TP53I11 -8.4

284723 SLC25A34 -21 254173 TTLL10 -7.4 255027 FU39599 -8.4

131669 UROC1 -22 55586 MIOX -7.4 8218 CLTCL1 -8.4

345 APOC3 -22 221421 C6orf206 -7.4 4720 NDUFS2 -8.4

79574 EPS8L3 -22 51050 PI15 -7.4 166929 MGC26963 -8.4

8431 NR0B2 -23 3991 LIPE -7.4 2155 F7 -8.4

3699 ITIH3 -23 389123 IQCF2 -7.4 84688 C9orf24 -8.5

10747 MASP2 -24 79713 U2AF1L4 -7.4 5738 PTGFRN -8.5

1 A1BG -28 54838 C10orf26 -7.4 29933 GPR132 -8.5

3083 HGFAC -30 89910 UBE3B -7.5 57149 LOC57149 -8.5

2875 GPT -31 284451 C19orfl9 -7.5 51110 LACTB2 -8.5

84292 MORG1 -7.5 2765 GML -8.5

1644 DDC -7.5 9982 FGFBP1 -8.6

25803 SPDEF -7.5 83886 PRSS27 -8.6

137835 TMEM71 -7.5 113828 FAM83F -8.6

4154 MBNL1 -7.5 27141 □ DEB -8.7

6397 SEC14L1 -7.5 2525 FUT3 -8.7

3460 IFNGR2 -7.5 3429 IFI27 -8.7

116337 PANX3 -7.5 22906 TRAK1 -8.7

8602 C4orf9 -7.5 2274 FHL2 -8.7

147746 HIPK4 -7.5 56169 MLZE -8.8

9962 SLC23A2 -7.5 93100 NAPRT1 -8.8

26103 LRRC21 -7.6 25953 PNKD -8.8

4049 LTA -7.6 143662 MUC15 -8.8

92305 TMEM129 -7.6 54742 LY6K -8.9

338321 NALP9 -7.6 169611 0LFML2A -8.9

WESTY222869914.1

331323-000880 56257 BCDIN3 -7.6 6865 TACR2 -8.9

90678 LRSAM1 -7.6 3579 IL8RB -8.9

11185 INMT -7.6 140700 SAMD10 -8.9

337880 KRTAPll-1 -7.6 50626 CYHR1 -8.9

5745 PTHR1 -7.6 116211 TM4SF19 -9

414062 CCL3L3 -7.6 148930 FU32011 -9.1

9027 NAT8 -7.6 25818 KLK5 -9.1

9145 SYNGR1 -7.6 5306 PITPNA -9.1

2516 NR5A1 -7.6 57449 PLEKHG5 -9.1

150248 C22orfl5 -7.6 149483 CCDC17 -9.2

27033 ZBTB32 -7.6 57124 CD248 -9.2

400696 LOC400696 -7.6 84225 ZMYND15 -9.2

9098 USP6 -7.7 1454 CSNK1E -9.2

5802 PTPRS -7.7 93643 TJAP1 -9.3

2151 F2RL2 -7.7 3918 LAMC2 -9.3

64403 CDH24 -7.7 5268 SERPINB5 -9.3

283298 0LFML1 -7.7 124565 GC15523 -9.3

1553 CYP2A13 -7.7 55615 PRR5 -9.3

59 ACTA2 -7.7 128272 ARHGEF19 -9.3

3097 HIVEP2 ^:7.7 100 ADA -9.5

54536 EXOC6 -7.7 203100 HTRA4 -9.5

115908 CTHRC1 -7.7 4056 LTC4S -9.5

64231 MS4A6A -7.7 8973 CHRNA6 -9.5

60681 FKBP10 -7.7 1991 ELA2 -9.5

3689 ITGB2 -7.8 8000 PSCA -9.6

84676 TRIM63 -7.8 128308 MRPL55 -9.6

1101 CHAD -7.8 64129 TINAGL1 -9.7

136371 ASB10 -7.8 151473 SLC16A14 -9.7

2260 FGFR1 -7.8 5524 PPP2R4 -9.8

6403 SELP -7.8 858 CAV2 -9.8

7809 BSND -7.8 5133 PDCD1 -9.8

50486 G0S2 -7.8 3772 KCNJ15 -9.8

389084 UNQ830 -7.8 9121 SLC16A5 -9.8

11093 ADA TS13 -7.8 11082 ESM1 -9.8

81472 OR2C3 -7.8 768 CA9 -9.9

4332 NDA -7.8 1116 CHI3L1 -9.9

10537 UBD -7.8 7075 TIE1 -10

23704 KCNE4 -7.8 10755 GIPCl -10

57876 MUC3B -7.8 8906 AP1G2 -10

283897 C16orf54 -7.8 284114 TMEM102 -10

51744 CD244 -7.9 83716 CRISPLD2 -10

115362 GBP5 -7.9 5623 PSPN -10

152065 C3orf22 -7.9 339789 LOC339789 -10

WEST\222869914.1

331323-000880 1513 CTSK -7.9 27019 DNAI1 -10

6707 SPRR3 -7.9 5349 FXYD3 -10

286256 LCN12 -7.9 200634 KRTCAP3 -10

3846 KRTAP5-9 -7.9 1365 CLDN3 -10

339883 C3orf35 -7.9 201799 FU32028 -10

6439 SFTPB -7.9 64288 ZNF323 -10

566 AZU1 -7.9 169166 MGC39715 -10

4855 N0TCH4 -7.9 84948 TIGD5 -11

126259 MGC23244 -7.9 4237 MFAP2 -11

9891 NUAK1 -7.9 9749 PHACTR2 -11

10578 GNLY -7.9 10614 HEXIM1 -11

5644 PRSS1 -8 6834 SURF1 -11

6847 SYCP1 -8 834 CASP1 -11

112802 KRT6IRS -8 4246 SCGB2A1 -11

4653 MYOC -8 128876 C20orfl28 -11

10661 KLF1 -8 4926 NUMA1 -11

6573 SLC19A1 -8 150135 C21orfl29 -11

8436 SDPR -8 2057 EPOR -11

114902 C1QTNF5 -8 1638 DCT -11

6036 RNASE2 -8 1018 CDK3 -11

5068 REG3A -8 9507 ADAMTS4 -11

55888 ZNF167 -8.1 201252 FU23754 -11

4295 MLN -8.1 56834 GPR137 -11

338398 TAS2R60 -8.1 51043 ZBTB7B -11

26476 OR10J1 -8.1 5957 RCV1 -11

1297 COL9A1 -8.1 10039 PARP3 -11

80834 TAS1R2 -8.1 23367 LARP1 -11

51314 TXNDC3 -8.1 244 ANXA8 -11

79843 FU22746 -8.1 164153 UBL4B -11

64405 CDH22 -8.1 4250 SCGB2A2 -11

929 CD14 -8.1 51475 CABP2 -11

3543 IGLL1 -8.1 2099 ESR1 -11

53828 FXYD4 -8.1 9022 CLIC3 -11

7380 UPK3A -8.2 2678 GGT1 -11

353134 LCE1D -8.2 8843 GPR109B -11

255022 FAM26C -8.2 125981 ASAH3 -11

5521 PPP2R2B -8.2 6755 SSTR5 -12

84618 NT5C1A -8.2 84033 OBSCN -12

1621 DBH -8.2 11322 TMC6 -12

285780 FU33708 -8.2 27134 TJP3 -12

84283 TMEM79 -8.2 4688 NCF2 -12

1261 CNGA3 -8.2 1056 CEL -12

23476 BRD4 -8.2 57111 RAB25 -12

487 ATP2A1 -8.2 114898 C1QTNF2 -12

149699 FAM112A -8.2 1003 CDH5 -12

1791 DNTT -8.2 84286 MGC4618 -12

9546 APBA3 -8.2 56300 IL1F9 -12

WEST\222869914.1

331323-000880 84225 ZMYND15 -8.3 57402 S100A14 -12

3681 ITGAD -8.3 221091 LOC221091 -12

140876 C20orfl75 -8.3 349236 LOC349236 -12

10962 MLLT11 -8.3 10850 CCL27 -12

7169 TPM2 -8.3 1776 DNASE1L3 -12

9961 MVP -8.3 1823 DSC1 -12

8930 MBD4 -8.3 3872 KRT17 -12

1742 DLG4 -8.3 259 AMBP -12

10044 SH2D3C -8.3 29984 RHOD -12

5320 PLA2G2A -8.3 404217 CTXN1 -12

23547 LIL A4 -8.3 1041 CDSN -12

147949 ZNF583 -8.3 3557 IL1RN -12

2205 FCER1A -8.3 80097 FU 14346 -12

64174 DPEP2 -8.4 4784 NFIX -13

3050 HBZ -8.4 124152 MGC35048 -13

53919 SLC01C1 -8.4 8839 WISP2 -13

2178 FANCE -8.4 79188 TMEM43 -13

2864 FFAR1 -8.4 9973 CCS -13

2687 GGTLA1 -8.4 5119 PC0LN3 -13

5552 PRG1 -8.4 150696 PR0M2 -13

79748 LMAN1L -8.5 3036 HAS1 -13

2251 FGF6 -8.5 2036 EPB41L1 -13

1003 CDH5 -8.5 2202 EFEMP1 -13

6014 RIT2 -8.5 26232 FBX02 -13

286887 KRT6E -8.5 55267 FU 10945 -13

8200 GDF5 -8.5 148741 ANKRD35 -13

10407 SPAG11 -8.5 80736 SLC44A4 -13

517 ATP5G2 -8.5 54106 TLR9 -13

55658 RNF126 -8.6 81607 PVRL4 -13

8993 PGLYRP1 -8.6 9290 GPR55 -13

6274 S100A3 -8.6 2769 GNA15 -13

4689 NCF4 -8.6 5045 FURIN -13

144347 FAM101A -8.6 4486 MST1R -13

142683 ITLN2 -8.6 822 CAPG -13

4130 MAP1A -8.6 23353 UNC84A -13

23187 PHLDB1 -8.7 4245 MGAT1 -13

137797 LYPD2 -8.7 977 CD151 -13

2533 FYB -8.7 1572 CYP2F1 -13

6444 SGCD -8.7 2886 GRB7 -14

3078 CFHR1 -8.7 388428 FU44861 -14

2745 GLRX -8.7 144501 LOC144501 -14

6504 SLAMF1 -8.7 2246 FGF1 -14

56920 SEMA3G -8.7 23553 HYAL4 -14

2028 ENPEP -8.7 9333 TGM5 -14

WEST\2228₆9914.1

331323-000880 124912 SPACA3 -8.7 1606 DGKA -14

6385 SDC4 -8.8 50604 IL20 -14

126432 FU45909 -8.8 85456 TNKS1BP1 -14

11189 TNRC4 -8.8 8581 LY6D -14

83795 KCNK16 -8.8 9362 CPNE6 -14

80341 BPIL1 -8.8 90199 WFDC8 -14

6688 SPI1 -8.8 195814 RDHE2 -14

79132 LGP2 -8.8 81875 ISG20L2 -14

2978 GUCA1A -8.8 29775 CARD10 -14

221188 GPR114 -8.8 3963 LGALS7 -14

3834 KIF25 -8.9 7462 LAT2 -15

25959 ANKRD25 -8.9 8710 SERPINB7 -15

348174 LOC348174 -8.9 83706 URP2 -15

8859 STK19 -8.9 9047 SH2D2A -15

6236 RRAD -8.9 79755 FU13841 -15

3770 KCNJ14 -9 7124 TNF -15

84617 TUBB6 -9 128178 EDARADD -15

168537 GIMAP7 -9 63970 P53AIP1 -16

285 ANGPT2 -9 104 ADARB1 -16

115352 FCRL3 -9 201294 UNC13D -16

11156 PTP4A3 -9 9051 PSTPIP1 -16

7318 UBE1L -9.1 10158 PDZK1IP1 -16

8857 FCGBP -9.1 124222 PAQR4 -16

93377 TME 10 -9.1 348094 ANKDD1A -17

6273 S100A2 -9.2 353135 LCE1E -17

55340 GIMAP5 -9.2 338785 KRT6L -17

2811 GP1BA -9.2 2709 GJB5 -17

30835 CD209 -9.2 114904 C1QTNF6 -17

56923 NMUR2 -9.2 81029 WNT5B -18

6569 SLC34A1 -9.2 118856 MMP21 -18

284756 FU33860 -9.2 64218 SEMA4A -18

1675 CFD -9.2 400931 FU27365 -18

359 AQP2 -9.3 5202 PFDN2 -18

347853 TBX10 -9.3 144568 A2ML1 -18

7305 TYROBP -9.3 3852 KRT5 -18

54674 LRRN3 -9.3 338442 GPR109A -18

254778 C8orf46 -9.3 3425 IDUA -18

340061 LOC340061 -9.4 27111 SDCBP2 -18

342666 FU43826 -9.4 55643 BTBD2 -18

84467 FBN3 -9.4 124220 LOC124220 -18

1638 DCT -9.4 339665 SLC35E4 -18

9536 PTGES -9.4 2867 FFAR2 -18

83638 Bles03 -9.5 4793 NFKBIB -19

3171 F0XA3 -9.5 340205 TRE L1 -19

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331323-000880 63934 ZNF667 -9.5 6820 SULT2B1 -19

6362 CCL18 -9.5 80781 C0L18A1 -19

7044 LEFTY2 -9.5 2810 SFN -19

55891 LENEP -9.5 1775 DNASE1L2 -19

54581 SCAND2 -9.5 149708 WFDC5 -19

23208 SYT11 -9.6 54471 FU20232 -19

9196 CNAB3 -9.6 7850 IL1R2 -19

27004 TCL6 -9.6 80818 ZNF436 -19

57678 GPAM -9.6 23650 TRIM29 -20

338323 NALP14 -9.7 57016 AKR1B10 -20

8572 PDLIM4 -9.7 162514 TRPV3 -20

6614 SN -9.7 1128 CHRM1 -21

146722 CD300LF -9.7 54578 UGT1A6 -21

10020 GNE -9.7 54905 CYP2W1 -22

1880 EBI2 -9.7 11259 D0C1 -22

10332 CLEC4M -9.7 2125 EV L -23

10077 TSPAN32 -9.7 1907 EDN2 -23

632 BGLAP -9.7 9028 RHBDL1 -23

1135 CHRNA2 -9.7 90019 SYT8 -23

257236 FU90575 -9.7 79413 ZBED2 -24

203074 UNQ9391 -9.8 55287 TMEM40 -24

5972 REN -9.8 284837 LOC284837 -24

2252 FGF7 -9.9 84985 FAM83A -25

4353 MPO -9.9 5753 PTK6 -25

5969 REGL -9.9 84951 TNS4 -25

962 CD48 -9.9 65094 JMJD4 -25

23263 MCF2L -10 3914 LAMB3 -26

57804 P0LD4 -10 23616 SH3BP1 -28

64600 PLA2G2F -10 3866 KRT15 -28

9121 SLC16A5 -10 9289 GPR56 -30

1586 CYP17A1 -10 116983 CENTB5 -40

4837 NNMT -10

676 BRDT -10

26191 PTPN22 -10

23550 PSD4 -10

10068 IL18BP -10

117156 SCGB3A2 -10

144124 OR10A5 -10

4063 LY9 -10

10344 CCL26 -10

11269 DDX19B -10

117159 DCD -10

9404 LPXN -10

157769 FAM91A1 -10

26236 C6orf54 -10

5995 RGR -10

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331323-000880 54020 SLC37A1 -10

4237 MFAP2 -10

30008 EFE P2 -10

7123 CLEC3B -10

4815 NINJ2 -10

2038 EPB42 -10

283807 FBXL22 -10

5799 PTPRN2 -10

114757 CYGB -10

91937 TIMD4 -10

131375 LYZL4 -10

286128 ZFP41 -10

6445 SGCG -10

25900 HOM-TES-103 -11

116835 HSPA12B -11

3857 KRT9 -11

253935 ANGPTL5 -11

147429 C18orfl6 -11

401040 FU45964 -11

148170 CDC42EP5 -11

1549 CYP2A7 -11

434 ASIP -11

10801 9-Sep -11

6582 SLC22A2 -11

1436 CSF1R -11

57088 PLSCR4 -11

79949 C10orf81 -11

220388 FU38159 -11

10395 DLC1 -11

83552 MFRP -11

83888 KSP37 -11

283598 FU25773 -11

90070 LACRT -11

64806 IL17E -11

5304 PIP -11

23543 RBM9 -11

2532 DARC -11

3888 KRTHB2 -11

5159 PDGFRB -11

9555 H2AFY -11

4284 MIP -11

4625 MYH7 -11

221472 FGD2 -11

115701 HAK -11

2215 FCGR3B -11

WEST\222869914.1

331323-000880 359710 C20orfl85 -11

4878 NPPA -11

140691 NF36 -11

23061 KIAA0676 -11

80380 PDCD1LG2 -11

84734 Clorf90 -11

2012 EMP1 -11

374872 C19orf35 -11

114899 C1QTNF3 -11

3671 ISLR -11

23436 ELA3B -11

26 ABP1 -12

3590 IL11RA -12

400931 FU27365 -12

126375 FU38451 -12

4056 LTC4S -12

56833 SLAMF8 -12

126308 M0BKL2A -12

4921 DDR2 -12

54739 BIRC4BP -12

6278 S100A7 -12

285676 ZNF454 -12

6776 STAT5A -12

2865 FFAR3 -12

284013 VM01 -12

10783 NEK6 -12

6358 CCL14 -12

5173 PDYN -12

27198 GPR81 -12

1293 COL6A3 -12

6356 CCL11 -12

5376 P P22 -12

3633 INPP5B -12

83401 EL0VL3 -12

26526 TSPAN16 -12

113622 ADPRHL1 -13

64333 ARHGAP9 -13

6348 CCL3 -13

148423 Clorf52 -13

221091 LOC221091 -13

388533 UNQ467 -13

1307 C0L16A1 -13

10669 CGREF1 -13

50507 N0X4 -13

23098 SARM1 -13

WESTY222869914.1

331323-000880 257203 DSCR9 -13

388218 UNQ5810 -13

81621 KAZALD1 -13

84766 MGC4266 -14

1805 DPT -14

284358 FU36070 -14

4222 ME0X1 -14

9253 NUMBL -14

56302 TRPV5 -15

338773 T EM119 -15

7054 TH -15

2214 FCGR3A -15

1760 DMPK -15

4939 OAS2 -15

29951 PDZRN4 -15

5368 PNOC -15

401459 FU46365 -15

81493 SYNC1 -15

170575 GIMAP1 -15

10335 MRVI1 -16

90226 UCN2 -16

3956 LGALS1 -16

56944 OLFML3 -16

9358 ITGBL1 -16

2569 GABRR1 -17

57124 CD248 -17

9002 F2RL3 -17

79652 C16orf30 -17

2694 GIF -18

23500 DAAM2 -19

55267 FU10945 -19

343035 Clorf36 -19

11117 EMI^'LINl -20

9450 LY86 -24

[0139] Although the invention has been described with reference to the above example, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

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Claims

What is claimed is:

1. A method of generating induced pluripotent stem (iPS) cells from cells of endoderm origin, comprising:

(a) introducing at least one pluripotent factor into the cells of endoderm origin under a first culture condition for a duration of less than about two weeks; and

(b) maintaining the cells of (a) under a second culture condition different from the first.

2. The method of claim 1, wherein the first culture condition comprises Williams'

Medium E, HGF, and EGF.

3. The method of claim 1 , wherein the first culture condition has a duration of less than about four days.

4. The method of claim 1, wherein the second culture condition comprises feeder cells and/or feeder-free matrigel-coated support.

5. The method of claim 1, wherein the second culture condition comprises hESC

medium.

6. An isolated population of human endoderm origin iPS cells according to the method of claim 1.

7. A somatic tissue derived induced pluripotent stem (iPS) cell having an equal or

greater in vivo engraftment potential as compared to an embryonic stem (ES) cell or an iPS cell derived from an embryonic tissue, wherein the somatic tissue has an endoderm origin.

8. The somatic tissue derived iPS cell of claim 7, wherein the somatic tissue comprises hepatocytes.

9. The somatic tissue derived iPS cell of claim 7, wherein the somatic tissue comprises human hepatocytes.

10. The somatic tissue derived iPS cell of claim 7, wherein the in vivo engraftment

potential is examined in liver.

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11. The somatic tissue derived iPS cell of claim 7, wherein Sodium Butyrate (NaB) is used to improve reprogramming efficiency.

12. The somatic tissue derived iPS cell of claim 7, wherein the cell has a differentiation potential to differentiate into cells of endoderm origin.

13. The somatic tissue derived iPS cell of claim 12, wherein the cells of endoderm origin are selected from the group consisting of hepatic cells, cells of pancreas, cells of gastrointestinal tract, cells of respiratory tract, cells of endocrine glands, cells of urinary system, and a combination thereof.

14. The somatic tissue derived iPS cell of claim 7, having a differentiation potential to differentiate into different hematopoietic cell types.

15. The somatic tissue derived iPS cell of claim 7, having a differentiation potential to differentiate into secondary neurospheres (SNS).

16. The somatic tissue derived iPS cell of claim 7, having a differentiation potential to differentiate into neurons, astrocytes, or oligodendrocytes.

17. The somatic tissue derived iPS cell of claim 7, wherein the somatic tissue derived PS cell has a distinct epigenetic pattern as compared to an ES cell derived from an embryonic tissue.

18. The somatic tissue derived iPS cell of claim 17, wherein the epigenetic pattern

comprises genomic DNA methylation.

19. The somatic tissue derived iPS cell of claim 7, wherein the somatic tissue derived iPS cell is a low-passage iPS cell.

20. The low-passage iPS cell of claim 19, having a passage between seven and twelve passages.

21. The low-passage iPS cell of claim 19, wherein no chromatin-modifying agents are used during passages.

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22. The somatic tissue derived iPS cell of claim 7, wherein the somatic tissue derived iPS cell has an equal or lower tumor-forming propensity as compared to an embryonic stem (ES) cell derived from an embryonic tissue.

23. An intermediate stage hepatic progenitor cell derived from the somatic tissue derived iPS cell of claim 7.

24. The intermediate stage hepatic progenitor cell of claim 23, displaying CYP450

enzyme activity when further differentiated into mature hepatocytes.

25. The intermediate stage hepatic progenitor cell of claim 23, wherein a step wise

differentiation protocol is used and the step wise differentiation protocol comprises:

(a) a definitive endoderm induction step;

(b) a hepatic progenitor induction and expansion step; and

(c) a hepatic maturation step.

26. The intermediate stage hepatic progenitor cell of claim 23, wherein the step wise differentiation protocol for further maturation uses a hepatocyte culture medium (HCM) comprising FGF-4, HGF, oncostatin M, and dexamethasone.

27. A system for studying drug metabolism or liver disease modeling, comprising the intermediate stage hepatic progenitor cell of claim 23.

28. A method for studying drug metabolism process or liver disease, comprising:

(a) obtaining a somatic tissue of endoderm origin;

(b) generating an iPS cell from the somatic tissue of endoderm origin of (a);

(c) generate an intermediate stage hepatic progenitor cell;

(d) contacting a test agent with the intermediate stage hepatic progenitor cell of (c) or a hepatocyte derived from the intermediate stage hepatic progenitor cell; and

(e) determining the effect of the test agent on expression of a gene or activity of a gene product in the cell.

29. A method for treatment of liver diseases to a subject, comprising:

administering an effective amount of the somatic tissue derived iPS cells of claim 7 to the subject, thereby treating the disease.

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30. A method for treatment of liver diseases to a subject, comprising:

administering an effective amount of the intermediate stage hepatic progenitor cells of claim 23 or hepatocytes derived from the intermediate stage hepatic progenitor cell to the subject, thereby treating the disease.

31. The method of claim 29 or 30, wherein administering is local or systemic.

32. A method for in vivo engraftment to a subject, comprising:

administering an effective amount of the somatic tissue derived iPS cells of claim 7 to the subject.

33. A method for in vivo engraftment to a subject, comprising:

administering an effective amount of the intermediate stage hepatic progenitor cells of claim 23 or hepatocytes derived from the intermediate stage hepatic progenitor cell to the subject.

34. A method for in vivo engraftment to a subject, comprising:

administering an effective amount of definitive endoderm stage cells derived from the somatic tissue derived iPS cell of claim 7 to the subject.

35. The method of claim 30, 33, or 34, wherein the hepatocytes or definitive endoderm stage cells express an endoderm marker CXCR4.

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