GB2434867A

GB2434867A - Genes that are up- or down-regulated during differentiation of human embryonic stem cells

Info

Publication number: GB2434867A
Application number: GB0708707A
Authority: GB
Inventors: Lawrence W Stanton; Ralph Brandenberger; Elisa Brunette; Joseph D Gold; John M Irving; Ramkumar Mandalam; Michael Mok; Sandra E Powell; Dawne Shelton
Original assignee: Geron Corp
Current assignee: Geron Corp
Priority date: 2003-03-13
Filing date: 2004-03-13
Publication date: 2007-08-08
Anticipated expiration: 2024-03-13
Also published as: GB2434867B; GB0708707D0

Abstract

Genes that are up- or down-regulated during differentiation provide important leverage by which to characterize and manipulate early-stage pluripotent stem cells. Over 35,000 unique transcripts have been amplified and sequenced from undifferentiated human embryonic stem cells, and three types of differentiated progeny. Statistical analysis of the assembled transcripts identified genes that alter expression levels as differentiation proceeds. The expression profile provides a marker system that has been used to identify particular culture components for maintaining the undifferentiated phenotype. The gene products can also be used to promote differentiation; to assess other relatively undifferentiated cells (such as cancer cells); to control gene expression; or to separate cells having desirable characteristics. Manipulation of particular genes can be used to forestall or focus the differentiation process, en route to producing a specialized homogenous cell population suitable for human therapy.

Description

GENES THAT ARE Up-OR DOwN-REGULATED

DURING DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS

BACKGROUND

A promising development in the fi&d of regenerative medicine has been the isolation and propagation of human stem cells from the early embryo. These cells have two very special properties: First, unlike other normal mammalian cell types, they can be propagated in culture almost indefinitely, providing a virtually unlimited supply. Second, they can be used to generate a variety of tissue types of interest as a source of replacement cells and tissues for use in therapy.

Thomson et at. (Science 282:114, 1998; U.S. Patent 6,200,806) were the first to successfully isolate and propagate embryonic stem cells from human blastocysts. Gearhart and coworkers derived human embryonic germ cell lines from fetal gonadal tissue (Shamblott et al., Proc. Nati. Acad. Sd. USA 95:13726, 1998;U.S. Patent 6,090,622).

International Patent Publication WO 99/20741 (Geron Corp.) describes methods and materials for the growth of primate-derived primordial stem cells. International Patent Publication WO 01 /51 616 (Geron Corp.) provides techniques for growth and differentiation of human plunpotent stem cells. An article by Xu et al. (Nature Biotechnology 19:971, 2001) describes feeder-free growth of undifferentiated human embryonic stem cells. Lebkowski et al. (Cancer J. 7 Suppl. 2:S83, 2001) discuss the culture, differentiation, and genetic modification of human embryonic stem cell for regenerative medicine applications. These publications report exemplary culture methods for propagating human embryonic stem cells in an undifferentiated state, and their use in preparing cells for human therapy.

Markers for identifying undifferentiated pluripotent stem cells include SSEA-4, Tra-1-60, and Tra-1-81 (Thomson et al. and Gearhart et al, supra). They also express human telomerase reverse transcriptase, and the POU transcription factor Oct 3/4 (WO 01/51616; Amit et at., Dev. Biol. 227:271, 2000; Xu et al, supra).

bring et al. (Restor. Neurol. Neurosci. 18:81, 2001) review gene expression profiles of embryonic stem cells and ES-derived neurons. Pesce et al. (Bioessays 20:722, 1998) comment on the potential role of transcription factor Oct-4 in the totipotent germ-line cycle of mice. Gajovic et at. (Exp.

Cell Res. 242:138, 1998) report that genes expressed after retinoic acid-mediated differentiation of embryoid bodies are likely to be expressed during embryo development. Zur Nieden et at. (Toxicol. in Vitro 15:455, 2001) propose certain molecular markers for embryonic stem cells. Henderson et at. (Stem Cells 20:329, 2002) report that pre-implantation human embryos and ES cells have comparable expression of SSEAs. Tanaka et at. (Genome Res. 12:1921, 2002) profile gene expression in mouse ES cells to identify candidate genes associated with pluripotency and lineage specificity. Draper et at. (J.

Anat. 299:249, 2002) review change of surface antigens of human embryonic stem cells upon differentiation in culture.

Kelly et at. (Mol Reprod. Dev. 56:113, 2000) report DNA microarray analyses of genes regulated during the differentiation of embryonic stem cells. Woltjen et al. (Nuci. Acids Res. 28:E41, 2000) report retro-recombination screening of a mouse embryonic stem cell genomic library. Monk et at. (Oncogene 20:8085, 2001) list human embryonic genes re-expressed in cancer cells. Tanaka et al. (Genome Res. 12:1921, 2002) discuss gene expression profiling of embryo-derived stem cefls, and candidate genes putatively associated with pluripotency and lineage specificity. Monk et al. report developmental genes identified by differential display (Reprod. Fertil Dev. 13:51, 2001). Natale et al. (Reprod. 122:687, 2001) characterize bovine blastocyst gene expression patterns by differential display RT-PCR.

Fan et al. (0ev. Biol. 210:481, 1999) propose that forced expression of the homeobox-containing gene Pem blocks differentiation of embryonic stem cells. Abdel-Rahman et al. (Hum. Reprod. 10:2787, 1995) report the effect of expressing transcription regulating genes in human preimplantation embryos.

Jackson et at. (J. Biol. Chem. 277:38683, 2002) describe the cloning and characterization of Ehox, a homeobox gene that reportedly plays a role in ES cell differentiation.

The following disclosure provides new markers and marker combinations that are effective means to identify, characterize, qualify, and control differentiation of pluripotent cells.

SUMMARY OF ThE INVENTION

This invention identifies a number of genes that are up-or down-regulated during the course of differentiation of early-stage pluripotent stem cells obtained from primates, exemplified by human embryonic stem cells. As a consequence, the genes are differentially expressed in undifferentiated versus differentiated cells. This property confers special benefit on these genes for identification, characterization, culturing, differentiation, and manipulation of stem cells and their progeny, and other cells that express the same markers.

One aspect of this invention is a system for assessing a culture of undifferentiated primate pluripotent stem (pPS) cells or their progeny, in which expression of one or more of the identified markers listed in the disclosure is detected or measured. The level of expression can be measured in isolation or compared with any suitable standard, such as undifferentiated pPS cells maintained under specified conditions, progeny at a certain stage of differentiation, or stable end-stage differentiated cells, such as may be obtained from the ATCC. Depending on whether the marker(s) are up-or down-regulated during differentiation, presence of the markers is correlated with the presence or proportion of undifferentiated or differentiated cells in the population.

An exemplary (non-limiting) combination suitable for qualifying cultures of pPS cells are markers of the undifferentiated phenotype selected from Cripto, gastrin-releasing peptide (GAP) receptor, podocalyxin-like protein, hTERT and/Oct 3/4 (POU domain, class 5 transcription factor), in various combinations. Other cell markers can be measured in conjunction, including any of the newly described differentiation markers listed in this disclosure, or traditional pPS cell markers like SSEA-4 and Tra-1-60.

In addition, markers of various differentiated cell phenotypes can be assayed as a measure of contaminating cells. Early stage cell types include stromal cells (marked by CD44 and Vimentin), tibroblasts, mesenchymal cells, and embryoid body cells. The markers can be detected or quantified at the mRNA level by PCR amplification, at the protein or enzyme product level by antibody assay, or by any

suitable technique.

The marker system of this invention can be used for quantifying the proportion of undifferentiated pPS cells or differentiated cells in the culture; for assessing the ability of a culture system or component thereof (such as a soluble factor, culture medium, or feeder cell) to maintain pPS cells in an undifferentiated state; for assessing the ability of a culture system or component thereof to cause differentiation of pPS cells into a culture of lineage-restricted precursor cells or terminally differentiated cells; or for any other worthwhile purpose. This invention includes kits and the use of specific reagents in order to measure the expression of the markers whenever appropriate.

This invention also provides a system assessing the growth characteristics of a cell population by detecting or measuring expression of one or more of the differentially expressed marker genes identified in this disclosure. This can be applied not only to various types of pPS cells and progenitor cells in various stages of differentiation, but also to clinical samples from a disease condition associated with abnormal cell growth. Renewed expression of markers of a relatively undifferentiated phenotype may be diagnostic of disease conditions such as cancer, and can serve as a means by which to target therapeutic agents to the disease site.

The marker system can also be used to regulate gene expression. Transcriptional control elements for the markers will cause an operatively linked encoding region to be expressed preferentially in undifferentiated or differentiated cells. For example, the encoding sequence can be a reporter gene (such as a gene that causes the cells to emit fluorescence), a positive selection marker (such as a drug resistance gene), or a negative selection marker. Vector constructs comprising recombinant elements linked in this fashion can be used to positively select or deplete undifferentiated, differentiated, or cancerous cells from a mixed population or in vivo, depending on the nature of the effector gene and whether transcription is up-or down-regulated during differentiation. They can also be used to monitor culture conditions of pPS cells, differentiation conditions, or for drug screening.

The marker system of this invention can also be used to sort differentiated cells from less differentiated cells. The marker can be used directly for cell separation by adsorption using an antibody or lectin, or by fluorescence activated cell sorting. Alternatively, these separation techniques can be effected using a transcription promoter from the marker gene in a promoter-reporter construct.

The marker system of this invention can be used to map differentiation pathways or influence differentiation. Markers suited for this purpose may act as transcription regulators, or encode products that enhance cell interaction in some fashion. pPS cells or their differentiated progeny are genetically altered to increase expression of one or more of the identified genes using a transgene, or to decrease expression, for example, using an antisense or siRNA construct. Alternatively, gene products involved in cell interaction or signaling can be added directly to the culture medium. The effect of this can be to help maintain the transfected cefi in the undifferentiated state, promote differentiation in general, or direct differentiation down a particular pathway.

Another aspect of the invention are methods for identifying these and other genes that are up-or down-regulated upon differentiation of any cell type. The methods involve comparing expression libraries obtained from the cells before and after differentiation, by sequencing transcripts in each of the libranes, and identifying genes that have statistically significant differences in the relative number of transcripts (as a percentage of transcripts in each library) at a confidence level of 67%, 95%, or 98%. The method can be enhanced by creating assemblies in which different sequences are counted for the same transcript if they are known to correspond to a single transcript according to previously compiled data.

Amongst the differentially expressed markers identified in this disclosure are 39 nucleotide sequences which are not present in their entirety in the UniGene database. These are listed in this disclosure as SEQ. ID NOs:l to 39. This invention includes novel nucleic acids consisting of or containing any of these sequences or the complementary sequences, and novel fragments thereof. This invention also includes novel polypeptides encoded in these sequences (made either by expressing the nucleic acid or by peptide synthesis), antibodies specific for the polypeptides (made by conventional techniques or through a commercial service), and use of these nucleic acids, peptides, and antibodies for any industrial application.

Also embodied in this invention are culture conditions and other cell manipulations identified using the marker system of this invention that are suitable for maintaining or proliferating pPS cells without allowing differentiation, or causing them to differentiate in a certain fashion. Culture conditions tested and validated according to this invention are illustrated in the example section.

Other embodiments of the invention will be apparent from the description that follows.

DRAWINGS

Figure 1 shows the profile of genes preferentially expressed in undifferentiated pluripotent stem cells, upon preliminary differentiation of the cells by culturing in retinoic acid or DM80. Level of gene expression at the mRNA level was measured by real-time PCR assay. Any of the genes showing substantial down-regulation upon differentiation can be used to characterize the undifferentiated cell population, and culture methods suitable for maintaining them in an undifferentiated state.

Figure 2 shows the level of expression of five genes in hES cells, compared with fully differentiated cells. This five-marker panel provides robust qualification of the undifferentiated phenotype.

Figure 3 show results of an experiment in which hES cells of the Hi line were maintained for multiple passages in different media. Medium conditioned with feeder cells provides factors effective to allow hES cells to proliferate in culture without differentiating. However, culturing in unconditioned medium leads to decreased percentage of cells expressing CD9, and the classic hES cell marker SSEA-4.

Figure 4 illustrates the sensitivity of hTERT, Oct 3/4, Cripto, GAP receptor, and podocalyxin-like protein (measured by real-time PCA) as a means of delermining the degree of differentiation of the cells.

After multiple passages in unconditioned medium, all five markers show expression that has been downregulated by 10 to 1 04-fold.

Figure 5 shows results of an experiment in which the tiES cell line Hi was grown on different feeder cell lines: mEF = mouse embryonic fibroblasts; hMSC = human rnesenchymal stem cells; ULSMC = uterine smooth muscle cells; WI-38 = human lung fibroblasts. As monitored using Cripto, the hMSC is suitable for use as feeder cells to promote tiES cell proliferation without differentiation.

Figure 6 shows results of an experiment in which different media were tested for their ability to promote growth of tiES cells without proliferation. The test media were not preconditioned, but supplemented with 8-40 ng/mL bFGFI with or without stem cell factor, F1t3 ligand, or LIE. Effective combinations of factors (Conditions 4 to 8) were identified by following the undifferentiated phenotype using the markers of this invention. Alterations in expression profiles were temporary and reversible, showing that the cells are still undifferentiated.

Figure 7 shows analysis of the undifferentiated hES cell markers SSEA-4, TR.A 1-60 and Oct-4 by antibody staining and flow cytometry. Oct4 is detected by permeabilizing the cells before staining.

Figure 8 shows the results of the immunocytochemical analysis for stromal cell markers C044, STRO-1 and Vimentin, which label cells in the hES cell culture that have undergone differentiation.

Figure 9 shows the relative gene expression levels for cell populations in which undifferentiated hES cells were mixed with BJ fibroblasts in increasing amounts.

DETAILED DESCRIPTION

The propensity of pluripotent stem cells to differentiate spontaneously has made it challenging for investigators to work with these cells. Consistent cultures of undifferentiated stem cells are required to compare results obtained from multiple experiments performed within or between laboratories.

Unfortunately, morphological characterization is subjective and especially difficult for cultures that often contain 10-20% differentiated cells. Nevertheless, having a set of standardized criteria will be important In qualifying these cells for use in clinical therapy.

The marker system identified in this disclosure provides the basis for establishing these standards. 148,453 different transcripts were amplified and sequenced from undifferentiated human embryonic stem cells, and three types of progeny. As a result of this sequencing effort, 532 genes were identified having substantially higher EST counts in undifferentiated cells, and 142 genes were identified having substantially higher EST counts after differentiation. Other thfterentially expressed genes were identified by microarray analysis of undifferentiated cells, compared with cells at the beginning of the differentiation process.

The system provided by this invention can be used not only to qualify populations of undifferentiated cells, but in other powerful ways of maintaining and manipulating cells described later in this disclosure. Culture systems have been identified and protocols have been developed to expand cultures of undifferentiated cells and produce commercially viable quantities of cells for use in research, drug screening, and regenerative medicine.

Definitions "Pluripotent Stem cells" (pPS cells) are pluripotent cells that have the characteristic of being capable under appropriate conditions of producing progeny of several different cell types that are derivatives of all of the three germinal layers (endoderm, mesoderm, and ectoderm), according to a standard artaccepted test, such as the ability to form a teratorna in 8-12 week old SCID mice. The term includes both established lines of stern cells of various kinds, and cells obtained from primary tissue that are pluripotent in the manner described. For the purposes of this disclosure, the pPS cells are not embryonal carcinoma (EC) cells, and are not derived from a malignant source. It is desirable (but not always necessary) that the cells be euploid. Exemplary pPS cells are obtained from embryonic or fetal tissue at any time after fertilization.

"Human Embryonic Stem cells" (hES cells) are pluripotent stem cells derived from a human embryo in the blastocyst stage, or human pluripotent cells produced by artificial means (such as by nuclear transfer) that have equivalent characteristics. Exemplary derivation procedures and features are provided in a later section.

tiES cell cultures are descnbed as "undifferentiated" when a substantial proportion (at least 20%, and possibly over 50% or 80%) of stem cells and their derivatives in the population display morphological characteristics of undifferentiated cells, distinguishing them from differentiated cells of embryo or adult origin. It is understood that colonies of undifferentiated cells within the population will often be surrounded by neighboring cells that are differentiated. It is also understood that the proportion of cells displaying the undifferentiated phenotype will fluctuate as the cells proliferate and are passaged from one cullure to another. Cells are recognized as proliferating in an undifferentiated state when they go through at least 4 passages and/or B population doublings whUe retaining at least about 50%, or the same proportion of cells bearing characteristic markers or morphological characteristics of undifferentiated cells.

A differentiated cell" is a cell that has progressed down a developmental pathway, and includes lineage-committed progenitor cells and terminally differentiated cells.

"Feeder cells" or "feeders" are terms used to describe cells of one type that are co-cultured with cells of another type, to provide an environment in which the cells of the second type can grow. hES cell populations are said to be "essentially free" of feeder cells if the cells have been grown through at least one round after splitting in which fresh feeder cells are not added to support the growth of pPS cells.

The term "embryoid bodies" refers to aggregates of differentiated and undifferentiated cells that appear when pPS cells overgrow in monolayer cultures, or are maintained in suspension cultures.

Embryoid bodies are a mixture of different cell types, typically from several germ layers, distinguishable by morphological criteria and cell markers detectable by immunocytochemistry.

A cell "marker" is any phenotypic feature of a cell that can be used to characterize it or discriminate it from other cell types. A marker of this invention may be a protein (including secreted, cell surface, or internal proteins; either synthesized or taken up by the cell); a nucleic acid (such as an mANA, or enzymatically active nucleic acid molecule) or a polysaccharide. Included are determinants of any such cell components that are detectable by antibody, lectin, probe or nucleic acid amplification reaction that are specific for the cell type of interest The markers can also be identified by a biochemical or enzyme assay that depend on the function of the gene product. Associated with each marker is the gene that encodes the transcript, and the events that lead to marker expression.

A marker is said to be "preferentially expressed" in an undifferentiated or differentiated cell population, if it is expressed at a level that is at least 10 times higher (in terms of total gene product measured in an antibody or PCA assay) or 10 times more frequently (in terms of positive cells in the population). Markers that are expressed 100, 1,000, or 10,000 times higher or more frequently are increasingly more preferred.

The terms "polynucleotide" and "nucleic acid" refer to a polymeric form of nucleotides of any length. Included are genes and gene fragments, rnRNA, cDNA, plasmids, viral and non-viral vectors and particles, nucleic acid probes, amplification primers, and their chemical equivalents. As used in this disclosure, the term polynucleotide refers interchangeably to double-and single-stranded molecules.

Unless otherwise specified, any embodiment of the invention that is a polynucleotide encompasses both a double-stranded form, and each of the two complementary single-stranded forms known or predicted to make up the double-stranded form.

A cell is said to be "genetically altered" or transfected when a polynucleotide has been transferred into the cell by any suitable means of artificial manipulation, or where the cell is a progeny of the originally altered cell that has inherited the polynucleotide.

A "control element" or "control sequence" is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. "Operatively linked" refers to an operative relationship between genetic elements, in which the function of one element influences the function of another element. For example, an expressible encoding sequence may be operatively linked to a promoter that drives gene transcription.

The term "antibody" as used in this disclosure refers to both polyclonal and monoclonal antibody.

The ambit of the term deliberately encompasses not only intact immunoglobulin molecules, but also such fragments and derivatives of immunogiobulin molecules that retain a desired binding specificity.

General Techniques Methods in molecular genetics and genetic engineering are described generally in the current editions of Molecular Cloning: A Laboratory Manual, (Sambrook et al.); Oligonucleotide Synthesis (M.J.

Gait, ed.); Animal Cell Culture (RI. Freshney, ed.); Gene Transfer Vectors for Mammalian Cells (Miller & Cabs, eds.); Current Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3rd Edition (F.M. Ausubel et al., eds.); and Recombinant DNA Methodology (A. Wu ed., Academic Press). Antibody production is described in Basic Methods in Antibody Production and Characterization (Howard & Bethell eds., CRC Press, 2000).

A survey of relevant techniques is provided in such standard texts as DNA Sequencing (A.E.

Barron, John Wiley, 2002), and DNA Microarrays and Gene Expression (P. Baldi et al., Cambridge U. Press, 2002). For a description of the molecular biology of cancer, the reader is referred to Principles of Molecular Oncology (M.H. Bronchud et al. eds., Humana Press, 2000); The Biological Basis of Cancer (R.G. McKinnel et al. eds., Cambridge University Press, 1998); and Molecular Genetics of Cancer (J.K.

Cowell ed., Bios Scientific Publishers, 1999).

Sources of Stem Cells This invention is based on observations made with established lines of hES cells. The markers are suitable for identifying, characterizing, and manipulating related types of undifferentiated pluripotent cells. They are also suitable for use with pluripotent cells obtained from primary embryonic tissue, without first establishing an undifferentiated cell line. It is contemplated that the markers described in this application will in general be useful for other types of pluripotent cells, including embryonic germ cells (U.S. Patents 6,090,622 and 6,251,671), and ES and EG cells from other mammalian species, such as non-human primates.

Embryonic Stem Cells Embryonic stem cells can be isolated from blastocysts of members of primate species (U.S. Patent 5,843,780; Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995). Human embryonic stem (hES) cells can be prepared from human blastoCySt cells using the techniques described by Thomson et al. (U.S. Patent 6,200,806; Science 282:1145, 1998; Curr. Top Dev. Biol. 38:133 if., 1998) and Reubinoff et al, Nature Biotech. 18:399, 2000. Equivalent cell types to hES cells include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, outlined in WO 01/51610 (Bresagen).

hES cells can be obtained from human preimplantation embryos. Alternatively, in vitro fertilized (IVF) embryos can be used, or one-cell human embryos can be expanded to the blastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989). Embryos are cultured to the blastocyst stage in Gi. 2 and G2.2 medium (Gardner et al., Fertil. Steril. 69:84, 1998). The zona pellucida is removed from developed blastocysts by brief exposure to pronase (Sigma). The inner cell masses are isolated by immunosurgery, in which blastocysts are exposed to a 1:50 dilution of rabbit anti-human spleen cell antiserum for 30 mm, then washed for 5 mm three times in DMEM, and exposed to a 1:5 dilution of Guinea pig complement (Gibco) for 3 mm (Solter et al., Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two further washes in DMEM, lysed trophectoderm cells are removed from the intact inner cell mass (1CM) by gentle pipetting, and the 1CM plated on mEF feeder layers.

After 9 to 15 days, inner cell mass derived outgrowths are dissociated into clumps, either by exposure to calcium and magnesium-free phosphate-buffered saline (PBS) with 1 mM EDTA, by exposure to dispase or trypsin, or by mechanical dissociation with a micropipette; and then replated on mEF in fresh medium. Growing colonies having undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and replated. ES-like morphology is characterized as compact colonies with apparently high nucleus to cytoplasm ratio and prominent nucleoli. Resulting ES cells are then routinely split every 1-2 weeks by brief trypsinization, exposure to Dulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase (-200 U/mL; Gibco) or by selection of individual colonies by micropipette. Clump sizes of about 50 to 100 cells are optimal.

Propagation of pPS Cells in an Undifferentiated State pPS cells can be propagated continuously in culture, using culture conditions that promote proliferation without promoting differentiation. Exemplary serum-containing ES medium is made with 80% DMEM (such as Knock-Out DMEM, Gibco), 20% of either defined fetal bovine serum (FBS, Hyclone) or serum replacement (US 20020076747 Al, Life Technologies Inc.), 1% non-essential amino acids, 1 mM L-glutamine, and 0.1 mM -mercaptoethanol. Just before use, human bFGF is added to 4 rig/mL (WO 99/20741, Geron Corp.).

Traditionally, ES cells are cultured on a layer of feeder cells,typically fibroblasts derived from embryonic or fetal tissue. Embryos are harvested from a CF1 mouse at 13 days of pregnancy, transferred to 2 mL trypsin/EDTA, finely minced, and incubated 5 miri at 37 C. 10% FBS is added, debris is allowed to settle, and the cells are propagated in 90% DMEM, 10% FBS, and 2 mM glutamine. To prepare a feeder cell layer, cells are irradiated to inhibit proliferation but permit synthesis of factors that support ES cells (-4000 rads y-irradiation). Culture plates are coated with 0.5% gelatin overnight, plated with 375,000 irradiated mEFs per well, and used 5 h to 4 days after plating. The medium is replaced with fresh hES medium just before seeding pPS cells.

Scientists at Geron have discovered that pPS cells can be maintained in an undifferentiated state even without feeder cells. The environment for feeder-free cultures includes a suitable culture substrate, particularly an extracellular matrix such as Matrigel or laminin. The pPS cells are plated at >15,000 cells cm2 (optimally 90,000 cm2 to 170,000 cm2). Typically, enzymatic digestion is halted before cells become completely dispersed (say, -5 mm with collagenase IV). Clumps of -10 to 2,000 cells are then plated directly onto the substrate without further dispersal. Alternatively, the cells can be harvested without enzymes before the plate reaches confluence by incubating -5 mm in a solution of 0.5 mM EDTA in PBS. After washing from the culture vessel, the cells are plated into a new culture without further dispersal. In a further illustration, confluent human embryonic stem cells cultured in the absence of feeders are removed from the plates by incubating with a solution of 0.05% (wt/vol) trypsin (Gibco) and 0.053 mM EDTA for 5-15 mm at 37C. The remaining cells in the plate are removed and the cells are tnturated into a suspension comprising single cells and small clusters, and then plated at densities of 50,000-200,000 cells cm2 to promote survival and limit differentiation.

Feeder-free cultures are supported by a nutrient medium containing factors that support proliferation of the cells without differentiation. Such factors may be introduced into the medium by culturing the medium with cells secreting such factors, such as irradiated (-4,000 rad) primary mouse embryonic fibroblasts, telomerized mouse fibroblasts, or fibroblast-like cells derived from pPS cells.

Medium can be conditioned by plating the feeders at a density of -5-6 x cm2 in a serum free medium such as KO DMEM supplemented with 20% serum replacement and 4 ng/mL bFGF. Medium that has been conditioned for 1-2 days is supplemented with further bFGF, and used to support pPS cell culture for 1-2 days. Alternatively or in addition, other factors can be added that help support proliferation without differentiation, such as ligands for the FGF-2 or FGF-4 receptor, ligands for c-kit (such as stem cell factor), ligands for receptors associated with gpl3O, insulin, transferrin, lipids, cholesterol, nucleosides, pyruvate, and a reducing agent such as 3-mercaptoethanol. Aspects of the feeder-free culture method are further discussed in International Patent Publications WO 99/20741, WO 01/51616; Xu et al., Nat.

Biotechnol. 19:971, 2001; and PCT application PCTIUSO2/28200. Exemplary culture conditions tested and validated using the marker system of this invention are provided below in Example 6.

Under the microscope, ES cells appear with high nuclear/cytoplasmic ratios, prominent nucleoli, and compact colony formation with poorly discemable cell junctions. Conventional markers for hES cells are stage-specific embryonic antigen (SSEA) 3 and 4, and markers detectable using antibodies Tra-1-60 and Tra-1-81 (Thomson et al., Science 282:1145, 1998). Differentiation of pPS cells in vitro results in the loss of SSEA-4, Tra-1-60, and Tra-1-81 expression, and increased expression of SSEA-1.

Markers of undifferentiated pPS cells and their differentiated progeny The tables and description provided later in this disclosure provide markers that distinguish undifferentiated pPS cells from their differentiated progeny.

Expression libraries were made from ES cells (WO 01/51616), embryoid bodies (WO 01/51 61 6), and cells differentiated towards the hepatocyte (WO 01/81 549) or neural cell (WO 01/88104) lineage.

mRNA was reverse transcribed and amplified, producing expressed sequence tags (ESTS) occurring in frequency proportional to the level of expression in the cell type being analyzed. The ESTs were subjected to automatic sequencing, and counted according to the corresponding unique (non-redundant) transcript. A total of 148,453 non-redundant transcripts were represented in each of the 4 libraries.

Genes were then identified as having a differential expression pattern if the number of EST counts of the transcript was statistically different between the libraries being compared.

In a parallel set of experiments, mRNA from each of the cell types was analyzed for binding to a broad-specificity EST-based microarray, performed according to the method described in WO 01/51616.

* Genes were identified as having a differential expression pattern if they showed a comparatively different signal on the microarray.

Significant expression differences determined by EST sequencing, microarray analysis, or other observations were confirmed by real-time PCR analysis. The mANA was amplified by PCR using specific forward and reverse primers designed from the GenBank sequence, and the amplification product was detected using labeled sequence-specific probes. The number of amplification cycles required to reach a threshold amount was then compared between different libraries.

Distinguishing markers fall into several categories. Those of particular interest include the following: * Markers characteristically expressed at a higher level in undifferentiated pPS cells than any of the differentiated cells, indicating down-regulation during differentiation. The gene products may be involved in maintaining the undifferentiated phenotype.

* Markers characteristically expressed at a higher level in the three differentiated cell types than in the undifferentiated cells, indicating up-regulation during differentiation. The gene products may be involved in the general differentiation process.

* Markers characteristically expressed at a higher level in one of the differentiated cell types.

The encoded genes may be involved in differentiation down restricted lineages.

Markers can also be classified according to the function of the gene product or its location in the cell. Where not already indicated, protein gene products can be predicted by referencing public information according to the GenBank accession number, or by translating the open reading frame after the translation start signal though the genetic code. Features of the markers listed can be determined by the descriptors give in the tables below, or by using the accession number or sequence data to reference public information. Marker groups of particular interest include the following: * Secreted proteins of interest, for example, because they can be detected by immunoassay of the culture supernatant, and may transmit signals to neighboring cells.

Secreted proteins typically have an N-terminal signal peptides, and may have glycosylation sites.

* Surface membrane protens -of interest, for example, because they can be used for cell-surface labeling and affinity separation, or because they act as receptors for signal transduction. They may have glycosytation sites and a membrane spanning region. A Markov model for predicting transmembrane protein topology is described by Krogh et al., J. Mol Biol. 305:567, 2001.

* Enzymes with relevant function. For example, enzymes involved in protein synthesis and cleavage or in apoptosis may influence differentiation. Glycosyltransferases decorate the cell membrane with distinguishing carbohydrate epitopes that may play a role in cellular adhesion or localization.

* Transcription regulatory factors -of interest for their potential to influence differentiation, as explained later in this disclosure. These factors sometimes have zinc fingers or other identifiable topological features involved in the binding or metabolism of nucleic acids.

Through the course of this work, the key signaling pathways Wnt, Sonic hedgehog (Shh), and Notch emerged as regulators of growth of pPS cells. Interestingly, these pathways have also been shown to play a rote in the growth of tumor cells of various kinds, and in embryonic development of lower species.

Now that genes have been identified that are up-regulated or down-regulated upon differentiation, a number of commercial applications of these markers will be apparent to the skilled reader. The sections that follow provide non-limiting illustrations of how some of these embodiments can be implemented.

Use of cell markers to characterize pPS cells and their differentiated oropeny The markers provided in this disclosure can be used as a means to identify both undifferentiated and differentiated cells -either a population as a whole, or as individual cells within a population. This can be used to evaluate the expansion or maintenance of pre-existing cell populations, or to characterize the pluripotent nature (or lineage commitment) of newly obtained populations.

Expression of single markers in a test cell will provide evidence of undifferentiated or differentiated phenotype, according to the expression pattern listed later in this disclosure. A plurality of markers (such as any 2, 3, 4, 5, 6, 8, 10, 12, 15, or 20 markers from Tables 2-3 or 5-9) will provide a more detailed assessment of the characteristics of the cell. Expression of genes that are down-regulated and/or tack of expression of genes that are up-regulated upon differentiation correlates with a differentiated phenotype. Expression of genes that are upregulated and/or lack of expression of genes that are down-regulated upon differentiation correlates with an undifferentiated phenotype. The markers newly identified in this disclosure may be analyzed together (with or without markers that were previously known) in any combination effective for characterizing the cell status or phenotype.

Exemplary combinations of markers are provided elsewhere in this disclosure. For determining the unddterentiated cell phenotype, combinations of markers like Cnpto, gastrin-releasing peptide (GRP) receptor, podocalyxin-like protein (PODXL), and human telomerase reverse transcriptase (hTERT) are effective, either alone, or in combination with cell surface markers like SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81, or intracellular markers like Oct 3/4. For determining differentiated cells, any marker can be used that is characteristic of contaminating cells that may be present. Depending on culture conditions, early stage non-specific hES cell differentiation generates cells having characteristics of stromal cells, tibroblasts, mesenchymal cells, embryoid body cells, and other cell types. Alternatively, a combination of markers characteristic of several types of cells can be used, as long as they are preferentially expressed in differentiated cells.

Tissue-specific markers can be detected using any suitable immunological technique -such as flow cytochemistry for cell-surface markers, or immunocytochemistry (for example, of fixed cells or tissue sections) for intracellular or cell-surface markers. Expression of a cell-surface antigen is defined as positive if a significantly detectable amount of antibody will bind to the antigen in a standard immunocytochemistry or flow cytometry assay, optionally after fixation of the cells, and optionally using a labeled secondary antibody or other conjugate to amplify labeling.

The expression of tissue-specific gene products can also be detected at the mRNA level by Northern blot analysis, dot-blot hybridization analysis, or by reverse trariscriptase initiated polymerase chain reaction (RT-PCR) using sequence-specific primers in standard amplification methods. See U.S. Patent No. 5843,780 for further details. Sequence data for particular markers listed in this disclosure can be obtained from public databases such as GenBank.

These and other suitable assay systems are described in standard reference texts, such as the following: PCR Cloning Protocols, 2' Ed (James & Chen eds., Humana Press, 2002); Rapid Cycle Real-Time PCR: Methods and Applications (C. Wittwer et al. eds., Spnriger-Vertag NY, 2002); Immunoassays: A Practical Approach (James Gosling ed., Oxford Univ Press, 2000); Cytometric Analysis of Cell Phenotype and Function (McCarthy et al. eds., Cambridge Univ Press, 2001). Reagents for conducting these assays, such as nucleotide probes or primers, or specific antibody, can be packaged in kit form, optionally with instructions for the use of the reagents in the characterization or monitoring of pPS cells, or their differentiated progeny.

Use of cell markers for clinical diagnosis Stem cells regulate their own replenishment and serve as a source of cells that can differentiate into defined cell lineages. Cancer cells also have the ability to self-renew, but lack of regulation results in uncontrolled cellular proliferation. Three key signaling pathways, Wnt, Sonic hedgehog (Shh), and Notch, are known growth regulators of tumor cells. The genomics data provided in this disclosure indicate that all three of these pathways are active in hES cells.

It is a hypothesis of this invention that many of the markers discovered to be more highly expressed in undifferentiated pPS cells can also be up-regulated upon dedifferentiation of cells upon malignant transformation. Accordingly, this disclosure provides a system for evaluating clinical conditions associated with abnormal cell growth, such as hyperplasia or cancers of various kinds. Markers meeting the desired criteria include those contained in Tables 2, 5, 7 and 9.

Expression of each marker of interest is determined at the mRNA or protein level using a suitable assay system such as those described earlier; and then the expression is correlated with the clinical condition that the patient is suspected of having. As before, combinations of multiple markers may be more effective in doing the assessment. Presence of a particular marker may also provide a means by which a toxic agent or other therapeutic drug may be targeted to the disease site.

In a similar fashion, the markers of this invention can be used to evaluate a human or non-human subject who has been treated with a cell population or tissue generated by differentiating pPS cells. A histological sample taken at or near the site of administration, or a site to which the cells would be expected to migrate, could be harvested at a time subsequent to treatment, and then assayed to assess whether any of the administered cells had reverted to the undifferentiated phenotype. Reagents for conducting diagnostic tests, such as nucleotide probes or primers, or specific antibody, can be packaged in kit form, optionally with instructions for the use of the reagents in the determination of a disease condition.

Use of cell markers to assess and manipulate culture conditions The markers and marker combinations of this invention provide a system for monitoring undifferentiated pPS cells and their differentiated progeny in culture. This system can be used as a quality control, to compare the characteristics of undifferentiated pPS cells between different passages or different batches. It can also be used to assess a change in culture conditions, to determine the effect of the change on the undifferentiated cell phenotype.

Where the object is to produce undifferentiated cells, a decrease in the level of expression of an undifferentiated marker because of the alteration by 3-, 10-, 25-, 100-and 1000-fold is progressively less preferred. Corresponding increases in marker expression may be more beneficial. Moderate decreases in marker expression may be quite acceptable within certain boundaries, if the cells retain their ability to form progeny of all three germ layers is retained, and/or the level of the undifferentiated marker is relatively restored when culture conditions are returned to normal.

In this manner, the markers of this invention can be used to evaluate different feeder cells, extracellular matrixes, base media, additives to the media, culture vessels, or other features of the culture as illustrated in WO 99/20741 and PCI application PCT/USO2/28200. illustrations of this technique are provided below in Example 6 (Figures 3 to 6).

In a similar fashion, the markers of this invention can also be used to monitor and optimize conditions for differentiating cells. Improved differentiation procedures will lead to higher or more rapid expression of markers for the differentiated phenotype, and/or lower or more rapid decrease in expression of markers for the undifferentiated phenotype.

Use of cell markers to regulate gene exoression Differential expression of the markers listed in this disclosure indicates that each marker is controlled by a transcriptional regulatory element (such as a promoter) that is tissue specific, causing higher levels of expression in undifferentiated cells compared with differentiated cells, or vice versa.

When the corresponding transcriptional regulatory element is combined with a heterologous encoding region to drive expression of the encoding region, then the expression pattern in different cell types will mimic that of the marker gene.

Minimum promoter sequences of many of the genes listed in this disclosure are known and further described elsewhere. Where a promoter has not been fully characterized, specific transcription can usually be driven by taking the 500 base pairs immediately upstream of the translation start signal for the marker in the corresponding genomic clone.

To express a heterotogous encoding region according to this embodiment of the invention, a recombinant vector is constructed in which the specific promoter of interest is operatively linked to the encoding region in such a manner that it drives transcription of the encoding region upon transfection into a suitable host cell. Suitable vector systems for transient expression include those based on adenovirus and certain types of plasmids. Vectors for long-term expression include those based on plasmid lipofection or electroporation, episomal vectors, retrovirus, and lentivirus.

One application of tissue-specific promoters is expression of a reporter gene. Suitable reporters include fluorescence markers such as green fluorescent protein, luciferase, or enzymatic markers such as alkaline phosphatase and -galactosidase. Other reporters such as a blood group glycosyltransferase (WO 02/074935), or Invitrogen's pDisplay, create a cell surface epitope that can be counterstained with labeled specific antibody or lectin. pPS cells labeled with reporters can be used to follow the differentiation process directly, the presence or absence of the reporter correlating with the undifferentiated or differentiated phenotype, depending on the specificity of the promoter. This in turn can be used to follow or optimize culture conditions for undifferentiated pPS cells, or differentiation protocols.

Alternatively, cells containing promoter-reporter constructs can be used for drug screening, in which a test compound is combined with the cell, and expression or suppression of the promoter is correlated with an effect attributable to the compound.

Another application of tissue-specific promoters is expression of a positive or negative drug selection marker. Antibiotic resistance genes such as neomycin phosphotransterase, expressed under control of a tissue-specific promoter, can be used to positively select for undifferentiated or differentiated cells in a medium containing the corresponding drug (geneticin), by choosing a promoter with the appropriate specificity. Toxin genes, genes that mediate apoptosis, or genes that convert a prodrug into a toxic compound (such as thymidine kinase) can be used to negatively select against contaminating undifferentiated or differentiated cells in a population of the opposite phenotype (WO 02/42445; GB 2374076).

Promoters specific for the undifferentiated cell phenotype can also be used as a means for targeting cancer cells -using the promoter to drive expression of a gene that is toxic to the cell (Wa 98/14593, WO 02142468), or to drive a replication gene in a viral vector (WO 00/46355). For example, an adenoviral vector in which the GRPR promoter (AY032865) drives the El a gene should specifically lyse cancer cells in the manner described in Majumdar et al., Gene Ther. 8:568, 2001.

Multiple promoters for the undifferentiated phenotype can be linked for improved cancer specificity (USSN 10/206,447).

Other useful applications of tissue-specific promoters of this invention will come readily to the mind of the skilled reader.

Use of markers for cell separation or purification Differentially expressed markers provided in this disclosure are also a means by which mixed cell populations can be separated into populations that are more homogeneous. This can be accomplished directly by selecting a marker of the undifferentiated or differentiated phenotype, which is itself expressed on the cell surface, or otherwise causes expression of a unique cell-surface epitope. The epitope is then used as a handle by which the marked cells can be physically separated from the unmarked cells. For example, marked cells can be aggregated or adsorbed to a solid support using an antibody or lectin that is specific for the epitope. Alternatively, the marker can be used to attach a fluorescently labeled antibody or lectin, and then the cell suspension can be subject to fluorescence-activated cell sorting.

An alternative approach is to take a tissue-specific promoter chosen based on its expression pattern (as described in the last section), and use it to drive transcription of a gene suitable for separating the cells. In this way, the marker from which the promoter is chosen need not itself be a cell surface protein. For example, the promoter can drive expression of a fluorescent gene, such as GFP, and then cells having the marked phenotype can be separated by FACS. In another example, the promoter drives expression of a heterologous gene that causes expression of a cell-surface epitope. The epitope is then used for adsorption-based separation, or to attach a fluorescent label, as already described.

Use of cell markers to influence differentiation In another embodiment of this invention, the differentially expressed genes of this invention are caused to increase or decrease their expression level, in order to either inhibit or promote the differentiation process. Suitable genes are those that are believed in the normal case of ontogeny to be active in maintaining the undifferentiated state, active in the general process of differentiation, or active in differentiation into particular cell lineages. Markers of interest for this application are the following: Transcription factors and other elements that directly affect transcription of other genes, such as Forkhead box O1A (FOXO1A); Zic family member 3 (ZIC3); Hypothetical protein FLJ20582; Forkhead box Hi (FOXH1); Zinc finger protein, Hsal2; KRAB-zinc linger protein SZF1 -1; Zinc finger protein of cerebellum ZIC2; and Coup transcription factor 2 (COUP-TF2). Other candidates include those marked in Tables 5 and 6 with the symbol U fl, and other factors with zinc lingers or nucleic acid binding activity.

* Genes that influence cell Interaction, such as those that encode adhesion molecules, and enzymes that make substrates for adhesion molecules * Genes encoding soluble factors that transmit signals within or between cells, and specific receptors that recognize them and are involved in signal transduction.

One way of manipulating gene expression is to induce a transient or stable genetic alteration in the cells using a suitable vector, such as those already listed. Scientists at Gerori Corp. have determined that the following coostitutive promoters are effective in undifferentiated hES cells: for transient expression CMV, SV4O, EF1c, UbC, and PGK; for stable expression, SV4O, EF1a, UbC, MND and PGK. Expressing a gene associated with the undifferentiated phenotype may assist the cells to stay undifferentiated in the absence of some of the elements usually required in the culture environment. Expressing a gene associated with the differentiated phenotype may promote early differentiation, and/or initiate a cascade of events beneficial for obtaining a desired cell population. Maintaining or causing expression of a gene of either type early in the differentiation process may in some instances help guide differentiation down a particular pathway.

Another way of manipulating gene expression is to alter transcription from the endogenous gene.

One means of accomplishing this is to introduce factors that specifically influence transcription through the endogenous promoter. Another means suitable for down-regulating expression at the protein level is to genetically alter the cells with a nucleic acid that removes the rnRNA or otherwise inhibits translation (for example, a hybridizing antisense molecule, ribozyrne, or small interfering RNA). Dominant-negative mutants of the target factor can reduce the functional effect of the gene product. Targeting a particular factor associated with the undifferentiated phenotype in this fashion can be used to promote differentiation. In some instances, this can lead to de-repression of genes associated with a particular cell type.

Where the gene product is a soluble protein or peptide that influences cell interaction or signal transduction (for example, cytokines like osteopontin and Cripto), then it may be possible to affect differentiation simply by adding the product to the cells -in either recombinant or synthetic form, or purified from natural sources. Products that maintain the undifferentiated phenotype can then be withdrawn from the culture medium to initiate differentiation; and products that promote differentiation can be withdrawn once the process is complete.

Since differentiation is a multi-step process, changing the level of gene product on a permanent basis may cause multiple effects. In some instances, it may be advantageous to affect gene expression in a temporary fashion at each sequential step in the pathway, in case the same factor plays different effects at different steps ol differentiation. For example, function of transcription factors can be evaluated by changing expression of individual genes, or by invoking a high throughput analysis, using cDNAs obtained from a suitable library such as exemplified in Example 1. Cells that undergo an alteration of interest can be cloned and pulled from multi-well plates, and the responsible gene identified by PCR amplification.

The effect of up-or down-regulating expression of a particular gene can be determined by evaluating the cell for morphological characteristics, and the expression of other characteristic markers.

Besides the markers listed later in this disclosure, the reader may want to follow the effect on particular cell types, using markers for later-stage or terminally differentiated cells. Tissue-specific markers suitable for this purpose are listed in WO 01/81549 (hepatocytes), WO 01/88104 (neural cells), PCT/USO2/20998 (osteoblasts and mesenchymal cells), PCT/US02122245 (cardiomyocytes), PCT/USO2/39091 (hematopoietic cells), PCT/USO2/39089 (islet cells), and PCT/USO2/39090 (chondrocytes). Such markers can be analyzed by PCR amplification, fluorescence labeling,or immunocytochemistry, as already described. Promoter-reporter constructs based on the same markers can facilitate analysis when expression is being altered in a high throughput protocol.

The examples that follow are provided for further illustration, and are not meant to limit the claimed invention.

EXAMPLES

ExamDle 1: An EST database of undifferentiated hES cells and their differentiated progeny cDNA libraries were prepared from human embryonic stem (hES) cells cultured in undifferentiated form. cDNA libranes were also prepared from progeny, subject to non-specific differentiation as embryoid bodies (EBs), or taken through the preliminary stages of established differentiation protocols for neurons (preNEU) or hepatocytes (preHEP).

The hES cell lines Hi, H7, and H9 were maintained under feeder-free conditions. Cultures were passaged every 5-days by incubation in 1 mg/mL collagenase IV for 5-10 mii at 37 C, dissociated and seeded in clumps at 2.5 to lOx cells/well onto MatrigelTM-coated six well plates in conditioned medium supplemented with 8 mg/mL bFGF. cDNA libraries were made after culturing for 5 days after the last passage.

EBs were prepared as follows. Confluent plates of undifferentiated hES cells were treated briefly with collagenase IV, and scraped to obtain small clusters of cells. Cell clusters were resuspended in 4 mL/well differentiation medium (KO DMEM containing 20% fetal bovine serum in place of 20% SR, and not preconditioned) on low adhesion 6-well plates (Costar). After 4 days in suspension, the contents of each well was transferred to individual wells pre-coated with gelatin. Each well was re-fed with 3 mL fresh differentiation medium every tWo days after replating. Cells were used for the preparation of cytoplasmic RNA on the eighth day after plating.

PreHEP cells were prepared based on the hepatocyte driferentiation protocol described in WO 01/81549. Confluent wells of undifferentiated cells were prepared, and medium was changed to KO DMEM plus 20% SR + 1% DMSO. The medium was changed every 24 h, and cells were used for preparation of cytoplasmic RNA on day 5 of DMSO treatment.

PreNEU cells were prepared based on the neural differentiation protocol described in WO 01/88104. hES cells of the H7 line (p29) were used to generate EBs as described above except that pM all-trans RA was included in the differentiation medium. After 4 days in suspension, EBs were transferred to culture plate precoated with poly-L-$ysine and laminin. After plating, the medium was changed to EPFI medium. Cells were used for the preparation of cytoplasmic RNA after 3 days of growth in EPFI.

Partial 5' end sequences (an expressed sequence tag, or EST) were determined by conventional means for independent clones derived from each cONA library. Overlapping ESTs were assembled into conjoined sequences.

TABLE 1: Non-redundant EST sequences I Lb Number i rary of ESTs hESC 37,081 EB 37,555 preHEP 35,611 preNEU 36,206 Total 148,453 All of the stem cell lines used for preparation of the expression libraries were originally isolated and initially propagated on mouse feeder cells. Accordingly, the libraries were analyzed to determine whether they were contaminated with murine retroviruses that had shed from the feeder cells and subsequently infected the stem cells. Three complete viral genomes were used in a BLAST search: Moloney munne leukemia virus, Friend murine leukemia virus, and murine type C retrovirus. No matches with a high score were found against any of the ESTs.

The sequences were then compared to the Unigene database of human genes. ESTs that were at least 98% identical, over a stretch of at least 150 nucleotides each, to a common reference sequence in Unigene, were assumed to be transcribed from the same gene, and placed into a common assembly.

The complete set of 148,453 ESTs collapsed to a non-redundant set of 32,764 assemblies.

Example 2: Selection of marker genes specific for undifferentiated and differentiated cells Candidate markers were selected from a database based on the imputed level of gene expression. The frequency of ESTs for any particular gene correlates with the abundance of that mRNA in the cells used to generate the cDNA library. Thus, a comparison of frequencies of ESTs among the libraries indicates the relative abundance of the associated rnRNA in the different cell types.

Candidate molecular markers were selected from the expressed gene (EST) database from their greater abundance in undifferentiated hES cells, relative to differentiated hES cells. Genes were identified as having a differential expression pattern (being up-or down-regulated) during the differentiation process, if the count of ESTs sequenced in the undifferentiated cells was substantially different from the sum of ESTs in the three differentiated libraries.

Oct 3/4 (a POU domain-containing transcription factor) and telomerase reverse transcriptase (hTERT) are known to be expressed preferentially in undifferentiated tIES cells (WO 01/51616). Other genes suitable for characterizing or manipulating the undifferentiated phenotype are those that are down.

regulated upon differentiation with a significance of p = 0.05, as determined by the Fisher Exact Test (explained below). 193 genes were found to have 4-fold more ESTs in hES cells, relative to each of the three cell types. 532 genes were found that were 2-fold greater hES cells, with a confidence of over 95% as determined by the Fisher Exact Test, relative to the sum of ESTs of the three cell types (minimum of 4 ESTs in hES cells). The following markers are of particular interest: TABLE 2: EST Frequency of Genes that are Down-regulated upon Differentiation of hES cells Geron ID Gen Bank ID Name EST counts ES EB preHEP preNEU GA_i 0902 NM_024504 Pr domain containing 14 (PRDM1 4) 12 1 0 0 GA_i 1893 NM_032805 Hypothetical protein FLJ1 4549 25 0 0 0 GA_12318 NM_032447 Fibrillin3 6 0 0 0 GA_1322 NM_000142 Fibroblast growth factor receptor 3 precursor 9 1 5 1 (FGFR-3) GA_34679 NM_002015 Forkhead box ola (FOXOia) 4 0 1 1 GA_1470 NM_003740 potassium channel, subfamily K, member 5 4 0 0 1 (KCNK5), mRNA GA_i 674 NM_002701 Octamer-Binding Transcription Factor 3a 24 1 2 0 (OCT-3A) (OCT-4) GA._2024 NM_003212 Teratocarcinoma-derived growth factor 1 20 1 0 0 (CR1 PTO) GA_2149 NM_00341 3 Zic family member 3 (ZIC3) 7 0 1 0 GA_2334 NM_000216 Kallmann syndrome 1 sequence (KAL1) 5 0 1 0 GA_23552 NM...152742 hypothetical protein DKFZp547M1O9 6 0 1 2 (DKFZp547M 109), mRNA GA_2356 NM_00285i Protein tyrosine phosphatase, receptor-type, 10 0 0 0 z polypeptide 1 (PTPRZ1), GA_2357 NM_001 670 Armadillo repeat protein deleted in 6 0 0 0 velo-cardlo-facial syndrome (ARVCF) GA_23578 BM454360 AGENCOURT_6402318 NIH_MGc_85 6 0 0 0 1-lomo sapiens cDNA clone IMAGE:5497491 TABLE 2: EST Frequency of Genes that are Down-regulated upon Differentiation of hES cells Geron ID GenBank ID Name EST counts ES EB preHEP preNEU 5', mRNA sequence GA 2367 NM_003923 Forkhead box Hi (FOXH1) 5 0 0 0 GA_2436 NM_004329 Bone morphogenetic protein receptor, type Ia 7 3 1 1 (BMPR1A) (ALK-3) GA_2442 NM_004335 Bone marrow stromal antigen 2 (BST-2) 13 0 2 3 GA_2945 NM_005232 Ephrin type-a receptor 1 (EPHA1) 5 1 1 1 GA_2962 NM_005314 Gastrin-releasing peptide receptor (GRP-R) 4 0 0 0 GA_2988 NM_005397 Podocalyxin-like (PODXL) 59 23 5 8 GA_3337 NM_006159 NELL2 (nel-like protein 2) 5 3 2 0 GA_3559 NM_005629 Solute carrier family 6, member B (SLC6A8) 5 1 0 1 GA_3898 NM_006892 DNA (cytosine-5-)-methyltransf erase 3 beta 49 2 3 1 (DNMT3B) GA_5391 NM_002968 Sal-like 1 (SALL1), 7 1 1 0 GA_33680 NM_016089 Krab-ziric finger protein SZF1-1 15 0 1 0 GA_36977 NM_020927 K1AA1576 protein 9 2 1 0 GA_8723 NM_152333 Homo sapiens chromosome 14 open reading 14 1 1 3 frame 69 (C14orf69), mRNA GA_9167 AF308602 Notch 1 (Ni) 6 2 1 0 GA_9183 NM_007129 Homo sapiens Zic family member 2 (odd-8 1 1 0 paired homolog, DrosophUa) (ZIC2), mRNA GA_35037 NM_004426 Homo sapiens polyhomeotic-like 1 34 9 5 4 (Drosophila) (PHC1), mRNA Only one EST for hTERT was identified in undifferentiated hES cells and none were detected from the differentiated cells, which was not statistically significant. Thus, potentially useful markers that are expressed at low levels could have been omitted in this analysis, which required a minimum of four ESTs.

It would be possible to identify such genes by using other techniques described elsewhere in this

disclosure.

Three genes were observed from EST frequency queries that were of particular interest as potentially useful markers of hES cells. They were Teratacarcinoma-derived growth factor (Cripto), Podocalyxin-like (PODXL), and gastrin-releasing peptide receptor (GRPR). These genes were not only more abundant in undifferentiated cells, relative to differentiated hES cells, but also encoded for proteins expressed on the surface of cells. Surface markers have the added advantage that they could be easily detected with immunological reagents. ESTs for Cripto and GRPR were quite restricted to hES cells, with one or zero ESTs, respectively, scored in any of the differentiated cells. PODXL ESTs were detected in all 4-cell types, but substantially fewer (2.5X -i2X) in differentiated cells. All three markers retained a detectable level of expression in differentiated cultures of hES cells. There may be a low level of expression of these markers in differentiated cells, or the expression detected may be due to a small proportion of undifferentiated cells in the population. GABA(A) receptor, Lefty B, Osteopontin, Thy-i co-transcribed, and Solute carrier 21 are other significant markers of the undifferentiated phenotype.

By similar reasoning, genes that show a higher frequency of ESTs in differentiated cells can be used as specific markers for differentiation. ESTs that are 2-told more abundant in the sum of all three differentiated cell types (EBs, preHEP and preNEU cells) and with a p-value = 0.05 as determined by the Fisher Exact Test, compared with undifferentiated hES cells are candidate markers for differentiation down multiple pathways ESTs that are relatively abundant in only one of the differentiated cell types are candidate markers for tissue-specific differentiation. The following markers are of particular interest: TABLE 3: EST Frequency of Genes that are Upregulated upon Differentiation Geron ID GenBank ID Name EST counts ES EB preHEP preNEU GA_35463 NM_024298 Homo sapiens leukocyte receptor cluster (LRC) 0 4 9 8 member 4 (LENG4), mANA GA_i 0492 NM_006903 Inorganic pyrophosphatase (PPASE) 0 5 5 6 GA_38563 NM_021 005 Homo sapiens nuclear receptor subfamily 2, 0 9 8 9 group F, member 2 (NR2F2), mANA GA_38570 NM_001844 Collagen, type II, alpha 1 (COL2A1), transcript 15 31 5 variant 1 GA_1476 NM_002276 Keratin type I cytoskeletal 19 (cytokeratin 19) 1 26 14 38 GA_34776 NM_002273 Keratin type II cytoskeletal 8 (cytokeratin 8) 9 71 144 156 (CK 8) GA_i 735 NM_002806 Homo sapiens proteasome (prosome, 1 7 7 8 macropain) 26S subunit, ATPase, 6 (PSMC6), mANA GA_i 843 NM_000982 SOS ribosomal protein 121 1 7 48 42 GA_35369 NM_003374 Voltage-dependent anion-selective channel 1 5 6 10 (VDAC-i) GA_231 17 NM_004772 P311 protein (Homo sapiens] 1 5 7 6 GA_2597 NM_i 38610 Homo sapiens H2A histone family, member V 1 5 5 14 (H2AFY), transcript variant 3, mANA GA_3283 NM_004484 Homo sapiens glypican 3 (GPC3), mRNA 1 6 7 12 TABLE 3: EST Frequency of Genes that are Upregulated upon Differentiation Geron ID GenBarik ID Name EST counts ES EB preHEP preNEU GA_3530 NM_002539 Homo sapiens omithirte decarboxylase 1 1 10 8 9 (ODd), mANA GA_4145 NM_002480 Protein phosphatase 1, regulatory(inhibitor) 1 6 6 6 subunit 12A (PPP1R12A) GA_5992 NM_01 4899 Homo sapiens Rho-related BIB domain 0 10 7 13 containing 3 (RHOBTB3), mRNA GA_6136 NM_01 6368 Homo sapiens myo-inositol 1-phosphate 1 7 5 16 synthase Al (ISYNA1), mRNA GA_6165 NM_01 5853 Orf (L0C51035) 1 5 9 5 GA_621 9 NM_01 6139 1 6.7Kd protein (LOC51 142), 1 5 13 14 GA_723 NM_005801 Homo sapiens putative translation initiation 1 14 15 19 factor (SUI1), mRNA GA_9196 NM_000404 Homo sapiens galactosidase, beta 1 (GLB1), 0 6 10 7 transcript variant 179423, mRNA GA_9649 NM_01 4604 Tax interaction protein 1 (TIP-i) 0 8 5 5 The relative expression levels were calculated as follows: (# ESTs of the gene in hES cells total unique genes in hES cells) es = (# ESTs of the gene in differentiated cells total unique genes in differentiated cells) -(# ESTs for the gene in hES cells 37,081) -(# ESTs for the gene in differentiated cells 111,372) The es value is substantially >1 for genes marking the undifferentiated phenotype, and <1 for genes indicating differentiation.

The Fisher Exact Test was used to determine whether changes were statistically significant.

S. Siegel & N.J. Castellan. Nonparametric Statistics for the Behavioral Sciences (2nd ed., McGraw-Hill NJ, 1988). This is a standard test that can be used for 2 x 2 tables, and is conservative in declaring significance it the data are sparse. For analysis of EST sequences, the tables were of the following form: TABLE 4: Fisher Exact Test for Statistical Analysis of Differential Expression Gene X AJI Other Genes Total a = number of sequences A = number of sequences N = a A Pool A in Pool A assigned to in Pool A NOT assigned total number of Gene X to Gene X sequences in Pool A b = number of sequences B = number of sequences M = b + B Pool B in Pool B assigned to in Pool B NOT assigned total number of GeneX toGeneX sequences in PoolB Total c=a b C=A+B N M=c C where Pool A contains the sequences derived from the undifferentiated hES cells and Pool B contains the sequences from the other three cell types (EB, preHep, preNeu). N is equal to the number of sequences derived from the undifferentiated hES cells (37,081) and M is equal to the sum of all ESTs from the three differentiated cell types (111,372). For any given pair of pool sizes (N, M) and gene counts (c and C), the probability p of the table being generated by chance is calculated where: p = [ N! M! c! C! ] / [(N + M)! a! b! A! B!) and where 0! by default is set to 1. The null hypothesis of a gene being equally represented in two pools is rejected when probability p = 0.05, where 0.05 is the level of statistical certainty. Thus, genes with p = 0.05 are considered to be differentially represented.

The following markers were identified as changing their expression levels significantly upon differentiation. The markers identified with the symbol "0" may play a role in the regulation of gene transcription.

TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_10021 NM_018124 hypothetical protein FLJ1O52O (FLJ1O52O) 1 0 3 10 es 4.51 p=0.02 GA_i 0053 NM_033427 cortactin binding protein 2 (CORTBP2) 4 0 0 0 4 es >4 p=0.00 GA_i 0057 ABO51 540 KIAA1 753 protein sequence 4 1 1 0 6 es 6.01 p=0.04 GA_i 0082 NM_030645 KIAA172O protein (KIAA1 720) 6 0 1 0 7 es 18.02 p=0.00 GA_i 01 53 NM_015039 chromosome 1 open reading frame 15 (don 15), 4 1 1 0 6 es 6.01 p=0.04 transcript variant 1 GA_i 02 NM_01 5043 KIAA0676 protein (K1AA0676) 6 4 0 1 11 es 3.60 p=0.03 GA_i 0252 NM_003376 vascular endothelial growth factor (VEGF) 5 2 0 2 9 Os 3.75 p=0.05 GA_i 0258 AK091 948 cDNA FLJ34629 fis, clone KIDNE2O1 551 5, highly 4 0 0 0 4 es >4 p=0.00 similar to NAD P-dependent (eukotriene b4 12-hydroxydehydrogenase (Ed 1.1.1.-) sequence GA_i 0308 NM_024046 hypothetical protein MGC8407 (MGC8407) 4 0 0 0 4 es >4 p=0.00 GA_i 0327 NM_024077 SECIS binding protein 2 (SBP2) 9 2 3 2 16 es 3.86 p=0.0i GA_i 0334 NM_024090 long-chain fatty-acyl elongase (LCE) 5 0 0 2 7 es 7.51 p=0.01 GA_i 0513 NM_033209 Thy-i co-transcribed (L0C94105) 7 2 2 1 12 es 4.20 p=0.01 GA_i 0528 NM_030622 cytochrome P450, subfamily IlS, polypeptide 1 6 0 1 0 7 es 18.02 p=0.O0 (CYP2S1) GA_i 053 NM_001618 ADP-ribosyltransferase (NAD+; poly (ADP-ribose) 25 13 14 9 61 es 2.09 p=0.01 polymerase) (ADPRT) GA_i 0531 NM_01 5271 tripartite motif-containing 2 (TRIM2) 6 2 0 2 10 es 4.51 p=0.02 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_i 0603 NM_025215 pseudouridylate synthase 1 (PUS1) 5 0 2 2 9 es 3.75 p=0.05 GA_10641 NM_025108 hypothetical protein FLJi39O9 (FLJ13909) 6 0 0 1 7 es 18.02 p=0.00 GA_i0649 NM_025082 hypothetical protein FLJ131i1 (FLJ13111) 8 3 0 0 ii es 8.01 p=O.OO GA_i 067 NM_020977 ankyrin 2, neuronal (ANK2), transcript variant 2 4 0 0 0 4 es >4 p=0.00 GA_i 0696 NM_024888 hypothetical protein FLJ11535 (FLJ11535) 5 2 0 0 7 es 7.51 p=0.01 GA_10713 NM_024844 pericentrin i (PCNT1) 8 1 1 0 10 es 12.01 p=0. 00 Ir' GA_i 076 NM_001 659 ADP-ribosylation factor 3 (ARF3) 19 8 5 4 36 es 3.36 p=0.00 GA_i 0831 NM_024619 hypothetical protein FLJ12171 (FLJ12i71) 4 0 1 1 6 es 6.01 p=0.04 GA_i 085 NM_000048 argininosuccinate Jyase (ASL) 6 2 0 0 8 es 9.Oi p=0.00 GA_i 0902 NM_024504 PR domain containing 14 (PRDM14) 12 1 0 0 i3 es 36.04 p=0.00 GA_i 0905 NM_022362 MMS19-like (MET18 homolog, S. cerevisiae) 10 5 4 1 20 es 3.00 p=0.02 (MMS1 9L) GA_10935 NM_032569 cytokine-like nuclear factor n-pac (N-PAC) 8 3 1 1 13 Os 4.81 p=0.Ol GA_ii047 NM_004728 DEAD/H (Asp-Glu-AIa-AsplHis) box polypeptide 21 18 9 3 5 35 es 3.18 p=0.00 (DDX21) GA_i 1103 NM_i 38347 hypothetical protein BC005868 (L0C90233) 4 0 2 0 6 es 6.01 p=0.04 GA_i 119 NM_001 217 carbonic anhydrase Xl (CAl 1) 5 i 2 1 9 es 3.75 p=0.05 GA_11368 NM_032147 hypothetical protein DKFZp434DO127 7 1 0 0 8 es 21.02 p=0.O0 (DKFZP434DO1 27) TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_11398 NM_015471 DKFZP56601646 protein (DC8) 5 1 1 0 7 es 7.51 p0.Oi GA_11528 NM_021633 keich-like protein C3IP1 (C3IP1) 5 1 0 1 7 es7.5i p=0.01 GA_i 1532 NM_024900 PHD protein Jade-i (Jade-i) 6 1 0 2 9 es 6.01 p=0.0i GA_i 1552 NM_024086 hypothetical protein MGC3329 (MGC3329) 6 3 0 1 10 es 4.51 p=0.02 GA_i 1577 AB058780 KIAA1 877 protein sequence 4 2 0 0 6 es 6.01 p=0.04 GA_1i60 NM_052988 cyclin-dependent kinase (CDC2-like) 10 (CDK1U), 4 0 1 1 6 es6.01 p=0.04 transcript variant 3 GA_i 1600 NM_002883 Ran GIPase activating protein 1 (RANGAP1) 12 7 3 5 27 es 2.40 p=0.03 GA_i 1656 NM_01 8425 phosphatidylinositol 4-kinase type II (PI4KII) 5 1 1 2 9 es 3.75 p=0.05 GA_il 773 NM_0251 09 hypothetical protein FLJ22865 (FL.J22865) 6 0 0 0 6 es >4 p=0.00 GA_i 1790 NM_Ol 3432 nuclear factor of kappa light polypeptide gene 5 2 0 0 7 es 7.51 p=0.0i enhancer in B-cells inhibitor-like 2 (NFKBIL2) GA_11868 NM_032844 hypothetical protein FLJI4813 (FLJ14813) 6 2 1 1 10 es 4.51 p=0.02 GA_11893 NM_032805 hypothetical protein FLJ14549 (FLJ14549) 25 0 0 0 25 es >4 p=0.00 GA_11964 NM_032620 mitochondrial GTP binding protein (GTPBG3) 5 1 1 2 9 es 3.75 p=0.05 GA_11971 NM_138575 hypothetical protein MGC5352 (MGC5352) 4 1 1 0 6 es 6.01 p=0.04 GA_i 2025 NM_020465 NDRG family member 4 (NDRG4) 4 1 0 0 5 es 12.01 p=0.02 GA_12064 4 1 0 0 5 esi2.01 p=0.02 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GonBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_1212 NM_001313 collapsin response mediator protein 1 (CRMP1) 7 1 1 2 11 es 5.26 p=0.Oi GA_12167 NM_i 38357 hypothetical protein BCO1 0682 (L0C90550) 4 0 0 0 4 es >4 p=0.00 GA....1217 NM_001316 CSE1 chromosome segregation 1-like (yeast) 23 7 5 2 37 os 4.93 p=O.00 (CSE1L) GA_12173 NM_021912 gamma-aminobutyric acid (GABA) A receptor, beta 4 0 0 0 4 es >4 p=0.00 3 (GABRB3), transcript variant 2 GA_12253 NM_032420 protocadherin 1 (cadherin-like 1)(PCDH1), 5 0 0 2 7 es 7.51 p=0.01 transcript variant 2 GA_i 2279 NM_033019 PCTAIRE protein kinase 1 (PCTK1), transcript 11 7 2 4 24 es 2.54 p=0.03 variant 3 GA_i 2318 NM_032447 fibrillin3 (KIAA1776) 6 0 0 0 6 es >4 p=0.00 GA_1236 NM_003611 oral-facial-digital syndrome 1 (OFDI) 4 0 1 0 5 es 12.01 p=0.02 GA_12367 NM_033317 hypotheticalgeneZD52Flo(ZD52F1O) 8 1 4 4 17 es2.67 p=0.05 GA_i 2386 AB002336 K1AA0338 sequence 4 1 0 0 5 es 12.01 p=0.02 GA_i 2440 NM_032383 Hermansky-Pudlak syndrome 3 (HPS3) 7 1 0 0 8 es 21.02 p=0.00 GA_i 2522 NM_052860 kruppel-like zinc finger protein (ZNF300) 6 2 2 1 11 es 3.60 p=O.03 GA_i 260 NM_000791 dihydrofolate reductase (DHFR) 15 4 2 4 25 es 4.51 p=0.00 GA_i 2630 NM_015356 scribble (SCRIB) 12 4 0 2 18 es 6. 01 p=0.00 GA_12635 NM_002913 replication factor C (activator 1)1, l45kDa (RFC1) 6 0 1 0 9 es 24.03 p=0.00 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST Counts Relative Expression ES EB prelIEP preNeu Total GA_12640 NM_004741 nucleolar and coiled-body phosphoprotein 1 16 9 7 6 38 os 2.18 p=0.02 (NOLC1) GA_i 265 NM_001 387 dihydropyriniidinase-Iike 3 (DPYSL3) 39 13 3 14 69 es 3.90 p=0.00 GAl 2672 D86976 similar to C.elegans protein (Z37093) sequence 5 2 0 1 8 es 5.01 p=0.03 GA_12767 NM_015360 KIAAOO52 protein (KIAAOO52) 8 2 2 1 13 es 4.81 p=0.01 GA_i 2899 8C039246 clone IMAGE:5278517 5 2 1 1 9 es 3.75 p=0.05 GA_i 2900 NM_003302 thyroid hormone receptor interactor 6 (TRIP6) 12 3 3 4 22 es 3.60 p=0.00 GA_i 2949 BC033781 PAX transcription activation domain interacting 4 0 0 1 5 es 12.01 p=0.02 protein 1 like sequence GA_12954 NM_003972 BTAF1 RNA polymerase II, B-TFIID transcription 7 3 2 0 i2 es 4.20 p=0.01 factor-associated, i7OkDa (Moti homolog, S. cerevisiae) (BTAF1) GA_i 322 NM_000142 fibroblast growth factor receptor 3 (achondroplasia, 9 1 5 1 16 es 3.86 p=0.01 thanatophoric dwarfism) (FGFR3), transcript variant GA_i 378 NM_000178 glutathiorie synthetase (GSS) 4 0 1 1 6 es 6.01 p=0.04 GA_i 386 NM_001 517 general transcription factor IlH, polypeptide 4 (52kD 8 1 2 2 13 Os 4.81 p=0.01 subunit) (GTF2H4) C,, GA_i 470 NM_003740 potassium channel, subfamily K, member 5 4 0 0 1 5 es 12.01 p=0.02 (KCNK5) GA_i 523 NM_002442 musashi homolog 1 (Drosophila) (MSI1) 4 1 0 0 5 es 12.01 p=0.02 GA_i 529 NM_172164 nuclear autoantigenic sperm protein (histone-58 7 32 15 112 es 3.23 p=0.00 bInding) (NASP), transcript variant 1 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST Counts Relative Expression ES EB prel-tEP preNeu Total GA_i 634 NM_002647 phosphoinositido-3-kinase, class 3 (PIK3C3) 5 1 1 2 9 es 3.75 p=0.05 GA_i 650 NM_002660 phospholipase C, gamma 1 (tormerly subtype 148) 10 4 4 1 19 es 3.34 p=0.01 (PLCG1) GA_i 662 AF1 951 39 pinin (PNN) gene, complete cds 23 9 7 5 44 es 3.29 p=0.00 GA_1665 NM_002691 polymerase (DNA directed), delta 1, catalytic subunit 9 6 2 1 18 es 3.00 p=0.02 125k0a (POLD1) GA_i 674 NM_002701 POU domain, class 5, transcription factor 1 24 1 2 0 27 es 24.03 p=0.00 (POU5F1) lr' GA_i 696 NM_000947 pnmase, polypeptide 2A, 58kDa (PRIM2A) 4 0 0 1 5 es 12.01 p=0. 02 GA_i 702 NM_002740 protein kinase C, iota (PRKCI) 8 2 2 1 13 Os 4.81 p=0.01 GA_17i BC013923 SimilartoSRY-boxcontaininggene2sequence 12 1 i 3 17 es7.21 p=0.00 GA_i 710 NM_002764 phosphoribosyl pyrophosphate synthetase 1 7 3 2 1 i3 es 3.50 p=0.02 (PRPS1) GA_i 752 NM_i 52881 PTK7 protein tyrosine kinase 7 (P1K7), transcript 15 14 5 3 37 es 2.05 p=0.04 variant 3 GA_i 777 NM_002862 phosphorylase, glycogen; brain (PYGB), nuclear 13 8 1 2 24 es 3.55 p=0.00 gene encoding mitochoncirial protein GA_1794 NM_003610 RAE1 RNA export 1 tiomolog (S. pombe) (RAE1) 5 0 0 2 7 es 7.51 p=O.01 GA_i 814 NM_002907 RecQ protein-like (DNA helicase 01-like) (RECOL), 4 2 0 0 6 es 6.01 p=0.04 transcript variant 1 GA_i820 NM_002916 replication factor C (activator 1)4, 37kDa (RFC4) 6 0 2 2 10 es 4.51 p=0.02 GA_i 865 NM_002949 mitochondrial ribosomal protein L12 (MRPL12), 4 0 0 2 6 es 6.01 p=0.04 nIulRar ncn Rnenclinn mitohnndriM nrntpin TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total nuclear gene encoding mitochondrial protein GA_i 909 NM_003012 secreted frizzled-related protein 1 (SFRPI) 12 8 1 7 28 es 2.25 p=0.05 GA_i 938 NM_003601 SWI/SNF related, matrix associated, actin 19 10 4 5 38 es 3.00 p=0.00 dependent regulator of chromatin, subfamily a, member 5 (SMARCA5) GA_i 942 NM_003076 SWI/SNF related, matrix associated, actin 10 3 3 3 19 es 3.34 p=O.01 dependent regulator of chromatin, subfamily d, member 1 (SMARCD1), transcript variant 1 GA_1962 NM_152826 sorting nexin 1 (SNX1), transcript variant 3 4 0 0 1 5 es 12.01 p=O.02 GA_1963 NM_003100 sorting nexin 2 (SNX2) 8 2 4 1 15 es 3.43 p=0.02 GA_2024 NM_003212 teratocarcinoma-derived growth factor 1 (TDGF1) 20 1 0 0 21 es 60.07 p=0.O0 GA_2031 NM_003234 transferrin receptor (p90, CD71) (TFRC) 13 9 3 4 29 es 2.44 p=O.02 GA_2066 NM_003283 troponin Ti, skeletal, slow (TNNT1) 5 1 1 0 7 es 7.51 p=O.Ol GA_2091 NM_001 069 tubulin, beta polypeptide (TUBB) 40 13 11 17 81 es 2.93 p=0 00 GA_2123 NM_003481 ubiquitin specific protease 5 (isopeptidase T) (USP5) 13 6 5 1 25 es 3.25 p=O.O0 GA_21 49 NM_00341 3 Zic family member 3 heterotaxy 1 (odd-paired 7 0 1 0 8 es 21.02 p=0.00 homolog, Drosophila) (ZIC3) GA_2175 NM_001 605 alanyl-IRNA synthetase (AARS) 23 6 1 3 33 es 6.91 p=0.00 GA_2178 NM_001104 actinln, alpha 3(ACTN3) 6 1 0 0 7 es 18.02 p=0. 00 GA_2234 NM_000107 damage-specific DNA binding protein 2, 48kDa 8 1 0 2 11 Os 8.01 p=0.00 (DOB2 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total (DDB2) GA_2235 NJM_001 356 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 15 13 7 3 1 24 es 3.55 p=0.00 (DDXI 5) GA_2240 NM_O01 384 diptheria toxin resistance protein required for 6 1 2 0 9 es 6.01 p=0.01 diphthamide biosynthesis-like 2 (S. cerevisiae) (DPH2L2) GA...2271 NM_001 533 heterogeneous nuclear ribonucleoprotein L (HNRPL) 10 1 4 5 20 es 3.00 p=0.02 GA_2289 NM_000234 ligase I, DNA, ATP-dependent (LIG1) 10 2 5 3 20 es 3.00 p=0.02 Ic, I0 GA_239 NM_000456 sulfite oxidase (SUOX), nuclear gene encoding 5 1 1 0 7 es 7.51 p=0.01 mitochonddal protetn GA_2323 NM_002164 indoleamine-pyrrole 2,3 dioxygenase (INDO) 6 0 0 0 6 es >4 p=0.00 GA_2334 NM_000216 Kallmann syndrome 1 sequence (KAL1) 5 0 1 0 6 es 15.02 p=0.00 GA_2337 NM_003501 acyl-Coenzyme A oxidase 3, pristanoyl (ACOX3) 4 0 0 1 5 es 12.01 p=0.02 GA_23430 NM_006474 lung type-I cell membrane-associated glycoprotein 5 2 1 0 8 es 5.01 p=0.03 (T1A-2) GA23457 AK055600 cDNA FLJ31 038 fis, clone HSYRA2000159 6 2 0 2 10 es 4.51 p=0.02 sequence GA_23467 AK092578 cDNA FLJ35259 fis, clone PR0ST2004251 4 0 0 0 4 es >4 p0.00 sequence GA_23468 6 2 0 2 10 es 4.51 p=0.02 GA_23476 5 0 2 0 7 es 7.51 p0.01 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_23484 43 0 1 0 44 es129.15 p0.O0 GA_23485 25 1 1 0 27 es 37.54 p0.O0 GA...23486 7 0 0 0 7 es >4 p=0.00 GA_23487 49 0 0 0 49 es >4 p=0.00 GA_23488 9 0 0 0 9 es >4 p=0.00 GA_23489 13 0 0 0 13 es>4 p=0.00 GA23490 12 1 1 0 14 esl8.02 pO.O0 GA_23514 5 1 0 2 8 es5.01 p=0.03 GA_23515 4 0 0 0 4 es>4 p=0O0 GA_23525 8 3 0 0 11 es 8.01 p=0.00 GA_2356 NM...002851 protein tyrosine phosphatase, receptor-type, Z 10 0 0 0 10 es >4 p=0.00 polypeptide 1 (PTPRZ1) GA_2357 NM...001 670 armadillo repeat gene deletes in velocardiofacial 6 0 0 0 6 es >4 p=0.00 syndrome (ARVCF) GA_23572 4 1 1 0 6 es 6.01 p=0.04 GA23577 4 2 0 0 6 es 6.01 p=0. 04 GA_23578 BM454360 AGENCOURT_6402318 NIH_.MGC85cDNA clone 6 0 0 0 6 es >4 p=0.U0 IMAGE:5497491 5' sequence GA23579 4 0 0 0 4 es >4 p=O.00 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID Gen Bank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_23585 8 0 1 1 10 es 12.01 p=0.00 GA_23596 4 0 1 0 5 es 12.01 p=0.02 GA_23612 NM_005762 tripartite motif-containing 28 protein; KRAB- 6 2 1 0 9 es 6.01 p=0.O1 associated protein 1; transcriptional inlermediary factor 1-beta; nuclear corepressor KAP-1 sequence GA_23615 4 1 0 0 5 es 12.01 p=0.02 GA_23634 4 1 0 0 5 es 12.01 p=0.02 GA_2367 NM_003923 forkhead box Hi (FOXH1) 5 0 0 0 5 es >4 p=0.00 GA_23673 5 1 0 0 6 as 15.02 p=O.00 GA_23683 4 1 1 0 6 es 6.01 p=0.04 GA_23981 AK057602cDNA FLJ33040 fis, clone THYMU2000382, weakly 4 0 0 0 4 as >4 p=0.00 similar to 60S RIBOSOMAL PROTEIN L12 GA241 8 NM_00431 7 arsA arsenite transporter, ATP-binding, homolog 1 6 3 1 1 ii es 3.60 p=0.03 (bacterial) (ASNAI) GA_2436 NM_004329 bone morphogenetic protein receptor, type Ia 7 3 1 1 12 es 4.20 p=O.01 (BMPR1A) GA_2442 NM_004335 bone marrow stromal cell antigen 2 (BST2) 13 0 2 3 18 es 7.81 p=0.O0 GA_2443 NM..004336 BUB1 budding uninhibited by benzimidazoles 1 10 5 4 2 21 es 2.73 p0.02 homolog (yeast) (BUB1) GA_2444 NM_004725 BUB3 budding uninhibited by benzlmidazoles 3 12 4 7 4 27 es 2.40 p=0.03 homolog (yeast) (BUB3) TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA2447 NM_004341 carbamoyl-phosphate synthetase 2, aspartate 11 8 2 1 22 es 3.00 p=0.01 transcarbamylase, and dihydroorotase (CAD), nuclear gene encoding mitochondrial protein GA_2467 NM_004804 WD4O protein Ciaol (CIAO1) 8 0 1 2 11 es 8.01 p=0.00 GA_2496 NM_004229 cof actor required for Spi transcriptional activation, 0 7 1 1 2 11 es 5.26 p=0.01 subunit 2, l5OkDa (CRSP2) GA_2501 NM_080598 HLA-B associated transcript 1 (BAT1), transcript 24 13 13 9 59 es 2.06 p=0.01 variant 2 , GA_2621 NM_0041 35 isocitrate dehydrogenase 3 (NADi-) gamma (IDH3G) 5 2 0 1 8 es 5.01 p=0.03 GA_2641 NM_.01 7522 low density lipoprotein receptor-related protein 8, 7 0 0 2 9 es 10.51 p=0.00 apolipoprotein e receptor (LRP8), transcript variant 3 GA_2643 NM...004635 mitogen-activated protein kinase-activated protein 6 0 1 2 9 es 6.01 p=0.01 kinase 3 (MAPKAPK3) GA_2644 NM_004526 MCM2 minichromosome maintenance deficient 2, 23 8 6 4 41 es 3.84 p=0.00 mitotin (S. cerevisiae) (MCM2) GA_2717 NM_004703 rabaptin-5 (RAB5EP) 5 1 1 0 7 es 7.51 p=0.01 GA...2728 NM_0041 68 succinate dehydrogenase complex, subunit A, 5 2 0 2 9 Os 3.75 p=0.05 flavoprotein (Fp) (SDHA), nuclear gene encoding mitochondrial protein GA_2751 NM_004596 small nuclear ribonucleoprotein polypeptide A 11 3 4 5 23 as 2.75 p=O.02 (SNRPA) GA_2762 NM_004819 symplekin; Huntingtin interacting protein I (SPK) 10 5 6 1 22 as 2.50 p=0.04 GA_2784 NM_004818 prp28, U5 snRNP 100 kd protein (U5-100K) 16 14 3 3 36 as 2.40 p=O.01 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_2791 NM_004652 ubiquitin specific protease 9, X chromosome (fat 10 2 2 1 15 as 6.01 p=0.00 facets-like Drosophila) (USP9X), transcript varIant 1 GA_2800 NMjD04629 Fanconi anemia, complementation group G 5 0 2 1 8 es 5.01 p=0.03 (FANCG) GA2840 NM..004960 fusion, derived from t(12:16) malignant liposarcoma 14 2 4 1 21 es 6 01 p=0.O0 (FUS) GA_2857 NM..004987 LIM and senescent cell antigen-like domains 1 5 2 0 1 8 es 5 01 p=0.03 (LIMS1) G&.2868 NM_005006 NADII dehydrogenase (ubiquinone) Fe-S protein 1, 6 1 2 2 11 es 3.60 p=0.03 75k0a (NADH-coerizyme Q reductase) (NDUFS1) GA2889 NM_005032 plastin 3 (1 isoform) (PLS3) 35 18 7 19 79 es 2.39 p=0.00 GA_2897 NM_005044 protein kinase, X-linked (PRKX) 6 3 0 1 10 as 4 51 p=0.02 GA....2898 NM_005049 PWP2 periodic tryptophan protein homolog (yeast) 6 0 1 2 9 es 6.01 p=0.01 (PWP2H) GA2937 NMJ)05207 v-crk sarcoma virus CT1 C) oncogene homolog 6 1 0 0 7 es 18.02 p=0.00 (aviari)-like (CRKL) GA_2945 NM..005232 EphAl (EPHAI) 5 1 1 1 8 es 5.01 p0.03 GA_2962 NM_005314 gastnn-releasing peptide receptor (GIRPR) 4 0 0 0 4 es >4 p=0.00 GA_2984 NM_.005474 histone deacetylase 5 (HDAC5), transcript variant 1 6 4 1 0 11 as 3.60 p=0.03 GA....2988 NM....005397 podocalyxin-like (PODXL) 59 23 5 8 95 es 4.92 p=0.00 GA_3017 NM_000098 camitine palmitoyltrarisferase II (CPT2), nuclear 4 1 1 0 6 as 6.01 p=0.04 gene encoding mitochondrial protein -a TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total C' GA_3024 NM_003902 far upstream element (FUSE) binding protein 1 13 4 6 3 26 es 3.00 p=0.01 (FUBP1) GA_3042 NM_005760 CCAAT-box-binding transcription factor (CBF2) 0 9 2 2 3 16 es 3.86 p=O.01 GA_3055 NM_005864 signal transduction protein (5H3 containing) (EFS2), 6 1 0 1 8 es 9.01 pO.0O transcript variant 1 GA_3112 NM005789 proteasome (prosome, macropain) activator subunit 12 2 6 2 22 es 3.60 p=0.00 3 (PA28 gamma; Ki) (PSME3) GA_31 18 NM_005778 RNA binding motif protein 5 (RBM5) 11 6 4 4 25 es 2.36 p=0.04 Ic GA_3130 NM_005785 hypothetical SBBIO3 protein (SBB1O3) 4 1 0 0 5 es 12.01 p=O.02 GA_31 34 NM_005877 splicing factor 3a, subunit 1, 1 2OkDa (SF3A1) 10 1 4 3 18 es 3.75 p=0.01 GA_31 37 NM005628 solute carrier family 1 (neutral amino acid 23 11 2 13 49 es 2. 66 p=0.0O transporter), member 5 (SLC1A5) GA_3144 NM..005839 serine/arginine repetitive matrix 1 (SRRM1) 16 6 5 8 35 es 2.53 p=0.01 GA_3150 NM....1 39315 TAF6 ANA polymerase II, TATA box binding protein 4 0 0 0 4 es >4 p=0.00 (TBP)-associated factor, 8OkDa (TAF6), transcript variant 2 GA_3175 NM005741 zinc finger protein 263 (ZNF263) 0 7 4 0 1 12 es 4.20 p=0.01 GA_3178 NM_006017 prominin-like 1 (mouse) (PROML1) 7 2 2 0 11 es 5.26 p=0.01 GA_3183 NM006035 CDC42 binding protein kinase beta (DMPK-like) 13 5 0 3 21 es 4.88 p0.O0 (CDC42BPB) GA....3219 NM_005928 milk fat globule-EGF factor 8 protein (MFGE8) 30 11 11 14 66 es 2.50 p=O.00 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_32806 BE568403 601341979F1 NIH_MGC.53cDNA clone 9 2 5 2 18 es3.00 p=0.02 IMAGE:3684283 5 sequence GA_32836 AK055259 cDNA FU30697 fis, clone FCBBF2000815, weakly 4 0 1 1 6 es 6.01 p=0.04 similar to ZYXIN GA_32842 8 3 0 0 11 es 8.01 p=0.00 GA_32860 7 0 0 0 7 es>4 p=O.O0 GA_32868 AK091598 cDNA FLJ34279 fis, clone FEBRA2003833 4 0 0 0 4 es >4 p=0.00 sequence GA...32887 NM.006141 dynein, cytoplasmic, light intermediate polypeptide 2 7 2 0 2 11 es 5.26 p0.01 (DNCLI2) GA_32895 5 4 0 0 9 es 3. 75 p=0.05 GA32908 AL832758 mRNA; cDNA DKFZp686C0927 (from clone 4 0 0 0 4 es >4 p=0.00 DKFZp686CO927) sequence GA..32913 4 0 0 0 4 es>4 p=0.00 GA_32917 4 0 0 0 4 es>4 p=0.00 GA_32926 7 0 0 0 7 es >4 p=0.00 GA_32947 4 0 2 0 6 es 6.01 p=0.04 G&..32979 4 0 0 0 4 es>4 p=O.00 GA_32985 4 0 0 0 4 es >4 p=0.0O GA_3321 NM_006345 chromosome 4 open reading frame 1 (C4orfl) 10 5 4 2 21 es 2.73 p=0.02 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_33423 NM_002537 ornithine decarboxylase antizyme 2 (OAZ2) 18 1 7 3 29 es 4.91 p=0.00 GA_3343 NM_006392 nucleolar protein 5A (56kDa with KKE/D repeat) 16 5 11 5 37 es 2.29 p=0.02 (NOL5A) GA..33455 NM_006047 RNA binding motif protein 12 (RBM12), transcript 17 4 3 4 28 es 4.64 p=0.00 variant 1 GA_33475 NM_004902 RNA-binding region (RNP1, ARM) containing 2 12 2 8 2 24 es 3.00 p=0.01 (RNPC2) GA_33503 NM_018135 mitochondrial ribosomal protein S18A (MRPS18A), 4 1 1 0 6 es 6.01 p=0.04 nuclear gene encoding mitochondrial protein GA_33528 NM_032803 solute carrier family 7 (cationic amino acid 4 0 1 0 5 es 12.01 p=0.02 transporter, y system), member 3 (SLC7A3) GA_33533 BC037428 Unknown (protein for MGC:46327) sequence 7 4 1 1 13 es 3.50 p=0.02 GA_33548 NM_01 5638 chromosome 20 open reading frame 188 7 3 0 1 11 es 5.26 p=0.Ol (C2Oorf 188) GA_33588 AL832967 mRNA; cDNA DKFZp6G6BO82 (from clone 5 0 2 1 8 es 5.01 p=0.03 DKFZp666BO82) sequence GA_33680 NM_01 6089 KRAB-zinc finger protein SZF1 -1 (SZF1) 0 15 0 1 0 16 es 45.05 p=0.00 GA_33684 NM005186 calpain 1, (mu/I) large subunit (CAPN1) 13 8 1 5 27 es 2.79 p=0.01 GA_33691 ALl 17507 mRNA; cDNA DKFZp434F1 935 (from clone 4 1 1 0 6 es 6.01 p=0.04 DKFZp434F1 935); partial cds GA_33704 AL833549 mRNA; cDNA DKFZp686N183 (from clone 4 1 1 0 6 es 6.01 p=0.04 DKFZp686N183) sequence GA_33730 AL832779 mANA; cDNA DKFZp686H157 (from clone 4 0 1 1 6 es 6.01 p=0.04 DKFZp686HI57) sequence TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_33747 NM_032737 lamin 82 (LMNB2) 11 8 3 3 25 es 2.36 p=0.04 GA_33755 NM_033547 hypothetical gene MGC16733 similar to CG12113 5 0 0 1 6 es 15. 02 p=0 00 (MGC1 6733) GA_33772 BF223023 7q27f09.xl NCI_CGAP_GC6cDNA clone 5 0 0 0 5 es >4 p=0.00 IMAGE:3699616 3 sequence GA_33816 NM_Ui 5850 fibroblast growth factor receptor 1 (fms-related 35 12 9 5 61 es 4.04 p=0.00 tyrosine kinase 2, Pfeiffer syndrome) (FGFRI), transcript variant 2 GA_33874 NM_01 7730 hypothetical protein FLJ20259 (FLJ20259) 19 6 4 4 33 es 4.08 p=0.O0 GA_33876 NM_i 48904 oxysterol binding protein-like 9 (OSBPL9), transcript 5 1 0 2 8 es 5.01 p=0.03 variant 1 GA....33877 NM_020796 sema domain, trarismembrane domain (TM), and 16 1 11 4 32 es 3.00 p=O.00 cytoplasmic domain, (semaphorin) GA (SEMA6A) GA_33959 NM_030964 sprouty homolog 4 (Drosophila) (SPRY4) 4 1 0 0 5 es 12.01 p=0. 02 GA_34010 AK000089 cONA FLJ20082 fis, clone C0L03245 8 0 3 0 11 es 8.01 p=0.00 GA_34047 NM_i 70752 chromodomain protein, V chromosome-like (CDVL), 8 1 1 1 11 es 8.01 p=0.00 transcript variant 3 GA_34061 NM_152429 hypothetical protein MGC39320 (MGC39320) 7 1 0 1 9 as 10.51 p=0.00 GA_3407 NM_006328 RNA binding motif protein 14 (RBM14) 16 3 4 3 26 es 4.81 p=0.00 GA_34077 NM_i 33457 likely ortholog of mouse type XXVI collagen 7 0 4 2 13 as 3.50 p=0.02 (COL26A1) GA_34137 NM_020314 esophageal cancer associated protein (MGC16824) 6 1 0 0 7 as 18.02 p=0.00 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression 41.

ES EB preHEP preNeu Total GA_34200 NM_005763 aminoadipate-semialdehyde synthase (AASS) 10 0 0 2 12 as 15.02 p=0.00 GA_34219 NM_O1 8449 ubiquitin associated protein 2 (UBAP2), transcript 6 2 1 0 9 es 6.01 p=0.O1 variant 1 GA_34245 NM_004922 SEC24 related gene family, member C (S. 10 6 0 1 17 es 4.29 p=O.O0 cerevisiae) (SEC24C) GA_34270 NM_i 52758 hypothetical protein FLJ31657 (FLJ31657) 5 2 1 0 8 es 5.01 p=0.03 GA_34280 NM_000702 ATPase, Na+/K+ transporting, alpha 2 (+) 4 0 0 0 4 es >4 p=0.OO polypeptide (ATP1A2) 1<0 GA_34320 NM_006461 sperm associated antigen 5 (SPAG5) 14 6 5 2 27 es 3.23 p=0.OO GA_34322 NM_023926 hypothetical protein FLJ12895 (FLJ 12895) 5 0 1 2 8 es 5.01 p=0.03 GA_3436 NM_018062 hypothetical protein FLJ10335 (FLJ10335) 5 1 3 0 9 es 3.75 p=0.05 GA_34419 NM_002952 ribosomal protein S2 (RPS2) 19 5 11 7 42 es 2.48 p=0.00 GA_34438 NM_006521 transcription factor binding to IGHM enhancer 3 5 2 0 2 9 es 3.75 p=O.05 (TFE3) GA_34480 NM_01 2218 interleukin enhancer binding factor 3, 9OkDa (ILF3), 41 26 13 20 100 es 2.09 p=0.00 transcript variant 1 GA_34503 NM_005762 tripartite motif-containing 28 (TRIM28) 13 6 5 2 29 es 2.44 p=0.02 GA_34505 NM_002065 glutamate-ammonia ligase (glutamine synthase) 21 1 8 2 32 es 5.73 p=0.00 (GLUL) 2 GA_34522 NM_000071 cystathionine-beta-synthase (CBS) 7 2 1 2 12 as 4.20 p=0.0l GA_34539 NM_002880 v-raf-1 murine leukemia viral oncogene homolog 1 14 7 3 0 24 es 4.20 p=0.O0 (RAF1) TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_34563 NM_007192 suppressorofTy 16 homolog (S. cerevisiae) 9 1 1 3 14 es 5.41 p=0 00 (SUPT1 6H) GA_34594 NM_004426 polyhomeotic-like 1 (Drosophila) (PHC1) 6 0 0 0 6 es >4 p=0.0O GA_34606 NM_01 5570 autism susceptibility candidate 2 (AUTS2) 7 0 0 2 9 es 10.51 p=O.00 GA34626 NM_00491 1 protein disulfide isomerase related protein (calcium-5 2 1 1 9 es 3.75 p=0.05 binding protein, intestinal-related) (ERP7O) GA_34655 X74794 P1 Cdc2l protein sequence 34 9 5 4 52 es 5.67 p=0.00 GA_34679 NM_002015 forkhead box O1A (rhabdomyosarcoma) (FOXO1A) 0 4 0 1 1 6 es 6.01 p=0.04 GA_3471 5 NM...002421 matrix metalloproteinase 1 (interstitial collagenase) 5 1 0 2 8 es 5.01 p=0.03 (MMP1) GA_34820 NM_024656 hypothetical protein FU22329 (FLJ22329) 5 1 1 1 8 es 5.01 p=0.03 GA_34875 NM..004459 fetal Alzheimer antigen (FALZ) 5 2 0 2 9 es 3.75 p=0.05 GA_35037 NM_004426 polyhomeotic-like 1 (Drosophila) (PHC1) 0 34 3 2 5 44 es 10.21 p=0.00 GA_35125 NM_005386 neuronatin (NNAT) 5 3 0 1 9 es 3.75 p=O.05 GA_351 41 NM_01 8555 zinc finger protein 331; zinc finger protein 463 0 13 2 5 2 22 es 4.34 p=0.00 (ZNF361) GA_35150 ABO1 4542 K1AA0642 protein sequence 5 1 2 1 9 es 3.75 p0.05 GA_35158 NM_015327 K1AA1089 protein (KIAA1O89) 10 6 2 2 20 es 3.00 p=0.02 GA_3520 NM_005915 MCM6 minichromosome maintenance deficient 6 12 5 5 2 24 es 3.00 p=0.01 (MIS5 homolog, S. pombe) (S. cerevisiae) (MCM6) TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_35206 NM_005678 SNRPN upstream reading frame (SNURF), 20 10 9 9 48 es 2.15 p=0.01 transcript variant 1 GA_35221 NM_020442 KIAA1 885 protein (DKFZP434L1 435) 6 0 0 0 6 es >4 p=0.00 GA_35231 NM_01 4389 proline and glutamic acid rich nuclear protein 14 11 3 1 29 es 2.80 p=0.01 (PELP1) GA_35233 NM.....1 38615 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 30 11 3 4 5 23 es 2.75 p=0.02 (DDX3O), transcript variant 1 GA_35239 NM_01 4633 KIAAO155 gene product (KIAAO155) 5 1 2 0 8 es 5.01 p=O.03 GA_35260 NM_004104 fatty acid synthase (FASN) 6 2 0 1 9 es 6.01 p=0.O1 GA_35393 NM_006861 RAB35, member RAS oncogene family (RAB35) 7 2 2 1 12 es 4.20 p=0.01 GA_35395 NM_024662 hypothetical protein FLJ10774 (FLJ10774) 6 4 0 1 11 es 3.60 p=0.03 GA_35405 12 8 3 1 24 es3.00 p=0.01 GA_35422 NM_02121 1 transposon-derived Busterl transposase-like protein 4 0 0 2 6 es 6.01 p=0.04 (L0C58486) GA_35457 AJ459424 JEMMA protein sequence 7 1 2 1 11 es 5.26 p=0.01 GA_35481 NM_006452 phosphoribosylaminoimidazole carboxylase, 36 14 13 9 72 es 3.00 p=0.00 phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) GA_35495 NM_003472 DEK oncogene (DNA binding) (DEK) 16 3 8 10 37 es 2.29 p=0.02 GA_35547 NM_032202 hypothetical protein KIAA1 109 (KIAA1 109) 4 0 0 2 6 es 6.01 p=0.04 GA_35558 AL831917 hypothetical protein sequence 6 1 1 1 9 es 6.01 p=0.01 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation 0 Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP proNeu Total GA_3559 NM_005629 solute carrier family 6 (neurotransmitter transporter, 5 1 0 1 7 es 7.51 p=0.01 creatine), member 8 (SLC6A8) GA_35606 NM_024586 oxysterol binding protein-like 9 (OSBPL9), transcript 4 1 1 0 6 es 6.01 p=Q.04 variant 6 GA_35607 AB002366 K1AA0368 sequence 8 4 2 3 17 es 2.67 p=0.05 GA..3561 5 NM_000251 mutS homolog 2, colon cancer, nonpolyposis type 1 16 6 6 0 28 es 4.00 p=0.0O (E. coli) (MSH2) GA_35687 NM_033502 transcriptional regulating protein 132 (TReP-132), 0 5 0 0 0 5 es >4 p=0.O0 transcript variant 1 GA_35693 NM...01 4782 armadillo repeat protein ALEX2 (ALEX2) 0 12 8 4 3 27 es 2.40 p=0.03 GA_35762 NM_020765 retinoblastoma-associated factor 600 (RBAF600) 12 4 3 1 20 es 4.51 p=0.00 GA_35833 NM_015878 omithine decarboxylase antizyme inhibitor (OAZIN), 17 8 10 6 41 es 2.13 p=0.02 transcript variant I GA_35852 AK056479 cDNA FLJ31 917 fis, clone NT2RP7004925, weakly 4 2 0 0 6 es 6.01 p=0.04 similar to VASODILATOR-STIMULATED

PHOSPHOPROTEIN

GA_35869 ABO1 1112 KIAAO54O protein sequence 5 2 1 0 8 es 5.01 p=0.03 GA_35905 NM_006640 MLL septin-like fusion (MSF) 28 25 6 6 65 es 2.27 p=O.00

-

GA 35913 NM_018265 hypothetical protein FU1O9O1 (FLJ1O9O1) 5 0 1 1 7 es7.51 p=0.01 GA_3593 NM000270 nucleoside phosphorylase (NP) 5 1 1 1 8 es 5.01 p=O.03 GA...35955 NM_022754 sideroflexin 1 (SFXN1) 5 1 1 0 7 es 7.51 p=0.01 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_35984 NM_01 5340 Ieucyl-tRNA synthetase, mitochondrial (LARS2), 4 0 2 0 6 es 6.01 p=0.04 nuclear gene encoding mitochondrial protein GA_36015 NM_015341 barren homolog (Drosophila) (BRRN1) 9 1 1 2 13 es 6.76 p=0.00 GA_36017 AK074137 FLJOO21O protein sequence 4 0 1 0 5 es 12.01 p=0 02 GA_36019 NM_01 2426 splicing factor 3b, subunit 3, l3QkDa (SF3B3) 11 3 2 3 19 es 4.13 p=0 DO GA_36080 NM_152333 chromosome 14 open reading frame 69 (C14orf69) 14 1 1 3 19 es 8.41 p=0.00 GA_36090 NM_020444 KIAA1191 protein (KIAA1191) 9 7 1 2 19 es 2.70 p=0.03 GA_361 1 NM_00121 1 BUBI budding uninhibited by benzimidazoles 1 13 4 4 4 25 es 3.25 p=0.00 homolog beta (yeast) (BUBI B) G&.36126 NM_004286 GTP binding protein 1 (GTPBP1) 4 1 0 0 5 as 12.01 p=0.02 GA_36127 NM_016121 NY-REN-45 antigen (NY-REN-45) 5 1 2 1 9 es 3.75 p=0.05 GA_36129 NM_018353 hypothetical protein FLJ1 1186 (FU1 1186) 10 0 3 3 16 es 5.01 p=0.0O GA_36133 NM_020428 CTL2 gene (CTL2) 9 6 0 0 15 es 4.51 p=0.OO GA_36137 NM_007363 non-POU domain containing, octamer-binding 39 12 22 14 87 es 2.44 p=0.OO (NONO) GA_36139 NM_004990 methionine-tRNA synthetase (MARS) 11 3 1 0 15 es 8.26 p=0.OO GA_36155 AB020719 KIAAO912 protein sequence 5 1 1 0 7 as 7.51 p=0.Ol GA_36183 NM..016333 serine/arginine repetitive matrix 2 (SRRM2) 23 21 9 1 54 es 2.23 p=0.00 GA_36184 NM...020151 START domain containing 7 (STARD7), transcript 17 6 0 1 24 es 729 p=0 00 vrInt 1 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total variant I GA_36219 NMj52392 hypothetical protein DKFZp564C236 7 1 2 1 Ii es 5.26 p=0.Oi (DKFZp564C236) GA_36221 NM_000966 retinoic acid receptor, gamma (RARG) 0 6 2 0 2 10 es 4.51 p=0.02 GA_36241 NM_01 8031 WO repeat domain 6 (WDR6), transcript variant 1 29 20 11 7 67 es 2.29 p=0.00 GA_36270 NM_003715 vesicle docking protein p115 (VOP) 12 5 4 2 23 es 3.28 p=0.Oi GA_3628 NM_006579 emopamil binding protein (sterol isomerase) (EBP) 7 1 3 0 11 es 5.26 p=0.Ol GA_36307 NM_01 5897 protein inhibitor of activated STAT protein PIASy 5 2 2 0 9 es 3.75 p=0.05 (PIASY) GA_36389 NM_025256 HLA-B associated transcript 8 (BAT8), transcript 11 5 6 2 24 es 2.54 p=0.03 variant NG36/G9a-SPI GA 36450 NM_003051 solute carrier family 16 (monocarboxylic acid 22 7 7 5 41 es 3.48 p=0.00 transporters), member 1 (SLC1 6A1) GA_36474 X87832 NOV 5 4 0 0 9 es 3.75 p=0.05 GA 36491 NM_02461 1 similar to NMDA receptor-regulated gene 2 (mouse) 6 4 0 1 11 es 3.60 p=0.03 (FLJ11896) GA_36526 NM_033557 similar to putative transmembrane protein; homolog 6 3 2 0 11 es 3.60 p=0.03 of yeast Golgi membrane protein Yif 1 p (YlpI p-interacting factor) (L0C90522) GA_36545 ABOI 4600 KIAAO700 protein sequence 8 4 1 3 16 es 3.00 p=0.04 GA_36581 NM_Ui 8071 hypothetical protein FLJ1 0357 (FLJ10357) 6 3 0 0 9 es 6.01 p=0.01 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_36592 AB002363 K1AA0365 sequence 6 1 0 1 8 es 9.01 p=0.O0 GA_36595 NM_024718 hypothetical protein FLJ1O1O1 (FLJ1O1O1) 8 4 2 3 17 es 2.67 p=O.05 GA_36643 NM_003918 glycogenin 2 (GYG2) 5 1 0 0 6 es 15.02 p=0.00 GA_36675 NM_003605 0-linked N-acetylglucosamine (GIcNAc) transferase 9 4 0 1 14 es 5.41 p=0.00 (UDP-N-acetylglucosamine:polypeptide-N-acetyiglucosaminyl transferase) (OGT) GA_36692 NM01 5902 progestin induced protein (DD5) 8 4 1 2 15 es 3.43 p=0.02 GA....36707 NM 021 627 sentrin-specific protease (SENP2) 4 0 1 0 5 es 12.01 p=0.02 GA_36730 AF164609 endogenous retrovirus HERV-K101, complete 5 0 0 0 5 es >4 p=0.00 sequence GA_36734 AF376802 neuroligin 2 sequence 6 3 0 0 9 es 6.01 p=0.01 GA_36771 NM_01 6238 anaphase-promoting complex subunit 7 (ANAPC7) 6 0 1 0 7 es 18.02 p=0.00 GA_36788 NM_0001 41 fibroblast growth factor receptor 2 (bacteria-9 5 1 2 17 es 3.38 p=0.02 expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pleitfer syndrome, Jackson-Weiss syndrome) (FGFR2), transcript variant 1 GA_36798 NM...000071 cystathionine-beta-synthase (CBS) 11 0 1 2 14 es 11.01 p=O.00 GA_36842 NM_006197 pencentriolar material 1 (PCM1) 6 3 1 1 11 es 3.60 p=O.03 GA_36897 NM_006773 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 18 7 3 2 1 13 es 3.50 p=0.02 (Myc-regulated) (DDXI 8) TABLE 5: EST Frequency of Genes that Down-regulate upon DifferentIation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_36933 NM_01 6424 cisplatin resistance-associated overexpressed 19 1 4 7 31 es 4.76 pO.0O protein (LUC7A) GA....36936 NM_149379 Williams Beuren syndrome chromosome region 20C 11 6 4 1 22 es 3.00 p=0.01 (WBSCR2OC), transcript variant 4 GA_36951 NM_005916 MCM7 minichromosome maintenance deficient 7 (S. 19 3 6 11 39 es 2.85 p=0.OO cerevisiae) (MCM7) GA_36957 NM_024642 UDP-N-acetyl-alpha-D-galactosamine:polypeptide 4 0 1 1 6 es 6.01 p=0.04 N-acetylgalactosaminyltransf erase 12 (GaINAc-Ti 2) (GALNT1 2) GA_36964 NG_001 332 T cell receptor alpha delta locus (TCRA/TCRD) on 16 2 0 0 18 as 24.03 p0.00 chromosome 14 GA_36974 AL834155 mRNA;cDNADKFZp76100611 (from clone 4 1 0 1 6 es6.01 p=0.04 DKFZp7610061 1) sequence GA_36977 NM....020927 KIAA1 576 protein (KIA.A1576) 9 2 1 0 12 es 9.01 p=O.00 GA_37071 NM_i 53759 DNA (cytosine-5-)-methyltransferase 3 alpha 9 2 1 1 13 es 6.76 p0.OO (DNMT3A), transcript variant 2 GA_37078 NM_01 4977 apoptotic chromatin condensation inducer in the 10 6 2 2 20 es 3.00 p=O.02 nucleus (ACINUS) GA_37079 NM_032156 EEG1 (EEG1), transcript variant S 7 0 0 0 7 es >4 p=O.OO GA_37094 AL832758 mRNA; cDNA DKFZp686C0927 (from clone 11 1 3 3 18 es 4.72 p=0.00 DKFZp686CO927) sequence GA_37215 NM_019023 hypothetical protein FU10640(FLJ1O64O) 7 1 3 0 11 es 5.26 p0.O1 GA_3723 NM_003750 eukaryotic translation initiation factor 3, subunit 10 30 15 6 17 68 es 2.37 p=0.OO theta, 150/l7OkDa (EIF3S1O) TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_37251 NM_000604 fibroblast growth factor receptor 1 (fms-related 7 1 5 0 13 es 3.50 p=0. 02 tyrosine kinase 2, Pfeiffer syndrome) (FGFR1), transcript variant 1 GA_3730 NM_003751 eukaryotic translation initiation factor 3, subunit 9 13 5 2 3 23 es 3.90 p=0.00 eta, 11 6kDa (E1F3S9) GA_3731 4 NM_0031 69 suppressor of Ty 5 homolog (S. cerevisiae) 14 6 1 1 22 es 5.26 p=0.00 (SUPT5H) GA_37354 NM_01 5726 H326 (H326) 5 1 1 0 7 es 7.51 p=0.Ol GA_37372 NM_024658 importin 4 (FLJ23338) 12 7 0 3 22 es 3.60 p=0.00 I-.4 GA_37389 NM_01 7647 FtsJ homolog 3 (E. coil) (FTSJ3) 13 7 5 1 26 es 3.00 p=O.Ol GA_37391 NM_004938 death-associated protein kinase 1 (DAPK1) 6 0 0 1 7 es 18.02 p=0.00 GA_37399 NM_i 48842 WII(iams-Beuren syndrome chromosome region 16 10 0 1 2 13 es 10.01 p=0.00 (WBSCR16), transcript variant 2 GA_37409 NM_021145 cyclin D binding myb-like transcription factor 1 5 1 0 2 8 es 5.01 p=0.03 (DMTF1) GA_37424 NM_i 52742 hypothetical protein DKFZp547M1O9 6 0 1 2 9 es 6.01 p=0.Ol (DKFZp547M1 09) GA_37431 NM_006034 p53-induced protein (PIG1 1) 7 4 1 0 12 es 4.20 p=0.01 GA_37478 NM_01 4670 basic leucine zipper and W2 domains 1 (BZW1) 24 13 11 9 57 es 2.18 p=0.Ol GA_37504 NM_i 53613 PISC domain containing hypothetical protein 5 1 0 3 9 es 3.75 p=0.05 (L0C254531) GA_37536 AK026970 cDNA: FLJ23317 fis, clone HEP12062, highly similar 5 2 1 0 8 es 5.01 p=0.03 to AFOOBg36syntaxin-168 mRNA TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation g Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_37538 NM_080797 death associated transcription factor 1 (DATF1), 0 6 0 1 0 7 es 18.02 p=0.00 transcript variant 3 GA_37589 AL834216 hypothetical protein sequence 4 0 1 0 5 es 12.01 p=0.02 GA_37595 NM_015062 K1AA0595 protein (K1AA0595) 7 3 0 1 11 es 5.26 p0.O1 GA_37606 NM_01 9012 phosphoinositol 3-phosphate-binding protein-2 4 2 0 0 6 es 6.01 p=0.04 (PEPP2) GA_37707 NM_022574 PERQ amino acid rich, with GYF domain 1 (PERQ1) 4 0 1 0 5 es 12.01 p=O.02 GA_37729 NM_005436 DNA segment on chromosome 10 (unique) 170 8 4 1 3 16 es 3.00 p=0.04 (D1OS 170) GA_37737 NM_003707 RuvB-Iike 1 (E. coli) (RUVBL1) 5 2 0 2 9 es 3.75 p=0.05 GA_37755 NM_015044 golgi associated, gamma adaptin ear containing, 13 5 0 2 20 es 5.56 p0.00 ARF binding protein 2 (GGA2), transcript variant 1 GA_37788 NM_i 33631 roundabout, axon guidance receptor, honiolog 1 7 4 1 0 12 es 4 20 p=0.Ol (Drosophila) (ROBO1), transcript variant 2 GA_37800 NM_032701 hypothetical protein MGC2705 (MGC2705) 4 1 0 1 6 es 6.01 p=0.04 GA_37805 NM_025222 hypothetical protein PR02730 (PR02730) 6 1 3 1 11 es 3.60 p=0.03 GA_37866 NM_I 38927 SON DNA binding protein (SON), transcript variant f 6 3 2 0 11 es 3.60 p=0.03 GA_37877 NM_01 2215 meningioma expressed antIgen 5 (hyaluronidase) 10 4 3 3 20 as 3.00 p=0.O2 (MGEA5) GA_37884 AB032993 KIAA1 167 protein sequence 5 2 1 0 8 es 5.01 p=0.03 GA_37904 NM_000478 alkaline phosphatase, liver/bone/kidney (ALPL) 4 1 1 0 6 es 6.01 p=0.04 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_37914 NM_i 53464 interleukin enhancer binding factor 3, 9OkDa (ILF3), 9 1 1 0 11 es 1352 p=0.0O transcript variant 3 GA_38001 NM_i 52312 hypothetical protein FLJ35207 (FLJ35207) 4 1 0 0 5 es 12.01 p=0.02 GA_38023 NM.015846 methyl-CpG binding domain protein 1 (MBD1), 7 0 1 0 8 es 21.02 p=0.00 transcript variant 1 GA_38029 4 1 0 0 5 es 12.01 p=0.02 GA_38084 NM_015658 DKFZP564C186 protein (DKFZP564C186) 13 5 3 5 26 es 3.00 p=0.Ol GA_3818 NM_006833 COP9 subunit 6 (M0V34 homolog, 34 kD) (COPS6) 8 1 1 6 16 es 3. 00 p004 GA_38225 NM_007152 zinc finger protein 195 (ZNFI95) 4 0 2 0 6 es 6.01 p=0.04 GA_38238 AL133439 mRNA full length insert cDNA clone EUROIMAGE 4 0 2 0 6 es 6.01 p=O.04 GA_38243 BM920378 AGENCOURT_6709352 NIH_MGCJ22cDNA 5 2 1 1 9 es 3.75 p=005 clone IMAGE:5750332 5' sequence GA_3826 NM_006875 pim-2 oncogene (PIM2) 5 0 1 0 6 es 15.02 p=0.00 GA_38266 NM_i 44504 junctional adhesion molecule 1 (JAM1), transcript 18 4 3 8 33 es 3.60 p=0.00 variant 5 GA...38278 NM_01 9852 methyltransferase like 3 (METTL3) 8 0 4 3 15 es 3.43 p=0.02 GA_36283 NM_01 341 1 adenylate kinase 2 (AK2), nuclear gene encoding 16 6 6 3 31 es 3.20 p=0.OO mitochondrial protein, transcript variant AK2B GA_38292 NM_005455 zinc finger protein 265 (ZNF265) 6 2 3 0 11 es 3.60 p=0.03 GA_38304 NM_002394 solute carrier family 3 (activators of dibasic and 4 0 1 0 5 as 12.01 p=O.02 neutral amino acid transport), member 2 (SLC3A2) TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_38370 NM_024923 nucleoporin 210 (NtJP21O) 8 0 2 1 11 es 8.01 p=O.OO GA_38371 NM_01 6003 uveal autoantigen with coiled-coil domains and 5 1 1 2 9 es 3.75 p=0.05 ankynn repeats (UACA) GA_38377 NM_033288 KRAB zinc finger protein KR18 (KR18) 0 5 2 1 0 8 es 5.01 p=0.03 GA_38426 NG_001 332 T cell receptor alpha delta locus (TCRAJTCRD) on 7 1 2 0 10 es 7.01 p=0.00 chromosome 14 GA_38431 NM 021 238 TERA protein (TERA) 26 5 2 8 41 es 5.21 p=0.OO GA_38500 AB040903 KIAA1 470 protein sequence 21 12 7 7 47 es 2.43 p=0.OO GA_3851 NM_006759 tJDP*glucose pyrophosphotylase 2 (UGP2) 17 4 5 2 28 es 4.64 p=0.OO GA_38548 AB033107 K1AA1281 protein sequence 6 2 0 3 11 es 3. 60 p=0.03 GA_3861 NM_006845 kinesin family member 2C (KIF2C) 9 1 4 1 15 es 4.51 p=0.00 GA_38627 AL831836 hypothetical protein sequence 5 1 1 2 9 es 3.75 p=0.0S GA_38635 NM_I 33370 KIAA1 966 protein (K1AA1966) 9 4 4 2 19 es 2.70 p=0.03 GA38666 BC0004O1 splicing factor 3b, subunit 2, 1 45kD sequence 16 9 9 6 40 es 2.00 p=0.04 GA_38677 NM_i 53280 ubiquitin-activating enzyme El (A1S9T and BN75 44 41 10 14 109 es 2.03 p=0.00 temperature sensitivity complementing) (UBE1), transcript variant 2 GA...38691 NM_004550 NADH dehydrogenase (ubiquinone) Fe-S protein 2, 9 1 2 6 18 es 3.00 p=0.02 49k0a (NADH-coenzyme 0 reductase) (NDUFS2) GA_387 AB020648 KIAAO841 protein sequence 4 1 1 0 6 es 6.01 p=0.04 TABLE 5: EST Frequency of Genes that Down-regulate upon Dfferentiaton Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_38786 NM.J 38769 mitochondrial Rho 2 (MIRO-2) 8 0 2 3 13 es 4.81 p=O.O1 GA_38804 NM018249 CDK5 regulatory subunit associated protein 2 5 3 1 0 9 es 3.75 p=0.05 (CDK5RAP2) GA_38826 NM_i 33171 engulfment and cell motility 2 (ced-12 homolog, C. 4 1 0 1 6 es 6.01 p=0.04 elegans) (ELMO2), transcript variant 1 GA_38854 NM_032228 hypothetical protein FLJ22728 (FLJ22728) 5 20 2 9 es 3.75 p=O.05 GA_38867 NM_01 8189 hypothetical protein FLJ1O713 (FLJ1O713) 34 2 6 1 43 es 11.35 p=0.00 GA_3897 NM_007015 chondromodulin I precursor (CHM-I) 4 0 1 0 5 es 12.01 p=0.02 GA_3898 NM_006892 DNP (cytoslne-5-)-methytransterase 3 beta 49 2 3 1 55 es 24.53 p=0.O0 (DNMT3B) GA_3899 NM_i 44733 El B-55kDa-associated protein 5 (El B-AP5), 23 16 6 7 52 es 2.38 p=0.O0 transcript variant 2 GA_3938 NM_006925 splicing factor, arginine/serine-rich 5 (SFRS5) 29 4 24 6 63 es 2.56 p=0.00 GA_3984 NM_0061 14 translocase of outer mitochondrial membrane 40 7 1 2 2 12 es 4.20 p=0.O1 homolog (yeast) (TOMM4O) GA_4038 NM_007223 putative G protein coupled receptor (GPR) 5 2 0 0 7 es 7.51 p=0.0l G&..4059 NM_007221 polyamine-modulated factor 1 (PMF1) 6 2 2 1 11 es 3.60 p=0.03 GA_4148 NM003826 N-ethylmaleimide-sensitive factor attachment 4 1 0 1 6 es 6.01 p=0.04 protein, gamma (NAPG) GA_4176 NM_004448 v-erb-b2 erythroblastic leukemia viral oncogene 15 ii 2 5 33 es 2.50 p=0.01 homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (ERBB2) TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total C.' GA_4247 NMOO1 975 enolase 2, (gamma, neuronal) (ENO2) 5 0 2 0 7 es 7.51 p=0.Ol GA_4251 NM_002528 nth endonuclease Ill-like 1 (E. coli) (NTHL1) 4 0 0 1 5 es 12.01 p=0.02 GA....4253 NM004761 RAB2, member RAS oncogene family-like (RAB2L) 6 3 2 0 11 es 3.60 p=0.03 GA_4255 NM_006929 superkiIer viralicidic activity 2-like (S. cerevisiae) 5 4 0 0 9 es 3.75 p=0.05 (SKI V2L) GA_4258 NM_08091 1 uracil-DNA glycosylase (LJNG), nuclear gene 9 3 6 0 18 es 3.00 p=0.02 encoding mitochondrial protein, transcnpt variant 2 GA_4263 NM_006247 protein phosphatase 5, catalytic subunit (PPP5C) 6 1 3 1 11 es 3.60 p=0.03 GA_4268 NM003852 transcriptional intermediary factor 1 (TIF1) 0 13 4 4 1 22 es 4.34 p=0.00 GA_4295 NM 005255 cyclin G associated kinase (GAK) 6 3 2 0 11 es 3.60 p=0.03 GA.. 4302 NM_005054 RAN binding protein 2-like 1 (RANBP2L1), transcript 4 0 0 1 5 es 12.01 p=0.02 variant 1 GA_4332 NM...01 9900 ATP-binding cassette, sub-family C (CFTRJMRP), 8 3 2 1 14 es 4.00 p=0.01 member 1 (ABCC1), transcript variant 5 GA_4446 NM_002388 MCM3 minichromosome maintenance deficient 3 (S. 38 4 8 7 57 es 6.01 p=0.00 cerevisiae) (MCM3) GA_4478 AK074826 cDNA FU90345 fis, clone NT2RP2002974, highly 0 4 0 0 0 4 es > 4 p=0.0O similar to HOMEOBOX PROTEIN SIX5 sequence GA_4551 NM007375 TAR DNA binding protein (TARDBP) 17 11 4 5 37 es 2.55 p=0.01 GA_4568 NM_01 21 00 aspartyl aminopeptidase (DNPEP) 8 1 1 1 11 es 8.01 p=0.00 GA_458 AF080158 1kB kinase-b sequence 4 0 0 0 4 es >4 p=O.00 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_461 9 NM_O1 2295 caicineurin binding protein 1 (CABIN1) 6 4 1 0 11 es 3.60 p=0.03 G&.4659 NM_i 34434 RAD54B homolog (RAD54B), transcript variant 2 4 0 2 0 6 es 6.01 p=0.04 GA_4689 NM_012470 transportin-SR (TRN-SR) 11 4 3 1 19 es 4.13 p=0.00 GA_4693 NM_012256 zinc finger protein 212 (ZNF212) 5 0 1 2 8 es 5.01 p=0.03 GA_4694 NM_012482 zinc finger protein 281 (ZNF281) 4 0 0 0 4 es >4 p=0.00 GA_4788 NM_016263 Fzrl protein (FZR1) 5 1 0 3 9 es 3.75 p=0.05 IC,' 10) GA_4802 AB033092 KIAA1266 protein sequence 9 4 2 0 15 es 4.51 p=0.00 GA_4973 NM_015503 SH2-B homolog (SH2B) 5 2 1 1 9 es 3.75 p=0.05 GA_5037 AB037847 K1AA1426 protein sequence 6 2 3 0 11 es 3.60 p=0.03 GA_5052 NM_015705 hypothetical protein DJ1O42K1O.2 (DJ1O42K1O.2) 9 2 2 1 14 es 5.41 p=0.00 GA_5301 NM_i 45251 serine/threonine/tyrosine interacting protein (STYX) 4 0 0 0 4 os >4 p=0.OO GA_5391 NM_002968 sal-like 1 (Drosophila) (SALL1) 7 1 1 0 9 es 10.51 p=0.00 GA_5470 NM_002610 pyruvate dehydrogenase kinase, isoenzyme 1 4 0 1 1 6 es 6.01 p=0.04 (PDK1), nuclear gene encoding mitochondrial protein GA_5475 NM_012280 FIsJ homolog 1 (E. coil) (FTSJ1) 6 0 1 0 7 es 18.02 p=0.00 GA_5493 NM_005415 solute carner family 20 (phosphate transporter), 6 1 0 3 10 es 4.51 p=0.02 member 1 (SLC2OA1) x TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_5504 NM_007318 presenilin I (Alzheimer disease 3) (PSEN1), 5 1 1 2 9 es 3.75 p=O.05 transcript variant 1-463 GA_5513 NM_01 4324 alpha-methylacyl-CoA racemase (AMACA) 4 0 1 0 5 es 12.01 p=0.02 GA_5534 NM_01 4316 calcium regulated heat stable protein 1, 24kDa 8 1 3 1 13 es 4.81 p=0.0i (CARHSP1) GA_5620 NM_014516 CCR4-NOT transcription complex, subunit 3 8 5 1 2 16 es 3.00 p=0.04 (CNOT3) GA_5622 NM_014434 NADPH-dependent FMN and FAD containing 5 0 1 0 6 es 15.02 p=0.OO oxidoreductase (NR1) GA_5665 NM_014264 serine/threontne kinase 18 (STK1B) 5 1 1 2 9 es 3.75 p=O.05 GA_5703 NM_i 34264 SOCS box-containing WD protein SWiP-1 (WSB1), 44 29 9 12 94 es 2.64 p=0.00 transcript variant 3 GA_5729 NM_015456 cofactor of BRCA1 (COBRA1) 7 2 2 0 11 es 5.26 p=0.Ol GA_5735 NM_01 5537 DKFZP586J1624 protein (0KFZP586J1624) 4 1 0 1 6 es 6.01 p=0.04 GA_551 1 NM_01 4669 K1AA0095 gene product (K1AA0095) 10 3 4 0 17 es 4.29 p=0.00 GA_5829 NM_014773 KIAAO14I gene product (KIAAO141) 8 1 2 3 14 es 4.00 p=0.Oi G&5836 NM_01 4865 chromosome condensation-related SMC-associated 12 5 4 2 23 Os 3.28 p=0.Ol -protein 1 (KIAAO159) GA_5906 NM_014675 K1AA0445 gene product (K1AA0445) 5 3 1 0 9 as 3.75 p=0.05 GA_591 1 NM_01 4857 KIAAO471 gene product (KIAAO471) 4 0 0 2 6 es 6.01 p=0.04

QO

GA_5954 NM_014871 KIAAO71O gene product (KIAAO71O) 5 2 0 0 7 as 7.51 p=0.Ol TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation 0 Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_5961 NM_014828 chromosome 14 open reading frame 92 (C14orf92) 7 3 0 3 13 es 3.50 p=0.02 GA5981 NM_014921 lectomedin-2 (K1AA0821) 11 5 0 1 17 es 5.51 p0.00 GA_6007 NMJ14962 BTB (POZ) domain containing 3 (BTBD3) 7 0 3 3 13 es 3.50 p=O.02 GA_6011 NM_014963 K1AA0963 protein (K1AA0963) 4 1 0 0 5 es 12.01 p=0.02 GA_6106 NM_015888 hooki protein (HOOK1) 5 0 0 1 6 es 15.02 pO.00 GA_6133 NM_016335 proline dehydrogenase (oxidase) 1 (PRODH), 5 1 2 0 8 es 5.01 p=0.03 nuclear gene encoding mitochondrial protein GA_6139 NM_01 6448 RAreguated nuclear matrix-associated protein 6 1 2 0 9 es 6.01 p=0.01 (RAMP) GA_6232 NM_016223 protein kinase C and casein kinase substrate in 5 1 1 1 6 es 5.01 p=0.03 neurons 3 (PACSIN3) GA_6271 NM_016518 pipecolic acid oxidase (PIPOX) 4 0 0 0 4 es >4 p=0.00 GA_6317 NM_O1 5935 CGI-01 protein (CGI-01) 7 2 1 3 13 es 3.50 p=0.02 GA_638 AB024494 huntingtin interacting protein 3 sequence 4 0 2 0 6 es 6.01 p=0.04 GA_6438 NM_002889 retinoic acid receptor responder (tazarotene 4 0 0 1 5 es 12.01 p=0.02 induced) 2 (RARRES2) GA_6445 NM.01 7424 cat eye syndrome chromosome region, candidate 1 10 2 2 4 18 es 3.75 p=0.Ol (CECR1) GA_6460 NM_017415 kelch-like 3 (Drosophila) (KLHL3) 4 0 0 0 4 es >4 p=0.00 GA_6649 NM_i 48956 Williams Beuren syndrome chromosome region 20A 4 0 0 0 4 es >4 p=0.00 (WBSCR2OA), transcript variant 1 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_6665 NM_01 8077 hypothetical protein FLJ10377 (FLJ10377) 7 0 2 3 12 es 4.20 p=0.Ol GA_6669 NM_018085 importin 9 (FU10402) 12 0 3 3 18 es 6.01 p=0.00 GA_6673 NM_01 8093 hypothetical protein FLJ10439 (FLJ10439) 5 2 0 2 9 es 3.75 p=0.05 GA_6731 NM_018182 hypothetical protein F1110700 (FLJ1O700) 7 0 2 1 10 es 7.01 p=0 00 GA_6742 NM_018198 hypothetical protein FLJ1 0737 (FU10737) 8 4 3 0 15 es 3.43 p=O.02 GA_6760 NM_01 8228 chromosome 14 open reading frame 115 13 1 0 0 14 es 39.05 p=O.00 I8 (Cl4ortll5) GA_6806 NM_01 8303 homolog of yeast Sec5 (SEC5) 5 1 1 1 8 es 5.01 p=0.03 GA_6905 NMO1 7722 hypothetical protein FLJ20244 (FLJ20244) 4 1 0 1 6 es 6.01 p=O.04 GA_6957 NM.017815 chromosome 14 open reading frame 94 (C14orf94) 4 0 0 1 5 es 12.01 p=O.02 GA_6975 NM_01 7840 mitochondrial ribosomat protein L16 (MRPL16), 6 0 2 2 10 es 4.51 p=0.02 nuclear gene encoding mitochondrial protein GAj078 NM_01 51 48 PAS domain containing serine/threonine kinase 5 0 0 0 5 es >4 p=0.00 (PASK) -GA_7155 NM_007098 c!athrin, heavy polypeptide-like 1 (CLTCL1), 4 0 1 0 5 es 12.01 p=O.02 transcript variant 2 GA_7158 NM_01 7489 telomeric repeat binding factor (NIMA-interacting) 1 14 3 2 3 22 es 5.26 p=O.OO (TERF1), transcript variant 1 GA_7170 NM_019013 hypothetical protein FLJ1OI56 (FLJ1O156) 7 1 3 2 13 es 3.50 p0.02 GA_7178 NM_01 9079 hypothetical protein FLJ10884 (FLJ10884) 34 2 4 1 41 es 14.59 p=0.00 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_7334 NM_020347 leucine zipper transcription factor-like 1 (LZTFL1) 0 6 2 1 0 9 es 6.01 p=0.01 GA_7382 AB040878 KIAA1 445 protein sequence 7 1 0 2 10 es 7.01 p=0.00 GA_7542 21 0 4 0 25 es 15.77 p=0.00 GA_7691 D42046 The ha3631 gene product is related to S.cerevisiae 4 1 1 0 6 es 6.01 p=0.04 protein encoded in chromosome VIII.

sequence GA_8100 NM_054013 mannosyl (alpha-i,3-)-glycoprotein beta-1,4-N5 1 1 2 9 es 3.75 p=0 05 acetyiglucosaminyltransferase, isoenzyme B (MGAT4B), transcript variant 2 GA_8103 NM_i 44570 HN1 like (HN1L) 14 2 4 4 24 es 4.20 p=0.00 GA_81 19 NM_C 12266 DnaJ (Hsp4O) homolog, subfamily B, member 5 4 1 0 1 6 es 6.01 p=0.04 (DNAJB5) GA_8152 AK095108 cDNA FLJ37789 fis, clone BRHIP3000081 6 2 1 0 9 es 6.01 p=0.01 sequence GA_82 NM_015545 K1AA0632 protein (KlAA0632) 5 1 1 1 8 es 5.01 p=0.03 GA_8484 AK026658 cDNA: FLJ23005 fis, clone LNG00396, highly similar 4 0 0 0 4 es >4 p=0.00 to AFO55O23clone 24723 mRNA sequence GA_8559 NM_022497 mitochondrial ribosomal protein S25 (MRPS25), 6 1 3 1 11 es 3.60 p=0.03 nuclear gene encoding mitochondrial protein GA_8603 NM_007175 chromosome 8 open reading frame 2 (C8orf2) 7 3 1 1 12 es 4.20 p=0.0l GA_8667 4 0 0 0 4 es >4 p=0.00 GA_6666 Z24725 mitogen inducible gene mig-2 sequence 10 3 0 3 16 es 5.01 p=0.00 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB prel-tEP preNeu Total GA_8730 AK098833 cDNA FLJ25967 fis, clone CBRO1 929 sequence 10 3 2 0 15 es 6.01 p=0.O0 GA_8803 NM_000533 proteolipici protein 1 (Pelizaeus-Merzbacher 6 3 0 0 9 es 6.01 p=0.01 disease, spastic paraplegia 2, uncomplicated) (PLP1) GA_8862 AK091593 cDNA FLJ34274 fis, clone FEBRA2003327 5 0 0 0 5 es >4 p=0.00 sequence GA_9014 6 0 1 1 8 es 9.01 p=0.00 GA_9162 AF31 1912 pancreas tumor-related protein sequence 7 1 0 4 12 Os 4.20 p=O.Ol GA_9163 NM_i 38639 BCL2-Iike 12 (proline rich) (BCL2L12), transcript 8 1 3 0 12 es 6.01 p=0.00 variant 1 GA_9167 AF308602 NOTCH 1 sequence 6 2 1 0 9 es 6.01 p=O.Ol GA_9183 NM....007129 Zic family member 2 (odd-paired homolog, 8 1 1 0 10 es 12.01 p=O.OO Drosophila) (ZIC2) GA_9257 NM_005088 DNA segment on chromosome X and V (unique) 4 1 0 1 6 es 6.01 p=0.04 expressed sequence (DXYS1 55E) GA9338 NM_020436 similar to SALL1 (sal (Drosophila)-like (L0C571 67) 11 2 3 0 16 es 6.61 p=O.00 GA_9365 NM_021 078 GCN5 general control of amino-acid synthesis 5-like 7 1 2 1 11 es 5.26 p=0.01 2 (yeast) (GCN5L2) GA_9384 NM_020997 left-right determination, factor B (LEFTB) 4 0 1 0 5 es 12.01 p=0.02 GA_9388 NM_021 643 GS3955 protein (GS3955) 7 1 0 2 10 es 7.01 p=0 00 GA_9486 NM_007372 RNA helicase-related protein (RNAHP) 12 7 1 6 26 es 2.57 p=0.O2 TABLE 5: EST Frequency of Genes that Down-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA...9571 NM_0221 30 golgi phosphoprotein 3 (coat-protein) (GOLPH3) 6 2 2 1 11 es 3.60 p=0.03 GA_9593 NM_022372 G protein beta subunit-like (GBL) 6 0 1 1 8 es 9.01 p=O.OO GA_96 NM_012297 Ras-GTPase activating protein SH3 domain-binding 19 9 6 8 42 es 2.48 p=0.00 protein 2 (KIAAO66O) GA_9664 NM_Ui 5339 activity-dependent neuroprotector (ADNP) 7 1 2 2 12 es 4.20 p=0.01 GA_9688 NM...022767 hypothetical protein FLJ12484 (FLJ12484) 14 3 1 3 21 es 6.01 p=0.00 GA_9697 NM_022778 hypothetical protein DKFZp434LO1 17 6 2 1 0 9 es 6.01 p=0.01 (DKFZP434LO1 17) GA_9784 NM_021 873 cell division cycle 25B (CDC25B), transcript variant 5 2 0 1 8 es 5.01 p=0.03 GA_9829 BM454622 AGENCOURT_6406365 NIH_MGC_92cDNA clone 6 1 1 0 8 es 9.01 p=0.00 IMAGE:5583082 5 sequence GA_9952 8C003542 Unknown (protein for IMAGE:361 1719) sequence 6 0 1 0 7 es 18.02 p=0.00 GA_9996 NM_00591 1 methionine adenosyltransferase II, alpha (MAT2A) 27 8 9 14 58 es 2.62 p=0.00

C

TABLE 6: EST Frequency of Genes that Up-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_i 0484 AK056774 unnamed protein product sequence 4 153 17 34 208 es 0.06 p=0.00 GA_i 0493 NM_023009 MARCKS-iIke protein (MLP) 6 7 15 32 60 es 0.33 p=0.01 GA_i 071 NM_001641 APEX nuclease (multifunctional DNA repair 5 13 15 12 45 es 0.38 p=0.04 enzyme) 1 (APEX1), transcript variant 1 GA_i 1334 NM_032272 homolog of yeast MAF1 (MAF1) 0 4 7 1 12 es 0.00 p=0.05 GA_11407 NM_015070 K1AA0853 protein (K1AA0853) 0 2 2 8 12 es 0.00 p=0.05 IC) I0 GA_12217 BC009917 Unknown (protein for MGC:2764) sequence 0 7 3 5 15 es 0.00 p=0.03 GA_1222 NM_001901 connective tissue growth factor (CTGF) 2 26 4 14 46 es 0.14 p=0.00 GA_12727 NM..004926 zinc finger protein 36, C3H type-like 1 (ZFP36L1) 3 8 12 22 45 es 0.21 p=0.00 GA_i 336 NM_002024 fragile X mental retardation 1 (FMR1) e 0 3 4 7 14 es 0.00 p=0.03 GA_i 353 NM_002051 GATA binding protein 3 (GATA3) 0 2 8 2 12 es 0.00 p0.05 GA_1403 NM_001530 hypoxia-inducible factor 1, alpha subunit (basic 4 22 5 8 39 as 0.34 p=0.04 helix-loop-helix transcription factor) (HIF1A) GA_i 432 NM_002166 inhibitor of DNA binding 2, dominant negative helix-1 3 17 4 25 es 0.13 p=0.01 loop-helix protein (1D2) GA_i 476 NM_002276 keratin 19 (KRTI9) 1 26 14 38 79 es 0.04 p=O.00 GA_1545 NM_002512 rion-metastatic cells 2, protein (NM23B) expressed 3 6 7 16 32 es 0.31 p=0.04 in (NME2), nuclear gene encoding mitochondriai protein TABLE 6: EST Frequency of Genes that Up-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES ES preHEP preNeu Total GA_i 556 NM_003633 ectodermal-neural cortex (with BTB-like domain) 1 5 2 28 36 Os 0.09 p=0. O0 (ENC1) GA_i 735 NM_002806 proteasome (prosome, macropain) 265 subunit, 1 7 7 8 23 es 0.14 p=0.03 ATPase, 6 (PSMC6) GA_1736 NM_002814 proteasome (prosome, macropain) 26S subunit, 0 4 10 5 19 as 0.00 p=0.0i non-ATPase, 10 (PSMD1O) GA_i 841 NM_000979 ribosomal protein L18 (RPL18) 4 6 36 35 81 es 0.16 p=0.00 GA_i 843 NM_000982 nbosomal protein 121 (RPL21) 1 7 48 42 98 es 0.03 p=O.00 GA_i 850 BC020169 clone IMAGE:3543815, partial cds 0 2 8 11 21 es 0.00 p=0.00 GA_1857 NM_000999 ribosomal protein L38 (RPL38) 1 2 12 10 25 es 0.13 p=0.01 GA_1866 NM_002950 ribophorinl(RPN1) 3 12 10 14 39 es0.25 p=O.01 GA_i 886 NM_O01 009 ribosomal protein S5 (RPS5) 8 14 46 30 98 es 0.27 p=0.00 GA_1977 NM_003134 signal recognition particle l4kDa (homologous Alu 1 4 18 12 35 es 0.09 p=O.00 RNA binding protein) (SRP14) GA_2014 NM_003564 transgelln 2 (TAGLN2) 5 31 8 28 72 es 0.22 p=0.00 GA_2039 NM_003246 thrombospondin 1 (THBS1) 0 3 2 7 12 es 0.00 p=0.05 GA_23018 NM_005336 high density lipoprotein binding protein; vigilin ii 37 17 21 86 es 0.44 p=0.01 sequence GA_23176 2 18 3 7 30 es0.21 p=0.02 GA_23180 ABOO9O1O polyubiquitin UbC, complete cds 7 16 23 26 72 es 0.32 p=0.00 x TABLE 6: EST Frequency of Genes that Up-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total C' GA_23653 NM_003289 tropomyosin 2 (beta) (TPM2) 2 14 7 8 31 as 0.21 p=0.01 GA_23969 0 1 181 20 202 es 0.00 p=0.00 GA_24037 0 1 6 5 12 as 0.00 p=0.05 GA_2524 NM_004415 desmoplakin (DPI, DPII) (DSP) 3 14 5 23 45 es 0.21 p=0.00 GA_2597 NM_i 38610 H2A tiistone family, member Y (l-I2AFY), transcript 1 5 5 14 25 es 0.13 p=0.01 variant 3 GA_2627 NM_004905 anti-oxidant protein 2 (non-selenium glutathione 3 6 11 17 37 es 0.27 p=0.01 peroxidase, acidic calcium-independent phospholipase A2) (AOP2) GA_2702 NM_000942 peptidyiprolyl isomerase B (cyclophilin B) (PPIB) 5 6 7 26 44 as 0.39 p=0.04 GA_2752 NM_0041 75 small nuclear ribonucleoprotein D3 polypeptide 0 1 9 4 14 as 0.00 p=0.03 1 8kDa (SNRPD3) GA_2782 NM_004786 thioredoxin-like, 32kDa (TXNL) 0 4 1 10 15 es 0.00 p=0.03 GA_2808 NM_001 154 annexin A5 (ANXA5) 2 14 4 11 31 as 0.21 p=0.0i GA_2968 BC007090 histidine triad nucleotide-binding protein, clone 0 1 11 9 21 es 0.00 p=0.00 MGC:14708 IMAGE:4250i72, complete cds GA_3016 NM_001 873 carboxypeptidase E (CPE) 1 8 4 9 22 es 0.14 p=0.02 GA_3026 NM_005722 ARP2 actin-related protein 2 tiomolog (yeast) 6 19 7 19 51 es 0.40 p=0.03 (ACTR2) GA_3033 NM_005717 actin related protein 2/3 complex, subunit 5, l6kDa 3 10 8 19 40 es 0.24 p=0.01 (ARPC5) TABLE 6: EST Frequency of Genes that Up-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_3036 NM_i 52862 actin related protein 2/3 complex, subunit 2, 34kDa 1 9 3 7 20 es 0.16 p=0.04 (AIRPC2), transcript variant 1 GA_3126 NM_005620 S100 calcium binding protein All (calgizzarin) 0 1 7 37 45 es 0.00 p=0.00 (S100A1 1) GA_3132 NM_005625 syndecan binding protein (syntenin) (SDCBP) 1 3 10 10 24 es 0. 13 p=0.02 GA_3260 NM_006004 ubiqulnol-cytochrome c reductase hinge protein 1 4 12 5 22 es 0.14 p=0.02 (UQCRH) GA...3283 NM_004484 glypican 3 (GPC3) 1 6 7 12 26 es 0.12 p=0.0l GA_3294 NM_006476 ATP synthase, H+ transporting, mitochondrial FO 0 1 3 11 15 es 0.00 p=0.03 complex, subunit g (ATP5L) GA...33625 NM058179 phosphoserine aminotraristerase (PSA), transcript 2 8 5 14 29 es 0.22 p=0.03 variant 1 GA_33660 BF528488 602043661 Fl NCI_CGAP..Brn67cDNA clone 0 7 7 2 16 es 0.00 p=0.02 IMAGE:41 81 462 5' sequence GA_33787 AL832673 mRNA; cDNA DKFZp313B1O17 (from clone 0 3 4 6 13 as 0.00 p=0.05 OKFZp31 3B1 017) sequence GA_3403 NM_006142 stratum (SFN) 0 2 1 14 17 es 0.00 p=0.01 GA_3431 NM_006294 ubiquinol-cytochrome c reductase binding protein 0 2 9 7 18 es 0.00 p=0.01 (UQCRB) GA_3435 NM_006472 thioredoxin interacting protein (TXNIP) 4 14 16 11 45 es 0.29 p=0.O1 GA_34569 NM_003299 tumor rejection antigen (gp96) 1 (TRA1) 3 9 27 20 59 es 0.16 p=0.00 GA_34776 NM_002273 keratin 8 (KRT8) 9 71 144 156 380 es 0.07 p=0.00 TABLE 6: EST Frequency of Genes that Up-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_3491 2 NM_006367 adenylyl cyclase-associated protein (CAP) 9 24 10 31 74 es 0.42 p=0.01 GA_34930 NMJ300700 annexin Al (ANXA1) 2 12 3 15 32 Os 0.20 p=0.0l GA_35086 NM002128 high-mobility group box 1 (HMGB1) 1 3 8 8 20 es 0.16 p=0.04 GA_35179 NM_001402 eukaryotic translation elongation factor I alpha 1 16 29 43 63 151 es 0.36 p=0.00 (EEF1A1) GA_3530 NM....002539 omithine decarboxylase 1 (ODC1) 1 10 8 9 28 es 0.11 p=0.01 GA_35369 NM...003374 voltage-dependent anion channel 1 (VDAC1) 1 5 6 10 22 es 0.14 p0.02 GA_35434 NM._006094 deleted in liver cancer 1 (DLC1) 0 8 1 5 14 es 0.00 p=0.03 GA_35463 NM_024298 leukocyte receptor cluster (LRC) member 4 0 4 9 8 21 es 0.00 p=0.00 (LENG4) GA3560 NM_003079 SWI/SNF related, matrix associated, actin 2 5 11 11 29 es 0.22 p=0.03 dependent regulator of chromatin, subfamily e, member 1 (SMARCE1) GA_35641 BC029424 similar to weakly similar to glutathione peroxidase 2 1 11 5 3 20 es 0.16 p=0.04 sequence GA_35978 NM_006830 ubiquinol-cytochrome C reductase (6.4kD) subunit 0 1 4 7 12 es 0.00 p=0.05 (UQCR) GA_3617 NM_000391 ceroid-lipofuscinosis, neuronal 2, late infantile 1 4 15 2 22 es 0.14 p=0.02 (Jansky-Bielschowsky disease) (CLN2) GA..36322 NM_00l 554 cysteine-,ich, angiogenic inducer, 61 (CYR61) 0 3 3 7 13 es 0.00 p=0.05 GA_36460 NM_001 300 core promoter element binding protein (COPEB) 0 6 2 7 15 es 0.00 p=0.03 TABLE 6: EST Frequency of Genes that Up-regulate upon Differentiation 0 Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_3652 NM_005556 keratin 7 (KRT7) 0 9 1 14 24 es 0.00 p0.00 GA....36638 NM_002954 ribosomal protein S27a (RPS27A) 3 5 37 35 80 es 0.12 p0.00 GA..36721 NM_005134 protein phosphatase 4, regulatory subunit 1 0 8 2 6 16 es 0.00 p0.02 (PPP4R1) GA_36891 NM_001019 ribosomal protein S15a (RPS15A) 0 2 50 32 84 es 0.00 p=0.00 GA_36932 NM_015338 K1AA0978 protein (K1AA0978) 0 5 3 5 13 es 0.00 p0.05 GA...3707 NM_003816 a disintegrin and metalloproteinase domain 9 0 8 1 3 12 es 0.00 p=0.05 (7% (meltrin gamma) (ADAM9) GA_37238 NM_021 019 myosin, light polypeptide 6, alkali, smooth muscle 0 2 2 12 16 es 0 00 p=0 02 and non-muscle (MYL6), transcript variant 1 GA_37377 NM_000516 GNAS complex locus (GNAS), transcript variant 1 0 12 16 27 38 93 es 0.44 pr0.01 GA_37494 NM_001305 claudin4(CLDN4) 1 2 10 12 25 es0.13 p=0.01 GA_37508 NM_000994 ribosornal protein L32 (RPL32) 2 6 26 35 69 es 0.09 p=0.00 GA....37557 NM.J 52437 hypothetical protein DKFZp761B128 1 7 13 3 24 es 0.13 p=0.02 (DKFZp761 Bi 26) GA_37660 NM_001 749 calpain, small subunit 1 (CAPNS1) 4 7 11 20 42 as 0.32 p=0.02 GA_37689 AK022962 cDNA FU12900 fis, clone NT2RP2004321 0 4 6 2 12 as 0.00 p=O.05 sequence GA_37776 NM.000366 tropomyosin 1 (alpha) (TPM1) 24 46 37 74 151 Os 0.46 p=0.00 GA_3782 NM_003968 ublquitln-activating enzyme E1C (UBA3 homolog, 0 1 5 6 12 es 0.00 p=0.05 yeast) (UBE1C) TABLE 6: EST Frequency of Genes that Up-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_3789 NM006818 ALL1-fused gene from chromosome lq (AF1Q) 0 17 1 11 29 es 0.00 p=0.00 GA._38037 NM_033480 F-box only protein 9 (FBXO9), transcript variant 2 0 4 4 4 12 es 0.00 p=O.05 GA_381 2 NM_006854 KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum 3 12 5 17 37 es 0.27 p=0.01 protein retention receptor 2 (KDELR2) GA_38124 NM_000269 non-metastatic cells 1, protein (NM23A) expressed 1 2 8 13 24 es 0.13 p=0.02 in(NME1) GA_38191 NM_000224 keratin 18(KRT18) 8 46 50 119 223 es0.11 p=000 GA....38341 NM_006931 solute carrier family 2 (facilItated glucose 28 49 45 85 207 es 0.47 p=0.00 transporter), member 3 (SLC2A3) GA_38503 NM_000612 insulin-like growth factor 2 (somatomedin A) (IGF2) 0 17 4 21 42 es 0.00 p=0.00 GA_38528 NM_012062 dynamin 1-like (DNM1L), transcript variant 1 0 5 4 3 12 es 0.00 p=0.05 GA..38545 NM_005801 putative translation initiation factor (SUI1) 1 14 15 19 49 es 0.06 p=0.00 GA_38563 NM_021 005 nuclear receptor subfamily 2, group F, member 2 0 9 8 9 26 as 0.00 p=O.00 (NR2F2) GA..3857 NM_006644 heat shock lO5kD (HSP1O5B) 1 11 3 7 22 es 0.14 p=0.02 GA_38570 NM_0331 50 collagen, type II, alpha 1 (primary osteoarthritis, 0 15 31 5 51 es 0.00 p=0.00 spondyloepiphyseal dysplasia, congenital) (COL2A1), transcript variant 2 C',, GA_38790 NM_001743 calmodulin 2 (phosphorylase kinase, delta) 15 23 36 37 111 es 0.47 p=0.00 (CALM2) GA_38817 NM_013341 hypothetical protein PTDOO4 (P10004) 0 4 5 3 12 es 0.00 p=0.05 TABLE 6: EST Frequency of Genes that Up-regulate upon Differentiation 0 Geron 1D GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total C.' GA_38830 NM006013 ribosomal protein L10 (RPL1O) 12 13 71 81 177 es 0.22 p=0.00 GA_3892 NMj06888 calmodulin 1 (phosphorylase kinase, delta) 1 3 11 9 24 es 0.13 p=0.02 (CALM1) GA_3973 NM_i 44497 A kinase (PRKA) anchor protein (gravin) 12 0 17 1 20 38 es 0. 00 p=0.00 (AKAP12), transcript variant 2 GA_3977 NM_005139 annexin A3 (ANXA3) 0 3 4 10 17 es 0.00 p=0.01 GA_4045 NM_003897 immediate early response 3 (IER3), transcript 1 14 2 4 21 es 0.15 p=0.04 variant short GA_4132 NM_002305 lectin, galactoside-binding, soluble, 1 (galectin 1) 0 5 2 7 14 es 0.00 p=0.03 (LGALS1) GA_4182 NM_001202 bone morphogenetic protein 4 (BMP4), transcript 0 7 6 4 17 es 0.00 p=0.01 variant 1 GA_4395 NM_003145 signal sequence receptor, beta (translocon-6 17 12 14 49 es 0.42 p=0.05 associated protein beta) (SSR2) GA_441 8 NM_004800 transmembrane 9 superfamily member 2 (TM9SF2) 0 7 2 8 17 es 0.00 p=0.01 GA_4615 NMj312286 MORF-relatedgeneX(MRGX) 10 22 16 23 71 esO.49 p=0.04 GA_4640 NM_01 2342 putative transmembrane protein (NMA) 1 8 3 10 22 es 0.14 p=0.02 GA_4914 NM_016282 adenylate kinase 3 like 1 (AK3L1) 0 2 6 4 12 es 0 00 p=0.05 GA_5243 NM_139207 nucleosome assembly protein 1-like 1 (NAP1L1), 7 19 28 25 79 es 0.29 p=O.00 transcript variant 1 GA_5387 NM_002047 glycyl-tRNA synthetase (GAAS) 8 9 34 34 85 es 0.31 p=0.00 GA_5557 NM_01 4211 gamma-aminobutyric acid (GABA) A receptor, pi 1 3 4 13 21 es 0.15 p0. 04 (C,ABRP'l TABLE 6: EST Frequency of Genes that Up-regulate upon Differentiation 0 Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total (GABAP) GA_5730 NM..01 5641 testis derived transcript (3 LIM domains) (TES), 0 2 2 9 13 os 0.00 p=0.05 transcript variant 1 GA5992 NM_01 4899 Rho-related BTB domain containing 3 (RHOBTB3) 0 10 7 13 30 Os 0.00 p=0.00 GA6118 NM_016403 hypothetical protein HSPC148 (HSPC148) 0 2 7 3 12 eso.00 p=0.05 GA6136 NM_016368 myo-inositol 1-phosphate synthase Al (ISYNA1) 1 7 5 16 29 Os 0.11 p=O.00 GA_6165 NM_01 5853 OAF (LOC51 035) 1 5 9 5 20 es 0.16 p=0.04 GA_621 9 NM...01 6139 1 6.7Kd protein (LOC51 142) 1 5 13 14 33 as 0.09 p=0.00 GA_6381 NM_0l 6641 membrane interacting protein of RGS16 (MIR16) 0 2 3 7 12 as 0.00 p=0.05 GA._6388 NM...016145 PTDOO8 protein (PTDOO8) 0 1 2 10 13 Os 0.00 p=0.05 GA_6437 NM_01 6732 RNA binding protein (autoantigenic, hnRNP-2 6 7 12 27 es 0.24 p=0.04 associated with lethal yellow) (RALY), transcript variant 1 GA_6481 NM..01 4360 nerve growth factor receptor (TNFRSF16) 1 4 8 17 30 as 0.10 p=0.00 associated protein 1 (NGFRAP1) GA.j280 NM_020199 HTGN29 protein (HTGN29) 0 6 2 6 14 as 0.00 p=0.03 0Aj286 NMj 72316 Meisi, myeloid ecotropic viral integration site 1 0 4 2 10 16 as 0.00 p=0.02 homolog 2 (mouse) (MEIS2), transcript variant h GA_749 BCO1 5794 Unknown (protein for MGC:8837) sequence 0 4 4 9 17 es 0.00 p=0.01 GA_7520 NM_003486 solute carrier famIly 7 (cationic amino acid 2 20 3 20 45 es 0.14 p=0.0O transporter, y+ system), member 5 (SLC7A5) TABLE 6: EST Frequency of Genes that Up-regulate upon Differentiation Geron ID GenBank ID Name EST counts Relative Expression ES EB preHEP preNeu Total GA_7635 NM_i 70746 selenoprotein H (SELH) 0 1 10 2 13 es 0.00 p=0.05 GA_8275 NM_01 2203 glyoxylate reductase/hydroxypyruvate reductase 0 3 2 12 17 es 0.00 p0.01 (GRHPR) GA_8627 NM_006868 RAB31, member RAS oncogene family (RAB31) 0 5 1 7 13 es 0.00 p0.05 GA_8674 NM_000598 insulin-like growth factor binding protein 3 (IGFBP3) 1 15 4 3 23 es 0.14 p=O.03 GA_8980 NM_005347 heat shock 7OkDa protein 5 (glucose-regulated 10 29 15 30 84 es 0.41 p0.01 protein, 7BkDa) (l-ISPA5) GA_9152 NM..005324 H3 histone, family 3B (H3.3B) (H3F3B) 20 26 57 49 152 es 0.46 p=0.00 GA_91 96 NM_000404 galactosidase, beta 1 (GLB1), transcript variant 0 6 10 7 23 es 0.00 p=0.00 GA_9251 NM_004373 cytochrome c oxidase subunit VIa polypeptide 1 0 3 7 8 18 es 0.00 p=0.01 (COX6A1), nuclear gene encoding mitochondrial protein GA_9266 NM_021 104 ribosomal protein L41 (RPL41) 6 9 70 75 160 es 0.12 p=0.00 GA_9649 NM_014604 Tax interaction protein 1 (TIP-i) 0 8 5 5 18 es 0.00 p=0.Ol GA_9734 NM..022908 hypothetical protein FLJ12442 (FLJ12442) 0 3 2 14 19 es 0.00 p=0.01

I

Exam le 3: Microarray analysis for other differentially expressed Qenes In another series of experiments, the level of gene expression was lested at the mRNA level in rnicroarrays.

Genes were selected from the non-redundant set of gene asser-nblies from the four cDNA libraries described in Example 1, based on their novelty and possible interest as markers. An additional 7,000 sequence-verified clones were obtained from Research Genetics (Huntsville AL) and incorporated into an array with a control set of -200 known housekeeping genes. Each clone was grown overnight in 96-well format and DNA purified using the Qiagen 96-well DNA kit. The DNA templates were PCR amplified in 100 lJL reactions. PCR product was then purified using the ArrayltTM PCR Purification Kit (Tetechem, Sunnyvale CA) according to manufacturer instructions. Product was dried down, resuspended in 50% DMSO and Arraylt1M Microprinting solution (Telechern, Sunnyvale CA) and arrayed onto GAPSIM amino sitane coated slides (Corning Inc., Acton MA) using a GMS 417 Arrayer (Affymetrix, Santa Clara, CA). After printing, slides were humidified and snap heated, baked at 80 for 4 h, then blocked with succinic anhydnde. Total RNA from undifferentiated ES cells, embryoid body cells (EB),

retinoic acid treated (preNeu), and DMSO treated (PreHep) cells S, EB, RA-treated, and DMSO-treated cells (10 pg, 15 pg, and 20 pg for sensitivity) was then reverse transcriptase labeled with Cy3 or Cy5 fluorophores, and competitively hybridized to the microarrays overnight at 42 C in 50% formarnide and Sigma hybridization buffer. Undifferentiated ES RNA was directly and indirectly compared with IRNA from all other cell types.

Experiments were repeated at least 5 times each, and dye reversed. Stratagene Universal Human Reference RNA (Cat. #740000) was used as the indirect comparator. Arrays were washed repeatedly and scanned using a GenepixTM 4000A microarray scanner (Axon Instruments, Fremont CA).

Image processing, data extraction and preliminary quality control were performed using GenePixTM Pro 3.0.6 (Axon Instruments). Quality control calculations involved quantifying overall signal intensities, statistical means and medians of pixel intensities and spot morphologies. Extracted data was further analyzed based on statistical algorithms of signal-to-noise, sensitivity range, and reproducibility.

Data was then loaded into the GeneSpringTM database and analysis prograr-n. Of particular interest were genes that showed reproducible expression differences of 2-fold in either direction, especially when the change occurred upon differentiation to all three differentiated cell types.

The following table lists genes that were identified as being downregulated or upregulated in their expression level upon differentiation into EB, preHEP, or preNEU cells. EST counts are provided from the data generated in the previous example.

TABLE 7: Microarray Analysis -Genes that Decrease Expression upon Differentiation Geron ID GenBank ID Name Fold Change EST Counts RA DMSO ES EB preHep preNeu GA_I 674 NM_002701P0U domain, class 5, transcription factor -3.61 -10.68 24 -1 2 -0 1 (POU5F1) GA_9384 NM_020997 left-right determination, factor B (LEFTB) -4.88 -5.48 4 0 1 0 GA_37788 NM_133631 undabout, axon guidance receptor, -7.93 -2.9 7 4 1 0 homolog 1 GA_12173 NM_021912 gamma-aminobutyric acid (GABA)A -3.37 -2.16 4 0 0 0 receptor, beta 3 (GABRB3) GA_37606 NM_01 9012 phosphoinositol 3-phosphate-binding -2.96 -9.99 4 2 0 0 protein-2 (PEPP2) GA_i 470 NM_003740 potassium channel, subfamily K, member -2.93 -2.47 4 0 0 1 (KCNK5) GA_2937 NM_005207 v-crk sarcoma virus CT1 0 oncogene -2.29 -3.78 6 1 0 0 homolog (avian)-like (CRKL) GA_10513 NM_033209 Thy-i co-transcribed (L0C94105) -2.21 -3.39 7 2 2 1 GA_36957 NM_024642 N-acetylgalactosaminyltransferase 12 -3.24 -5.05 4 0 1 1 (Gal NAc-Ti 2) (GALNT1 2) GA_36420 NM_001064 transketolase (Wemicke-Korsakoff -2.25 -2. 28 14 17 11 17 syndrome) (TKT) GA_i 677 NM_003712 phosphatidic acid phosphatase type 2C -2.46 -2.71 1 0 0 0 (PPAP2C) GA_36793 NM_152295 threonyl-tRNA synthetase (TARS) -2.18 -3.5 8 4 1 6 GA_7151 NM_017488 adducin 2 (beta) (ADD2), transcript -4.21 -2.03 4 2 2 0 variant beta-4 GA_i2053 NM_001986 ets variant gene 4 (E1A enhancer binding -2.76 -2.04 0 1 0 4 protein, E1AF) (ETV4) GA_i 798 NM_000964 retinoic acid receptor, alpha (RARA) -2.76 -3.3 3 2 0 0 GA_5617 NM_014502 nuclear matrix protein NMP200 related to -2.19 -2.33 5 3 4 2 splicing factor PRP19 (NMP200) GA_2753 NM_000582 secreted phosphoprotein 1 (osteopontin) -3.78 -3.32 3 6 2 39 (SPP1) GA_7151 NM_017486 adducin 2 (beta) (ADD2), transcript -3.34 -2.13 4 2 2 0 vanant beta-6a GA_36775 NM_000918 procollagen-proline, thyroid hormone -2.01 -2.65 12 28 10 22 binding protein p55) (P4HB) GA_1086 NM_133436 asparagine synthetase (ASNS), transcript -2.27 -2.53 6 5 3 13 variant i GA_2928 NM_005163 v-akt murine thymoma viral oncogene -2. 79 -3.45 2 10 2 5 homolog 1 (AKT1) GA_33799 NM_003250 thyroid hormone receptor (THRA) -4.28 -4.44 0 2 0 1 GA_37861 NM_021784 forkhead box A2 (FOXA2), transcript -3.56 -2.99 2 0 0 0 variant 1 TABLE 7: Microarray Analysis -Genes that Decrease Expression upon Differentiation Geron ID GenBarik ID Name Fold Change EST Counts RA DMSO ES EB preHep preNeu GA_34109 NM_002026 fibronectiri 1 (FN1), transcript variant 1 -2.91 -2.01 17 166 5 27 GA_38641 NM._004309 Rho GDP dissociation inhibitor (GDI) -2. 72 -2.35 7 8 9 14 alpha (ARHGD1A) GA_33829 NM_002081 gtypican 1 (GPC1) -2.61 -2.32 3 9 4 1 GA_5549 NM_014600 EH-domain containing 3 (EHD3) -2.39 -2.81 1 5 1 1 GA_9269 NM_021074 NADH dehydrogenase (ubiquinone) -2.26 -2.01 0 0 9 6 flavoprotein 2, 24kDa (NDUFV2) GA_2934 NM_0051 80 B lymphoma Mo-MLV insertion region -2.11 -3.24 1 2 0 1 (mouse) (BMI1) GA_3522 NM_00241 5 macrophage migration inhibitory factor -2.04 -2.05 4 2 8 9 (glycosylation-inhibiting factor) (MIF) GA_2465 NM_004364 CCAAT/enhancer binding protein -2.79 -4 0 1 0 0 (C/EBP), alpha (CEBPA) GA_36793 NM_152295 threonyl-tANA synthetase (TARS) -5.34 -2.98 8 4 1 6 GA_9259 NM_005539 inositol polyphosphate-5-phosphatase, -4.37 -6.54 1 0 0 2 4OkDa (INPP5A) GA_2232 NM_001348 death-associated protein kinase 3 -2.9 -3.56 3 3 1 2 (DAPK3) GA_37240 NM_007029 stathmiri-like 2 (STMN2) -4.37 -2.37 0 4 0 1 GA_461 7 NM_012289 Ketch-like ECH-associated protein 1 -11.88 -2.59 2 4 2 2 (KEAP1) GA_38021 NM_0021 11 huntingtin (Huntington disease) (HD) -10.84 -2.16 1 5 0 2 GA_9227 NM_001552 insulin-like growth factor binding protein 4 -6.13 -3.06 5 4 0 2 (IGFBP4) GA_267 NM_007041 arginyltransferase 1 (ATE1) -3.03 -3.22 1 1 0 2 GA_38392 NM_006597 heat shock 7OkDa protein 8 (HSPA8), -8.8 -2.7 39 20 48 62 transcript variant 1 GA_1829 NM_002936 nbonucleaseHl (RNASEH1) -2.81 -2.11 1 0 1 2 GA_9228 NM_001664 ras homolog gene family, member A -3.21 -2.48 11 18 8 17 (ARHA) GA_i 495 NM_002347 lymphocyte antigen 6 complex, locus H -2.33 -2.57 0 0 0 1 (LY6H) GA_3840 NM_006749 solute carrier family 20 (phosphate -5.4 -2.83 0 1 1 3 transporter), member 2 (SLC2OA2) GA_i 045 NM_001 105 activin A receptor, type I (ACVR1) -2.7 -2.37 0 3 1 3 GA_36361 NM_020636 zinc finger protein 275 (ZNF275) -4.09 -2.07 0 0 0 3 TABLE 7: Microarray Analysis -Genes that Decrease Expression upon Differentiation Geron ID GenBarik ID Name Fold Change EST Counts RA DMSO ES EB preHep preNeu GA_2445 NM_004337 chromosome 8 open reading frame 1 -3.02 -2.2 1 0 0 0 (C8orf 1) GA_4652 NM_012228 pilin-like transcription factor (PILB) -2.73 -2.46 0 0 1 0 GAJ 0567 NM_025195 phosphoprotein regulated by mitogenic -4.74 -3.64 0 2 0 pathways (C8FW) GA_9258 NM_005393 plexin B3 (PLXNB3) -3.56 -3.04 0 2 0 0 GA_35992 NM001402 eukaryotic translation elongation factor 1 -5.55 -2.22 419 467 454 428 alpha 1 (EEF1A1) GA 33537 NM_133259 leucine-rich PPR-motif containing -2.47 -3.41 8 7 5 3 (LRPPRC) GA..6367 NM_016354 solute carrier family 21 (organic anion -2.08 -3.26 0 0 0 transporter), member 12 (SLC2 1 Al 2) GA_667 AB028976 mRNA for KIAA1 053 protein, partial cds -7.55 -3.52 0 2 0 2 BQ023180 NCI_CGAPYI6 cDNA clone UI-i -BB1 p. -2.96 -2.1 aui-g-05-0-Ul 3' sequence AA41 9281 Soares ovary tumor NbHOT cDNA clone -3.36 -2.59 IMAGE:755641 3 sequence NM_006604 ret finger protein-like 3 (RFPL3) -2.69 -2.5 NM_012155 echinoderrn microtubule associated -9.82 -6.65 protein like 2 (EML2) NM...000160 glucagon receptor (GCGR) -3.94 -2.18 NM_0031 81 T, brachyury homolog (mouse) (1) -9.15 -2.11 NM 014620 homeo box C4 (HOXC4), transcript -9.54 -2.1 variant 1 NM_005583 lymphoblastic leukemia derived sequence -4.36 -2.79 1 (LYL1) NM_014310 RASD family, member 2 (RASD2) -2.72 -3.13 NMjD12467 tryptase gamma 1 (TPSG1) -2.63 -2.55 NM_000539 rhodopsin (opsin 2, rod pigment) (retinitis -4.84 -5.53 pigmentosa 4, autosomal dominant) (RHO) NM_021076 neurofilament, heavy polypeptide (200kD) -2.03 -2.41 (NEFH) NM_012407 protein kinase C, alpha binding protein -5.44 -2.56 (PRKCABP) NM_000201 intercellular adhesion molecule 1 (CD54), -2.18 -2.06 human rhinovirus receptor ((CAM 1) TABLE 8: Microarray Analysis -Genes that Increase Expression upon Differentiation Geron ID GenBank ID Name Fold Change EST Counts RA DMSO ES EB preHep preNeu GA_1055 NM_001134 alpha-fetoprotein (AFP) 8.02 5.07 0 4 0 0 GA_1055 NM_001134 alpha-fetoprotein (AFP) 6.45 3.71 0 4 0 0 GA_1055 NM_001134 aipha-fetoprotein (AFP) 2.58 2.67 0 4 0 0 GA_1213 NM_001884 cartilage linking protein 1 (CRTL1) 4.57 8.71 3 1 17 3 GA_1476 NM_002276 keratin 19 (KRT19) 2.09 5.21 1 26 14 38 GA_8674 NM_000598 insulin-like growth factorn binding protein 3.16 3.59 1 15 4 3 3 (IGFBP3) GA_3283 NM_004484 glypican 3 (GPC3) 2.6 3.29 1 6 7 12 GA_37735 NM_058178 neuronal pentraxin receptor (NPTXR) 3.77 4.04 1 0 0 1 GA_1280 NM_001957 endothelin receptor type A(EDNRA) 3.05 6.37 2 2 1 0 GA_37308 NM_003068 snail homolog 2 (Drosophila) (SNAI2) 2.24 4.68 4 3 0 0 GA_5909 NM_014851 K1AA0469 gene product 2.77 2.03 3 3 0 1 GA_23450 XM_027313 ATP synthase mitochondrial Fl complex 2.48 3.55 3 1 1 1 assembly factor 1 (ATPAF1), GA_7286 NM_0201 19 likely ortholog of rat zinc-finger antiviral 2.5 3.55 1 0 0 0 protein (ZAP) Example 4: Specificity of expression confirmed by real-time PCR To verify the expression patterns of particular genes of interest at the mRNA level, extracts of undifferentiated hES cells and their differentiated progeny were assayed by real-time PCR. Cells were cultured for 1 week with 0.5% dimethyl sulfoxide (DMSO) or 500 nM retinoic acid (RA). The samples were amplified using sequence-specific primers, and the rate of amplification was correlated with the expression level of each gene in the cell population.

TaqmanTM RT-PCR was performed under the following conditions: 1 x RT Master Mix (ABI), 300 nM for each primer, and 80 nM of probe, and 10 pg to 100 ng of total RNA in nuclease-free water.

The reaction was conducted under default RT-PCR conditions of 48 C hold for 30 miri, 95 C hold for 10 mm, and 40 cycles of 95 C at 15 sec and 60' C hold for 1 mm. ANA was isolated by a guanidinium isothiocyanate method (RNAeasyTM kit, Qiagen) according to manufacturer's instructions, and subsequently DNAse treated (DNAfreeTM kit, Ambion). Gene-specific primers and probes were designed by PrimerExpressTM software (Ver. 1.5, ABI). Probe oligonucleotides were synthesized with the fluorescent indicators 6-carboxyfluorescein (FAM) and 6-carboxy-tetrarnethylrhodamine (TAMRA) at the 5' and 3' ends, respectively. Relative quantitation of gene expression between multiple samples was achieved by normalization against endogenousl8S ribosomal RNA (primer and probe from ABI) using the MC1 method of quantitation (ABI). Fold change in expression level was calculated as 2 The table below shows the results of this analysis. Since the cells have been cultured in RA and DMSO for a short period, they are at the early stages of differentiation, and the difference in expression level is less dramatic than it would be after further differentiation. Of particular interest for following or modulating the differentiation process are markers that show modified expression within the first week of differentiation by more than 2-fold (a), 5-fold (**), 10-fold (***), or 100-fold (****) TABLE 9: Quantitative RT-PCR analysis of gene expression in hESC differentiation Geron ID GenBank ID Name Fold Change

RA DMSO

A. GA_10902 NM_024504 Pr domain containing 14 (PRDM14) ** -1.9 -8.3 GA_11893 NM_032805 Hypothetical protein FLJ14549 -2.3 -10.0 GA_12318 NM_032447 Fibrillin3 GA_1322 NM_000142 Fibroblast growth factor receptor 3 precursor 1.5 2.3 (FGFR-3) * GA 1329 NM_00201 5 Forkhead box ol a (foxol a) * -1.6 -2.9 GA_i 470 NM_003740 Potassium channel subfamily k member 5 (TASK-2) -1.6 1.0 GA_i 674 NM_002701 Octamer-binding transcription factor 3a (OCT-3A) -3.7 -7.7 (OCT-4) ** GA_2024 NM_00321 2 Teratocarcinoma-derived growth factor 1.4.0 -12.5 (CRIPTO) GA_2149 NM_00341 3 Zic family member 3 (ZIC3) ** -1.7 -5.3 GA2334 NM_000216 Kallmann syndrome 1 sequence (KAL1) -1.1 -2.5 GA_23552 BC027972 Glypican2 (cerebroglycan) -1.5 -1.2 GA_2356 NM_002851 Protein tyrosine phosphatase, receptor-type, z -1.7 -3.3 polypeptide 1 (PTPRZ1) * GA_2367 NM_003923 Forkhead box hi (FOXH1) -1.8 -5.6 GA_2436 NM_004329 Bone morphogenetic protein receptor, type Ia -2.4 -2.4 (BMPR1A) (ALK-3) * GA_2442 NM_004335 Bone marrow stromal antigen 2 (BST-2) 1.1 -1.9 GA_2945 NM_005232 Ephrin type-a receptor 1 (EPHA1) -i.3 -1.9 GA_2962 NM_005314 Gastrin-releasing peptide receptor (GAP-A) ** -6.3 -9.1 GA_2988 NM_005397 Podocalyxin-like (PODXL) a -2.6 -4.3 GA_3337 NM_006159 Ne112 (NEL-like protein 2) -1.3 -1. 3 GA_3559 NM_005629 Solute camer family 6, member 8 (SLC6A8) -1.1 -1.1 TABLE 9: Quantitative RT-PCR analysis of gene expression in hESC differentiation Geron ID GenBank ID Name Fold Change

RA OMSO

GA_420 X98834 Zinc finger protein, HSAL2 * -1.4 -2.8 GA_5391 NM_002968 Sal-like 1 (SAul), 1.4 -1.3 GA_6402 NM_016089 Krab-zinc finger protein SZF1 -1 * -1.8 -3.1 GA_9167 AF308602 Notch 1 (Ni) 1.3 1.0 GA_9183 AF1 93855 Zinc finger protein of cerebellum ZIC2 * 1.0 -2.9 GA_9443 NM_004426 Early development regulator 1 (polyhomeotic 1 -1.8 -5.6 homolog) (EDR1) ** B. GA_9384 NM_020997 Left-right determination, factor b (LEFTB) -16.7 -25.0 GA_12173 BCO1 0641 Gamma-aminobutyric acid (GABA) A receptor, -2.8 -5.6 beta 3 ** GA_i 0513 NM_033209 Thy-i co-transcribed *** -12.5 -11.1 GA_i 83i NM_002941 Roundabout, axon guidance receptor, homolog 1 1. 1 1.0 (ROBO1), GA_2753 NM_000582 Secreted phosphoprotein 1 (osteopontin) *** -3.8 -i 0.0 GA_329 19 NM_i 33259 130 kDa leucine-rich protein (LAP 130) -1.9 -1.9 GA_28290 AK055829 FU31 267 (acetylglucosarrnyltransferase-like -2.3 -4.5 protein) * C. GA_28053 T24677 EST <-ioo <-100' GA_26303 NM_I 38815 Hypothetical protein BC018070 -3.2 -10.0 GA_2028 NM_00321 9 Telomerase reverse transcriptase (TEAT) * -2.1 -2.3 ExamDle 5: Selection of markers for monitoring ES cell differentiation Genes that undergo upor down-regulation in expression levels during differentiation are of interest for a variety of different commercial applications, as descnbed earlier. This experiment provides an example in which certain genes were selected as a means to monitor the ability of culture conditions to maintain the undifferentiated cell phenotype -and hence, the pluripotent differentiation capability of the cells.

Particular genes were chosen from those identified as having differential expression patterns, i 0 because they are known or suspected of producing a protein gene product that is expressed at the cell surface, or is secreted. These attributes are helpful, because they allow the condition of the cells to be monitored easily either by antibody staining of the cell surface, or by immunoassay of the culture supernatant. Genes were chosen from the EST database (Groups 1), microarray analysis (Group 2), and other sources (Group 3).

TABLE 10: Additional Genes analyzed by real-time PCR GenBank or Name ID No. Group 1 Bone marrow stromal antigen NM_004335 Podocalyxin- like NM_005397 Rat GPCI glypican-2 (cerebroglycan) TA..541 6486 Potassium channel subfamily k member 5 (TASK-2) NM_003740 Notch 1 protein AF308602 Teratocarcinoma-derived growth factor 1 (Cripto) NM.....00321 2 Nell like / NELL2 (Nel-like protein 2) NM_006159 Gastrin releasing peptide receptor NM_0053 14 Bone morphogenetic protein receptor NM_004329 ABCG2-ABC transporter AY017168 Solute earner family 6, member 8 (SLC6A8) NM_005629 hTERT NM_003219 Oct 3/4 octamer-binding transcription factor 3a (oct-3a) (oct-4) NM_002701 Group 2 Left-right determination factor b (LEFTB) NM_020997 Secreted phosphoprotein 1 (osteopontin) NM000582 Gamma-aminobutyric acid (GABA) A receptor, beta 3 NM_021912 Roundabout, axon guidance receptor, homologue 1 (ROBO1), NM_002941 Glucagon receptor NM_O0l 60 Leucine-rich PPR-motif hum 130 kDa humi 3Oleu 1 3Okd Leu M92439 Thy-i co-transcribed NM_033209 Solute earner family 21 NM_0l 6354 LY6H lymphocyte antigen 6 complex locus H NM.002347 Plexin (PLXNB3) NM... 005393 ICAM NM_000201 Group 3 Rhodopsin NM 000539 Kallmann syndrome 1 sequence (KAL1) NM 000216 Armadillo repeat protein deleted in velo-cardio-facial syndrome 1670 (ARVCF) NM_O0 Ephnn type-a receptor 1 (EPHA1) NM_005232 Figure 1 shows the decrease in expression of the genes in Group I (Upper Panel) and Group II (Lower Panel) in H9 hES cells after culturing for 7 days with PA or DM. Gene expression of rhodopsiri and ICAM was below the limit of detection in differentiated cells. KAL1 and EPHA1 were not tested.

Besides hTERT and Oct 3/4, three other genes were selected as characteristic of the undifferentiated hES cell phenotype. They were Teratocarcinoma-derived growth factor (Cripto), Podocalyxin-like (PODXL), and gastrin-releasing peptide receptor (GRPR).

Figure 2 compares the level of expression of these five genes in hES cells with fully differentiated cells: BJ fibroblasts, BJ fibroblasts transfected to express hTEPT (BJ-51A), and 293 (human embryonic kidney) cells. The level of all markers shown was at least 10-fold higher, and potentially more than 1 02, 1 1 1 0, or 1 06-fold higher in plunpotent stem cells than fully differentiated cells. All five markers retained a detectable level of expression in differentiated cultures of hESC. It is not clear if there is lower level of expression of these markers in differentiated cells, or if the detectable expression derived from the undifferentiated cells in the population. The one exception observed in this experiment was the hTERT transgene, expressed at an elevated level as expected in the BJ-5TA cells.

High-level expression of Cripto, GRPR and PODXL in undifferentiated hES cells reveals interesting aspects of the biology of these cells. Cripto has been implicated in normal mammalian development and tumor growth. Cnpto encodes a glycosylphosphoinositol anchored protein that contains an EGF repeat and a cystelne rich motif, which makes it a member of the EGF-CFC family. It has been demonstrated that Cripto serves as a co receptor for Nodal, which is essential for rnesoderm and endoderm formation in vertebrate development (Yeo et al., Molecular Cell 7:949, 2001). The finding that Cripto is expressed preferentially on undifferentiated hESC suggests that Nodal is an important signaling molecule for stem cells, perhaps to promote survival and/or proliferation.

PODXL encodes for transmembrane sialoprotein that is physically linked to the cytoskeleton.

PODXL is suspected to act as an inhibitor of cell-cell adhesion and has been implicated in the embryonic development of the kidney podocyte. The anti-adhesion properties of PODXL when expressed on undifferentiated hESC may be an important feature related to stem cell migration.

The receptor for gastrin releasing peptide (GAP) is a G-protein coupled receptor that mediates numerous biological effects of Bombesin-like peptides, including regulation of gut acid secretion and satiety. A critical role has also been established for GRP and GRPR in control growth of cultured cells and normal mammalian development. GAP and GRPR may be oncofetal antigens that act as morphogens in normal development and cancer.

Exam le 6: Use of cell markers to modify ES cell culture conditions This example illustrates the utility of the differentially expressed genes identified according to this invention in the evaluation of culture environments suitable for maintaining plunpotent stem cells.

Figure 3 show results of an experiment in which hES cells of the Hi line were maintained for multiple passages in different media. Medium conditioned with feeder cells provides factors effective to allow hES cells to proliferate in culture without differentiating. However, culturing in unconditioned medium leads to loss of the undifferentiated phenotype, with an increasing percentage of the cells showing decreased expression of CD9 (a marker for endothelial cells, fibroblasts, and certain progenitor cells), and the classic hES cell marker SSEA-4.

Figure 4 illustrates the sensitivity of hTERT, Oct 3/4, Cnpto, GRP receptor, and podocalyxin-like protein (measured by real-time PCR assay) as a means of determining the degree of differentiation of the cells. After 4 passages in unconditioned XVlVOTM 10 medium containing 8 ng/mL bFGF, all 5 markers show expression that has been downregulated by about 10-fold. After 8 passages, expression has decreased by 102, or 104-fold.

Figure 5 shows results of an experiment in which the hES cell line Hi was grown on different feeder cell lines: mEF = mouse embryonic fibroblasts; hMSC = human mesenchymal stem cells; UtSMC = human uterine smooth muscle cells; Wl-38 = an established line of human lung fibroblasts. As monitored by RT-PCR assay of Cripto, Oct 3/4, and hTERT, at least under the conditions used in this experiment, the hMSC are better substitutes for mEF feeders than the other cell lines tested.

Figure 6 shows results of an experiment in which different media were tested for their ability to promote growth of hES cells without differentiation. Expression of Podocalyxin-like protein, Cripto, GFP Receptor, and hTERT were measured by RT-PCR. The test media were not preconditioned, but supplemented with the growth factors as follows: TABLE 11: Growth Conditions Tested for Marker Expression Standard conditions: DMEM preconditioned with mEF+ bFGF (8 ng/mL) Condition 3 XVlVOTM 10 + bFGF (8 ng/mL) Condition 4 XVlVOTM 10+ bFGF (40 ng/mL) Condition 5 X.VIVOTM 10 + bFGF (40 ng/mL) + stem cell factor (SCF, 15 ng/mL) Condition 6 X.VIVOTM 10 + bFGF (40 ng/mL) + Flt3 ligand (75 ng/mL) Condition 7 XVlVOTM 10 + bFGF (40 ng/mL) + LIF (100 nglmL) Condition 8 OBSF-60 + bFGF (4Ong/mL) Tho results show that the markers selected to monitor the undifferentiated phenotype showed similar changes in each of these culture conditions. By all criteria, XVIVO 1 DIM supplemented according to Condition 6 was found to be suitable for culturing hES cells without having to be preconditioned. As shown on the right side, when cells were put back into standard conditioned medium after 8 passages in the test conditions, expression of all four markers returned essentially to original levels. This shows that alterations in expression profiles in media Conditions 4 to B are temporary and reversible -consistent with the cells retaining full pluripotency.

Examnle 7: Measuring undifferentiated cell markers by flow cvtomety Cells from the undifferentiated hES cell line Hi were grown in mEF conditioned medium in Matngel coated 6-well plates. Cells were harvested using 3.0 mL of 0.5 mM EDTA and resuspended in PBS containing 5% fetal calf serum and 0.05% NaN3 at a concentration of 5 x 106 cells/mL. For SSEA-4 and TRA1-60 staining, 1 ig of antibody (Chemicon International) was used. Cells were incubated for a period of 30 mm on ice followed by one wash with 2.0 mL of PBS-FCS buffer. Cell pellets were resuspended in 100 jil of tluorochrome conjugated secondary antibody. For intracellular Oct-4 staining, the cells were fixed with 2% PEA (final concentration) for 15 mm at room temperature. After one wash, cells were resuspended in a permeabilization buffer (PBS-FCS plus 90% cold methanol) followed by 15 mm in ice, washed again, and then resuspended the cell pellet in blocking solution (20% goat serum in permeabilization buffer). 0.5 x 106 or 1.0 x 106 permeabilized cells were stained with 1 tg of anti-Oct-4 antibody (Santa Cruz Biotechnology) in 10 xL of blocking solution, incubated on ice for 30 mm. After rewashing, the cells were stained with labeled secondary antibody.

Figure 7 shows that SSEA-4, TRA 1-60 and Oct-4 markers were all strongly expressed on undifferentiated cells under these conditions. Solid areas in each panel indicate background staining observed with the respective isotype-matched controls. In fact, greater than 85% of hES cells expressed all three markers.

Example 8: Measuring differentiated cells usina strornal markers The extent of differentiation can be determined by detecting or measuring markers for undifferentiated cells, in combination with markers for differentiated cells of the type expected in early differentiation cultures -either by antibody staining, or by PCR amplification (TaqmanTM), or by a combination of techniques.

In this example, screening of useful stromal cell markers was done by immunocytochemistry of hES cells cultured in XVIVO 1OTM with bFGF, or medium conditioned using mouse embryonic fibroblasts.

Antibodies were obtained from commercial sources as follows: TABLE 12: Primary Antibody for Measuring Differentiated Cells Marker Vendor Catalog No. STRO-1 RnD Systems MAB 1038 Human Thymus Stroma BD Pharmingen 555825 CD44 BD Pharmingen 5509B8 Pharmingen 555593 CD1O5 (Endoglin) Chemicon MAB21 52 CD1O6 (VCAM-1) BD Pharmingen 555645 Vimentin Sigma V 5255 Figure 8 shows the results of the immunocytochemical analysis. CD44, STRO-1 and Vimentin stain stromal-like cells in the hES cell populations cultured with mEF conditioned medium.

Exam le 8: Sensitivity of the assay for undifferentiated cells Real-time PCR assays were performed using mixtures of undifferentiated hES cells and BJ fibroblasts, to determine the sensitivity of the assay for the presence of differentiated cells.

Freshly harvested cells were combined to a total of 2 x 106 cells in 10% increments of each cell type. Total RNA was isolated (Roche isolation kit), and then treated with DNAse 1 to remove potential DNA contaminants. (Ambion kit). Amplification mixtures were made up in QRT- PCR master mix buffer (P/N 4309169) to a final volume of 25 liL at a concentration of 10 pM forward pnmer, 10 pM reverse primer, 10 pM probe, and -100 ng RNA. Data analysis was performed using the comparative Ct method using 18S rRNA endogenous control. (Other suitable housekeeping genes for standardization can be used instead, such as acidic ribosomal protein, -actin, cyclophilin, G3P dehydrogenase, or f32-microglobulin).

Figure 9 shows the relative change of gene expression measured in mixtures of differentiated (BJ) and undifferentiated hES cells, compared with undifferentiated liES cells alone. These five markers are able to rank 10% changes in the proportion of undifferentiated cells.

SEQUENCE DATA

Sequences Referred to in this Disclosure

SEQ. ID NO: Designation Reference hTERT mRNA sequence GenBank Accession NM_003129 blEAT protein sequence GenBank Accession NM_0031 29 Oct 3/4 rnRNA sequence GenBank Accession NM_002701 Oct 3/4 protein sequence GenBank Accession NM_002701 Cnpto mRNA sequence GenBank Accession NM_003212 Cripto protein sequence GenBank Accession NM_003212 podocalyxin-like protein mRNA sequence GenBank Accession NM_005397 podocalyxrn-like protein amino acid sequence GenBank Accession NM_005397 GAP receptor mRNA sequence GenBank Accession NM_005314 GRP receptor proteins sequence GenBank Accession NM_005314 Primers & probes for real-time PCR assay This disclosure Human telomeric repeats U.S. Patent 5,583,016 1 Geron sequence designation GA_i 2064 This disclosure 2 Geron sequence designation GA_231 76 This disclosure 3 Geron sequence designation GA_23468 This disclosure 4 Geron sequence designation GA_23476 This disclosure Geron sequence designation GA_23484 This disclosure 6 Geron sequence designation GA_23485 This disclosure 7 Geron sequence designation GA_23486 This disclosure 8 Geron sequence designation GA_23487 This disclosure 9 Geron sequence designation GA_23488 This disclosure Geron sequence designation GA_23489 This disclosure ii Geron sequence designation GA_23490 This disclosure 12 Geron sequence designation GA_23514 This disclosure 13 Geron sequence designation GA_23515 This disclosure 14 Geron sequence designation GA_23525 This disclosure Geron sequence designation GA_23572 This disclosure 16 Geron sequence designation GA_23577 This disclosure 17 Geronsequence designation GA_23579 This disclosure

Sequences Referred to in this Disclosure

SEQ. ID NO: Designation Reference 18 Geron sequence designation GA_23585 This disclosure 19 Geron sequence designation GA_23596 "This disclosure Geron sequence designation GA....23615 This disclosure 21 Geron sequence designation GA_23634 This disclosure 22 Geron sequence designation GA_23673 This disclosure 23 Geron sequence designation GA_23683 This disclosure 24 Geron sequence designation GA..23969 Thisdisclosure Geron sequence designation GA_24037 This disclosure 26 Gerori sequence designation GA_32842 This disclosure 27 Geron sequence designation GA_32860 This disclosure 28 Geron sequence designation GA...32895 This disclosure 29 Geron sequence designation GA_32913 This disclosure Geron sequence designation GA_32917 This disclosure 31 Geron sequence designation GA_32926 This disclosure 32 Geron sequence designation GA_32947 This disclosure 33 Geron sequence designation GA_32979 This disclosure 34 Geron sequence designation GA_32985 This disclosure Geron sequence designation GA_35405 This disclosure 36 Geron sequence designation GA_38029 This disclosure 37 Geron sequence designation GAj542 This disclosure 38 Geron sequence designation GA_8667 This disclosure 39 Geron sequence designation GA_9014 This disclosure LOCUS TERT 4015 bp mRNA hnear PRI 31-OCT-2000 DEFINITiON Homo sapiens telornerase reverse transcriptase (TERT), mRNA.

ACCESSION NM_003219 AUTHORS Nakaniura,T.M.. Morin,G8., Chapman,I(.8., Weinrich,S.L., Aridrews,W.H. . Lingner,J. , Harley,C.B. and Cech,T.R.

TITLE Telonierase catalytic subunit homologs from fission yeast and human JOURNAL Science 277 (5328), 955-959 (1997) CDS 56. .3454 LOCUS POU5F1 1158 bp mRNA linear PRI 31-DCT-2000 DEFINITION Homo sapiens POU domain, class 5, transcription factor 1 (POU5F1), mRNA.

ACCESSION NM_002701 AUTHORS Takeda,J. Seino,S. and Bell,G.I.

TITLE Human Oct3 gene family: cDNA sequences, alternative splicing, gene organization, chromosomal location, and expression at low levels in adult tissues JOURNAL Nucleic Acids Res. 20 (17), 4613-4620 (1992) CDS 102. .899 LOCUS TDGFI 2033 bp mRNA linear PRI O5-NOV-2002 DEFINITION Homo sapiens teratocarcinoma-derived growth factor 1 (TDGF1), mRNA.

ACCESSION NM_003212 AUTHORS Dono,R., Montuori,N., Rocchi,M., De Ponti-Zilli,L., Ciccodicola,A.

and Persico,M.G.

TITLE Isolation and characterization of the CRIPTO autosomal gene and its X-linked related sequence JOURNAL Am. J. Hum. Genet. 49 (3), 555-565 (1991) COS 248. .814 LOCUS PODXL 5869 bp niRNA linear PRI O1-NOV-2000 DEFINITION Homo sapiens podocalyxin-like (PODXL), mRNA.

ACCESSION NM_O05397 AUTHORS Kershaw,D.B., Beck,S.G., Wharram,B.L., Wiggins,J.E., Goyal,M., Thomas,P.E. and Wiggins.R.C.

TITLE Molecular cloning and characterization of human podocalyxin-like protein. Orthologous relationship to rabbit PCLP1 and rat podocalyxin JOURNAL J. Biol. Chem. 272 (25), 15708-15714 (1997) CDS 251. .1837 LOCUS GRPR 1726 bp niRNA linear PR! O5-NOV-2002 DEFINITION Homo sapiens gastrin-releasing peptide receptor (GRPR), mRNA.

ACCESSION NM_005314 AUTHORS Xiao,D., Wang,J., Hanipton,L.L. and Weber,H.C.

TITLE The human gastrin-releasing peptide receptor gene structure, its tissue expression and promoter JOURNAL Gene 264 (1), 95- 103 (2001) GUS 399. .1553 * * * * * * * * * * The subject matter provided in this disclosure can be modified as a matter of routine optimization, without departing from the spirit of the invention, or the scope of the appended claims.

SEQUENCE LISTING

<110> Geron Corporation Stanton, Lawrence Ralph, Brandenberger Brunette, Elisa Joseph, Gold 0.

Irving, John Mandalam, Ramkumar 110k, Michael Powell, Sandra Shelton, Dawne <120> Genes that are Up-or IJown-Regulated During Differentiation of Human Embryonic Stem Cells <140> [to be assigned] <141> 2004-03-12 <150> US 10/388,578 <151> 2003-03-13 <160> 39 <170> Custom <210> 1 <211> 769 <212> DNA <213> Homo sapiens <400> 1 catcagtata gagaacgtta gcctgtggag ctgtgaatgt gatggagaca agatttagtg 60 tatagctctg ctacctgcct ggtgttcctt tgagtttctt tatccttaga tttgacagct 120 gagaaatcta ggtggattca tattcgtaat cattgattaa catgcacatt tgggtttgca 180 catttttgtt tatcatacat ttttctccgt tttctattaa agaacatgct ctaggggaac 240 tattaatagc ccaccagtcg ggtaggcagc attcaatcct tctatgcctt ctttcgccac 300 ctgttgaggt ctttcttctg aaacaaagaa gaaatagaca aatcagactt gccctcttgg 360 aaatgtggtc cagatttctc tactcccaag ctccaaaaaa ggcatacatt ggatgggcta 420 gatcaactcc tcctgagagc cataaatccg ccaagagttg ttttccatgt aagggtgtgg 480 tacaatgggg aacgcctgat gttggaggaa agcaggagga ctttagagtg gagttgcatt 540 ctaatctctc tgccgcttca actatgtgac ctggggcaaa tgatataeac tctatgagcc 600 tctttcctta tctttaaaat gaagagaagt aatacctacc ttgtagggct gttgtgagga 660 ttaaatgaag taatgcatac agtgcctaac aaagtattta acatcatatt ttttaaaagc 720 tcatgaaata ttagtttttC ttccttcccC tctttctatt ttctctcct 769 <210> 2 <211> 1683 <212> DNA <213> Homo sapiens <400> 2 ggcctccaag cacctcccgc ctgcccatca tcgatgtggs ccccttggac gttggtgccc 60 cagaccagga attgaataca aaaccaccaa gacctcccgc ctgcccatca tcgatgtggc 120 ccccttggac gttggtgccc cagaccagga attcggcttc gacgttggcc ctgtctgctt 180 cctgtaaact ccctccatcc caacctggct ccctcccacc caaccaactt tccccccaac 240 ccggaaacag acaagcaacc caaactgaac cccctcaaaa gccaaaaaat gggagacaat 300 ttcacatgga ctttggaaaa tatttttttc ctttgcattc atctctcaaa cttagttttt 360 atctttgacc aaccgaacat gaccaaaaac caaaagtgca ttcaacctta ccaaaaaaaa 420 aaaaaaaaaa aaaagaataa ataaataact ttttaaaaaa ggaagcttgg tccacttgct 480 tgaagaccca tgcgggggta agtccctttc tgcccgttgg gcttatgaaa ccccaatgct 540 gccctttctg ctcctttctc cacacccccc ttggggcctc ccctccactc cttcccaaat 600 ctgtctcccc agaagacaca ggaaacaatg tattgtctgc ccagcaatca aaggcaatgc 660 tcaaacaccc aagtggcccc caccctcagc ccgctcctgc ccgcccagca cccccaggcc 720 ctgggggacc tggggttctc agactgccaa agaagccttg ccatctggcg ctcccatggc 780 tcttgcaaca tctccccttc gtttttgagg gggtcatgcc gggggagcca ccagcccctc 840 actgggttcg gaggagagtc aggaagggcc aagcacgaca aagcagaaac atcggatttg 900 gggaacgcgt gtcaatccct tgtgccgcag ggctgggcgg gagagactgt tctgttcctt 960 gtgtaactgt gttgctgaaa gactacctcg ttcttgtctt gatgtgtcac cggggcaact 1020 gcctgggggc ggggatgggg gcagggtgga agcggctccc cattttatac caaaggtgct 1080 acatctatgt gatgggtggg gtggggaggg atcactggt gctatagaaa ttgagatgcc 1140 cccccaggcc agcaaatgtt cctttttgtt caaagtctat ttttattcct tgatattttt 1200 cttttttttt tttttttttt ggggatgggg acttgtgaat ttttctaaag gtgctattta 1260 acatgggagg agagcgtgtg cggctccagc ccagcccgct gctcactttc caccctctct 1320 ccacctgcct ctggcttctc aggcctctgc tctccgacct ctctcctctg aaaccctcct 1380 ccacagctgc agcccatcct cccggctccc tcctagtctg tcctgcgtcc tctgtccccg 1440 ggtttcarag acaacttccc aaagcacaaa gcagtttttc cccctagggg tgggaggaag 1500 caaaagactc tgtacctatt ttgtatgtgt ataataattt gagatgtttt taattatttt 1560 gattgctgga ataaagcatg tggaaatgac ccaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620 aaaaaaaaaa aaaaaaaaaa aaaaoaaaaa aaaaaaaaaa accccaaaaa aaaaaaaagg 1680 ggg 1683 <210> 3 <211> 377 <212> DNA <213> Momo sapiens <400> 3 cgcgtccggg cggctcccgc gctcgcaggg ccgtgccacc tgcccgcccg cccgctcgct 60 cgctcgcccg ccgcgccgcg ctgccgaccg ccagcatgct gccgagagtg ggctgccccg 120 cgctgccgct gccgccgccg ccgctgctgc cgctgctgcc gctgctgctg ctgctactgg 180 gcgcgagtgg cggcggcggc ggggcgcgcg cggaggtgct gttccgctgc ccgccctgca 240 cacccgagcg cctggccgcc tgcgggcccc cgccggttgc gccgcccgcc gcggtggccg 300 cagtggccgg aggcgcccgc atgccatgcg cggagctcgt ccgggagccg ggctgcggct 360 gctgctcggt gtgcgcc 377 <210> 4 <211> 844 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (108). .(109) <223> any nucleotide <220> <221> misc_feature <222> (113). .(115) <223> any nucleotide <400> 4 cccacgcgtc cgcccacgcg tccgggtcgc cctccgtcgt ggtctggcgt gtattccgag 60 csttggtgtc tggcggtttc cgagcgttgg tgtctggcgg tttccganng ttnnngaccg 120 ttggtgtctg gcggtttccg accgttggtg tctggcacgc gccaccctct cttgctttgg 180 ttgcgccatg ccgatgtacc agacaagaag acaagaaaat gatttgagga cagcttcaat 240 cgcggtgtga agaagaaagC agcaaaacga ccactgaaaa caacgccggt ggcaaaatat 300 ccaaagaaag ggtcccaagc ggtacatcgt catagccgga aacagtcaga gccaccagcc 360 aatgatmttt tcaatgctgc gaaagctgcc aaaagtgaca tgcagggatg tccttcctga 420 qatccgtgct atctgcattg aggaaattgg gtgttggatg caaagctaca gcacgtcttt 480 cctcaccgac agctatttaa aatatattgg ttggactctg catgataagc accgagaagt 540 ccgcgtgaag tgcgtgaagg ctctgaaagg gctgtacggt aaccgggacc tgaccgcacg 600 cctggagctc ttcactggcc gcttcaagga ctggatggtt tccatgatcg tggacagaga 660 gtacagtgtg gcagtggagg ccgtcagatt actgatactt atccttaaga acatggaagg 720 ggtgctgatg gacgtggact gtgagagcgt ctaccccatt gtgtaggcgt ctaattgagg 780 cctggcctct gctgtgggtg aatttctgta ctggaaactt ttctaccctg agtgcgagat 840 aaga 844 <210> 5 <211> 3357 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (1554).. (1554) <223> any nucleotide <220> <221> misc_feature <222> (1789)..(1789) <223> any nucleotide <220> <221> misc_feature <222> (1794). .(1794) <223> any nucleotide <220> <221> misc_feature <222> (2053). .(2053) <223> any nucleotide <400> 5 ggccccctgt ggtgcccaac cccatacact cttttgtcct saataccttc ctycacwact 60 cactattccg tgcytgatct taaagatgct tttttcacta ttcccctgca yccctcrtyc 120 cagcctctcy ttgctttcac ttrgactgac cckgrcaccc attaggctca gcaaattacc 180 aaggctgtac tgccrCaagg cttcayagac agcccccatt acttcagtca agcccaaatt 240 tcatcctcat ctgttaccta tytcggcata attctcmtaa aaacacacrt gctttccctg 300 ctgatcgtgt ccgattaatc tcccaaacct caatccctta caaaacaaca actcctttcc 360 ttcctaggca tggttmgtgc ggtcagaatt cttaniacaag agccaggact gaaccctgta 420 gcctttctgt ccaaacaact tgaccttact gttttagcct agccctcagg tctgcgtaca 480 gaggctgccg ctgctttaat acttttagag gccctaaaaa tcacaaacta cgctcaactc 540 actctctaca tttctcataa cttccaaaat ctattttctt cctcatacct gacgcatata 600 ctttctgctc cccggctcct tcagctgtac tcactctttc ttaagtccca caattaccgt 660 tgttcctggc cgggacttca atctggcctc ccacattatt cctgatacca cacctgaccc 720 ccacgattgt atctctctga tccacctgat attcacccca tttccccata tttccttctt 780 tcctgttcct caccctgatc acacttgatt tattgatggc agttccacca ggcctaatcg 840 ccacatacca gcaaaggcag gctatgctat agtacaagcc actagcccgc ctctcagaac 900 ctctcatttc ctttccatca tggaaatcta tcctcaagga aataacttcc cagtgttcca 960 tctgctattc tactactcct cagggattat tcaggccccc tcccttccct acacatcaag 1020 ctcraggatt tgcccccacc caggactggc aaaytagctt tactcaacat gcctgagtca 1080 ggaaactaaa atacctctta gtctaaatag acactttcac tgaataagta aaggcctttc 1140 ctacagggtc tgagaaggcc tccgcagtca tttcttccat tctgtcagac ataattcctc 1200 agtttagcct tcccacctca atacagtctg ataacagatg agcctttatt agtcaaatca 1260 gccaagcagt ttttcaggct cttagtattc agtgaaacct ttatatccct tacrgtcctc 1320 crtcttcaag aaargtagaa tggactraag gtcttttaaa aacacacctc accaagctca 1380 gccaccaaaa aggactggac aatactttta ycactttccc ttctcagaat tcaggcctgt 1440 cctcggaatg ctacarggta cagcccattt aagctcctgt atagaygctc ctttttatta 1500 ggccccagtc tcattccaga caccrgacca acttagactg tgcccccaaa aaancttgtc 1560 atccctacta tyttctgtct agtcatactc ctattywccr ttctcaacta ctcatacatg 1620 ccctgctctt gtttacactg ccggtttaca ctgtttytcc aagccatcac agctgatatc 1680 tcctggtgct atccccaaac ygccactctt aactcttgaa gtaaataaat aatctttgct 1740 ggcaggacta tgctgaatct ccttaggcac tctctaatca gatrtcctng gtcntcccaa 1800 ttcttagacc ttttatacct gtttttctcc ttctgttatt ccatttagtt tytcaattca 1860 tmcaaaaccg tatccaggcc atcaccaatc attctatacr acaaatgttt cttctaacaw 1920 ccccacaata tcacccctta ccacaagacc tcccttcagc ttaatctctc ccactctagg 1980 ttcccacgcc gcccctaatc ccgcttgaag cagccctgag aaacatcgcc cattctctct 2040 ccataccacc ccncaaaaat tttcgccgcc ccaacacttc aacactattt tgttttattt 2100 ttcttattaa tataagaagg caggaatgtc aggcctctga gcccaagcca agccatcgca 2160 tcccctgtga cttgcacgta taygcccaga tggcctgaag taactraaga atcacaaaag 2220 aagtgaatat gccctgcccc accttaactg atgacattcc accacaaaag aagtgtaaat 2280 ggccrgtcct tgccttaast gatgacatta ccttgtgaaa gtccttttcc tggctcatcc 2340 tggctcaaaa agcaccccca ctgagcacct tgcgaccccc actcctrccc gccagagaac 2400 aaacccCctt tgactgtaat tttcctttac ctacccaaat cctataaaac ggccccaccc 2460 ttatctccct tcgctgactc tcttttcgga ctcagcccgc ctgcacccag gtgaaataaa 2520 cagccttgtt gctcacacaa agcctgtttg gtggtctctt cacacagacg cgcatgaaag 2580 ggaagacata caaaaacaag gtaaataagt aaactacgtt atatgtttga taatggtgat 2640 gttaagggtg gggaaagaag aaagcaaaga aggataagaa atgggagggg gcaattctag 2700 aaaccatagt cagggaagac ctcactgaga aggtgacatt tgagttatac ctgagagatg 2760 tgagtatctg agggaaagat attccaggaa gggcaaacgt taagtgcaaa ggcactgagt 2820 gggagtgtgc ctggcaggtt caatctattg aaccatgaca ctggggaggg atggtggcta 2880 ctcttggctt tgctggctgg ccactggtga atgagagacg taataaagca ttcaaattaa 2940 agatattaat gcctagtctt caggcactta gacatctgat gtggagtctg aagttgcagt 3000 aacttgagag aagaccatac ataactggat agatgcatag atagataaat ggatgaatgg 3060 aattgcctta tggccatact gagacacagc aaagccaact cgaatcacgc acggggtacc 3120 atggcatagg ggaaagcact ctatgtcatc tcagcaacac agctgtgtgc ctgggataag 3180 tttccttcCg gagctttcat tcttccacag acaagataag aataacatcC ttaagtggtt 3240 ggtacaccac aggttaaatg ttcaatgttt gttatatgcc aggctacgtg tattaatacg 3300 aatttactta atccttacag gcctctgagg taggtactac tgagacagcc aggtggg 3357 <210> 6 <211> 1252 <212> DNA <213> Homo sapiens <400> 6 tcaatcccct gtcctcctgc tctttgctcc atgagaaaga tccacctacg acctcgggtc 60 ctcagaccga ccagcccaag aaacatctca ccaatttcaa atccggtata tgcccagatg 120 gcctgaagta actgaagaat cacaaaagaa gtgaatatgc tttgtcccac cttaactgat 180 gacattccat cacaaaagaa gtgtaaatgg ccggtccttg ccttaactga tgacattacc 240 ttgtgaaagt ccttttcctg gctcatcctg gctcaaaaag cacccccact gagcaccttg 300 tgacccccac tcctgcccac tgagcacctt gcgaccccca ctcctaccca ccagaaaaca 360 aacccccttt gactgtaatt ttcctttacc twcccaaatc ctataaaacg gccccaccct 420 tatctccgtt tgctgactct tttcggactc agcccgcctg cacccaggtg aaataaacag 480 cctcgttgct cacacaaagc ctgtttggtg gtctcttcac acggacgcgc atgaaatttg 540 gtgccgtgac tcggatcggg ggacctccct tgggagatca atcccctgtc ctcctgctct 600 ttgctccgtg agdaagatcc acctacgacc tcaggtcctc agaccaacca gcccaagaaa 660 catctcacca atttcaaatc cggtaagcgg cctcttttt ctctgttctc caacctccct 720 cactatccct caacctcttt ctcctttcaa tcttggcgcc acacttcaat ctctcccttc 780 tcttaatttc aattcctttc attttctggt agagacaaaa gagacatgtt ttatccgtga 840 acccaaaact ccggcgccgg tcacggactg ggaaggcagt cttcccttgg tgtttaatca 900 ttgcagggac gcctctctga tttcacgttt cagaccacgc agggatgcct gccttggtcc 960 ttcaccctta gcggcaagtc ccgctttcct ggggcagggg caagtacccc tcaacccctt 1020 ctccttcacc cttagcggca agtcccgctt ttctggggca ggggcaagta cccctcaacc 1080 ccttctcctt cacccttagc agcaagtccc gctttcctag ggggcaagaa ccccccaatc 1140 gcttattttc acgccccaac ctcttatctc tgtgccccaa tcccttattt ccacgcccca 1200 atctcttatc tctgcgcccc aatcccttat ttctgtgccc caaccccttc tc 1252 <210> 7 <211> 1501 <212> DNA <213> Homo sapiens <400> 7 caaagcctgt ttggtggtct cttcacatgg atgcgcatga aatttggtgc ggtgactcgg 60 atcgggggac ctcccttggg agatcaatcc cctgtcctcc tgttctttgc tccgtgagaa 120 agagccacct acgacctcag gtCctcagac caaccaggcc aagaaacatc tcaccaattt 190 caaatccggc tgctcctcgc caggccgagc tagttcccaa ttcttcctca gcctctcctc 240 ctccaccctr taatcttttt atcacctccc ctcctcacac ctggtccgrc ttacagtttc 300 gttcygtgac tagccctccc ccwcctgccc agcaayttac tcttraaaak gtggckggag 360 ccaaaggcat agtcaaggtt aatgctcctt tttctttatc ccaaatcrga tagygtttag 420 gctctttttc atcaaatata aaaayccagc ccagttcatg rcttgttysg cagcaaccct 480 gagacrcttt acagccctag accctaaaar gtcaaaaggc crtcttattc tcaaaataca 540 ttttattacc caatctkctc ccgacattar ataaaactcc aaaaattaaa ttccrgccct 600 caaaccccac aacaggattt aattaacctc gccttcaagg tgtacmataa tagaaaaaag 660 ttgcaattcc ttgcctccac tgtgagacaa accccagcca catctccagc acacaagaac 720 ttccaaacgc ctgaaccgca gckgccaggs gttcctccag aacctcctcc cmcakgagct 780 tgctacatgt gccggaaatc tggccactgg gccaaggaak gcccgcagcc ygggattcct 840 cctaagccgy gtcccatctg tgtgqgaccc cactgaaaat cggactgttc aactcacctg 900 gcagccactc ccagagcccc tgqaactctg gcccaaggct ctctgactga ctccttccca 960 gatcttctcg gcttascggy tgaagactga cactgcccga tcrcctcgga agccccctag 1020 accatcacga acgccgagct ttgggtaact ctcacagtga aaggcccatc catctggcag 1080 agaaagggat gctcaggaca cagaacaacc atgctacctt aacaagactt ccgtgagcac 1140 caactttgga tgcggtctac tctctacaga ggtctctggc aacctcacaa cctgcagttc 1200 cttgCcctca tgcagcactt cctgagaggc agagacgtgg actaggagaa acctgagaga 1260 cacggtctcg ctctacacct caggctggag tgcagtggca caaacacagc tcagtgtaat 1320 ctagaactcc tgggctcaag agatcttcct gccttagcct ccggagtagc caggactaca 1380 ggtatgcacc accacatcca gctgagaata tgcagtcctg ctaggatgta atgaaaatgg 1440 tactttatct tggtggtatt cctccaaaaa acatacaact ccaggttaac catgagagaa 1500 a 1501 <210> 8 <211> 5507 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (2144). .(2144) <223> any nucleotlde <220> <221> misc_feature <222> (3562). .(3562) <223> any nucleotide <220> <221> misc_feature <222> (4983). .(4983) <223> any nucleotide <400> 8 tttttttttt tggaaaataa aaatttattt ttaagtcaaa gtatgcaaca aataaaccta 60 cagaaaacat tttcccatcc caatttgttg ctttaccaaa taatattttg aaaacacatt 120 ccttcagtca ttataaagtt tttaaaatac aaaagaaatt aaatttgtaa gaaagtttag 180 tagaccagat gctgttgtca agacttgtaa ggtggggttt ttgctttcag tacatcccac 240 gccatccacc tccactcatg ccgccttgag aacaaacccc ctttgactgt aatttttttt 300 tacytaccca aatcctrtaa aacggccccm cccttatytc ccttcgctga ctytyttttc 360 ggactcagcc crcctgcacc caggtgaaat aaacagccwt gttgCtcaca caaagcctgt 420 ttggtggtct cttcacasgg acgcgcatga aatttggtgy cgtgactcgg atcgggggac 480 ctcccttrgg agatcaatcc cctgtcctcc tgctctttgc tccgtgagaa agatccacct 540 acgacctcag gtcctcagac cgaccagccc aagaaacatc tcaccaattt caaatccggt 600 aagcggcctc tttttactct cttctccarc ttccctcact atccctcaac ctctttctcc 660 tttcaatctt ggygccacac ttcaatctct cccttctctt aatttcaatt cctttcattt 720 tctggtagag acaaaggaga cacrttttat ccgtggaccc aaaactcygg cgycggtcac 780 ggactgggaa ggcagccttc ccttggtgtt taatcattgc aggggcrcct ctctgattat 840 tccccacgt ttcadaggty tcagaccacg cagggaygcy tgccttggtc cttcaccctt 900 agcggcaagt cccgcttttc tggggaaggg gcaagtaccc caaccccttc tctccttgtc 960 tctacccctt ctctgctttt ctgggggagg gacaagtacc cctcaacccc ttctccttca 1020 cccttaatgg caagtcccgc ttttctgggg gaggggcaag tacccctcaa ccccttctcc 1080 ttcaccctta gtggcaagtc cygykttyct agggggcaag aacccccaat cccttatttc 1140 cgcaccccaa cctcttatct ctgtgcccta attccttatt tccatgcccc aaccctttct 1200 ctgcttttct ggagggcaar aaacccctac cgcttctccg tgtctctact cttttctctg 1260 ggcttgcctc cttcactatg ggcaagtttc caccttccat tcctccttct tctcccttag 1320 cctrtattct taagaactta aaacctcttc aaytctcacc tgacctaaaa tctaagcrtc 1380 ttattttctt ctgcaatgcc gcttgacccc aatacaaact cgacagtagt tccaaatagc 1440 yrgaaaaygg cactttcaat ttttccatcc trcaagatct aaataattct tgtwgtaaaa 1500 tgggcaaatg gtctgaggtg cctgacrtcc aggcattctt ttacacatca gtcccytcct 1560 agtctctgtg cccagtgcaa ctcstcccaa atcttcyttc tttccctccc kcctgtcccc 1620 tcagtaccaa ccccaagtgt cgctgagtct ttctaatctt ccttttctac agacccatct 1680 gacctctccc ctcctcgaca ggctgagcta ggtcccaatt cttcctcagc ctccactcct 1740 ccaccctata atctttttat cgcctcccct cctcacaccy gktcyrgctt acagtttcrt 1800 tccgtgacya gccctccccc acctgcccag caatttaytc ttaaaaggt ggctggagcc 1860 aaagtcataa tcaaggtgaa tgctcctttt tctttatccc aaatcagata gcgtttaggc 1920 tctttttcat caaatataaa aatccagccc agttcatgac ttgtttggca gcaaccctga 1980 gacgctttac agccctggac cctaaaaggt caaaaggctg tcttattctc aatatacgtt 2040 ttattaccca atctgctycc gayattaaat aaaactccaa aaattrgaat ctggccctca 2100 aaccccacaa caggatttaa ttaacctcrc cttcaaggtg tacnataaya gaaaaaagtt 2160 gcaattcctt gcctccwctg tgagacaaac cccagccaca tctccarcac acaagaactt 2220 ccaaacgcct raaccgcagc rgccaggcgt tcctccagaa cctcctcccm caggagcttg 2280 ctacaygtgc cggaaatctg gccacygggc caaggaatgc ccgcagscyg ggattcctcc 2340 taagcygygt cccatctgtg tgggacccca ctgaaaatcg gactgttcaa ctcacctggc 2400 agccaytccc agagcccctg gaactctggc ccargsctct ctgactgact ccttcccaga 2460 tcttctcggc ttagcggctg aagacygaca ctgccsgatc acctcggaag ccccstagac 2520 catyatggaC gccragcttt rggtaactct cacagtggaa ggtargcccr tccccttctt 2580 aatcaatayg gaggctaccc actccacatt accttctttt caagggcctg tttcccttgc 2640 ctccataact gttgtgggta ttgacagcya ggcttctaaa cytcttaaaa ctccccaact 2700 ctggtgccaa cttagacaat actcttttaa gcactccttt ttagttaycc ccacctgccc 2760 agttccctta ttaggctgag acactttaac taaattatct gcttccctga ctattcctgg 2820 gctacagcca cacctcattg ctgccttttc ccccartyca aagcctcctt crcatcctcc 2880 ccttgtatcy ccccacctta acccacaagt ataagatacc tctactccct ccttrgcgac 2940 cgaccatgcr ccccttacca tctcattraa acctaatcac cyttaccyca ctcaacgcca 3000 atatcccatc ccgcagcacg ctttaaaaag attaaagcct gttatcactc gcctgctaca 3060 gcatggcctt ttaaagccta taaactctcc ttacaattcc cccattttac ctgtcctaaa 3120 accagacaag ccttacaagt tagttcagga tctgcrcctt atcaaccaaa ttgttttgcc 3180 tatccacccc gtggtgccaa acccatatac tctcctatcc tcaatacctg cctcyacaac 3240 ccattattct gttctagatc tcaaacatgc tttctttact attcctttgc acccttaatc 3300 ccagcctctc ttcgctttca cttggactga ccctgacacc catcaagctc agcaaattac 3360 ctaggctgta ctgcygcaaa gcttcacaga cagcccccat tacttcaatc aagcccaaat 3420 ttcttcctca tctgttacct atctcggcat aattctcata aaaacacacg tgctctccct 3480 gccaatcgtg tcygactgat ctctcaaacc cmagcacctt ctacaaaaca acaactcctt 3540 tccttcctag gcatggttag cntggtcaga attcttacac adgagccagg accacaccct 3600 gtagcctttc tgtccaaaca acttgacctt actgttttag cctagccctc atgtctgcgt 3660 gcagcrgctg ccrctgcttt aatactttta gaggccctca aaatcacaaa ctatgctcaa 3720 ctcactctct acagttctca taacttccaa aatctatttt cttcctcata cctgacrcat 3780 atactttctg cttcccggct ccttcagctr tactcactct ttgttgagtc tcccacaatt 3840 accattgttc ctggcccrga cttcaatccg gcctcccaca ttattcctga taccacacct 3900 gacccccatg actgtatctc tctgatccac ctgacattca ccccatttcc ccaaatttcc 3960 ttctttcctg ttcctcaccc tgatcacrct tgatttattg atggcggttc caccaggcct 4020 aatcgccaca caccagcaaa ggcaqgttat gctatagtac aaqccactaq cccgcctctt 4080 agaacctctc atttcctttc catcgtggaa atctatcctc aaggaaataa cttctcagtg 4140 ttccatctgc tattctacta ctcctcagqg attattcagg ccccctccct tccctacaca 4200 tcaagctcra ggatttgccc cacecaggac tggcaaatta gctttactca acatgccctg 4260 agtcniSataa ctaaaatacc tcttagtcta ggtagatact ttcactggat agrtasaggc 4320 ctttcctaca gggtytgaga aggccaCcrc agtcatttct tccrttctgt cagacataat 4380 tcctcagttt agccttccca cctcaataca gtctgataac agacsagcct ttattagtca 4440 aatcagccaa gcagtttttc aggctcttag tttcagtga aacctttata tcccttatgg 4500 tcctccgtct tcaagaaaag tagaatggac taaaggtctt ttaaaaacac acctcaccaa 4560 gctcagccac caacttaaaa aggactggac aatactttta ccactttccc ttctcagaat 4620 tcaggcctgt cctcrgaatg ctacagggta cacccattt aagctcctgt atagacgctc 4680 ctttttatta ggccccagtc tcattccaqa caccagacca acttagactg tgccccmaaa 4740 aaacttgtca tccctactat cttctgtcta gtcatactcc tattcccgt tctcaactac 4800 tcatacatgc cctgctcttg tttacactgc yggtttacac tgtttttcca agccatcaca 4860 gctgatatct cctggtgcta tccccaaact gccactctta actcttgaag taaataaaya 4920 atctttgctg gcaggactat gctgaatctc cttargcact ctctaatyag atrtcctrrg 4980 tcntcccaat tcttagacct tttatacctg tttttctcct tctgttattc catttagttt 5040 ytcaattcat ccaaaaccrt atccaggcca tcaccaatca ttctatayga caaatgtttc 5100 ttctaacatc cccacaatat caccccttac cacaagacct cccttcagct taatctctcc 5160 cactctaggt tcccacrccg cccctaatcc cgcttgaagc agccctgaga aacatcgccc 5220 attctctctc cataccaccc cccaaaaatt ttcrccgccc caacacttca acactatttt 5280 gttttrtttt tcttattaat ataagaaggc rggaatgtca ggcctctgag cccaagccaa 5340 gccatcgcat cccctgtgaC ttgcayrtat acryccagat ggcctgaagt aactgaagaa 5400 tcacaaaaga agtgaatatg ccctgcccca ccttaactga tgacattcca ccacaaaatg 5460 gccggtattt atttattcca ctggtaaatg gccgggcctt gccttaa 5507 <210> 9 <211> 1997 <212> DNA <213> Homo sapiens <220> <221> mscjeature <222> (1063). .(1063) <223> any nucleotide <220> <221> nisc_feature <222> (1090).. (1090) <223> any nucleotide <400> 9 gacccacgcg tccgcccacg cgtccgcccc actcaatgcc aatatcccat cccgcagcac 60 actttaaaaa gattaaagcc tgttatcact cgcctgctac agcatagtct tttaaagcct 120 ataaactctc cttacaattc ccccatttta cctgtcctaa aaccagacaa gccttacaag 180 ttagttcagg acctgcacat tatcaatcaa attgttttgc ctatcgaccc tgtggtgccc 240 aacccataca ctcttttgtc ctcaatacct tcctccacaa ctcactattc cctgcttgat 300 cttaaagatg cttttttcac tattcccctg cacccctcgt cccagcctct ctttgctttc 360 atttggactg accctgacac catcaagctc agcaaactac ctaggctgta ctgccgcaaa 420 gcttcacaga cagcccccat tacttcaatc aagcccaaat ttcttcctca tctgttacct 480 atctyggcat aattctcata aaaacacacg tgctctccct gccaatcgtg tccgactgat 540 ctctcaaacc cnarcacctt ctacaaaaca acaactcctt tccttcctrg gcatggttag 600 cacagtcaga attcttacac aagarccagg accacaccct gtagcctttc tgtccaaaca 660 acttgacctt actgttttag ccyagccctc atgtctgygt gcagcggctg ccrctgcttt 720 aatactttta raggccctca aaatcacaaa ctrtgctcaa ctcactctct acagttctca 780 taacttccaa aatctatttt cttcctcata cctgacgcat atactttctg cttcccggct 840 ccttcagctg taCtCactct ttgttragtt cccacaatta ctgttgttcc tgrcccagac 900 ttcaatccgg cctcCcact tattcctgat accacacctg acccccatga ctgtatctct 9b0 stgatccacc tgacattcac cccatttccc caaatttcct tctttcctgt tcctcacyct 1020 gatcacgctt gatttattga tggtggttcc accaggccta atngccacac accagcaaag 1080 gcaggttatn ctatagtaca agccactagc cyrcctctta gaacctctca tttcctttcc 1140 atcgtggaaa tctatcctca aggaaataac ttctcagtgt tccatctgct attctactac 1200 tcctcaggga ttattcaggc cccctycctt ccctacacat Caagctcgag gatttgcccc 1260 acccaggact ggcaaattag ctttactcaa catgccctga gtcagataac taaaatacyt 1320 cttagtctag gtagatactt tcactrgata ggtagaggCC tttcctacag ggtctgagaa 1380 rgccaccaca gtcatttctt cccttctgtt agacataatt cctcagttta gccttcmgca 1440 cctcaatasa gtctgataac agatgagcct ttattagtca aatcagscaa gcagtttttc 1500 aggctcttag tattcagtga aacctttata tcccttacgg kcctccrtct tcaagaaaag 1560 do tagaatggac taaaggtctt ttaaaaacac acctyaccaa gctcagycac caacttaaaa 1620 aggactggac aatactttta ccactttccc ttctcagaat tcaggcctgt cctyggaatg 1680 ctacagggta cagcccattt aagctgctgt atagacataa cttggcccat gatagctagt 1740 attcagttct tccttttatg cacaaccaca gccagcagga agctaccaga gaatatgcac 1800 cagtgaaata aggtgtgtaa ataaaaaaga tatgcaatcc atgaaacaga acatccagcc 1860 aaggatcata acagcaaatg ccagctctgg tgagcacgtt atattgaaaa gggtgtgact 1920 gtggtgaaag acttgccaca aatcatgaaa caaaaccaac cagcactgac agatcattta 1980 aaatgtttaa atacttg 1997 <210> 10 <211> 1920 <212> DNA <213> Homo sapiens <400> 10 ccgcctgcac ccaggtgaaa taacagccat gttgcttaca cacagcctgt ttggtggtct 60 cttcacatgg acgcgcatga aatttggtgc cgtgactcgg atcgggggaC ctcccttgct 120 agatcaatcc cccgtcctcc tgctctttgc tccgtgagaa agatccaccc acgacctcag 180 gtcctcagac caaccagccc aaggaacatc tcaccaattt taaatcagat cttctcggct 240 tagcggctga agactgrcac tgccssatcr cctyggaagc cccctagacc rtcacwgacg 300 ccgagcttca ggtaactctc acagtggaag gtaagcccgt cyccttctta atcaatacrg 360 aggstaccca ctccacrtta ccttcttttc aagggcctgt ttcccttgcc tccataactg 420 ttgtgggtat tgacrgccag gcttctaaac ctcttaaaac tccccaactc tggtgccaac 480 ttagacaata ctcttttaag cactcctttk tagttatccc yacctgccca gttcccttat 540 taggctgaga cactttaact aaattatctg cttccctgac tattcctgga ctacagctat 600 atctcattgc cgcccttctt cccaatccaa agcctccttt gcgtcctcct cttgtatccc 660 cccaccttaa cccacaagta taagatacst ctactccctc cttggygacc gatcatgcac 720 cccttaccat ctcattaaaa cctaatcacc cttacccyac tcaacgccaa tatcccatcc 780 cgcagcacrc tttaaaaaga ttaaagcctg ttatcactck yctgctacag catggccttt 840 taaagcctat aaactcycct tacaattcyc ccattttacc tgtcctaaaa ccrgacaagc 900 cttacaagtt agttcmggat ctgtgcctta tcaaccaaat tgttttgcct atccacccyg 960 tggtgccaaa cccrtatmct ctcctatcct caatacctsc ctctacwacc cattaktctg 1020 ttctagawct caaacatgct ttctttacta ttcctttgca cccttcatcc cagcctctct 1080 yyrctttcac ttrgactsac cctgacacys atyargctca gcaaattacc trggctgtac 1140 tgccrcaarg cttcacagac agcccccatt acttcartca agcccaaatt tcwtcctcat 1200 ctgttaccta tctcggcata attctcataa aaacacacgt gctytccctg cyratcgtgt 1260 ccgaytratc tcycaaaccc aakcccttta caaaacaaca actcctttcc ttcctaggca 1320 tygttagcgc ggtcagaatt cttacacaag agccaggacc acaccctgta gcctttctgt 1380 ccaaacaact tgaccttact gktttagcct agccctcatg tctgcgtgca ginggctgccg 1440 ctgctttaat acttatagag gccctcaaaa taagtagagg cctttcctac agggtctgag 1500 aaggccaccg cagtcatttc ttcccttctg tcagacataa ttcctcagtc tagccttccc 1560 acctcaatac agtctgataa cagacgagcc tttattagtc aaatcagcca agcagttttt 1620 caggctctta gtattcagtq aaacctttat atcccttata gtcctccatc ttcaagaaaa 1680 cacrncctcac caogctcagc caccaactta aaaaggactg gacaatactt ttaccacttt 1740 cccttctcag aattcaggcc tgtcctcaga atgctacagg gtacagccca tttaaggtcc 1800 tgtatagatq ctccttttta ttaggcccca gtctcattcc agacaccaga ccaacttaga 1860 ctgtgcctca aaaaaaaaaa aaaaaaaaaa aaaactcgag actagttctc tctctctccc 1920 <210> 11 <211> 1943 <212> DNA <213> Homo sapiens <400> 11 gggagagaga gagagagaga gagagagaga gagagagaga gagagagaga gagagagaga 60 gagagagaga gagagagaga gagagagaga gagagagaga gagagagaga gagagagaga 120 gagagagaga gagcgtgtct ctactctttt ctctgggctt gcctccttca ctatgggyaa 180 gyttccacct tccattcctt tcttctccct tagcmtgtrt tctyaaraay twaaaayctc 240 ttcaactcwc acctgaccta aaayctaary gycttatttt cttctgcaat gccrcttgac 300 cccaatacaa actcracagt agttccaaat agccagaaaa tggcacttts aatttttcca 360 mcctrcaara tctaaataat tcttgkcrtaaaatrggcaa atggtgtgag gtgcctgacg 420 tccaggcatt cttttacaca tcagtccctt cctagtcyct gtgcccagtg caactcgtcc 480 caaatcttcc ttctttccct cccgcctgtc ccctcagtac caaccccaag cgtcactgag 540 tctttctaat cttccttttc tacagaccca tctgacctct cccttcctcc ccaggctgct 600 ccttgccagg ccgagctagg tcccaattct tcctcagcct ctgctcctcc accctataat 660 ctttttatca cctcccctcc tcacacctgc tccggcttac agtttcattc cgtgactagc 720 cctccccgac ctgcccagca atttattctt aaaaaggtgg ctggagctaa acgcatagtc 780 aaggttaatg ctcctttttc tttatcccaa atcagatagt gtttaggctc tttttcatca 840 aatataaaaa tctagcccag ttcatggctc gtttggCagc aaccctaaga cactttacag 900 ccctagcccc taaaaggtca aaaggccatc ttattCtcaa tatacatttt attacccaat 960 ctgctcccga cattaaataa aactccaaaa actggaatct ggccctcaaa ccccacaaca 1020 cj ggacttaatt aacctcacct tcaaggtgtg aaataacaga daaaagttgc aaytccttgc 1080 CtCcactgtg agaCaaaCCC cagccacatc tccagcacac aagaacttcc aaacgcctga 1140 actgtagcag ccagacgttt ctccagaacc tcctccccca ggaacttgct acacatgccg 1200 gaaatctggc cactgggcca aggaacgccc gcagcccggg attcctccta agccgcgtcc 1260 catctgtgtg ggaccccact gaaaatcgga ctgttcaact cacctggcag ccactcccag 1320 agctcctgga actctggccc aaggttctct gactgactcc ttcttggctt actggctgaa 1380 gactgacgct gcctgatcgc ctcagaagcc ccgcagacca tcatggacgc cgagctttag 1440 cccgcctgca cccaggtgaa ataaacagcc ttgttgctca cacaaagcct gtttggtggt 1500 ctcttcacac agacgcgcat gaaagggaag acatacaaaa acaaggcctc tgaggtaggt 1560 actactgaga cagccaggtg ggaaggactc cttggcaaaa ctccaaccag cctgtacact 1620 gggaggaatg tgcactggga tggagccata gaagtttgtg tcgtttgcag tggggaggag 1680 cctggtccct cctcttcCtg tgaggaacct ggaattCaat ctgtgaggaa cttcttgaaa 1740 gacccatcaa ttcttcaata qaaagcatca aaggacaatt tacaCcctaa gactgaaccc 1800 ctgacctcaa aatctttccc ttgctatgtt caccaacctc aocagaaata ttaggattct 1860 tacctgatcc tagccaagcc ccctccctca tctcccatta aagggtccat cttcaaccaa 1920 acttaagtct caataaatat ctg 1943 <210> 12 <211> 2286 <212> DNA <213> Homo sapiens <400> 12 gggtgagccc cgtgcccggc ccaatttttg tatttttagt agagacgggt tcaccatgtt 60 ggccaggcta gtcttgaaCt cctgacctca ggtgatctgc ctacctcagc ctcccgagta 120 gctgggatta caggtqcctg ccaccaCgcc tggctaattt tttgtatttt tagtagagaa 180 ggggtttcac catattagcc acaatggtct caatctCctg acctcgtgat ccatctgccc 240 cgccctccca gagtgctggg attacaggcg tcagccaccg tgaccggctc agactgtact 300 cttatagcca tctgaaatac gttttctagg tagagataga ttgtgtaagg gtacagttgt 360 gaggataaca gaaacatggc agattattta aaatcatcct gaaagtggtg ctttatctga 420 tgaaagtgat tgtaatccat aggaaaatgt ttcaacgtgc gcaagagttg cggcggcggg 480 cagaggacta ccacaaatgc aaaatccccc cttctgcaag aaaggctctt tgcaactggg 540 taagtttgct tgttttcctt gcttttggac atagtctgcc aggtcaggac atggatacat 600 ttttctccct acagctctgt gctcaagccc tgcagaggga gatggcagag agaaaggctg 660 cctacaagca tcacagtccc atccctgtkg gkaaccgtgt tgygcaaaaa caccttcatc 720 cccacccagt ggggcccctg atctaatatt ctaagtgtca gaggttccgt atttgtaata 780 gcaratgggc cctgactgta aaytagtgaa gagtgaatgt aacttattac ccacagggac 840 aattccaaat garggcctta aatgatgctc agctaagctg gttcttgtgt ggcctctgta 900 ccttcaaaag ctgccgagtc ctatgattgc acgcgatggg acttgtacac ttgaagtgaa 960 acacagtttt aaaacttgct ttgtttagaa ttcccacctc atttttccat ggacaaaagt 1020 attctttatg tcctagtgca cttacaattt ggtattacct gggagtgaaa agaaatatta 1080 cagccatgcc taastgactt cttgaggtaa gattgttctg tcagaaaacc ctctcccagt 1140 tcccctgcag ctcttcagga atccacatct ctccagagct ctttgttctc atgggtggca 1200 cctccagagt gaagaagatC ctttgtcaag aagggaaaca gaggggaaat gagagggtcc 1260 tgcaggcaga gctggaatca acttccactc tgcctcttgc aagctgtgtg accctgggca 1320 caatttctcc ttcctctgga aacctctgtt ttcttagatt tggagcaggr tggtcacact 1380 gaccttgcag agttctgaga atcagagaca gaacataaaa ggcctggaaa acattctcca 1440 aaaagaagct gcaacatgtg tggacaatgg gcttttcatg cctctcttac tgtctcttac 1500 tgkctattga cctggtgcaa gaaacatgct ctggtgatgg ctgtgaggga ggaatgagga 1560 tagacataga cactcctgtg tctcaaacat gcttctttat tactctgtta tgactctgtc 1620 ttccctgggg caggacccca gcctgcctac atttgcagac agacacagtg gcatgtggag 1680 acaacagtgt gtcccartga cttttcttta ccccc.yagct gtcggcagta ctcagtggaa 1740 gggtgatatg acactgayac tgctattttg aaacctggag gatggaaagg tgcaaaaatc 1800 tatcaccagc aacagaaggt gcagactgtg ttggtggcgg taattttgtc catcaaatga 1860 atatgtgtga aaacattccc tcctttggcc ctacaggtca gaatggcggc agyrgagcat 1920 cgtcattctt caggattgcc ctrctggccc tacctcacag ctgaaacttt aaaaaacagg 1980 atgggccacc agccacctcc tccaactcaa caacattcta taattgataa ctccctgagc 2040 ctcaagacac cttccgagtg tgtgctctat ccccttccac cctcagcgga tgataatctc 2100 aagacacctc ccgagtgtct gctcactccc cttccaccct cagctctacc ctcagcggat 2160 gataatctca agacacctgc CgagtgCctg ctctatcccC ttccaccctc agcggatgat 2220 aatctcaaga cacctcccga gtgtctgctc actccccttc caccctcagc tccaccctca 2280 gcggat 2286 <210> 13 <211> 1280 <212> DNA <213> Homo sapiens <400> 13 cagcattcag attgcctttt ctctcaacca ggatctttaa agtcgatgac aagagttcca 60 gtcctgaatc atggcaaagt gcagtagtga actgcggggt tattctggaa ggatctctct 120 atggctgatg gtctcagttc cggcatcagc ctctgactga gaatcaggtc tcacacagga 180 ggagtcagat gaggagcaat cctctgcttc cgatggagtt agttgtgatg aattggtgag 240 gtctggtttt tcacactgaa ctaaaatgag ctttcgctgt gtcaagcaca agactgaccc 300 cagagacaca catagtgcac ctcatagaag cttttaatag tctttatatt tactaaagaa 360 taggactaac tatggaacta tgaagatgag ctggaaatga caggtgactt gccagcaggc 420 cagagtgtga yttttttttg tccctcaatg ggaggtgtcy attctccctt ygsttgtgag 480 aatcagttgg ttcatttgtg ggaaggttgc aggggggatc tttgaatcac agccttcaga 540 tgccagaagg gcagagggaa tcccacacgg gctggtggat catgtgtgtg catttctctc 600 ccttctartc tgaggaaact aagcrtgaaa gaaygtgagc aygsagaaaa ggagaggcag 660 gtrtcagagg cagaggaaaa ygggaaattg gatatgaaag aaatacacac ctacaagtga 720 gttcagaaac tgaaccccac cctcytggga aacgcccatt ggagtgttgt ttttaaccty 780 tgtacaatgt ttagacccag taaatgcaga aatagaaaca aatggtcaga agacatatcg 840 tgagagagag agagagagtt cacaaaacag aaaacaaagt accttaatat ttaccagtga 900 ccaaaagatg tgaagcagca aaaggtctcc tgaccccatt gccagctaga ctgtgtagaa 960 actcggttca taccagccat tctaggggtg gggtgagttt gttgtcatcc ttaggaaagt 1020 gtgttgttgt aggatcaacc acatccttca aaaggactat gcctgtttat aagcccagct 1080 gtttctgccc tgtgaaacac ggtaaggata ttaatacaaa gagaatacag ctttatgata 1140 aaagatgctc agtgaaggat gaattaggga tatactgaga atggggaagg aaactatcat 1200 ctcagaagtc agcaggcagt aagcaagagg aggaatcaat atagcaacag tttggatcag 1260 actgtacagt ttttttttgt 1280 <210> 14 <211> 2247 <212> DNA <213> Homo sapiens <400> 14 ggcgtgaggc gccgcccggg tgtccccgcg gcgcaggagg cggtggagcg cagagcgggc 60 gagcgcgaaa aatcactacc aatataatgg attttatata tcagattgct ttattctgga 120 tatcatggta acaatacaga aagctcctac gtgtacctgg agggccgctg cctcaattgc 180 agcagcggct ccaagcgagg gcggtgggct gcacgtacgt tcagcaacaa gacactggtg 240 ctggatgaga ccaccacatc cacgggcagc gcaggcatgt gactggtgct gcggcggggc 300 gtgctgcggg acggcgaggg atacaccttc acgctgacgg tgctgggccg ctctggcgag 360 gaggagggct gcgcctccat ccccctgtcc cccaaccgcc cgccgctggg gggctcttgc 420 cgcctcttcc cactgggcgc tgtgcacgcy ctcaccacca aggtgcactt cgaatgcayg 480 ggctggcatg acgcggagga tgctggcgcc ccgctggtgt acgccctgct gctgcagcgc 540 tgtcgccagg gccactgcga ggagttctgt gtctacaagg gcagcctctc cggctacgga 600 gccgtgctgc ccccgggttt caggccacac ttcgaggtgg gcctggccgt ggtggtgcag 660 gaccagctgg gagccgctgt ggtcgccctc aacaggtctc tggccatcac cctcccagag 720 cccaacggca gcgcaatggg gctcacagtc tggctgcacg ggctcaccgc tagtgtgctc 780 ccggggctgc tgcggcaggc cgatccccag cacgtcatcg agtactcgct ggccctggtc 840 actgtgctga acgagtacga gcgggccctg gacgtggcgg cagagcccaa gcacgagcgg 900 cagcgccgag cccagatacg caagaacatc acggagactc tggtgtccct gagggtccac 960 actgtggatg acatccagca gatcgctgct gcgctggccc agtgcatggg gcccagcagg 1020 gagctcgtat gccgctcgtg cctgaagcag acgctgcaca agctggaggc catgatgcgc 1080 atcctgcagg cagagaccac cgcgggcacc gtgacgccca ccgccatcgg agacagcatc 1140 ctcaacatca caggagacct catccacctg gccagctcag acgtgcgggc accacagcgc 1200 tcagagctgg gagCCgagtC accatcgcgg atggtggcgt cccaggccta caacctgacc 1260 tctgccctca cgcccatcst cacgcgctcc cgcgtgctca acgaggagcc cctgacgctg 1320 gcgggcttts agsagggccc cggscaacct crgtgaygtg gtgcagctca tctttctggt 1380 ggactccaat ccctttccct ttggctatat cagcaactac accgtctcca ccaaggtggc 1440 ctcgatggcg ttccagacac aggccggcgc ccagatcccc atcgagcggc tggcctcaga 1500 gcgcgcctca ccgtgaaggt gcccaacaac tcggactggg ctgcccgggg ccaccgcagc 1560 tccgccaact ccgttgtggt ccagccccag gcctccgtcg gtgctgtggt caccctggac 1620 agcagcaacc ctgcggccgt gctgcatctg cagctcaact atacgctgct ggacggtgca 1680 tgcagcggtt ggggcacacg cggccccctg gccttgttct tggggggaag gcgtttctcg 1740 tagggcttcc atgggtgtct ctggtgaaat ttgctttctg tttcatgggc tgctgggggc 1800 ctggccggag aggagctggg ggccacggag aarcaggccg ctacctgtct gaggaacccg 1860 agccctacct ggcagtctac ctgcactcgg agccccggcc caatgagcgc aactgctcgg 1920 ctagcaggag gatccgccca gagtccctcc agggtgccga ccaccggccc tacaccttct 1980 tcatttcccc ggggaccaga gacccagtgg ggagttaccg tctgaacctc tccagccact 2040 tccgctggtc ggcgctggag gtgtccgtgg gcttgtacac gtccctgtgc cagtacttca 2100 gcgaggagga cgtggtgtgg cggacagagg ggctgctgcc cctggaggag acctcgcccc 2160 gccaggccgt ctgcctcacc cgcacctcac cggcttcggc accagcctct tcatgccccc 2220 aagccatgta cgcttttgtg tttcctg 2247 <210> 15 <211> 684 <212> DNA <213> Homo sapiens <400> 15 ggccggcagg cagcgatggc ggccgtacgg ggcctgcggg tgtcggtgaa ggcggaggcc 60 ccggcggggc cggccctggg gctcccgtcc cctgaggcgg agtccggtgt tgaccgtggc 120 gagccggagc ccatggaggt ggaggagggc gagctggaaa tcgtgcctgt gcggcgctcg 180 ctcaaggaac tgatcccgga cacgagcaga agatatgaaa acaaggctgg cagcttcatc 240 actggaattg atgtcacctc caaggaagca attgaaaaga aagagcagcg agccaagcgc 300 ttccattttc gatcggaagt aaatcttgcc caaagaaatg tagccttgga ccgagacatg 360 atgaagaaag caatccccaa ggtgagactg gagacaatct atatttgcgg agtagatgag 420 atgagcaccc aagatgtctt ttcctatttt aaagaatatc ctccagctca catcgaatgg 480 ttggatgata cctcctgtaa tgtagtttgg ctggatgaaa tgacagccac acgagcactt 540 atcaatatga gctccctgcc tgcacaggat aagatcagaa gcagggatgc cagtgaggac 600 aagtcagctg agaaaaggaa aaaagacaag caggaagaca gttcagatga tgatgaagct 660 gaagaaggag aggttgaaga tgag 684 <210> 16 <211> 613 <212> DNA <213> Homo sapiens <400> 16 ggcggtgcca cccctccccc cggcggcccc gcgcgcagct cccggctccc tcccccttcg 60 gatgtggctt gagctgtagg cgcggagggc cggagacgct gcagacccgc gacccggagc 120 agctcggagg cggtgaagtc ggtggctttc cttctctcta gctctcgctc gctggtggtg 180 cttcagatgc cacacgcgtc ccgggggccc ggttctccgc tcccctcccc tccccttctc 240 gccggacccc gcgccgggag ctgcgggaag gagtggaggg tcgggcggtg gcctcgcggc 300 tggcctggcg cgcggccagc gccggtagtt agtgggggga ctgctctgcc ctcgaggggg 360 tagggdgctg tggcgacggt tgccccattt cgagacaaag cgcatttccc cctcccctcc 420 cccacccgcg ttccggcgga ggcgccccct cccccagccg ccacgcgggg ctgggtcgag 480 acttgggcct cccggagggc ggcgcgtggt cccgcgtccg cgaggcctgg cggcgcgcgg 540 ccggctgtcc cgaggctgcg gcgaccgccc agttaacgtg gccgccgcgg gggtaggcgc 600 gtgcggtgtg gcg 613 <210> 17 <211> 1006 <212> DNA <213> Hono sapiens <400> 17 caagcaatag cgcaaaattt aggagacagg atccttgcaa atttaaaagg tgaatgtagt 60 gagggggatg gcaagtggct ggtacaggct gtggtgattc cttttactca agggtttttg 120 tggagtatag ggagaagggg ttgatattta tggacaccta tgtgtcaggc actgtgcatc 180 attttatcct tacaggdtgt tgtgaggtag gtattattgt tttcattttt acaggtgaag 240 aaagcaggtc tcagagggac taaaatcctg cccaaggtta gtggtagagc tgggatccaa 300 aaatctgtca gaatcctgag actgcgctgt tccactgtgc cacgcagaca gttcattcag 360 tttagatgtc acatagtcaa gagggaactc tatgcatcct ttaatttttt agactatgat 420 attcttttta aaaattagcc tttattttct aactaccaaa agaaatatga aagcattaca 480 gaaacactgg aaaatagaaa agaaaaaata aaatcactta caaccacttt ttgttttttg 540 gagtctcgct ttgccaccca ggctggagtg cagtggtgtg atcatggctc attgtagcct 600 caacctccca ggctcaggta atcctcctgt ctcagcctcc tgaatagctg gaaccacaca 660 cacacacgca cacacaggtg tgtgccacca cacccagcta tttttttgta ttttcttttg 720 taaagacaag gtttcaccat gttgcccagg ctggtctcag agtcctgagc tcaaacgatc 780 tgcctgcctt ggcctcccaa aatgttggga ttacaggcat gagccaccac atctgaccta 840 caaccacttt ttaatgtgwg acttaaaaat cttagataaa taaggctgtg aagcaaaacc 900 agggattttt ttgtttgttt ttgatttgca aaacaagtga ctgacaatta ttgagaaatt 960 aaagatagct atgtgtaggt cttgcccctg cgggtttgga ggtttc 1006 <210> 18 <211> 1916 <212> DNA <213> Homo sapiens <400> 18 cccacgcgtc cgcacgaaag aagtgccttt tgcctcccgt catgattctg

aggcctcccc 60 agccatgtgg aactgtttga ggcacagagc tgtatataca ataacagtga aattgatccc 120 actactaatt atgacaaaaa tgatcttcca cgtaaacagg tggtgaagct ccttatggtc 180 ctgaccctac agttcctgtc ccatgaccag ggccagatca ccaaggagct gcagcagttc 240 gtcgtcagtg gcagccccat gcgagcaccc gaggaaggca agtacgtggg tgatatattc 300 ctgtattctt ggacaagtac actggtgaca tgtagctgta ttcagagtca caggtgccca 360 ggccggagtg cagtggcgtg atctcggctc gctacaacca ccacctccta gcagcctgcc 420 ttggccttcc aaagtgctga gattgcagcc tctgcccrgc cgccaccccg tctgggaagt 480 gaggagcgtc tctgcctggc cgcccatcgt ctgggatgtg aggagcccct ccgcccagca 540 gccgccccgt ctgagaagtg aggagcccct cagcccggca gccaccccat ctgagaagtg 600 aggagcccct ccacctggca gccaccccgt ctgggagggc tgkgaccgtc tatgacaagc 660 cagcatcttt ctttcaagag acctctggac ctgcagcacc aactcttcat gaagctgggc 720 ggcacgcact ctccgttcag ggcctgaacc tgaggaccca gacacggagc ggtcggcctt 780 catggagcgg gatgctggga gcgggctggt gatgcgcctc cgcgagcggc cagccctgct 840 ggtcagcagc acaggctgga cagaggacga agacttctcc atctgctggc agctttagaa 900 agagtttgaa caactgactc ttgatggaca caaccttcct tctctcgtct gtgtgataac 960 aggcaaaggg cctccgaggg agtattacag ccgcctcatc caccagaagc atttccagca 1020 catccaggtC tgcacccctt ggctggaggc cgaggactaC ccccgcttct agggtcggtg 1080 gatctgggtg tctgtctgca cacgtcctgc agtggcctgg acctgcccat gaaggtggtg 1140 gacatgttcg ggtgctgttt gcctgtgtgt gccgtgaact tcaagtggca ggagcagaac 1200 ccgaatcttt ctggggatag cttcacagat ccaccgctga ggaggaaaca gtgcagagcg 1260 agctgcccac agtgaggccc tgctcctggt ttacatgagc tggkgaaaca tgaagaaaat 1320 ggcctggtct ttgaggactc agaggaactg gcagctcagc tgcaggtgct tttctcaaac 1380 tttcctgatc ctgcgggcaa gctaaaccag ttccggaga acctgcggga gtcgcagcag 1440 ctccgatggg attagagctg ggtgcagact gtgctccctt tggttatgga cacataactc 1500 ctgggccaga ggctaaacc ccgggacccc tgctgtcctt cccacagctt cttctcagag 1560 tctcagggca aatcctttcg agcagcgcct cccagtggcc agaagctgaa atgatggcag 1620 tagtgccacc tggtgaatga attggttctg tgacccggga agctgtgctt ggctctgatt 1680 tcttttctgg aggctcggaa acacttcctc tcttcttctg ttcttcacgc cccatgcccc 1740 tgctagcgta ttactgttct gtgacttccc tgtgacctct gcagtdctcc tcatcctgcg 1800 tttggtctcc aggtgtcacc tttctgccgt gttcctaaca ttttgattcc tgtcttgaaa 160 aaagcacctg ctgcaccata agcccaggga tgtggcagct gcagcgggct tggctt 1916 <210> 19 <211> 1168 <212> DNA <213> Horno sapiens <400> 19 ctgccatcct ctgggcctga ggctgcctgg cccagcccct cctaactccc tggactcttc 60 cacggtgtct tcaggcccct acaccatcct ttgtgtaagg ggaggtggca gcatagagat 120 gatgggggaa ctgccccatg tgccaaggaa agctcaccca tctgtgcgaa atgctctggt 180 tgacattggg tttttgcgca ccaaactggg ccatgaccaa ggtttataac caaggtgtct 240 ccgggcatgg gcactttggc tcttgtagaa accaccccac tggcaqgaga cggcggtagc 300 tgtggtcatt gaaaacaagc tcctgctgat aaatctcaga caccagacac agaagaacct 360 ggagaccctg ccagagagct tgaggcaaat ggatggactg ttggagcagc tgagggtgaa 420 gcagcacaaa ctcctcaaag ttgaatagca aagcagccac cagagatgga caagaaaaat 480 gaacaaagaa aattagcaga aatcaaaggc agatgctaaa gcagtgcaaa atcattcatt 540 caatgataga aatgaaattg atgaaggagt ctggaaaatg aatgacagaa gagaattaaa 600 cagcagtgac catagtaagg tcctgacgat tctggtccac tgaatcccat catccctaag 660 acagtaaata tcatcacagt caccaccmgc aagttaccac cacagcattt cctgtttgtt 720 ccaaaatgaa taaagatgat tctcatcaca agggcaaata caaagtagtt tagtatgttt 780 ttaactaaac ttcaggtgtt tggtttactt tttctaagtt ctcataattc tgaaaatgca 840 gttgacactt gtgtggctca tgatgttttt aatagtctaa tgctacttga attgttcaaa 900 aaccactgta ttttaaatta agatgaataa acggtccttt gaaaactggc acaaggcaag 960 gatgccctct gtcaccactc ctattcaaca cagtattgga agttctggcc agggcaatca 1020 ggcaagggaa agcaatacag cgtatcaaaa taggaagaga ggaagtcaaa ttgtctctgt 1080 ttgcagatga catgattgca tatttagaaa accccatctt ctcagcccaa aacctcctta 1140 agctgataag ccaccttcag cagtctca 1158 <210> 20 <211> 475 <212> DNA <213> Homo sapiens <400> 20 ctgtggggaa gcggggccgc tggtccggag gtagcggtgc cggccgaggg ggtcggggcg 60 gctggggCgq tcggggccgg cgtcctcggg cccagcggtc tccatcccgg ggcacgctgg 120 acgtagtgtc tgtggacttg gtcaccgaca gcgatgagga aattctggag gtcgccaccg 180 ctcgcggtgc cgcggacgag gttgaggtgg agcccccgga gcccccgggg ccggtcgcgt 240 cccgggataa cagcaacagt gacagcgaag gggaggacag gcggcccgca ggacccccgc 300 gggagccggt caggcggcgg cggcggctgg tgctggatcc gggggaggcg ccgctggttc 360 cggtgtactc ggggaaggtt aaaagcagcc ttcgccttat cccagatgat ctatccctcc 420 tgaaactcta ccctccaggg gatgaggaag aggcagagct ggcagattcg agtgg 475 <210> 21 <211> 1770 <212> DNA <213> Homo sapiens <400> 21 gggttcttcc ttttctctta qcgactcctg tgtgtgtctg ctgaggtgcc ctgtccgctg 60 gtgctgtgct ctgacttact aacccagccc ctactaaccc tgttttctct tcttactaac 120 cccagccCtg ccgagctctg g9CtCCCCCC gggggctggt ccccctCctt ttggcaagca 180 gatgacctgg ggctactggc cctgtagaca gatgtcccac tttgctgccc catattggct 240 gtaagatcag agtccactgg gccaggtcta aggcagggga tggccctatt aacaagactc 300 agaggaggaa gaggtggtcc tgtggatgtg ggaggctgga ctctgagtat gacatctctc 360 ctatgtgcag aagtctggtt gccactggga gtaggtggga ccagggaaat ctctgggacg 420 tgagtgtgga ggcctgttgg tctagactct agactgtgga gctctgagct tttgtgtcct 480 ctggaaggaa gctggggaag aatcctctcc attgttaagt gacagggata gaagctgtCC 540 tgcacaggaa gtcacgaggg gggcgtatcc cacgaggaag gcaggagggg gcgtgcccct 600 caccggaaat tagcagaggg gcgtgtccca cacaggaagt cagaaagcgg agcctttctt 660 acaccggaag tcaatgaagc gggtctttcc tacgctaaaa accactgagt ggagtattta 720 gtacacagga agtcggccag agaaacattt ctcatatttg aaggccggaa agagggacat 780 ttctgacacc ggaagtcagt gagaggactc tttccCaCac aggaagtcag ctagagagcc 840 gtctcccctC tCtggagccg agagaggccg tttcCcCCa ccgkaagtag acgtggggcc 900 gtgaccggaa gtccttggga aagatccgty ccattcccgg aagctagagg gcgttagttg 960 tcgggttgaa aaggggtgtg gggaggggaa gcagctttac cccgggctcg gagtttgcag 1020 gagagagaag tggggagcaa gaagtgacc tcaggggctc acagggttcc cgcagatgct 1080 caggccggcc aggaatgcat ctctggctct ctgttccCac ggacgtcact gcctcagcca 1140 gcctccccca gagcccgcca gccgctaagC cggggccaca cctgggggtg atttcatgcc 1200 tcacctccag taggcacctt ggtttctttg ggctaatctc tggctccctt gcgctaactc 1260 ttgctctcac ccagctaatc cctgcctcac cctgactgcc ccaggggctg accactaaca 1320 accaacctgg ccctgtytgg gggttccagg ctcctggcct ggccctgacc agttcttaat 1380 taacctttcc ttcaccttga ctaactcctg ccttcctggt ctgttccttt cagcagaaac 1440 taatggtttg tggatttttt tctgactaac aacagqtcta acattcctcg ttactgttaa 1500 cagcttggat gtcggcatgg ctgggaaggg gctaacacag ctttgaaCtt ggctaacaca 1560 ggtttgaact tggctaacac aggtttgaac ttgactaaca cagggaaaag catagctaac 1620 aattttgggC gtggtggctg ctctgagtca gaacaatcag aagtcggtaa agatggtagt 1680 tttctaaagg aggtgccagg gctctggtgt ggaccaagcc tgatggagca gtggtaccca 1740 ccaaggtggg qtcagaagta tagccagtct 1770 <210> 22 <211> 1579 <212> DNA <213> Homo sapiens <400> 22 cccgtgtcat gagggatggt catcatcttg tgtgatcctt ggagatggca ggaagccctg 60 gacatacatg gtgtgggggc tcctccagag gctgttggga tcctcctgga tgtggtgtgg 120 gcatggaagg aaggccagtg gagaCaatgg atgatcttgt tcttagcaga tcactggatg 180 tggcagggag tcctaggaca tgtgtggtgt gggcttcttc aggtgctgca cactcgtatt 240 tccgctgcac ttcccaggtg gtgttggcat gaggaaagga ggtatcttcg agggacaatc 300 ttcttcttgt gcgatccttg gagatgccat gaggcccctg gacacatgtg gtgtgggctc 360 ctttggaggc tgttgtatcC cttctgaatg tggcgtgggc atagaaggaa ggccagtggc 420 cacgagggac aatcttggtC ttgggagatc ctggaaatga tagggagtCC cttgatatgt 480 gtggcatggg ctccttcagg tgctagcgga ttccttagga tgggacaaac actgtgcgtg 540 gatcgatgat gacttccata tatacattcc ttggaaagct gaacaaaatq agtgaaaact 600 ctataccgtC atcctcgtcg aactgaggtc cagcacatta ctccaaCagg ggctagacag 660 agagggccaa catcygtttt ttgacatggg ttataccaag gcatccgttc aggcttagga 720 tggggtcttt tatgggtgat gggggtcaca ggagagtggt ggctcccatg tataggaaat 780 ttcttgtttg aaggactgtc agtgagggtg ggtaacacat gcattgtCtg caggactagg 840 tgaatgtcCa tgtggcctag caagagttag ctggtagccc gcctctggtt gccaatttgt 900 tcttgagtcc ttgttctgag ttctggaag gaaacagatt tgtctggtt ggaggagaat 960 acaaggccac atctttgtcg tttgttggct actttgtcc ttggttgagg acattagagt 1020 tttggtcacc aggcatagcc tatgtgcctg tgtgcccgtg ttgtatccca tgtgtttggg 1080 ggacatgtac attgcatgaa ctagtgagct cctgctcatt gcttctgata cccaaggagt 1140 ccctggctta tcctaaaccc aatataggtt aaagcctttc tcattagggq cccagggtcc 1200 caaggctttt gtgagtatca ttgtaggtat tgaagcaacg atgttgagaa ggatgctgaa 1260 catgctcttt gtgggatga cgtactctga oggctcctga cccccagatg agcatccttg 1320 tgtccgttaa cttctgtgtt tatgacagg tgaggccaga gcaggcaga cagcagatgt 1380 attgcaggga gctggatgac atggcccttg gaacctgtgc acatgcctgC ctttctgatg 1440 cacgtCcatg ttttctCtgc acctCccCgg tggtgttggt dtaaaaagca ggcttacatc 1500 agcaagggat gattgtcgtc tcatgcgatc ctgggagatq gcagaagtcC cgggacacat 1560 ggagtgtggg ctctttcgg 1579 <210> 23 <211> 1595 <212> DNA <213> Homo sapiens <400> 23 acctcagcac agacccttta tgggtgtcgg gctcggggac ggtcaggtct ttctcatccc 60 acgaggccac ttttcagact atcacatggg gagaaacctt ggacaataaa cggctttcaa 120 gggcagggct ccctgcagct ttccacagtg tatcgtgccc ctggtttatt gagactagag 180 aatggcgatg acttttacca agtatactgc ttggaaacat cttgttaaca aggcatgtcc 240 tgcacagtcc tagatccctt aaaccttgat ttcctacaac acatgttttt gtgagcttca 300 ggttgggtca aagtggctgg ggcaaagcta cacattaaca acatctcagc aaagcaattg 360 ttgaaagtac aggtcttttt caaaatggag tctcttatgt ctttcctttc tacatagaca 420 cagtaacagt ctgatcgctc tttcttttgc ctacactcac tgaactgccc ttcccctttg 480 ctgggccatg accacgggga acaggtccac tgtcctccct gcgtggtgca cgatggatgc 540 tcagactcca tcctcaaggc tggcaagaag acacgttgag acatgtgcct cctgatacag 600 gtgatggctg tggagccCaC aggactggaa cctcacactg cagggctgga ggcacagacc 660 atttactgtt ctgtgccctg gggggctcaa ggcacagagc tcctcattag ccaaagtcac 720 ccaagttccc caacctctta aagatttcct catcatcatg caagaagaag agaaaagtga 780 gtgtccatac aagctttggg gctcttcctc taatcaggag aaagctggtg tgtattcttc 840 rcttctttct ttkcttttta aasatccc tgctttaatt ttcatctttt attrtgggaa 900 aatataccay gtataaatat taaaaattat aaatatatat tagtkcatat agaatggcca 960 gtataaacat ttacartttc cactsttttt cagtttacag tttmatgaca ttaartaygt 1020 tcacattgtt tagcaaccat caccgycatc rtctccggaa cagttttaty tttcaaaatg 1080 gaaattgcam ccattcrcca agctctccc tcctctctct ygccyacccc tgggggccac 1140 ctttctagtt tgcaactcta kgagtytaac tactctagac acttgataga taagtggaat 1200 cataccgtgt ttaatttttt tttttagagg tagaatcttt ctctgtcacc caggctggag 1260 tgcagtgqcg tgatctcggc tcactgcaac ttccacttcg ggggctcaag caattcttat 1320 gtctcagtct cccgagtagc tgggattaca ggcgtgcgct atcatgcCCa gctaattttt 1380 gtatttttaa tagagacgag ctttcaccat attggccagg ctggtctcga actcctgagc 1440 ttaagggatc cacctgtctc agcctcccaa aatgctgggg ttacaggtgt gagccactga 1500 gcctgggcat gtttatcctt ttgggattta tttatttcac tgacgataat gtcttcaagg 1560 gtcatccatg ttgcggcctg catcaaaagt gcctg 1595 <210> 24 <211> 1459 <212> DNA <213> Homo sapiens <400> 24 cgggagtcta acacgtgcgc gagtcggggg ctcgcacgaa agccgccgtg gcgcaatgaa 60 ggtgaaggcc ggcgcctagc agccgactta gaactggtgc ggaccagggg aatccgactg 120 tttaattaaa acaaagcatc gcgaaggccc gcggcgggtg ttgacgcgat gtgatttctg 180 cccagtgctc tgaatgtcaa agtgaagaaa ttcaatgaag cgcgggtaaa cggcgggagt 240 aactatgact ctcttaaggt aqccaaatgc ctcgtcatct aattagtgac gcgcatgaat 300 ggatgaacga gattcccact gtccctacct actatccagc gaaaccacag ccaagggaac 360 gggcttggcg gaatcagcgg ggaaagaaga ccctgttgag cttgactcta gtctggcacg 420 gtgaagagaC atgagaggtg tagaataagt gggaggcccc cggcgccccc ccggtgtccc 480 cgcgaggggc ccggggcggg gtccgccggc cctgcgggcc yccggtgaaa taccactact 540 ctgatcgttt tttcactgac ccggtgaggc gggggggcga gccccgaggg gctctcgctt 600 ctggcgccaa gcgcccggcc gcgcgccggC cgggcgcgac ccgctccggg gacagtgcca 660 ggtggggagt ttgactgggg cggtacacct gtcaaacggt aacgcaggtg tcctaaggcg 720 agctcaggga ggacagaaac ctcccgtgga gcagaagggc aaaayctcgc ttgatcttga 780 ttttcagtac gaatacagac cgtgaaagcg gggcctcacg atccttctga ccttttgggt 840 tttaagcagg aggtgtcaga aaagttacca cagggataac tggcttgtgg cggccaagcg 900 ttcatagcga cgtcgctttt tgatccttcg atgtcggctc ttcctatcat tgtgaagcag 960 aattcaccaa gcgttggatt gttcacccac taatagggaa cgtgagctgg gtttagaccg 1020 tcgtgagaca ggttagtttt accctactga tgatgtgttg ttgccatggt ddtcctgctc 1080 agtacgagag gaaccgcagg ttcagacatt tggtgtatgt gcttggctga ggagccaatg 1140 gggcgaagct accatctgtg gqattatgac tgaacgcctc taagtcagaa tcccgcccag 1200 gcggaacgat acggcagcgc cgcggagcct cggttggcct cggatagccg gtcccccgcc 1260 tgtccccgcc ggcgggccgc ccccccctcc acgcgccccg cqcgcgcggg agggcgcgtg 1320 ccccgccgcg cgccgggacc ggggtccggt gcggagtgcc cttcgtcctg ggaaacgggg 1380 cgcggccgga aaggcggccg ccccctcgcc cgtcacgcac cgcacgttcg tggggaacct 1440 ggcgctaaac cattcgtag 1459 <210> 25 <211> 2071 <212> DNA <213> Horno sapiens <400> 25 cgcgtccgat taaattacat acttagtaaa tagatattaa ttattttttg aaactcttgt 60 tagtgggaag aatatggtaa attttttgtt aaataaaata gacccttatg tttagcattt 120 tgtttttaga gaactattct ggtactatca gaacaaatac ataaaataac ttcccataga 180 gaacaggata tagcaataat actccttag atactcagtg gcttctgaCt ccaatcaagg 240 tcttgttgat attatatagt aaaaataaaa ccaaaaataa atattattca agtggctctt 300 ctaagcatgt gaatcatgaa gcactqaaat atgtatttta atgatgatct tatttattcc 360 catttttgcc cttagttaaC atttactggt gctcacctag gattggctat tctgagggat 420 tgcatagaaa ccaagctcca cttgctgtcc ttgggaaggt tataactgaa tgcagctctt 480 tatttrgact aaagtgtCag gatatgcatt agattctCtc ctgaaccaaa aacacaacag 540 tcattatctg tgaaccataa tttaaaaatc tttctagaat aacaacagca gactccactc 600 ttgtttgtCt aaaagagCCC tactgggtat gqatcattCt gatgacagat ttatacaaaa 660 tgattcaaaC cagtaactta gtaaaattga ccttcgcaaa acctcactgg gggagtgcct 720 tgtagagCtg tgggtgggaC tgcacattct tctcctctta gtaaaagata ggcccacttt 780 attccaagaa taacacttag cacataaact cttcttcCag ctcgttagca gcattagcac 840 cttctgaatt ccaccctCtC agaagaatcc acagtgtttg aacaatttgc ataaaggtca 900 gctagcatcC tgctgcCaag ccaCtgCata gcatttgtga taagaaggac caactctagg 960 ctcaatatga agggatttag ttctgtaagc agcaaaaaag cttctttatc aagtcatctt 1020 acctctaatt cttttccagt rtgccaactc caaagtcaac attaaaaatg taaatggacc 1080 tgtgtaaata tcacagagag cttttcctta tacatctcaa tgctgagagt taaaatattc 1140 ccaggttaaa tttttttaa agtaccaata atagagctaa atacaatgac atttgctttt 1200 aaaaggtgga tattttattt ctgctttttg aaaatactta tttagtattg acttggaagc 1260 caatttggtc ctttaataag taaagaaaat aatatgttta aaaatgtaaa tgktttacaa 1320 atttgaaact ttcataattg tattaatcag aaaacaagca cattgccatt ctttgaaact 1380 catgtttcta gacatgacag cagtaataaa aggatgaaaa caagtgtctt cactaagcgt 1440 atggccaata aatgggaccc aaacgttCaa tctgttcagt ttaccaaggt tcagaaatac 1500 gtaatttagc aggaaactat aaataccagt gctatcaCag ccacacatac acacacacag 1560 acataaaata accaaacatc tcatttctag gaaagagata acactaaagg catcataggt 1620 ttaactgaaa tacgttatat gaagttttac aaaaaggtca acagaaagct catttgtgaa 1680 aacatactct catgggagct tctttaacat tagttcagag gttaatatat ttcctggagg 1740 tgttttccta gaattgattg cactattgca tggtaataac atttaattgt taaggaaaca 1800 ttatatatag gttcaaatta tcccttaatg ttgatttctc cccttttcca tggattttga 1860 tactaagaaa caaaatgctt tgagattttg gtaactattt tgattttgat aaaacatgtt 1920 aaaatagaag gacatgatat ttttctatag tttccatcag gaagagtaca tcagaaactt 1980 ctccataagg aaagaaaact gactctctct tgaactaggt gttgataaaa tacactaatg 2040 gctttcttaa ttttatttta ttaggagaaa a 2071 <210> 26 <211> 477 <212> DNA <213> Homo sapiens <400> 26 gggaggttac ggccgaggcg gcggcggcgg cgagcCcggg ggcgaggcgc ggacgggaac 60 aggaaaagcC tccggcgCC cctgcgggcg gcggcgcagc cacggccgcg ctccgaggtg 120 aagcCgCgCg cggagaggaa gcgggtgttt tcccctctgc ctttcggccc ccgcccttcc 180 tttcagtttc tgcccgctcg ctcggaagtt ggcggttgac aaaaatggca ggagccgggg 240 cccgggCCgg ttgccgcagC gccgcgggga ccttctgagt tggcccggtg gcagggagac 300 tcgtgcaggg gcgtccgatg cgcggggccc ggggcctCgg gagagctcag ctgctgcggg 360 ccccagaCga ggcgacaggg atggacttgc gtagacagCC agcgccgggc cgccgggcgc 420 gcggtctggg agggcgtgcc gccgcggcgc cgggccgcgc tctgtgaacc ggcgagg 477 <210> 27 <211> 1446 <212> DNA <213> Homo sapiens <400> 27 taatccccag gtccctggga ggggtgctca tgctttgggt gggggaagca atggtgacag 60 gtctggtggg cctgatctca gggcatcagg gtgtgcagag ctccaggagg tagtaggcag 120 ggcaggcagt ctgtggtgtt ggttgtggag agcctgacct ctgggctggt gctagagtgt 180 ggtgatcctg ctgttgagta tgggtggggt tgctatcagt ggtcccctgc agggagctct 240 caggttctga ggggtgtaCa ctttcaactc tggcagtagc agtgtccaca gtggtgtgtg 300 tgaagagcCt gcactcatga catgcactag agcacagagg ccatgctttt gaagggggca 360 gggttgctat tcagagcccc aaacaggcac ttctcagttt ctgggtagtg tttgctttgt 420 ctcctggctg cagtcagtga ctgctatcat gttcaaaggg gtcagatgga tcctgccttt 480 ctgggtgtga actcaagcac agaggctgtg ttgttggtgg gaatggggtt actatttgca 540 tcctcagaca ggcagctgtc aggctcactc actttggctc cccgtggcag cagcactatt 600 gtgatatgca gaaaggggaa gggatccatt ttcacatgag cccaagtact gagaacatac 660 tgctaatagg gatgtggtta ctgtttacat acccagactc tcagatttaa ggtttgcttg 720 ctttggcttt cagaggcagcagtggctgca rcartgtgga gagttgggga agggatcttg 780 acctctgtgc ataagctaga gcacaaaggc catgctgcta gttagggcag ggtggttccc 840 tgccctaatg gtaccaggta ccattggtat cattatacca ggcagggagc tcttgggttc 900 tgccaagcac atgcactggt tccctttgtC tcaggagaag cctccttgat gtactgcgct 960 atcatttcct tgaggagttg tactccctgt gggttagagt gctggggacc ccacaacacc 1020atcgggtcca gccaccattg tgccactgaa gccctccagg tggatgccag ggaattctac 1080 tgggggttca cagggtgtga agatgtggaa ttgttggttc tcagaagagg atgcagtctg 1140 gtggaagctg gactctggCC atagtgccct actgcagctg cttatgtctt gctatgtgat 1200 gc, gtggtgcaag tttcccgctt gcagcaatgc cctggcaggc ctctagatca ccacgctgta 1260 gagtccccac ctatgctaat ctcagagctg tatagatgga agaggtctcc tgtggttagg 1320 attgcagtag tctaaggtaa gactgtgtac ccctaacggc tcacactgac cctttcccta 1380 tdatdgggag ccgttccgg atcccagctg gtcctggctg dgctagctgc tagcttcctc 1440 tccttc 1446 <210> 28 <211> 472 <212> DNA <213> HomO sapiens <400> 28 gagggcgcat tcggccccgg acgaaggtac tcgcagcact tggagcgcag aaccggccgc 60 gcccgatcct ccgagcggcg gcgacggctg ttgctaaggg aggggacgcg cgaggaagcg 120 cgacccgggC ggcagacggc acccagcgcc accagccgag cggcgccccc tccccaggac 180 ccttaacCgC gccgcgtccc ggtcgcgccc gccgcccttt gaaggagaag cadgtgccgt 240 ccccaccccc ggaaggcgcc cccaggagcc ggagcgacct cggagcgcca ctcggatttt 300 ggatttcggt ctcgcattcc gcggccggga ctttctcgag gaggacgcgc gctgctccgc 360 gcccccgagt gcccggagga cccggcatcc ggggagcctc tcgcccctgt cccggaggcg 420 cggcgaggat tggcggcgcc cgccgccccc agccccccag cgcgcgccgg gg 472 <210> 29 <211> 1102 <212> DNA <213> Homo sapiens <400> 29 ttcggcaca gggtggggcC caagagggaa gatgaagcga ggatgccsr gaccagtggg 60 agacgccagg acttcggaag ctcttctgcg ccacggtggg tggtgagggc ggctgggaaa 120 gtgagctcca gggccccagg agcagcctgc tcgtgggtgc ggaaggaaaa aggcacaggg 180 gcttggtgtg ggcggctttt ggctgggaga agtttgcacg tagggagaat agtagccagt 240 gtttgcagag cacttactat gcaggaaggc ctgtcctaag tattgtaagt gtattacatc 300 atgtacaagt gtctgtgatt aaccccgtct tgcagagaag gaaacaaaag tacaaacaga 360 aaatgtaact aagcatgcaa ttaatdaaa gggaccaggt tttgaacgcg agcaatctgg 420 ctcaagaatc tgcgcccaaC caccggctcc tgttcttaga gatgaacgtg gagtcctqga 480 gactqctCaa cattgtgact tgactgtgag cgtacgcgct ccctgtcccc aggagacaga 540 tttccagtgc aatcatagaa agtgcctgtg tgggcttcgg gagatgtgtc tgccttgggg 600 agaattttcc ttttcagcta gagccaggcC caggatgttg acgtcagtga gacgctggtg 660 acgttctctg ctccagtggc tgatgagaaa agttcctcca agccagctca gttgagaaga 720 attaagttct ctgggtccca ctggcttcac ctacagatgc caactttgag gccagtgaac 780 tgtgaggCCa gctgggctga ttgccatggC aacaggaatt ggaccaaagt caccggagga 840 tggagaggga agacacagtg gtggcttccC caggtcttgg accacaaggc acagccgtgg 900 cctCCaggaa ccctgagata acccgttagt gggtcctgca ctccaacaga gctcatgcaa 960 tcagcctctg gtcctcaccc tcctcccatt ggtggccgtt gtgctctcta acattgacat 1020 tgagcagtga gtgctccaga tcttgttcca ctgatttttt ccactggtct ccagtctagc 1080 actttctgaa attcatccaa ge 1102 <210> 30 <211> 1243 <212> DNA <213> Homo sapiens <400> 30 gcgtccgaca ctggtgacat gttgctgtat gcttggatga gtacgctggt gacacgttgc 60 tgtattcttg ggcgtgtaca ctggtgacat gttgctgtat tcttgtgtga atacgctggt 120 gacatattgc tgtattcttg ggcgtgtaca ctggtgacat attgctatgt tcttaggcaa 180 gtacattgtt gacatgttgc tgcattctta ggcaagtacg tgggtgatat attcctgtat 240 tcttggacaa gtacactggt gacatgtagc tgtattcaga ggtgagtaca ctggtgatgt 300 attgctgtat tctagggtga gtacactgtt gaaatgttgc tgtattctta ggtgagtaca 360 ctggtgacat attyctatat tcttgttctt cgtgtctagc aactcataca tgtttaccag 420 aatattccta aaggttcatt ttcaccatca attctaccca aaactcggtt agccctttta 480 acaggcagat tcagcttttc ctttgtttca ggaaattttc tttttttgtg cttaatcacg 540 gcctctcctc catctacctc ttttcctccc cctgaaactc ctatgttatt tgcacctgat 600 gtcctgggtc tgttttcaaa tcttttctct catgttttca atttctttgt attcctgtca 660 attcaagatt tttcttctac ttaatctttg aggccattaa tttgaatctt aatgatcacc 720 ttcaattcat ttgcaaccgt ttttcagtag gctttatttt ttggaacaat ttctgcttca 780 cagcaaaatt aagcagaaag tgcaaagagc tcccataacc acctgacccc acacatgcac 840 agcctctcct actatcagca tgccacacct actatcaaca tgccacacca gagcagtaca 900 ttgcttacaa tcaatgggcc cgtgtggaca catcataatc accccaagtc cattgtccac 960 attggagtta acattccgtg ttgtacattt ttttggattt tgataagtat aatgggaaga 1020 ggacagacac tgatcttcac tgtgttctgt ggctctttgt ggtccaagtt tttcttcaga 1080 cccatcacat tCcaatcttc tcccagacca tggtctccaa tgctgttacc caagttctat 1140 cccacCCdga gtttcaagtg aagcctaaaa ccttatCCaC aaccttaCga cctctctgcc 1200 cactgtgctg cagagcagag gctgaaatgg gttggagtga aag 1243 <210> 31 <211> 764 <212> DNA <213> Homo sapiens <400> 31 ggcagaggag aaggggagga gcgcgattgc gcccgggatg ggttgccaga ccagctgggg 60 cggtggtggt ccagaggccc gaggtcggcg ggacctgatc gaaggcagcg ccgcgtcgac 120 caccccggga gccggacgCt tgggagcccc agcccggcag cggcgcccgg tcactgaagt 180 tgcgcccca ctcccagccg cctccaagct tctcgagcta agtttcctga cccctccaag 240 ggagtctcac agagctcggt ggccctcggc cttgccaacg tcactttaac tgtttggaac 300 tcgtgagcaa gaaccgagaa gtggagagcc cagccgggga gttttcagct tttctgtttc 360 acttcgggct tcttctattc aaatggctct gcgctggcca ccgaatcctg aatgaggcgg 420 ggctcctctg ccccaactcc agcagcggga acttggttcc cctgggcagc cggggcaggg 480 ggcgccaagg ccgtggcgat aatgaaggct gagacggcca aggccagcgg gtcggcgcgg 540 ggcactctcg ggccggagtg gccatcggcc ggagttcagg aggtctgtga caagcaggga 600 acaaggcaac ggacggcgce rcccagcccc ggctgacgga cgctggcgac tcagacatgg 660 acagtagctg ccacaacgcg actaccaaaa tgttagcgac tgctccagct cggggcaaca 720 tgatgagcac gtccaaacCC ttggctttct ccattgaacg aatc 764 <210> 32 <211> 486 <212> DNA <213> Homo sapiens <400> 32 ggaggcagag ttcggqgaaa gcgtcggagt tcgggagacc agggtccagc atgggtttca 60 gcacagcaga cggcgggggC ggcccaggcg cccgggatct ggaatctctt gatgcctgta 120 tccagaggac gctctctgcc ttgtacccac cgtttgaagc cacggcagcc acggtgctct 180 ggcagctgtt cagcgtggcc ggaggtgcc acggtgggga cgggctgcac tgcctcacca 240 gcttcctcct cccagccaag agggccctgc agcacCtgca gcaggaagcc tgtgccaggt 300 acagggytct ggtcttcctg cacccaggct ggccgctgtg cgcccatgag aggtggtgg 360 tgcagctggc gtccctgcac ggagtcaggc tccgcccgg ggacttctac ctgcaggtca 420 cgtcggcggg gaagcagtca gctagactgg tcttgaaatg cctgtcccgg ctgggaagag 480 gcacag 486 <210> 33 <211> 1238 <212> DNA <213> Homo sapiens <400> 33 ccccgcgtcc gcacctggcc aggtccaaag tattaaagga tggataggat gttaggtaaa 50 gatacaaagt tcaatttgtg gagatgcata gtaacttcca caggcatcaa gtggaagagt 120 gagaatgggt cgtaatgtta gtttgttact cagcagatgc cagctgtttt aattatacat 180 aaacgctact ggcagtaaag ggagagcttg aacagatgtc cacgtgaaac tccagggaga 240 ggagcatggg agtcagagtc agttacctga cctcaCtgag cctgtttctc ctgtgaaatg 300 ggtaatgagg ctgcttactc acagtggtgg caagactcag agatggttac cacctgcaca 360 gcatttagga ctctggagaa gtgtttgtga gccdttttgg aggggtgaac ctttgtcctt 420 caagaggggc tggatttttg gcaggacctg aagaaccaag gatgaccgca cagtcacaag 480 ctgtctccct gggctcaagg tggctcccac tgagggagg ggacggaggt atcagccagt 540 gcatcaggac ctggggtcgt cactcccaag gggccattac cctgttcagt ctccgtggcc 600 actctggggg agggaggtaa acctttacag gtaaggccca gagtgaggcc cagagacaga 660 gtcatttgtg agcacgccag gctgatgagc ggcaggggga aaattcaaat ctggggaggg 720 tctgacccca aagtccaaca tctctggagc ctcctgccca tgtcaggtgt ttggattaat 780 gggatatccC aqaaatagtg tgtgcagcct cccaggggac aacttctgct gtcagccacc 840 cagaCCagtC agccgcggag agcagcagcc tgcagatggg acaccagtgC tgagtgggac 900 aggtgCtggC ttggccttgg gatgtcacat gcataccctc ccagtggacg tgaggattcc 950 aggggctcat gggatctgcc tgctgcaccc acaggtgtgg caggcgtgct tgtgggacac 1020 ccgtttgaca cggtcaaggt gagtctcatc gctgcttttt tttcctcggc gcgtacattg 1080 gagagaggct cacagggttg gggtggcttg gaagcctgtt tccgtgtaca gccccaggtg 1140 ggcagcttgc ttttacacca ggccgggttg aaccttcctc actgctttgt cctggcatct 1200 cccagctggg gctgatccac atgctgggtt catggcca 1238 <210> 34 <211> 1205 <212> DNA <213> Homo sapiens <400> 34 ttgcaaaatt aaaaaaaaat Ctcaacagta cagcatgttc tttatatatt atctgaaaga 60 taattttcag aaaaaggtra aacaatgact tgcaccaaga tattaaaata cacaactctt 120 aaagatttta ttttacacat rtgatagaag ggaactaggc agatgttaga aatagtttaa 180 aggaaaagtg aaaacaatac aaatttatat ggagtaaagg aattttgaaa tgagttgcaa 240 atggaaagaa aactttttta tttatttatt ttcaaatttt ttacaggaga aagaagccag 300 taaaaatCaC tactagacag ggcagaagat agatagatag atagatagat agataqatag 360 atcgatctat gtctatatat ctccatcagt tacctgcaat ttgcaaagaa ttgtaaaata 420 gttcaaagac aatgaacaac ccagaagtat gtgttacagt tttccattga aatacatttt 480 ttaaacatat ctaataggta tgtcttaact agcgaattCa caccactctt cagtgagagg 540 dctdtttatt gatcatctgc ctgtgtgttg caggttgctg tctacctttt tcaaatttga 600 agcaaagatt ttcattaaaa gattttcact agaattaatt aaaaatcaaa gcccaaatca 660 aaacagaata cacagcaagc tgtgctagtg acatggatga caacttctcc tggggattac 720 aactctcagg gtgacatccg tgtagatgat tctgtaactg ttaaaatgaa aaactcccac 780 cctgtgggaa cagagccggg tgagccctgg cttccacaca gtgccaccct gagaaggcga 840 ggkctcccca gcgtctgtct gcagtgcagc cagggcrgag gaatgaagtg tcacagcagg 900 aagcagatgg ctgcatttgc agataatcaa tctagagact tgcagccctg agtttcaggg 960 gaacttgtct aagtagcatc ctgtcgctgg aaggcatcta atgaactaag ttactggtgt 1020 tcttgcttgt cagatagccc tggaacactg tctggatttt ataatcattt tcttgagatt 1080 gacaaagtct aaattcttgc tgatcattga cgagtctaag ttgtaaagaa tgctacccat 1140 ggatggaact ttttgcttaa acttaagaaa gggaggagaa ataacagcag cggtgccccg 1200 tgaag 1205 <210> 35 <211> 1414 <212> DNA <213> Homo sapiens <400> 35 cgcgtccgct gggagctcag gaaggaagga gcgcccagaa gcagggacag ggagctggtt 60 ggggaggacC agaaatcagg ttatcaatac tctggctgac catcatcatc gtgggactga 120 ctttggtgga agtccttggt tacatgtcat tattgcgttt ccgacaagtt ataaagttgt 180 cattaccctc tggatagttt acctttgggt gtctctcctg aagactatct tctggtctcg 240 aaatggacat gatggatcca cggatgtaca gcagagagcc tggaggtcca accgccgtag 300 acaggaaggg ctgaggtcca tttgtatgca cacaaagaaa agagtttctt cctttcgagg 360 aaataaaatt ggcctgaaag acgtcattac tctacggaga catgtggaaa caaaagttag 420 agctaaaatc cgtaagagga aggtgacaac gaaaatcaac catcatgaca aaatcaatgg 480 aaagaggaag accgccagaa aacagaaaat gtttcaacgt gcgcaagagt tgcggcggcg 540 rgcagaggac taccacaaat gcaaaatccc cccttctgca agaaaggctc tttgcaactg 600 ggtcagaatg gcggcagcgg agcatcgtca ttcttcagga ttgccctact ggccctacct 660 cacagctgaa actttaaaaa acaggatggg ccaccagcca cctcctccaa ctcaacaaca 720 ttctataact gataactccc tgagCctCaa gacacctccc gagtgtctgC tcactcccct 780 tccaccctca gcggatgata atctcaagac acctcccgag tgtgtgctca ctccccttcc 840 accctcagcg gatgataatc tcaagacacc tcccgagtgt gtgctcactc cccttccacc 900 ctcagcggat gataatctca agacacctcc tgagtgtctg ctcactcccC ttccaccctc 960 agcggatgat aatctcaaga cacctcccga gtgtctactc actccccttc caccctcagc 1020 tctaccctCa gctccaccCt cagcggatga taatctcaag acacgtgccg agtgtctgct 1080 ccatcccctt ccaccctcag cggatgataa tctcaagaca ccttccgagc gtcagctcac 1140 tccccttcca ccctcagctc caccctcagc agatgataat atcaagacac ctgccgagcg 1200 tctgcggggg ccgcttcCaC cctcagcgga tgataatctc aagacacctt ccgagcgtca 1260 ctcactccc cttccaccct cagctccacc ctcagcagat gataatatca agacacctgc 1320 cgagcgtctg cgggggccgc ttccaccctc agcggatgat aatctcaaga caccttccga 1380 gcgtcagctc actccccttc caccctcagc tcca 1414 <210> 36 <211> 1218 <212> DNA <213> Honlo sapiens <400> 36 gagacggagt ctcgctctgt cacccaggct ggagtgcagt ggcgggatct cggctcactg 60 caagctccgc ctcccgggtt cacgccattc tcctgcctca gcctcccaag tagctgggac 120 tacaggcgcc cgccactacg cccggctaat tttttgtatt tttagtagag acggggtttc 180 accgttttag ccgggatggt ctcgatctcc tgacctCgtg atccgcccgc cctcggcctc 240 ccaaagtgct gggattacag gcgtgagcca ctgcgcccgg ccacatttca cttcttaagt 300 cttctgtgtt tttgggtatc aaatattccc ggagagatgc tcttgaggat ctaagatcca 360 gctgtgggat gaggtgtact tcccaccctg ccacaatcac tgggcctgcc cagacgggca 420 gaggccctgt gcgccccacc tgcctctctC acgtggactc tgggggtcag agctgggtgg 480 ggtgtgccgc gtgtgggtcc tgagtggcca gggcagggtc ogcagcaCag gaagctgccc 540 agggggtcct tgcaagcgtg ggctctggcC agcgtctggg ggaggctgtg ctaggcgggg 600 cctcccgtgg gcatgtccct ggagctcaca ggctggcgcc ctatgcccat ctccagatag 660 (0( cctgggctgg aagctcttCt acgtcacagg ctgcCtgttt gtggctgtgc araacttgga 720 ggactgggag gtaaggccgg ctcgggtgcg ggacagagtc cagggctgtt cagctcctgg 780 gttttttgCd atgggaatga aaggaggagg aagggccctg ggtggcctag cgcctCccCg 840 tcctgaagcg ttggtccctg cttggaggtC tccgttcatc aggacatggc ccctgcactc 900 atctgggaCC gttcttggcc aaggaattcC ccgdaggcat ttttctctta gaagctctcc 960 atgactatCt tcaccaaagt gctttcttCC cagagttgcc acaatgggat gcgagtcagc 1020 tttccccgtg gccggccctc ccacctcgga gcccctcatg agtcctttca gcctggccca 1080 gtgctgccCt ctgacCtCCa tgccctcgtt tgctggttcc actgcctccc tgcacttgtt 1140 ttgcctgcag gggtggagCd agcgcctgct gcacctgCCC acctctccat ttcccaacag 1200 gagtcgggtt ggctgccg 1218 <210> 37 <211> 2588 <212> DNA <213> Honio sapiens <400> 37 ggaagaatgt taaccccaga ggcaacaaaa gaaattaaat tagtggaaga aaaaattcag 60 tcagcgcaaa taaatagaat agatccctta gccccactcc arcttttgat ttttgccact 120 gcacattctc caacaggcat cattattcaa aatactgatc ttgtggagtg gtcattcctt 180 cctcacagta cagttaagaC ttttacaytg tacttqgtc aaatrgctac attaatyggt 240 cagacaagat tacgaataat aaaattatgt ggaaatgacc magacaaaat agttgtccct 300 ttaaccaagg aacaagttag acaagccttt atcaattCtg gtgcatggca gattggtctt 360 gctaattttg tgggaattat tgataatcat tacccaaaaa caaagatCtt ccagttctta 420 aaattgacta cttggattct aCctaaaatw accagacgtg aaCctttaga aaatgctcta 480 acagtattta ctgatggttc cagcaatgga aaagcagctt acacagggcc gaaagaacga 540 gtaatcaaaa ctcctatCa atcggctcaa agagCagagt tggttgcagt cattacagtg 600 ttacaagatt ttgaccaacc tatcaatatt atatcagatt ctgcctatgt agtcaggct 660 acaagggatg ttgagaCrgc tctaattaaa tatagcatgg atgatcagtt aaaccagcta 720 ttcaetttat tacaacaaac tgtaagaaaa agaaatttcc cattttatat tactcatatt 780 cragcacaca ctaatttacc agggcctttg actaaagcaa atgaacaagc tgacttactg 840 gtatcatctg cactcataaa agcacaagaa cttcatgctt tgactcatgt aaatgcagca 900 ggattaaaaa acaaatttga tgtcacatgg aacaggcaa aagatattgt acaacattgc 960 acccagtgtc aagtcttaca cctgcccact caagaggcag gagttaatcc cagaggtctg 1020 tgtcctaatg cattatggca aatggatgtc acgcatgtac cttcatttgg aagattatca 1080 tatgttcatg taacagttga tacttattca catttcatat gggcaacttg ccaaacagga 1140 gaaagtactt cccatgttaa daaacattta ttgtcttgtt ttgctgtaat gggagttcca 1200 gaaaaaatca aaactgacaa tggaccagga tattgtagta aagctttcca aaaattctta 1260 agtcagtgga aaatttcaca tacaacagga attccttata attcccaagg acaggccata 1320 gttgaaagaa ctaatagaac actcaaaact caattagtta aacaaaaaga agggggagac 1380 agtaaggagt gtaccactcc tCagatgcaa cttaatctag cactctatac tttaaatttt 1440 ttaaacattt atagaaatCa gactactact tctgcagaac aacatcttac tggtaaaaag 1500 aacagcccac atgaaggaaa actaatttgg tggaaagata ataaaaataa gacatgggaa 1560 atagggadgg tgataaCgtg ggggagaggt tttgcttgtg tttcaccagg agaaaatcag 1620 cttcctgttt ggatacCcaC tagacatttg aagttctaca atgaacccat cggagatgca 1680 aagaaaaggg cctccgcgga gatggtaaca ccagtcacat ggatggataa tcctatagaa 1740 gtatatgtta atgatagCga atgggtacct ggccccacag atgatcgctg ccctgccaaa 1800 cctgaggaag aagggatgat gataaatatt,'tccattgggt atcgttatcc tcctatttgc 1860 ttagggacag cac&aggatg tttaatgcct gcagtccaaa attggttggt agaagtacct 1920 attgtcagtC'CGatcagtag attcacttat cacatggtaa gcgggatgtc actcaggcca 1980 cgggtaaatt atttacaaga ctttycttat caaagatcat taaaatttag acctaaaggg 2040 aaaccttgcc ccaaggaaat tcccaaagaa tcaaaaaata cagaagtttt agtttgggaa 2100 gaatgtgtgg ccaatagtgc ggtgatatta caaaacaatg aattcggaac tattatagat 2160 tgggcacctc gaggtcaatt ctaccacaat tgctcaggac aaactcagtc rtgtccaagt 2220 gcacaagtga gtccagctgt tgatagcgac ttaacagaaa gtttagacaa acataagcat 2280 aaaaaattgC agtctttsta cccttgggaa tggggagaaa aaggaatctc taccccaaga 2340 ccaaaaatar taagtcctgt ttCtggtCCt gaacatccag aattatggag gcttaytgtg 2400 gcctcacacc acattagaat ttggtctgga aatcaaactt cagaaacaag agatcgtaag 2460 ccattttata ctatcgacct aaattccagt ctaacggttc ctttacagag ttgcgtaaag 2520 cccccttata tgctagttgt aggaaatata gttattaaac cagactccca aactataacc 2580 tgtgaaaa 2588 <210> 38 <211> 1863 <212> DNA <213> Homo sapiens <400> 38 cccacgcgtc cgtggtctct tcacatgqac gtgcatgaaa tttggtgccg tgactcagat 60 tgggggacct cccttcggag atcaatcccc tgtcctcctg ctctttgctc cgtgagaaag 120 (oJ-atccacctac gacctcaggt cctcagaccg accagcccaa gaaacatctc accaatttca 180 aatccagact ccactggaaa tcggactgtt caactcacct ggcagccact cccagagccc 240 ctggaactct ggcccaaggc tctctgactg actccttctt ggcttagcgg ctgaagactg 300 atgctgcctg atcgcctcgg aagccccgta gaccatcacg gatgccgagc tttaggtaac 360 tctcacagcg gaaggtatac gcccagatgg cctgaactaa ctgaagaatc acaaaagaag 420 tgaaaatgcc ctgccccacc ttaactgatg acattccacc acaaaagaag tgtaaatggc 480 cggtccttyC cttaagtgat gacattacct tgtgaaagtc cttttcctgg ctcatcctgg 540 ctcaaaaagC acccccactg agcaccttgc gacccccrnct Cctrcycgcc agagaacaaa 600 ccccctttga ctgtaatttt cctttaccta mccaaatcct ataaaacggc cyyaccctta 660 tctcccttcg ctgactctct tttcggacty agcccgcctg Cacccaggtg aaataaacag 720 cctcgttgct cacacaaagc ctgtttggtg gtctcttcac acggacgcgc atgaaatttg 780 gtgccgtgaC tcggatcggg ggacctccct tgggagatCa atcccctgtc ctcctgctct 840 ttgctccgtg agaaagatcc acctacgacc tcaggtcctc agaccaacca gcccaagaaa 900 catctcacca atttcaaatc cggaacttgc tacacatgcc ggaaatctgg ccactgggcc 960 aagqaacgcc cgcagcccgg gattcctcct aagccgcgtc ccatctgtgt gggaccccac 1020 tgaaatcgg actgttcaac tcacctggca gccactccca gagctcctgg aactctggcc 1080 caaggttctc tgactgctc cttcttggct tactggctga agactgacgc tgcctgatcg 1140 cctcagaagc cccgcagacc atcatggacg ccgagcttta gcccgcctgc acccaggtga 1200 aataaacagc cttgttgctc acacaaagcc tgtttggtgg tctcttcaca cagacgcgca 1260 tgaaagggaa gacatacada aacaaggcct ctgaggtagg tactactgag acagccaggt 1320 gggaaggact ccttggcaaa actccaacca gccwgtgcac attcctccca gtgtacaggc 1380 tggttggaat gtgcactggg atggagccat ataagtttgt gtcgtttgca gtggggagga 1440 gcctggtccc tcctcttcct gtgaggaacc tggaattcaa tctgtgaggt tgttctggag 1500 atgttctggg gagactgcat taaacacagc ttcgcaccat tgaataaact cagcaacaag 1560 ccaatgcata aaagtaatct atgcttcagg tcacagaagc ttcaagggga aaaaaacaga 1620 atactctagg gccattgttc acaaactcat ctgaaaacat cctggaaaaa ttttcccaaa 1680 cacatggaaa gaaagagagg aaaaaagaag atatctgaat aatgtggact agaataaaga 1740 gctgccagga gctgtttatt taaaaacagt actttcttct ctggctgagt ccctggtatt 1800 ctctgctgca atctgtagct gtagaatttt gaaaaatgca attaaattca aatggtttga 1860 tga 1863 <210> 39 <211> 717 <212> DNA <213> Home sapiens <400> 39 tcgacccacg cgtccgggcg gccgggaggg acgcggagcc acagcccgac gcacggacgg 60 agggacgccg gagcccgcct gaccatgtgg aagctgggcc ggggccgagt gctgctggac 120 gagccccccg aggaggagga cggcctgcgt ggggggccgc caccggccgc cgccgccgcc 180 gcccaggcgc aggttcaggg agcaagtttc cgaggttgga aagaagtgac ttcactgttt 240 aacaaagatg atgagcagca tctcctggaa agatgtaaat ctcccaagtc caaaggaact 300 aacttacgat taaaagaaga gttgaaggca gagaagaaat ctggattttg ggacaatttg 360 gttttaaaac agaatataca gtctaaaaaa ccagatgaaa ttgaaggttg ggagcctcca 420 aaacttgctC ttgaagacat atcggctgac cctgaggaca ccgtgggtgg ccacccatcc 480 tggtcaggct gggaggatga cgccaagggC tcgaccaagt acaccagcct ggccagctct 540 gccaacagct ccaggtggag cctgcgcgcg gcagggaggc tggtgagcat ccgacggcag 600 agtaaaggcC acctgacaga tagcccggag gaggcggagt gaggggggct gtgtggcaag 660 tgtgccccgacatggtggCC ttttatgagt ataccatgta gttgttgagt cttttcc 717 (o,

Claims

CLAIMS

1. A method for assessing a culture of undifferentiated primate pluripotent stem (pPS) cells or their progeny, comprising detecting or measuring expression of three or more of the markers in any of

Tables 5 to 9.

2. The method of claim 1, comprising measuring expression of three or more of the markers in Tables 2, 7, and 9(C), and correlating the expression measured with the presence of undifferentiated embryonic stem (ES) cells in the culture.

3. The method of claim 1, comprising measuring expression of three or more of the markers in Tables 3 and 8, and correlating the expression measured with the presence of differentiated cells in the culture.

4. The method of claim 1, comprising detecting or measuring expression of one or more of the following markers: bone marrow stromal antigen; Podocalyxin-like; Rat GPC/ glypican-2 (cerebroglycan); Potassium channel subfamily k member 5 (TASK-2); Notch 1 protein; Teratocarcinoma-deriVed growth factor 1 (Cripto); Nel 1 like I NELL2 (Nel-like protein 2): Gastrin releasing peptide receptor; Bone morphogenetic protein; ABCG2-ABC transporter, Solute carrier family 6, member 8 (SLC6A8); hTERT; Oct 3/4 Octamer-binding transcription factor 3a (Oct-3a) (Oct-4); Left-right determination factor b (LEFT); Secreted phosphoprotein 1 (osteopontin); Gamma-aminobutyiic acid (GABA) A receptor, beta 3; Roundabout, axon guidance receptor, homologue 1 (ROBO1),; Glucagon receptor; Leucine-rich ppr-motif hum 130 kDa huml30leu 1 3Okd eu; Thy-i co-transcribed; Solute carrier family 21; LY6H lymphocyte antigen 6 complex locus H; Plexin (PLXNB3); Armadillo repeat protein deleted in velo-cardio-facial syndrome; and Ephrin type-a receptor 1 (EPHA1).

5. The method of claim 1, comprising detecting or measuring expression of three or more of the markers in any of Tables 2, 7, and 9(C), and two or more of the markers in any of Tables 3 and 8 6. The method of claim 1 further comprising detecting or measuring expression of hTERT and/or Oct 3/4.

7. A method for assessing a culture of undifferentiated human embryonic stem (hES) cells or their progeny, comprising detecting or measuring two or more markers selected from Cripto, gastrin-releasing peptide (GAP) receptor, podocalyxin-tike protein (PODXL), and human telomerase reverse transcriptase (hTERT).

8. The method of claim 7, further comprising detecting or measuring one or more markers selected from Oct 3/4, SSEA-3, SSEA-4, Tra- 1 -60 and Tra-i -81.

9. The method of claim 7, comprising detecting or measuring Cripto, hTERT, and Oct 3/4.

10. A method for assessing a culture of undifferentiated human embryonic stem (hES) cells or their progeny, comprising detecting or measuring two or more markers preferentially expressed in undifferentiated hES cells, and one or more markers expressed preferentially after differentiation of the hES cells.

11. The method of claim 10, wherein at least one of the markers preferentially expressed in undifferentiated hES cells is selected from Cnpto, gastrin-releasing peptide (GRP) receptor, podocalyxin-like protein (PODXL), and human telomerase reverse transcriptase (hTERT).

12. The method of claim 11, wherein at least one of the markers preferentially expressed in undifferentiated hES cells is selected from Oct 3/4, SSEA-4, Tra-1-60 and Tra-1-81.

13. The method of claim 10, comprising detecting or measuring hTERT, Oct 3/4, and a marker selected from Cnpto, SSEA-4, Tra-1-60 and Tra-1-81.

14. The method of claim 10, wherein at least one of the markers expressed preferentially after differentiation of the hES cells is a stromal cell markers.

15. The method of claim 14, wherein the stromal cell marker is selected from CD44, CD1O5 (endoglin), CD1 06 (VCAM-1), CD9O (Thy-i), STRO-1, Vimentin, and Human Thymus Stroma.

16. The method of claim 10, wherein expression of hTERT, Oct 3/4, Cripto, GAP receptor, PODXL, CD44, CD1O5, CD1O6, or CD9O is detected or measured at the rnRNA level by PCR amplification.

17. The method of claim 10, wherein expression of SSEA-4, Tra-1-60, Tra-1-81, Cripto, Oct 3/4, CD44, CD1 05, CD1 06, CD9O, STRO-1, Vimentin, or Human Thymus Stroma is detected or measured at the antigen expression level by antibody assay.

18. A kit for assessing a culture of pPS cells according to claim 1, comprising polynucleotide probes and/or primers for specifically amplifying a transcript for three or more markers in any of Tables 5 to 9, accompanied by written instructions for assessing the pPS cells according to the expression of said markers measured using the probes or primers in the kit.

19. A kit for assessing a culture of pPS cells according to claim 1, comprising an antibody specific for the gene product of three or more markers in any of Tables 5 to 9, accompanied by written instructions for assessing the pPS cells according to the expression of said markers measured using the antibody in the kit.

20. A kit for assessing a culture of undifferentiated human embryonic stem (hES) cells or their progeny according to claim 10, comprising antibody or PCR amplification primers specific for three or more markers, of which at least two are expressed preferentially in undrfferentiated hES cells, and at least one is expressed preferentially in stromal cells.

21. The kit of claim 20, comprising antibody or PCA amplification primers specific for at least two markers selected from Cripto, gastrin-releasing peptide (GRP) receptor, podocalyxin-like protein (PODXL), human telomerase reverse transcnptase (hTERT) Oct 3/4, SSEA-4, Tra-1-60, Tra-1-81, CD44, CD1O5 (endoglin), 00105 (VCAM-1), CD9O (Thy-i), STRO-1, Vimentin, and Human Thymus Stroma.

22. Use of antibody or PCR amplification primers specific for three or more markers selected from Cripto, gastrin-releasing peptide (GRP) receptor, podocalyxin-like protein (PODXL), human telomerase reverse transcriptase (hTERT) Oct 3/4, SSEA-4, Tra-1-60, Tra-1-81, CD44. CD1O5 (endoglin), CD1O6 (VCAM-1), CD9O (Thy-i), STRO-1, Vimentin, and Human Thymus Stroma, for assessing a culture of undifferentiated human embryonic stem (hES) cells or their progeny.

23. The method of claim 1, which is a method for quantifying the proportion of undifferentiated pPS cells or differentiated cells in the culture.

24. The method of claim 1, which is a method br assessing the ability of a culture system or component thereof to maintain pPS cells in an undifferentiated state.

25. The method of claim 24, which is a method of assessing the ability of a soluble factor to maintain pPS cells in an undifferentiated state.

26. The method of claim 24, which is a method of assessing the ability of a culture medium to maintain pPS cells in an undifferentiated state.

27. The method of claim 24, which is a method of assessing the ability of a preparation of feeder cells to maintain pPS cells in an unditterentiated state.

28. The method of claim 1, which is a method for assessing the ability of a culture system or component thereof to cause differentiation of pPS cells into a culture of lineage-restricted precursor cells and/or terminally differentiated cells.

29. The method of claim 1, which is a method for assessing the suitability of a pPS cell culture for preparing cells for human administration.

30. The method of claim 1, wherein the level of the markers of undifferentiated hES cell markers is determined to be at least 100-fold higher than in BJ fibroblasts.

31. A method for assessing the growth characteristics of a cell population, comprising detecting or measuring expression of three or more of the markers in any of Tables 5 to 9.

32. A method for assessing the growth characteristics of a cell population,, comprising detecting or measuring two or more markers selected from Cripto, gastrin-releasing peptide (GAP) receptor, podocalyxin-like protein (PODXL), and human telomerase reverse transcriptase (hTERT).

33. The method of claim 32, wherein the cell population has been obtained by culturing cells from a human blastocyst.

34. The method of claim 32, which is a method for determining whether the cell population is pluripotent.

35. The method of claim 31, wherein the cell population has been obtained from a human patient suspected of having a clinical condition related to abnormal cell growth.

36. The method of claim 31, which is a method for assessing whether the patient has a malignancy.

37. A method for maintaining pPS cells in a pluripotent state, comprising causing them to express one of the following markers at a higher level.

Forkhead box O1A (FOXO1A); Zic family member 3 (ZIC3); Hypothetical protein FU20582; Forkhead box Hi (FOXHI); Zinc finger protein, Hsal2; KRAB-zinc finger protein SZF1-1; and Zinc finger protein of cerebellum ZIC2.

38. The method of claim 37, wherein the cells are caused to express the marker by genetically altering it with a gene that encodes the marker.

39. A method for causing pPS cells to differentiate into a particular tissue type, comprising causing them to express one of the following markers at an altered level.

* Forkhead box O1A (FOXO1A); Zic family member 3 (ZIC3); Hypothetical protein FLJ20582; Forkhead box Hi (FOXH1); Zinc finger protein, Hsal2; KRAB-zinc finger protein SZF1 -1; Zinc finger protein of cerebellum ZIC2; and Coup transcription factor 2 (COUP-TF2).

40. The method of claim 39, wherein the cells are caused to express the marker by genetically altering it with a gene that encodes the marker, or with an antisense nucleic acid that binds to mANA encoding the marker.

41. A method for causing an encoding sequence to be preferentially expressed in undifferentiated pPS cells, comprising genetically altering pPS cells with the encoding sequence under control of a promoter for one of the markers listed in any of Tables 2, 7, and 9(C).

42. The method of claim 41, which is a method for selecting undifferentiated cells, and the encoding sequence is a reporter gene (such as a gene that causes the cells to emit fluorescence), or a positive selection marker (such as a drug resistance gene).

43. The method ot claim 41, which is a method for depleting undifferentiated cells from a population of differentiated cells, and the encoding sequence is a negative selection marker (such as a gene that activates apoptosis or converts a prodrug into a compound that is lethal to the cell).

44. A method for causing an encoding sequence to oe preferentially expressed in differentiated cells, comprising genetically altering the pPS cells with the encoding sequence under control of a promoter for one of the markers listed in Table 3 or Table 8.

45. The method of claim 44, which is a method for selecting differentiated cells, and the encoding sequence is a reporter gene (such as a gene that causes the cells to emit fluorescence), or a positive selection marker (such as a drug resistance gene).

46. The method of claim 44, which is a method for depleting differentiated cells from a population of undifferentiated cells, and the encoding sequence is a negative selection marker (such as a gene that activates apoptosis or converts a prodrug into a compound that is lethal to the cell).

47. A method for sorting differentiated cells from less differentiated cells, comprising separating cells expressing a surface marker in any of Tables 5 to 9 from cells not expressing the marker.

48. The method of claim 47, wherein the cetls are sorted using an antibody or lectin that binds the marker or product thereof on the cell surface.

49. A method for causing pPS cells to proliferate without differentiation, comprising culturing them in a culture system assessed according to the method of claim 7.

50. A method for causing pPS cells to proliferate without differentiation, comprising culturing them with human mesenchymal stem cells.

51. A method for identifying genes that are up-or down-regulated during differentiation of pPS cells, comprising: a) sequencing transcripts in an expression library from undifferentiated pPS cells; b) sequencing transcripts in one or more expression libraries Irom one or more cell types that have differentiated from the same line of pPS cells; C) determining the frequency of transcripts from each gene sequenced in each of the libraries; d) identifying the gene as being up- or down-regulated during differentiation of the pPS cells if the frequency of transcripts in the library from the undifferentiated pPS cells is different from the frequency of transcripts in one or more libraries from the differentiated cell types at a statistical probability of at least 95%.

52. The method of claim 51, further comprising assessing a culture of pPS cells depending on the expression level measured in cells from the culture of the marker identified in d).

53. The method of any of claims 1-52, wherein the pPS cells are obtained from a human blastocyst, or are the progeny of such cells.

54. The method of claim 53, wherein the pPS cells are human embryonic stem cells. (OCI

Amendments to claims have been filed as follows *1

CLAIMS

1. A method for assessing a culture of undifferentiated primate pluripotent stem (pPS) cells, comprising detecting or measuring expression of podocalyxin-like protein (PODXL).

2. The method of claim 1, wherein the method further comprises measuring or detecting a marker in any one of Tables 5 to 9.

3. The method of claim 1 or claim 2, wherein the podocalyxin-like protein (PODXL) expression level is measured at the mRNA level.

4. The method of claim 3, wherein the podocalyxin-like protein (PODXL) expression level is measured at the mRNA level by PCR amplification.

5. The method of claim 1 or claim 2, wherein the podocalyxin-like protein (PODXL) expression level is measured at the protein level. I... * * ***.

6. The method of claim 5, wherein the podocalyxin-like protein (PODXL) expression * level is measured at the protein level by antibody assay. S. S * .* * **

** 7. The method of claim 1 or claim 2, wherein the podocalyxin-like protein (PODXL) expression level is measured using flow cytometry.

8. A method for assessing a culture of undifferentiated primate pluripotent stem (pPS) cells substantially as hereinbefore described with reference to the Examples.