US 2011 0306129A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0306129 A1 Nistor (43) Pub. Date: Dec. 15, 2011
(54) STEM CELL GROWTH MEDIA AND Publication Classification METHODS OF MAKING AND USING SAME (51) Int. Cl. (76) Inventor: Gabriel Nistor, Placentia, CA (US) CI2N 5/0735 (2010.01) (21) Appl. No.: 13/151,163 (52) U.S. Cl...... 435/366 (22) Filed: Jun. 1, 2011 (57) ABSTRACT Related U.S. Application Data The invention provides media formulations. A complete media formulation of the invention includes, for example, the (63) typics, S; 3. 69,120, filed on following components: albumin, an iron carrier, glutamine, a • - s s vs. - V - - - glycosidase or hydrolase, fibroblast growth factor (FGF), a (60) Provisional application No. 60/883,281, filed on Jan. salt or mineral, and essential amino acids at an osmolarity of 3, 2007. about 220-330 mOsm/Liter.
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an Cassic, conditioned media (CM) unting tassic conditioned media with hyalurodase aira Nowel media, conditiored on ?troblasts autheat Novemea Patent Application Publication Dec. 15, 2011 Sheet 2 of 15 US 2011/0306129 A1
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Figure 9
Existent Expansion in Differentiation to pluripotent stock ectodermal the target biased media population: neurons, glia
Expansion in Differentiation to Initial derivation mesodermal the targeted biased media population: cardiomyocytes, blood vessels
Expansion in Differentiation to Existing lineage endodermal the targeted biased stem biased media population: Cells hepatocytes, islet Cells Patent Application Publication Dec. 15, 2011 Sheet 14 of 15 US 2011/0306129 A1
Figure 10A
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STEM CELL GROWTH MEDIA AND 0006 Complete and incomplete media formulations METHODS OF MAKING AND USING SAME include formulations with components (ingredients) in amounts compatible with survival or proliferation of cells. STATEMENT OF PRIORITY AND RELATED Exemplary cells include mammalian cells, such as embry APPLICATIONS onic, tissue specific, germinal and adult stem cells. Stem cells include multipotent, totipotent and pluripotent stem cells. In 0001. This application is a continuation of U.S. Utility particular aspects, components are in amounts compatible patent application Ser. No. 1 1/969,120, filed Jan. 3, 2008, with survival or proliferation of stem cells without substantial which claims priority to U.S. Application Ser. No. 60/883, differentiation. 281, filed on Jan. 3, 2007, which is expressly incorporated 0007 Albumin is a particular example of a component of herein by reference as if fully set forth herein. a media formulation of the invention. A non-limiting amount of albumin, is a concentration of about 5 to 100 grams/Liter. INTRODUCTION 0008. An iron carrier is a particular example of a compo 0002 The typical conditions for the growth of the cells nent of a media formulation of the invention. Iron carriers such as stem cells involve a base media (DMEM or DMEM: include transferrin. A non-limiting amount of transferrin, is a FI2) supplemented with serum or serum replacement. Stem concentration of about 5 to 100 ug/ml. cell colonies are grown on mouse embryonic fibroblasts (or 0009 Glutamine is a particular example of a component of fibroblasts from other species, including humans) mitotically a media formulation of the invention. Glutamine can be pro inactivated by treatment with a cytostatic or by irradiation. vided as a peptide, Such as a di-, tri-, tetra-, etc.-peptide. A Alternatively, growth media can be exposed to Supportive non-limiting example of a di-peptide is glutamine-alanine. A cultures, collected, sterile filtered and used to fBCd the stem non-limiting amount of glutamine, is a concentration of about cell cultures. Typically the media is supplemented with basic 10 to 40 mg/Liter. FGF. As a result of exposure to non-human tissue, stem cells 0010. A glycosidase or hydrolase is a particular example or their derivatives cultured as above cannot be used in clini of a component of a media formulation of the invention. A cal, human applications, due to the possibility of Xeno-con particular non-limiting example is a hyaluronidase (e.g., tamination. hyaluronidase type 1). Additional particular non-limiting 0003. Other groups have reported growth of stem cells examples are a glycosidase or hydrolase enzyme set forth in using serum frEC media supplemented with B27 (Invitrogen) Table 1. A non-limiting amount of a glycosidase or hydrolase, and large amounts of bRGF (40-100 ng/ml), or using high is a concentration of about 1 to 100 ug/ml. density cultures in the absence of Supportive cells. A mixture 0011. A fibroblast growth factor (FGF) is a particular of growth factors including neurotrophins has also bECn example of a component of a media formulation of the inven tested with relative success. When low amounts of growth tion. Particular non-limiting examples are basic FGF and factors were used in serum frEC media, a spontaneous differ acidic FGF. Additional particular non-limiting examples are entiation tendency towards ectodermal lineage was observed. FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, Mixtures or high concentrations of growth factors led to FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, increased incidences of chromosomal abnormalities. FGF18, FGF19, FGF20, FGF21, FGF22 and FGF23. A non 0004) Hyaluronic acid (HA) is a nonsulfated linear gly limiting amount of an FGF, is a concentration of about 5 to cosaminoglycan (GAG), one of the principal extra cellular 100 ng/ml. FGF can optionally be produced or provided by matrix components in nearly all tissues involved in the regu feeder cells. lation of cell proliferation, adhesion and migration. Addi 0012. A salt or mineral is a particular example of a com tional GAGs of physiological significance are hyaluronic ponent of a media formulation of the invention. Particular acid, dermatan Sulfate, chondroitin Sulfate, heparin, heparan non-limiting examples are sodium, potassium, calcium, mag sulfate, and keratin sulfate. Each of these GAGs has a pre nesium, copper, manganese, molybdenum, Selenium (e.g., dominant disaccharide. Hyaluronic acid is unique among the Sodium selenite), iron, and zinc. A non-limiting amount of GAGS in that it does not contain any Sulfate and is not found sodium, is a concentration of 130-160 mg/Liter. A non-lim covalently attached to proteins as a proteoglycan. It is, how iting amount of potassium, is a concentration of 3 to 6 mg/Li ever, a component of non-covalently formed complexes with ter. A non-limiting amount of calcium, is a concentration of 7 proteoglycans in the extra-cellular matrix. Hyaluronic acid to 12 mg/Liter. A non-limiting amount of magnesium, is a polymers are very large (with molecular weights of 100,000 concentration of 1 to 4 mg/deciliter. Non-limiting amounts of 10,000,000) and can displace a large volume of water. copper, manganese, molybdenum, selenium, iron, and Zinc, is a concentration of 1 pg/deciliter to 1 lug/deciliter. SUMMARY 0013. One or more essential amino acids is a particular example of a component of a media formulation of the inven 0005. The invention provides media formulations. Media tion. Particular examples are arginine, cystine, histidine, iso formulations may be sterile or non-sterile. In one embodi leucine, leucine, methionine, phenylalanine, threonine, tryp ment, a complete media formulation includes: albumin, an tophan, tyrosine and valine. A non-limiting amount of an iron carrier, glutamine, a glycosidase or hydrolase, fibroblast essential amino acid, is a concentration of about 0.5 to 10 growth factor (FGF), a salt or mineral, and essential amino nmol/Liter. An additional non-limiting amount of an essential acids, at an osmolarity of about 220-330 mOsm/Liter. In amino acid, is a concentration of about 2.5 nmol/Liter. another embodiment, a complete media formulation 0014 Media formulations include formulations having includes: albumin, an iron carrier, glutamine, a glycosami desired or target osmolarities, for example, when the media noglycan degradation product, fibroblast growth factor comprises a liquid. A non-limiting osmolarity is about 240 (FGF), a salt or mineral, and essential amino acids, at an 300 mOsm/Liter. An additional non-limiting osmolarity is osmolarity of about 220-330 mOsm/Liter. about 250-270 mOsm/Liter. US 2011/0306129 A1 Dec. 15, 2011
0015. A globulin is a particular example of a component of example, labels, as well as instructions, for example, instruc a media formulation of the invention. Particular non-limiting tions for maintaining Survival or proliferation of cells (e.g., examples of globulins include alpha, beta and gamma globu stem cells). lins. An additional non-limiting example of a globulin is an 0023 Kits and containers can include a plurality of com antibody (e.g., IgG, IgA, IgM, IgE and Ig|D). A non-limiting plete or incomplete media formulations. A particular non amount of globulin, is a concentration of about 0.1 to 20 limiting example of a kit includes first and second containers, grams/Liter. An additional non-limiting amount of globulin, in which the first container has a media formulation with the is a relative ratio of globulin to albumin of about 1:2, or less following components: an iron carrier, a salt or mineral, and than 1:2. essential amino acids; and the second container has following 0016. Media formulations include formulations that are components: albumin, glutamine, a glycosidase or hydrolase, pH buffered. Particular non-limiting examples of buffering and fibroblast growth factor (FGF). Kits and containers agents are bicarbonates, phosphates, ethanolamines, trietha include media formulations that, when combined, provide a nolamines and trometamols. Particular non-limiting complete media formulation. Kits and containers include examples of pH, are ranges between about 6.8-7.8 when media formulations that, when combined, produce a com present in a 2-20% oxygen environment, in a 5-15% carbon plete media formulation having a desired or target osmolarity, dioxide environment, or in a 0.04-0.06% carbon dioxide and for example, an osmolarity of about 220-330 mOsm/Liter. 20-21% oxygen environment. 0024. The invention provides methods of producing media 0017. One or more energy sources is a particular example formulations. In one embodiment, a complete media formu of a component of a media formulation of the invention. lation is produced by combining albumin, an iron carrier, Particular examples are a mono-saccharide (e.g., glucose) or glutamine, a glycosidase or hydrolase, fibroblast growth fac poly-saccharide. A non-limiting amount of glucose, is a con tor (FGF), a salt or mineral, and essential amino acids. In centration of about 10 to 1000 mg/Liter. An additional non another embodiment, a complete media formulation is pro limiting example of an energy source is pyruvate. duced by combining albumin, an iron carrier, glutamine, a glycosaminoglycan degradation product, fibroblast growth 0018. Additional components of a media formulation of the invention include, for example, one or more of a non factor (FGF), a salt or mineral, and essential amino acids. essential amino acid, a hormone, a growth factor, Vitamin, Such complete media formulations, when combined, have a heparin, heparin Sulfate or a glycosaminoglycan degradation desired or target osmolarity, for example, in a non-limiting product. Particular examples of non-essential amino acids are example, the media formulation is a liquid that has an osmo alanine, asparagine, aspartate, glutamine, glycine, proline larity of about 220-330 mOsm/Liter. and serine. Particular examples of hormones are insulin, insu 0025. The invention provides cell cultures that include a lin-like growth factor, a thyroid hormone (e.g., thyroxine (T4) complete media formulation of the invention. Non-limiting and triiodothyronine (T3)), and a progesterone. A non-limit examples of cell cultures include mammalian cells, for ing amount of insulin or insulin-like growth factor, is a con example, mammalian primary, secondary or passaged cells, centration between about 5 to 40 ug/ml. A non-limiting and immortalized cells. Additional non-limiting examples of amount of thyroid hormone, is a concentration between about cell cultures include mammalian stem cells, for example, 5 to 40 ng/ml. Particular examples of glycosaminoglycan embryonic, tissue specific, germinal and adult stem cells. degradation products include a hyaluronic acid degradation Further non-limiting examples of cell cultures include mam product. Glycosaminoglycan degradation products can one malian multipotent, totipotent and pluripotent stem cells. Cell or more of di-, tri-, tetra-, penta-, hexa-, hepta-, octa-saccha cultures of the invention include other cells, such as feeder ride, or larger saccharide polymers. cells (e.g., FGF or proteoglycan producing feeder cells). 0026. The invention provides methods for culturing cells, 0019. Additional components of a media formulation of including mammalian cells Such as mammalian primary, sec the invention include, for example, a Substrate. A non-limit ondary or passaged cells, and immortalized cells; embryonic, ing example of a Substrate is an adhesion molecule. Adhesion tissue specific, germinal and adult, multipotent, totipotent molecules include, for example, laminin, fibronectin, or a and pluripotent stem cells. In one embodiment, a method proteoglycan (e.g., hyaluronic acid, chondroitin, chondroitin includes growing or incubating cells in a complete media Sulfate or a mucin). Adhesion molecules Such as proteogly formulation of the invention for a period of time allowing cans can be produced or provided by feeder cells. cells to proliferate, for example, increase in numbers by 25%, 0020. An anti-microbial is a particular example of a com 50%, 75%, 100% or more. In another embodiment, a method ponent of a media formulation of the invention. Particular includes growing or incubating cells in a complete media non-limiting examples of anti-microbials include anti-bacte formulation of the invention for at least about 30, 60,90, 120, rials, anti-virals, anti-mycoplasmas or anti-fungals. 240 minutes or more. In an additional embodiment, a method 0021 Complete and incomplete media formulations includes growing or incubating cells in a complete media include dry and liquid formulations. Volumes of media can be formulation of the invention for at least about 1, 2, 3, 4, 5, 6, convenient for handling or for shipment. A non-limiting 7, 8, 10, 12, 16, 20, 24, 36, or 48 hours or more. In a further example of a liquid media, is a volume of about 100-250 ml, embodiment, a method includes growing or incubating stem 250-500 ml, or 500-1000 ml. cells in a complete media formulation of the invention under 0022. The invention provides kits and containers that conditions in which stem cells survive or proliferate without include media formulations, either sterile or non-sterile. Kits substantial differentiation. Methods for culturing cells and containers include, for example, packaging material Suit include maintaining pluripotency of stem cells, or a majority able for liquid or dry complete or incomplete media formu of stem cells in the culture (e.g., 50%, 60%, 70%, 80% or lations (e.g., suitable for a volume of media of about 100-250 more remain pluripotent), for a plurality of passages, e.g., 2. ml, 250-500 ml, or 500-1000 ml). Kits also include, for 3, 4, 5 or more passages. Methods for culturing cells also US 2011/0306129 A1 Dec. 15, 2011
include co-cultures, for example, culturing the cells with factor (FGF), a salt or mineral, and essential amino acids, at other cells, such as feeder cells (e.g., FGF or proteoglycan an osmolarity of about 220-330 mOsm/Liter. Media formu producing feeder cells). lations include formulations compatible with survival or pro liferation of cells. BRIEF DRAWING DESCRIPTIONS 0038 A complete media formulation is a mixture of com ponents which, when used under appropriate conditions (e.g., 0027 FIG. 1 is a graph representing cell growth curves in at appropriate concentrations or dilutions, pH, temperature, the presence of the indicated media. % CO or % O.) are compatible with survival or proliferation 0028 FIGS. 2A-2D illustrates stem cell colonies devel of cells. Such media formulations are sufficient to maintain or oped in the indicated media. sustain cell viability for at least a period of time, whether the 0029 FIGS. 3A-3D illustrates new stem cell lines derived cells proliferate or not, or whether the cells differentiate or in absence of feeder cells, using the indicated media. not. The terms “media composition or media preparation' can 0030 FIG. 4 illustrates growth of colonies over multiple be used interchangeably with “media formulation.” The term days and passages in exemplary invention media formulation “culture media' can also be used to refer to a media formu and conditioned media. lation that is able to maintain or sustain viability of one or a 0031 FIG. 5 illustrates sustained enhanced growth of plurality of cells for at least a period of time. A “cell culture' hESCs in exemplary invention media containing 20 ng/ml as used herein refers to one or a plurality of cells whose cell bFGF and 1 Lug/ml hyaluronidase over three passages (arrows viability is maintained or sustained for at least a period of indicate passage). time. Not all cells are required to survive or proliferate in a 0032 FIGS. 6A-6F illustrate morphology of stem cell complete media formulation of the invention and, in fact a colonies after multiple passages in the exemplary invention Small or even a large number of cells may die or senesce. media formulation: A) stem cell colony grown in conditioned Likewise, not all cells of a given cell culture are required to media 4x (control); B) two days after 4" passage in exem survive or proliferate in a complete media formulation of the plary invention media, with no addition of hyaluronidase 4x; invention. C) two days after 2" passage in exemplary invention media 0039. “Components’ refer to particular compounds or 4x; D) Two days after the 3' passage in exemplary invention ingredients that are present or make up a media formulation. media 4x; E) Two days after the 5" passage in exemplary Such components can be used in the media to Sustain or invention media 4x; and F) stem cells inside of a colony after maintain cell survival, viability or proliferation. Such com 5" passages in exemplary invention media appear very ponents can be unrelated to cell survival, viability or prolif healthy with minimal floating debris, large nucleus with eration, but may serve another purpose, such as a preserva prominent nucleolus (20x). tive, dye or coloring agent (e.g., to indicate pH of the media). 0033 FIGS. 7A-7C illustrate that “sternness” was pre served after 5 passages: A) Oct4 labeling of a stem cell 0040. A media formulation can be complete or incom colony; B) nuclear counterstain (bisbenzimide); and C) plete. A “complete' media formulation includes appropriate Imposed: pink Oct4 labeled stem cells, blue differentiated types and amounts of components adequate to be compatible cells. with survival or proliferation of cells. An “incomplete' media 0034 FIGS. 8A-8C illustrate in vitro differentiation of formulation typically lacks one or more components as com cultures grown in exemplary invention media Supplemented pared to a complete media formulation, although lack of a with hyaluronidase: A) Nestin positive cells specific for ecto particular component does not necessarily make an incom derm; B) alpha feto-protein positive cells for endodermal plete media formulation inadequate or insufficient to be com lineages; and C) Smooth muscle actin positive cells for meso patible with survival or proliferation of cells. dermal lineages. 0041 Media formulations of the invention include com 0035 FIG. 9 illustrates that instead of expanding the stem ponents in amounts compatible with Survival of cells such as cells in a fully balanced and pluripotent media, the use of a stem cells, tissue specific, germinal or adult, whether totipo “biased media is possible when certain embryonic germ tent, multipotent or pluripotent. Media formulations of the layer lineages are targeted. invention also include components in amounts compatible with survival of stem cells without substantial differentiation 0036 FIGS. 10A-10B show that A) when differentiationis of the stem cells. The term “without substantial differentia initiated in a typical differentiation media, there is an initial tion, when used in reference to stem cells, means that no drop in the number of cells which reduces the efficiency of the more than about 20%, +/-5%, of the total number of stem differentiation; and B) when exemplary invention media is cells in a given stem cell population have begun to differen used to expand the stem cells for multiple passages, the initial tiate or have differentiated. This term can be used to refer to drop in the cell population is avoided significantly increasing one or a plurality of passages, e.g.,2,3,4, 5 or more passages, yield and efficiency of differentiation. of a cell culture that includes stem cells. DETAILED DESCRIPTION 0042 Stem cells are characterized typically by morphol ogy as well as the presence of characteristic markers. For 0037. The invention provides media formulations. In one example, morphology of a stem cell is typically dense, well embodiment, a complete media formulation includes the fol delimited Small cells with a large nucleus representing about lowing components: albumin, an iron carrier, glutamine, a 80 to 95% of the total cellular volume. Stem cell differentia glycosidase or hydrolase, fibroblast growth factor (FGF), a tion can result in a phenotypic change—the most commonly salt or mineral, and essential amino acids, at an osmolarity of observed change is in cell morphology. For example, the about 220-330 mOsm/Liter. In another embodiment, a com proportion of nucleus to cytoplasm is reduced, cells acquire plete media formulation of the invention includes the follow migratory capability, and the colony edges become less ing components: albumin, an iron carrier, glutamine, a gly defined. Stem cell differentiation can also result in a loss of cosaminoglycan degradation product, fibroblast growth stem cell markers (e.g., OCT4, SSEA4, TRA1-81) or telom US 2011/0306129 A1 Dec. 15, 2011
erase activity. Stem cell differentiation can further result in combination with N-acetylcysteine promotes the synthesis of acquiring markers or morphologies characteristic of one or glutathione, an antioxidant. Glutamine can be provided as a more of the three embryonic germ layers—ectoderm, meso monomer, or as a di-, tri-, tetra-, penta-hexa-hepta-, or larger derm or endoderm. Under certain conditions, stem cells can peptide. One non-limiting example of a glutamine di-peptide grow outside of stem cell colonies and their number and the is glutamine-alanine. growth can be determined by immunolabeling with markers 0049 Amounts or concentrations of glutamine appropri characteristic of stem cells. ate in media formulations of the invention are from about 10 0043 Spontaneous differentiation of stem cells is normal to about 50 mg/mL. Additional non-limiting examples of and reflects normal functioning stem cells. Spontaneous dif amounts or concentrations of glutamine appropriate in media ferentiation results in a cellular mass-stroma—which fills the formulations of the invention are from about 10 to about 40 space between the colonies. The proportion between the mg/mL, 15 to about 30 mg/mL, or about 25 mg/mL. stroma representing differentiated cells and colonies repre Glutamine stock solution is typically stored frozen, but can be senting non-differentiated cells can vary, as long as the stem stored for short periods of time at refrigeration temperatures. cell colonies are properly defined (delimitation, dense, typi 0050 Media formulations of the invention can include, for cal cellular content). Stem cells can be a single colony in a example, a glycosidase or hydrolase. As used herein, the culture dish (which can be 0.1% of the total cell number) to terms glycosidase or hydrolase refer to an enzyme that is virtually 100% with a complete absence of stromal cells. The capable of cleaving a GAG glycosidic linkage (O- or S-gly proportion between stroma (differentiated cells) and colonies cosyl) thereby hydrolyzing GAGs. Glycosidases and hydro (stem cells) in media can be regulated by other factors unre lases vary with respect to origin, Substrate specificity, and lated to the media composition, for example the ratio that mechanism of action. Glycosidases and hydrolases include cells are split when passaged. soluble forms and membrane bound forms. A soluble glycosi 0044) Media formulations of the invention can include, for dase or hydrolase means that the enzyme is at least partially example, albumin. Albumins play a role in various functions, soluble in the media formulation, and membrane bound gly Such as transporting fatty acids, thyroid and steroid hormones cosidase or hydrolase means that the enzyme is tethered or and other Substances. Albumins also contribute to maintain anchored to a lipid, such as a lipid bilayer of a cell membrane. ing osmotic pressure of extracellular fluid. Non-limiting 0051. Non-limiting examples of glycosidases or hydro examples of albumins include mammalian albumins, such as lases include hyaluronidases, which can cleave hyaluronic primate (e.g., human) and bovine serum albumin (BSA), goat acids or chondroitin Sulfates. Mammalian-type hyalu serum albumin (GSA), rabbit serum albumin (RSA). Addi ronidases (e.g., Hyall, Hyal2, Hyal3, Hya4 and PH204) are tional specific examples of albumins include probumin endo-beta-N-acetylhexosaminidases with produce tetrasac (Chemicon) and ICPBio albumin, highly purified forms of charides and hexasaccharides as end products. Mammalian BSA that are virus/endotoxin free. type hyaluronidases typically have hydrolytic and transgly 0045 Amounts or concentrations of albumin appropriate cosidase activities, and can degrade hyaluronan, chondroitin in media formulations of the invention are from about 1 to and chondroitin Sulfates. Mammalian hyaluronidases include about 100 g/L. Additional non-limiting examples of amounts enzymes active at neutral pH and enzymes active at acid pH. or concentrations of albumin appropriate in media formula Mammalian hyaluronidases Hyal1, Hyal2 and Hyal3 cleave tions of the invention are from about 1 to about 50 g/L, 1 to the glycosidic linkage between the glucuronic acid compo about 25g/L, or 1 to about 5 g/L. Albumins are susceptible to nent and the N-acetylgalactosamine component of the HA to pH below 6, exposure to light and temperatures that cause produce tetra and hexasaccharides. Hyall is the prototypical protein denaturation. Albumin Stock solutions can be frozen acid-active enzyme and PH204 is the prototypical neutral at -20°C. A concentrated solution of 10-40% can be frozen active enzyme. Hyall has been reported to lack detectable for long periods of time. activity in vitro above pH 4.5. Hyal4 is a chondroitinase and 0046 Media formulations of the invention can include, for lacks activity towards hyaluronan. Hyal2 is an acid active example, an iron carrier. Iron is an essential trace element for enzyme. cells but can be toxic in the free form. An iron carrier is 0.052 Bacterial hyaluronidases degrade hyaluronan and, typically a ligand for transferrin receptor. A non-limiting and to various extents, CS and DS. They are endo-beta-N- example of an iron carrier is therefore transferrin. Iron carri acetylhexosaminidases that operate by a beta elimination ers can be mammalian, Such as primate (e.g., human) or reaction that yields primarily disaccharide end products. ungulate (e.g., bovine, goat, equine or porcine). Hyaluronidases (EC 3.2.1.36) from leeches, other parasites, 0047 Amounts or concentrations of iron carrier appropri and crustaceans are endo-beta-glucuronidases that generate ate in media formulations of the invention are from about 5 to tetrasaccharide and hexasaccharide end products through about 100 ug/mL. Additional non-limiting examples of hydrolysis of the beta 1-3 linkage. amounts or concentrations of iron carrier appropriate in 0053 Non-limiting examples of glycosidases or hydro media formulations of the invention are from about 10 to lases also include N-glycosidase F, which can cleave derma about 50 ug/mL, 15 to about 25 ug/mL, or about 20 ug/mL. tan Sulfate. Transferrin stock solutions can be frozen long term or refrig 0054 Non-limiting examples of glycosidases or hydro erated for shorter time periods. Transferrin can withstand lases also include chondroitinases, which can cleave chon repeated freezing and thawing. Transferrin is sensitive to pH droitin Sulfate through an endoglycosidase reaction. Specific changes and temperatures greater than 60° C. examples of chondroitinases include chondroitinase ABC 0.048 Media formulations of the invention can include, for (Suzuki, et al., J. Biol. Chem., 243:1543 (1968)), chondroiti example, glutamine, which can also be referred to as nase AC (Yamagata et al., J. Biol. Chem. 243:1523 (1968)), glutamate. Glutamine is involved in a variety of cell meta chondroitinase AC II (Hiyama and Okada, J. Biol. Chem., bolic processes. Glutamine can be converted to glucose, 250:1824 (1975), hyaluronidase ACIII (Miyazono et al., which can be used as a carbon energy source. Glutamine in Seikagaku, 61:1023 (1989)), chondroitinase B (Michelacci US 2011/0306129 A1 Dec. 15, 2011
and Dietrich, Biochem. Biophys. Res. Commun., 56:973 (0103 EC 3.2.1.55 Cl-N-arabinofuranosidase (1974), and chondroitinase C (Miyazono, et al., Seikagaku, 0104 EC 3.2.1.56 glucuronosyl-disulfoglucosamine glu 61:1023 (1939)). curonidase 0055. Non-limiting examples of glycosidases or hydro 0105 EC 3.2.1.57 isopullulanase lases further include heparanase or glycosidase, which can 0106 EC 3.2.1.58 glucan 1.3-3-glucosidase cleave heparin or heparan Sulfate. Heparanase is an endo-B- 0107 EC 3.2.1.59 glucan endo-1,3-O-glucosidase D-glucuronidase that cleaves the B-1,4-glycosidic bond 0.108 EC 3.2.1.60 glucan 1,4-O-maltotetraohydrolase between a D-glucuronate and a D-glucosamine in heparan 0109 EC 3.2.1.61 mycodextranase sulfate. 0110 EC 3.2.1.62 glycosylceramidase 0056. Non-limiting examples of glycosidases or hydro 0111 EC 3.2.1.63 12-O-L-fucosidase lases additionally include keratan Sulfate hydrolases, which 0112 EC 3.2.1.642,6-B-fructan 6-levanbiohydrolase can cleave keratan Sulfate. Specific examples of keratan Sul 0113 EC 3.2.1.65 levanase fate hydrolases include endo-B-N-acetylglucosaminidase. 0114 EC 3.2.1.66 quercitrinase 0057. Further specific non-limiting examples of glycosi 0115 EC 3.2.1.67 galacturan 1,4-O-galacturonidase dases or hydrolases areas set forth in Table 1: 0116 EC 3.2.1.68 isoamylase 0117 EC 3.2.1.70 glucan 1,6-O-glucosidase Table 1 0118 EC 3.2.1.71 glucan endo-1,2-f-glucosidase 0058 EC 3.2.1 class Glycosidases/Hydrolases that hydro 0119 EC 3.2.1.72 xylan 1,3-B-xylosidase lyze O- and S-glycosyl GAGs 0120 EC 3.2.1.73 licheninase 0059 EC 3.2.1.1 C-amylase I0121 EC 3.2.1.74 glucan 1,4-B-glucosidase 0060 EC 3.2.1.2 B-amylase 0.122 EC 3.2.1.75 glucan endo-1,6-B-glucosidase 0061 EC 3.2.1.3 glucan 1,4-O-glucosidase (0123 EC 3.2.1.76 L-iduronidase 0062 EC 3.2.1.4 cellulase 0.124 EC 3.2.1.77 mannan 1.2-(1,3)-O-mannosidase 0063 EC 3.2.1.6 endo-1.3(4)-f-glucanase 0.125 EC 3.2.1.78 mannan endo-1,4-B-mannosidase 0064 EC 3.2.1.7 inulinase (0.126 EC 3.2.1.80 fructan B-fructosidase 0065 EC 3.2.1.8 endo-1,4-B-xylanase (O127 EC 3.2.1.81 B-agarase 0066 EC 3.2.1.10 oligo-1,6-glucosidase I0128 EC 3.2.1.82 exo-poly-CL-galacturonosidase 0067 EC 3.2.1.11 dextranase I0129 EC 3.2.1.83 K-carrageenase 0068 EC 3.2.1.14 chitinase 0.130 EC 3.2.1.84 glucan 1.3-O-glucosidase 0069 EC 3.2.1.15 polygalacturonase I0131 EC 3.2.1.85 6-phospho-?3-galactosidase 0070 EC 3.2.1.17 lysozyme I0132 EC 3.2.1.86 6-phospho-?3-glucosidase (0071 EC 3.2.1.18 exo-O-Sialidase 0.133 EC 3.2.1.87 capsular-polysaccharide endo-1,3-C.- 0072 EC 3.2.1.20 C-glucosidase galactosidase 0073 EC 3.2.1.21 B-glucosidase I0134 EC 3.2.1.88 B-L-arabinosidase 0074 EC 3.2.1.22 C.-galactosidase 0.135 EC 3.2.1.89 arabinogalactan endo-1,4-B-galactosi 0075 EC 3.2.1.23 B-galactosidase dase 0.076 EC 3.2.1.24 C-mannosidase 0.136 EC 3.2.1.91 cellulose 1,4-B-cellobiosidase 0077 EC 3.2.1.25 B-mannosidase 0.137 EC 3.2.1.92 peptidoglycan B-N-acetylmuramidase 0078 EC 3.2.1.26 B-fructofuranosidase 0.138 EC 3.2.1.93 O.C.-phosphotrehalase 0079 EC 3.2.1.28 O.C.-trehalase 0.139 EC 3.2.1.94 glucan 1,6-O-isomaltosidase 0080 EC 3.2.1.31 B-glucuronidase 0140 EC 3.2.1.95 dextran 1,6-O-isomaltotriosidase 0081 EC 3.2.1.32 Xylan endo-1,3-B-xylosidase 01.41 EC 3.2.1.96 mannosyl-glycoprotein endo-B-N- 0082 EC 3.2.1.33 amylo-1,6-glucosidase acetylglucosaminidase 0083 EC 3.2.1.35 hyaluronoglucosaminidase 0142 EC 3.2.1.97 glycopeptide Cl-N-acetylgalac 0084 EC 3.2.1.36 hyaluronoglucuronidase tosaminidase I0085 EC 3.2.1.37 xylan 1,4-B-xylosidase 0.143 EC 3.2.1.98 glucan 1,4-O-maltohexaosidase I0086 EC 3.2.1.38 B-D-fucosidase 0.144 EC 3.2.1.99 arabinan endo-1,5-O-L-arabinosidase 0087 EC 3.2.1.39 glucan endo-1,3-B-D-glucosidase (0145 EC 3.2.1.100 mannan 1,4-mannobiosidase 0088 EC 3.2.1.40 C-L-rhamnosidase 0146 EC 3.2.1.101 mannan endo-1,6-O-mannosidase I0089 EC 3.2.1.41 pullulanase 0147 EC 3.2.1.102 blood-group-substance endo-1,4-B- 0090 EC 3.2.1.42 GDP-glucosidase galactosidase 0091 EC 3.2.1.43 B-L-rhamnosidase 0148 EC 3.2.1.103 keratan-sulfate endo-1,4-B-galactosi 0092 EC 3.2.1.44 fucoidanase dase 0093 EC 3.2.1.45 glucosylceramidase 0149 EC 3.2.1.104 steryl-3-glucosidase 0094 EC 3.2.1.46 galactosylceramidase O150 EC 3.2.1.105 strictosidine B-glucosidase 0095 EC 3.2.1.47 galactosylgalactosylglucosyl 0151 EC 3.2.1.106 mannosyl-oligosaccharide glucosi ceramidase dase 0096 EC 3.2.1.48 sucrose C-glucosidase 0152 EC 3.2.1.107 protein-glucosylgalactosylhydroxyl 0097 EC 3.2.1.49 C.-N-acetylgalactosaminidase ysine glucosidase 0098 EC 3.2.1.50 C-N-acetylglucosaminidase O153 EC 3.2.1.108 lactase 0099 EC 3.2.1.51 C-L-fucosidase 0154 EC 3.2.1.109 endogalactosaminidase 0100 EC 3.2.1.52 B-L-N-acetylhexosaminidase 0155 EC 3.2.1.110 mucinaminylserine mucinaminidase 0101 EC 3.2.1.53 B-N-acetylgalactosaminidase 0156 EC 3.2.1.111 1,3-O-L-fucosidase 0102 EC 3.2.1.54 cyclomaltodextrinase (O157 EC 3.2.1.112 2-deoxyglucosidase US 2011/0306129 A1 Dec. 15, 2011
0158 EC 3.2.1.113 mannosyl-oligosaccharide 1.2-C.- 0185 EC 3.2.1.140 lacto-N-biosidase mannosidase 0186 EC 3.2.1.141 4-O-D-(14)-C-D-glucano trehalose 0159 EC 3.2.1.114 mannosyl-oligosaccharide 1,3-1,6-O- trehalohydrolase mannosidase 0187 EC 3.2.1.142 limit dextrinase 0160 EC 3.2.1.115 branched-dextran exo-1,2-O-glucosi 0188 EC 3.2.1.143 poly(ADP-ribose) glycohydrolase dase (0189 EC 3.2.1.1443-deoxyoctulosonase 0161 EC 3.2.1.116 glucan 1,4-O-maltotriohydrolase 0.190 EC 3.2.1.145 galactan 1.3-3-galactosidase 0162 EC 3.2.1.117 amygdalin B-glucosidase 0191 EC 3.2.1.146 B-galactofuranosidase 0163 EC 3.2.1.118 prunasin B-glucosidase (0192 EC 3.2.1.147 thioglucosidase 0164 EC 3.2.1.119 vicianin B-glucosidase (0193 EC 3.2.1.148 now EC 4.4.1.21 0.165 EC 3.2.1.120 oligoxyloglucan B-glycosidase (0194 EC 3.2.1.149 B-primeverosidase 0166 EC 3.2.1.121 polymannuronate hydrolase 0.195 EC 3.2.1.150 oligoxyloglucan reducing-end-spe 0167 EC 3.2.1.122 maltose-6-phosphate glucosidase cific cellobiohydrolase 0168 EC 3.2.1.123 endoglycosylceramidase 0169 EC 3.2.1.1243-deoxy-2-octulosonidase 0.196 EC 3.2.1.151 xyloglucan-specific endo-B-1,4-glu 0170 EC 3.2.1.125 raucaffricine B-glucosidase CaaSC 0171 EC 3.2.1.126 coniferin B-glucosidase 0.197 EC 3.2.1.152 mannosylglycoprotein endo-B-man 0172 EC 3.2.1.1271,6-O-L-fucosidase nosidase 0173 EC 3.2.1.128 glycyrrhizinate B-glucuronidase (0198 EC 3.2.1.153 fructan B-(2,1)-fructosidase 0.174 EC 3.2.1.129 endo-O-Sialidase (0199 EC 3.2.1.154 fructan B-(2,6)-fructosidase 0175 EC 3.2.1.130 glycoprotein endo-O-1,2-mannosi 0200 EC 3.2.1.155 xyloglucan-specific exo-f-1,4-gluca dase a SC 0176 EC 3.2.1.131 xylan C-1,2-glucuronosidase 0201 EC 3.2.1.156 oligosaccharide reducing-end xyla 0177 EC 3.2.1.132 chitosanase a SC 0.178 EC 3.2.1.133 glucan 1,4-O-maltohydrolase 0202 EC 3.2.1.157 L-carrageenase (0179 EC 3.2.1.134 difructose-anhydride synthase (0203 EC 3.2.1.158 C-agarase 0180 EC 3.2.1.135 neopululanase 0204 EC 3.2.1.159 C.-neoagaro-oligosaccharide hydro 0181 EC 3.2.1.136 glucuronoarabinoxylan endo-1,4-B- lase Xylanase 0205 EC 3.2.1.160 deleted, identical to EC 3.2.1.155 0182 EC 3.2.1.137 mannan exo-1,2-1,6-O-mannosidase 0206 EC 3.2.1.161 B-apiosyl-f-glucosidase 0183 EC 3.2.1.138 now EC 4.2.2.15 Sequences of particular glycosidases or hydrolases are as 0184 EC 3.2.1. 139 C-glucuronidase follows:
HYAL1 HUMAN Hyaluronidase-1 - Homo sapiens (Human). MAAHLLPICALFLTLLDMAOGFRGPLLPNRPFTTVWNANTOWCLERHGVDVDVSVFDV
WANPGQTFRGPDMTIFYSSOLGTYPYYTPTGEPVFGGLPONASLIAHLARTFODILAAIPA
PDFSGLAVIDWEAWRPRWAFNWDTKDIYRORSRALVOAOHPDWPAPOVEAVAODOF
QGAARAWMAGTLOLGRALRPRGLWGFYGFPDCYNYDFLSPNYTGQCPSGIRAQNDQL
GWLWGOSRALYPSIYMPAVLEGTGKSOMYWOHRVAEAFRVAVAAGDPNLPVLPYVOI
FYDTTNHFLPLDELEHSLGESAAOGAAGVWLWWSWENTRTKESCOAIKEYMDTTLGPFI
LNWTSGALLCSQALCSGHGRCVRRTSHPKALLLLNPASFSIOLTPGGGPLSLRGALSLED
OAOMAVEFKCRCYPGWOAPWCERKSMW; HYAL1 BOVIN Hyaluronidase-1 - Bos taurus (Bovine). MRPFSLEVSLHLPWAMAAHLLPVCTLFLNLLSMTOGSRDPVVPNOPFTTIWNANTEWC
DVDISIFDVVTNPGQTFRGPNMTIFYSSOLGTYPYYTSAGEPVFGGLPONASL
NAHL.ARTFODILAAMPEPRFSGLAVIDWEAWRPRWAFNWDTKDIYRORSRALVOKOH
PDWLAP RVEAAAODOFEGAAEEWMAGTLKLGOALRPOGLWGFYNFPECYNYDFKSPN
YTGRCP LNICAONDOLGWLWGOSRALYPSIYLPAALEGTKKTOMFWOHRVAEAFRVAA
GAGDPK LPVLPYMOLFYDMTNHFLPAEELEHSLGESAAOGAAGVWLWWSWLSTSTKES
COAIKEYWDTTLGPSILNWTSGARLCSOVLCSGHGRCARRPSYPKARLILNSTSFSIKPTP
GGGPLT LOGALSLEDRLRMAVEFECRCYRGWRGTRCEOWGMW; HYAL1 MOUSE Hyaluronidase-1 - Mus musculus (Mouse). MLGLTO HAOKVWRMKPFSPEVSPGSSPATAGHLLRISTLFLTLLELAQVCRGSVVSNRP
FITVWNGDTHWCLTEYGVDVDVSVFDVVANKEOSFOGSNMTIFYREELGTYPYYTPTG
US 2011/0306129 A1 Dec. 15, 2011
- Continued
WSATDIEYLAKWTFEESAKAFMKETIKLGIKSRPKGLWGYYLYPDCHNYNWYAPNYSGS
CPEDEWLRNNELSWLWNSSAALYPSICWWKSLGDSENILRFSKFRWHESMRISTMTSHD
YALPVFWYTRLGYRDEPLFFLSKODLVSTIGESAALGAAGIVIWGDMNLTASKANCTKV
KOFWSSDLGSYIANVTRAAEWCSLHLCRNNGRCIRKMWNAPSYLHLNPASYHIEASEDG
EFTWKGKASDTDLAVMADTFSCHCYOGYEGADCREIKTADGCSGVSPSPGSLMTLCLL
LLASYRSIQL.
0207 For cells such as stem cells, a glycosidase or hydro cesses including mesoderm induction, antero-posterior pat lase can be added periodically to media or to cells in a culture terning, neural induction, angiogenesis, axon extension and media. A glycosidase or hydrolase can be added to media or limb formation. to cell cultures, for example, hourly, daily, or when adding 0210. Non-limiting examples of a fibroblast growth factor fresh media or a media Supplement to a cell culture, Such as a acidic FGF (aFGF, also referred to as FGF-1) and basic FGF stem cell culture. (bFGF, also referred to as FGF-2). FGF1 is a heparin-binding 0208 Glycosidase or hydrolase amounts or concentra growth factors, which has angiogenic activity in vivo and is a tions appropriate in media formulations of the invention are potent mitogen for a variety of cell types in vitro. FGF1 binds from about 1 to about 100 ug/ml. Additional non-limiting FGFR2 and forms a ternary complex containing 2 molecules examples of amounts or concentrations of glycosidase or each of FGFR2 and FGF1 for 1 heparin molecule. FGF2 hydrolase appropriate in media formulations of the invention promotes the endothelial cell proliferation and the physical are from about 1 to about 50 ug/ml, 1 to about 25 ug/ml, 1 to organization of endothelial cells into tube-like structures. It about 10 ug/ml. Glycosidases and hydrolases (e.g., hyalu thus promotes angiogenesis, the growth of new blood vessels ronidases) typically become slowly inactivated in solution. from the pre-existing vasculature. As well as stimulating Glycosidase and hydrolase powder and stock solutions are blood vessel growth, FGF2 participates in wound healing, for typically frozen at about -20° C. for long term storage. example, it stimulates proliferation of fibroblasts that give Repeated freeze/thaw cycles typically result in loss of activ rise to granulation tissue, which fills up a wound space/cavity ity. After reconstitution, glycosidases and hydrolases are early in the wound healing process. FGF2 in vitro has mito stable in solution at 4°C. for about 7-30 days, and may be genic activity, stimulating proliferation of various cell types. used for up to one week. 0211 Additional specific examples of FGF include FGF3, 0209 Media formulations of the invention can include, for FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, example, a fibroblast growth factor (FGF). FGF promotes or FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, sustains fibroblast cell viability, survival, growth or prolifera FGF19, FGF20, FGF21, FGF22 and FGF23. Sequences of tion. FGFs are associated with many developmental pro these and other FGFs are as follows:
FGF1 HUMAN Heparin-binding growth factor 1 - Homo sapiens (Human) . MAEGEITTFTA LTEKFNLPPGNYKKPKILLYCSNGGHFLRILPDGTVDGTRDRSDOHIOLO
SAESWGEWYI KSTETGOYLAMDTDGLLYGSOTPNEECLFLERLEENHYNTYISKKHAE
KNWFVGLKKNGSCKRGPRTHYGOKAILFLPLPWSSD; FGF1 MOUSE Heparin-binding growth factor 1 - Mus musculus (Mouse) . MAEGEITTFAA LTERFNLPLGNYKKPKILLYCSNGGHFLRILPDGTVDGTRDRSDOHIOLO
SAESAGEWYI KGTETGOYLAMDTEGLLYGSOTPNEECLFLERLEENHYNTYTSKKHAE
KNWFVGLKKNGSCKRGPRTHYGOKAILFLPLPWSSD; FGF2 HUMAN Heparin-binding growth factor 2 - Homo sapiens (Human) . MAAGSITTLPA PEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRWDGWREKSDPHI
KLOLOAFEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSR
KYTSWYVALKRTGOYKLGSKTGPGOKAILFLPMSAKS; FGF2 MOUSE Heparin-binding growth factor 2 - Mus musculus (Mouse). MAASGITSLPALPEDGGAAFPPGHFKDPKRLYCKNGGFFLRIHPDGRWDGWREKSDPHW
KLOLOAFEERGVVSIKGVCANRYLAMKEDGRLLASKCVTEECFFFERLESNNYNTYRSR
KYSSWYVALKRTGOYKLGSKTGPGOKAILFLPMSAKS;
US 2011/0306129 A1 Dec. 15, 2011 11
Continued GKPDGTSKECWFIEKVLENNYTALMSAKYSGWYWGFTKKGRPRKGPKTRENOODVHF
MKRYPKGOPELOKPFKYTTWTKRSRRIRPTHPA; FGF19 HUMAN Fibroblast growth factor 19 - Homo sapiens (Human). MRSGCWWWHWWILAGLWLAWAGRPLAFSDAGPHWHYGWGDPIRLRHLYTSGPHGLSS
CFLRIRADGWWDCARGOSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMOGLLOY
SEEDCAFEEEIRPDGYNVYRSEKHRLPWSLSSAKOROLYKNRGFLPLSHFLPMLPMVPEE
PEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; FGF20 HUMAN Fibroblast growth factor 20 - Homo sapiens ( Human) . MAPLAEWGGFLGGLEGLGQQVGSHFLLPPAGERPPLLGERRSAAERSARGGPGAAQ L.A.
HLHGILRRROLYCRTGFHLOILPDGSWOGTRODHSLFGILEFISWAVGLVSIRGWDSGLYL
GMNDKGELYGSEKLTSECIFREOFEENWYNTYSSNIYKHGDTGRRYFVALNKDGTP RD
GARSKRHOKFTHFLPRPVDPERVPELYKDLLMYT; FGF21 HUMAN Fibroblast growt h factor 21 - Homo sapiens ( Human) . MDSDETGFEHSGLWWSWLAGLLLGACQA HPIPDSSPLLOFGGOVRORYLYTDDAQQ TE
AHLEIREDGTWGGAADQSPESLLOLKAL KPGVIQILGWKTSRFLCORPDGALYGSL
EACSFRELLLEDGYNWYOSEAHGLPLHL PGNKSPHRDPAPRGPARFLPLPGLPPAL PEPP
GILAPOPPDWGSSDPLSMVGPSOGRSPSYAS FGF22 HUMAN Fibroblast growth factor 22 - Homo sapiens (Human) . MRRRLWLGLAWLLLARAPDAAGTPSASRGPRSYPHLEGDWRWRRLFSSTHFFLRWDPG
GRVOGTRWRHGODSILEIRSVHVGVVVIKAVSSGFYVAMNRRGRLYGSRLYTVDCRFR
ERIEENGHNTYASORWRRRGOPMFLALDRRGGPRPGGRTRRYHLSAHFLPVLVS and FGF23 HUMAN Fibroblast growth factor 23 - Homo sapiens (Human) . MLGARLRLWWCALCSVCSMSVLRAYPNASPLLGSSWGGLIHLYTATARNSYHLOIHKN
GHVDGAPHOTIYSALMIRSEDAGFWWITGVMSRRYLCMDFRGNIFGSHYFDPENCRFOH
OTLENGYDWYHSPOYHFLVSLGRAKRAFLPGMNPPPYSOFLSRRNEIPLIHFNTPIPRRHT
RSAEDDSERDPLNWLKPRARMTPAPASCSOELPSAEDNSPMASDPLGVWRGGRVNTHA
GGTGPEGCRPFAKFI.
0212 FGF homologous factors (FHF) can also function as activity decreases with time in a cell culture media for FGF since they share substantial sequence homology as FGF. example, after 24 hours in culture media, FGF2 activity Kaposi's sarcoma cells secrete a homologue of FGF (FHF) decreases significantly. FGF is typically added to media alone called the K-FGF proto-oncogene. Additional FHFs are or in combination with a fresh media to cultured cells when FHF1-FHF4, which are also known as FGF11-FGF14. Pair cells are fed. wise comparisons between the FHF1-FHF4 show between 0215 FGF can be provided by feeder cells. Feeder cells 58% and 71% amino acid sequence identity, but each FHF produce FGF, which in turn can be added as a component of shows less than 30% identity when compared with other a media formulation of the invention. Thus, FGF in media can FGFS comprise FGF without feeder cells or FGF producing feeder 0213 Amounts or concentrations of FGF appropriate in cells, such as FGF feeder cells in a cell culture that includes a media formulations of the invention are from about 1 to about media formulation of the invention. Thus, a media formula 500 ng/ml. Additional non-limiting examples of amounts or tion of the invention may or may not include cells that pro concentrations of FGF appropriate in media formulations of duce FGF, referred to as FGF feeder cells. FGF feeder cells the invention are from about 5 to about 100 ng/ml, 5 to about are usually a mixture or derived from fibroblastic or connec 50 ng/ml, 10 to about 20 ng/ml. tive tissue. Specific non-limiting examples of feeder cells 0214 FGF2 is susceptible to degradation by repeated include embryonic fibroblasts (human or other species—ro freeze? thaw cycles or exposure to plastic, filters and similar dent, for example) and fetal fibroblasts (from fetal annexes surfaces. FGF is typically stored at about -20°C. or less, with resulting at birth placenta, umbilical cord, for example). a carrier protein (for example albumin) at concentrations Specific examples of feeder cells also include adult tissue, greater than 1 mg/ml total protein. FGF can be stored as a Such as skin (foreskin) and peritoneum (resulting from exci liquid at 4°C. for up to about a week or so. FGF biological sions of hernial sacs). Specific non-limiting examples of US 2011/0306129 A1 Dec. 15, 2011
established feeder cell lines include, for example, immortal 0223 Amounts or concentrations of globulins appropriate ized BJ-TERT fibroblasts, CHO and STO cell lines. in media formulations of the invention are from about 0.1 to 0216. Media formulations of the invention can include, for about 25 g/L. Additional non-limiting examples of amounts example, salts or minerals. Minerals and salts include, for or concentrations of globulin appropriate in media formula example, sodium, potassium, calcium, magnesium, copper, tions of the invention are from about 0.5 to about 20 g/L, 1 to manganese, molybdenum, selenium, Silicon, iron, Zinc, Vana about 10 g/L, or 1 to about 5 g/L. As with albumins, globulins dium, boron, cobalt, iodine, chromium and tin. Minerals or are susceptible to pH below 6, exposure to light and tempera salts can be provided as organic (organic acids) or inorganic tures that cause protein denaturation. Globulin stock solu salts (e.g., chlorides, Sulfates, phosphates or nitrates). In a tions can be stored at -20°C. Concentrated globulin solutions non-limiting example, a selenium salt is sodium selenite. of 10-40% can be frozen for long periods of time. 0217. Amounts or concentrations of minerals or salts will 0224 Relative concentration ratios of globulin to albumin depend upon the particular mineral or salt. A particular non appropriate in media formulations of the invention are about limiting example for sodium is a concentration of about 130 1:2, or less than about 1:2. Additional non-limiting examples 160 mg/Liter. A particular non-limiting example for potas of concentration ratios of globulin to albumin appropriate in sium is a concentration of 3 to 6 mg/Liter. A particular non media formulations of the invention are from about 1:0.5, limiting example for calcium is a concentration of 7 to 12 1:0.75, 1:1, 1:1.5, 1:2.5, or 1:3. mg/Liter. A particular non-limiting example for magnesium 0225. The synthesis of HA occurs in membrane structures. is a concentration of 1 to 4 mg/deciliter. For trace elements HA, with different levels of polymerization (or fragmenta Such as copper, manganese, molybdenum, selenium, iron, or tion), may have various effects on cell Survival, proliferation, Zinc, a non-limiting concentration is about 1 pg/deciliter to 1 adhesion or migration. Although not wishing to be bound by ug/deciliter. Mineral and salt solutions are stable and there theory, CD44 is the major receptor for HA and a ubiquitous fore can be stored at room temperature, refrigerated or frozen membrane protein with multiple functions. After binding to if desired. CD44, HA could form a surrounding capsule with beneficial 0218. Media formulations of the invention can include, for or detrimental effects on the cell biology. For example, encap example, essential amino acids. Essential amino acids Sulating a more complex structure of a blastocyst could be include, for example, arginine; cystine; histidine; isoleucine; beneficial by creating a trap for the autocrine secretion of the leucine; methionine; phenylalanine; threonine; tryptophan; trophoblast or by creating a protection envelope for the tyrosine; and valine. embryo up to the stage of hatching and implantation. In this 0219. Amounts or concentrations of essential amino acids regard, the concentration of HA is a good indicator of the can vary and may depend in part upon the particular amino viability, maturation and implantation of the blastocyst. The acid. An amount or concentration of an essential amino acid HA concentration is 0.05 to 3 mg/ml in the adult tissue, in the appropriate in media formulations of the invention is about cumulus ooforus complex is 0.5-1 mg/ml, and about 50 ng/ml 0.5 to 10 mmol/Liter. A more particular non-limiting amount in the follicular fluid. or concentration is about 2.5 nmol/Liter. Amino acid solu 0226 Media formulations of the invention can include, for tions are stable and can be stored at 4°C. Amino acid solu example, a glycosaminoglycan or a glycosaminoglycan deg tions should be protected from light. radation product. In various embodiments, glycosaminogly 0220 Media formulations of the invention typically have cans or fragments of glycosaminoglycans, such as HA, can be an osmolarity Such that when the media is in contact with the used as a Supplement in addition to a glycosidase or hydrolase cells, the osmolarity will be compatible with cell survival or in a media formulation of the invention, or can be used in proliferation. Exemplary osmolarity for a liquid media for place of a glycosidase or hydrolase in a media formulation of mulation can range from about 240-300 mOsm/Liter, or from the invention. about 250-270 mOsm/Liter. 0227 Glycosaminoglycan degradation products can be 0221 Osmolarity is measure of osmoles of solute per litre produced by glycosidases or hydrolases. A non-limiting of solution. Media osmolarity can be measured by various example of a glycosaminoglycan degradation product is a methods including freezing-point depression osmometry (us degradation product of hyaluronic acid (HA). Additional non ing the variations in the freezing temperature of the aqueous limiting examples of a glycosaminoglycan degradation prod liquids at different osmotic pressures), vapor pressure uct is a degradation product of chondroitin, chondroitin Sul oSmometry (determine the concentration of osmotically fate or a mucin (e.g., mucin1, mucin2, mucin3, mucin4. active particles that reduce the vapor pressure of a solution) mucin5AC, mucin5B, mucinó, mucin7, mucin8 or mucin9). and oncometry using a semipermeable membrane and a ref Glycosaminoglycan degradation products include, for erence solution to measure the oncotic pressure (given by example, di-, tri-, tetra-, penta-, hexa-, hepta-, octa-saccha large molecules—for example proteins, carbohydrates). ride, or larger saccharide polymers. Commercially available osmometers can accurate measure 0228. Media formulations of the invention therefore can osmolarity between 0 and 4000 mOsm/KgHO. include one or more Supplements. A Supplement refers to a 0222 Media formulations of the invention can include, for component or ingredient that can be added to a complete or example, globulins. Globulins play a role in various func incomplete media formulation. Thus, a Supplement of an tions, such as transporting fatty acids, thyroid and steroid incomplete media formulation can be a component of a com hormones and other substances. Globulins also contribute to plete media. For example, where an incomplete media lacks maintaining osmotic pressure of extracellular fluid. Non-lim albumin, a Supplement can be albumin which, when added to iting examples of globulins include alpha-, beta- and gamma the incomplete media results in a complete media. Similarly, globulin and antibodies (e.g. IgG, IgA, IgM, IgE and Ig)). where an incomplete media lacks an iron carrier, glutamine, a Globulins can be mammalian, Such as primate (e.g., human) glyocidase or hydrolase, FGF, a salt or mineral or essential or ungulate (e.g., bovine, goat, equine or porcine). amino acids, a Supplement for each such incomplete media US 2011/0306129 A1 Dec. 15, 2011 could be, respectively, an iron carrier, glutamine, a glyocidase examples are a pH between about 7.0–7.8, when present in a or hydrolase, FGF, a salt or mineral and essential amino acids. 2-20% oxygen environment, a 5-15% carbon dioxide envi 0229. Additional specific non-limiting examples of ronment, or a normal atmospheric environment (e.g., atmo Supplements include energy sources such as mono- or poly spheric carbon dioxide concentration is between about 0.03 saccharides (e.g., glucose or pyruvate); non-essential amino and 0.06% and normal atmospheric oxygen concentration is acids (e.g., alanine, asparagine, aspartate, glycine, proline or about 20%). serine); hormones (e.g., insulin, insulin-like growth factor, a 0236 Complete and incomplete media formulations can thyroid hormone such as thyroxine (T4) or triiodothyronine be a liquid, Solution, Suspension, powder, tablet, capsule, (T3), or a progesterone); cytokines and growth factors (e.g. crystals, granules, cake, paste, lyophilized or freeze-dried. epidermal growth factor (EGF), keratinocyte growth factor The powder, tablet, capsule, crystals, granules, cake, paste, (KGF), hepatocyte growth factor (HGF), insulin like growth lyophilized or freeze-dried forms can be reconstituted by factor-1 and -2 (IGF-1, IGF-2), nerve growth factor (NGF)); mixing in liquid to produce a reconstituted liquid, e.g., a interleukins and interferons; vitamins (e.g., A, B, B2, BB, liquid media formulation. Powdered media typically have a C, D, E, K, biotin); heparin, heparin sulfate, buffers or salts longer shelf live than liquid media (e.g., Earle's salts, Hanks' salts, Puck’s salts, etc.), gly 0237 Liquid culture media can be provided ready-to-use cosaminoglycan degradation products, and co-factors. Addi or require Supplementation with one or more components or tional Supplements include, for example, B-mercaptoethanol, ingredients prior to use, if desired, and the formulation can be Leukemia Inhibitory Factor (LIF, ESGROTM), or serum sub optimized for particular cell types. Liquid media may be stitutes, such as KNOCKOUTSR, an FBS substitute for stem Supplemented prior to packaging, shipment or use in a cell cell culture media. culture with more labile components to produce a media 0230 Supplements also include, for example, animal sera, formulation of the invention. For example, glutamine, trans Such as bovine sera (e.g., fetal bovine, newborn calf or normal ferrin, albumin, FGF, a glycosidase or hydrolase (e.g., hyalu calf sera), human sera, equine sera, porcine sera, monkey ronidase), globulin, serum (calf), amino acids (e.g., sera, or ape Sera, typically at a concentration of about 1-25% glutamine), transferrin, hormones (e.g., progesterone, insu (e.g., about 5-15% or about 10%); attachment factors or lin, thyroid hormones), cytokines, growth factors, and lipids extracellular matrix components, such as collagens, laminins, (e.g., phospholipids, fatty acids) are all at least somewhat proteoglycans, fibronectin, and vitronectin; and lipids, Such labile and may added as Supplements to liquid media. as phospholipids, cholesterol, fatty acids, and sphingolipids. 0238 Liquid or powder complete or incomplete media 0231. Amounts or concentrations of these and other formulations may be stored at temperatures below ambient Supplements are typically determined by the particular temperature in order to inhibit degradation of media compo media, growth conditions and cell types cultured in the media. nents or ingredients. Supplements for addition to complete or For glucose, a particular concentration is about 10 to 1000 incomplete media formulations may also be stored at tem mg/Liter. For insulin or insulin-like growth factor a particular peratures below ambient temperature in order to inhibit deg concentration is between about 5 to 40 ug/ml. For a thyroid radation. Such temperatures include refrigeration (e.g., from hormone such as thyroxine (T4) or triiodothyronine (T3) a about 0-18°C.) to or freezing (e.g., about 0°C. or less, for particular concentration is between about 1 to 40 ng/ml. example, -20°C., or less). 0232 Media formulations of the invention can include, for 0239 Non-liquid (e.g., powdered) media formulations are example, anti-microbials. Anti-microbials are any an anti typically produced by admixing dried individual components bacterial (cidal or static), e.g., gram positive or gram negative, or sets of components in amounts or concentrations according anti-viral (cidal or static), anti-mycoplasma (cidal or static) or to the media formulations set forth herein, via a mixing pro anti-fungal (cidal or static) molecule. Specific non-limiting cess, e.g., ball-milling (also referred to as Fitzmilling), or by examples include antibiotics Such as amplicillin, penicillin, lyophilizing/freeze-drying a liquid culture media. Such non geneticin, streptomycin, kanamycin, gentamycin; and anti processed powders often produce dust when used, or dissolve fungals such as mycostatin (Nystatin) and amphotercin B poorly or slowly in liquid. Powdered media formulations can (Fungizone). therefore be prepared using fluid bed technology (i.e., 0233 Media formulations of the invention can include, for “agglomeration'), via tumble granulation, or spray-drying. example, a Substrate. Substrates include adhesion molecules Agglomerated or spray-dried powders are substantially dust useful for cell attachment. Non-limiting examples of adhe free or dissolve rapidly. sion molecules include one or more of laminin or fibronectin. 0240 Fluid bed technology is a process of producing An additional non-limiting example of an adhesion molecule agglomerated powders having altered characteristics (par is a proteoglycan, such as hyaluronic acid, chondroitin, chon ticularly, for example, solubility) from the starting materials. droitin Sulfate or a mucin (e.g., mucin1, mucin2, mucin3. In brief, powders are suspended in an upwardly moving col mucina, mucin5AC, mucin5B, mucinó, mucin7, mucin8 or umn of a gas (e.g., atmospheric air or an inert gas Such as mucin9). nitrogen) while at the same time a controlled and defined 0234 Media formulations of the invention when used for amount of liquid is injected into the powder stream to produce Sustaining or maintaining cells are typically pH stabilized or a moistened powder. The volume of liquid introduced into the buffered. Exemplary buffers are bicarbonate, phosphate etha dry powder will depend at least in part upon the mass of media nolamine, triethanolamine (Tris), trometamol and HEPES to be agglomerated. Typical volumes of solvent, per 500 based buffers. grams of media, are about 5-100 ml, or about 10-50 ml, or 0235 Exemplary media formulations of the invention, about 25-50 ml (e.g., about 35 ml). Liquid introduction rates, when in a liquid form, are in the physiological pH range. per 500 grams of media, are a rate of about 1-10 ml/min, or Physiologic pH is typically greater than about 4 and less than about 2-8 ml/min, or about 4-8 m/min (e.g., about 6 ml/min). about 9. Other exemplary pH ranges are about 4.0 to 5.0, 5.0 In some situations, it may be desirable to cycle between to 6.0, 6.0 to 7.0, 7.0 to 8.0, 8.0 to 9.0. Non-limiting particular adding liquid for a period of time (e.g., about 1 minute) and US 2011/0306129 A1 Dec. 15, 2011 then not adding liquid for a period of time (e.g., about 1 than all of the components of complete media and therefore, minute), so as to inhibit clumping of the powder during prior to all components or ingredients being present in a agglomeration. Mild heat is then used to dry the material, complete media formulation of the invention. producing an agglomerated powder. Typical temperatures for drying of agglomerated powder are about 50-80°C., or about 0247 Liquids suitable for media include solvents or solu 55-75° C., or about 60-65° C. Powder is typically dried in tions compatible with the media formulation. The term “com about 3-10 minutes (e.g., for about 5-7 minutes), per 500 patible,” when used in reference to a liquid for a media for grams of powder. mulation means that the solvent or Solution does not induce 0241 Apparatuses for producing or processing materials irreversible deleterious changes in the performance charac by fluid bed technology are available commercially (e.g., teristics of the media, Such as breakdown or aggregation of from Niro, Inc., Columbia, Md.). Such apparatuses have been the components or ingredients of the media formulation or used to prepare agglomerated powders of various materials. destroying the compatibility of media with cell survival or 0242 Powdered media formulations can also be produced proliferation. Non-limiting examples of suitable solvents are by tumble granulation, which also produces an agglomerated water (e.g., distilled or deionized water), embryo tested water material. In such a process, dry powder media is introduced (e.g., Sigma-Aldrich), aqueous buffer Solutions and serum into a tumble granulator or a tumble blender Such as those (e.g., bovine, such as fetal bovine serum or calf serum or commercially available from Gemco (Middlesex, N.J.) and human serum). Patterson Kelley (East Stroudsburg, Pa.). A liquid (e.g., water, 0248 One or more components or ingredients of a com buffered saline, or other solvent) is introduced into the pow plete or incomplete media formulation may be included in the der under controlled conditions in the tumble granulator and liquid solvent used for dissolving a powdered media. One or the batch is then dried according to the manufacturer's speci more Supplements may be included in the liquid solvent used fications to form a granulated powder media formulation. for dissolving a powdered media. 0243 Powdered media formulations can additionally be produced by spray-drying. Media in a liquid form is placed 0249 Media formulations that are included in the inven into a spray-drying apparatus and are then converted into a tion include, but not limited to, DMEM, MEM, F-12, RPMI corresponding powder by spraying the solution into a cham 1640, MDEM, M199, IMDM, MCDB (105, 131), M199 ber in the apparatus under appropriate conditions to produce McCoy's 5A, Williams' media, Lebovitz's L-15, and combi the powders, such as under controlled temperature and nations thereof (e.g., DMEM:F-12). Media formulations that humidity, until powder is formed. are also included in the invention include KO (knockout) 0244. In a typical spray-drying approach, a liquid media is media, which lack a particular component or ingredient. aspirated into the apparatus and atomized into a spray with a These formulations, and components and ingredients for pro rotary- or nozzle-type atomizer. The atomized spray is then ducing the media formulations of the invention, are available mixed with a gas (e.g., nitrogen or air) and sprayed into a commercially (e.g., Sigma-Aldrich, St Louis Mo.; Chemi drying chamber under conditions that promote production of con, Temecula, Calif.; Invitrogen, Carlsbad, Calif.) or can be a powdered product. Typical spray conditions are a spray rate obtained using methods known in the art. of about 25-100 g/min, or about 30-90 g/min, 35-85 g/min, 0250 Components that make up the complete and incom 40-80 g/min, 45-75 g/min, 50-75 g/min, 55-70 g/min, or plete media formulations may but are not required to be 60-65 g/min, or about 65 g/min. Typical inlet air temperatures purified. The term “purified used as a modifier of a compo in the atomizer is about 100-300° C., or about 150-250° C., or sition, such as a media formulation component, refers to a about 200° C., with a typical outlet temperature of about composition free of most or substantially all of the materials 50-100° C. or about 60-80°C., or about 70° C. Airflow in the with which it typically associates within nature. For example, atomizer is typically set at about 50-100 kg/hr, or about 75-90 purified albumin is typically removed from components nor kg/hr, or about 80.0 kg/hr, at a nozzle pressure of about 1-5 mally present in the serum milieu. Purified does not require bar, or about 2-3 bar, or about 2.0 bar. Under such conditions, absolute purity and is context specific. Furthermore, a “puri the solvent in the liquid evaporates in a controlled manner, fied’ composition can be combined with one or more other thereby forming free-flowing particles (i.e., powder). Follow molecules, such as components of a media formulation. Thus, ing drying, the powder is discharged from the drying cham the term “purified” does not exclude combinations of purified ber, passed through one or more filters and collected for components. further processing (e.g., Sterilization, packaging, etc.). 0251 Typically, media formulations are sterilized so as to 0245 Apparatuses for producing particulate materials by inhibit or prevent microbial growth. Sterilization methods spray-drying are commercially available (e.g., from Niro, include gamma or ultraviolet irradiation; liquid media is often Inc., Columbia, Md.). According to the manufacturer, these sterilized by filtration, and powdered media can be sterilized apparatuses have been used to prepare powders of various by ethylene oxide permeation after formulation. Total doses materials. of gamma irradiation are typically about 10-100 kilograys 0246 The resulting powdered media may then be dis (kGy), or about 15-75 kGy, 15-50 kGy, 15-40 kGy or 20-40 solved in a reconstituting volume of liquid with or without kGy, or a total dosage of about 20-30 kGy, or about 25 kGy. further supplementation. Such media can be reconstituted for about 1 hour to about 7 days, or about 1 hour to about 5 under Sterile conditions and, following reconstitution, be days, 1 hour to about 3 days, about 1-24 hours or about 1-5 stored at temperatures below ambient temperature (e.g., 4 to hours, or about 1-3 hours. A slow dose of gamma irradiation 10°C.). As with liquid media, for a powdered media, one or is a total dosage of about 25-100 kGy over a period of about more labile components may be added at Subsequent time 1-5 days. During irradiation, the media can be stored at a point to the powder, for example, prior to sterilization, pack temperature of about -70° C. to about room temperature aging, shipmentoruse in a cell culture. Thus, such mixing and (about 20-25°C.), or about -70°C. Of course, radiation dose processing of media may occur with incomplete media or less and exposure times can be adjusted according to bulk or mass US 2011/0306129 A1 Dec. 15, 2011
of the irradiated material. Media formulations may be steril maintain or be suitable to maintain media or components ized prior to or following packaging, for example, in a kit or sterilely, and can be made of material commonly used for container. Such purposes. A kit or container can include a label or pack 0252 Complete media formulations, compositions and aging insert with appropriate instructions, for example. The preparations and components therein are generally described instructions may be on "printed matter, e.g., on paper or herein in terms of amounts to be used for cell culture, which cardboard within the kit or container, or on a label affixed to can be referred to as a “1x media formulation” or a “working the kit or container. Instructions may comprise audio or video concentration' or “working amount.” A 1x complete media medium and additionally be included on a computer readable formulation or a working amount is that which is appropriate medium, Such as a disk (floppy diskette or hard disk), optical for Sustaining or maintaining cell viability, Survival or prolif CD such as CD- or DVD-ROM/RAM, magnetic tape, elec eration of a cell culture. trical storage media such as RAM and ROM and hybrids of 0253 Media formulations, compositions and preparations these such as magnetic/optical storage media. also include concentrated and diluted forms. When the con 0257 Specific non-limiting examples of kits and contain centrated media is diluted the media can maintain cell viabil ers include containers Suitable for a liquid, such as bottles, ity, and when diluted forms concentrated the media can main flasks, jars, vials, tubes, and ampules. Materials Suitable for tain cell viability. Thus, invention media formulations include bottles, flasks, jars, vials, tubes, and ampules include glass or concentrated and diluted media formulations. Such concen a polyolefin. Exemplary polyolefins include, or example, trated and diluted media may be diluted or concentrated, as polystyrene, polypropylene, polyethylene, and polybutylene. appropriate, to working concentrations or amounts prior to Additional specific non-limiting examples of kits and con use. Exemplary concentrated forms of a media formulation, tainers include pouches, boxes, cartons and drums. Such kits media composition or media preparation are 5x, 10x, 20x, and containers include a packaging material Suitable for a 50x, 100x, 150x, 200x or more concentrated, which means volume of media of about 100-250 ml, 250-500 ml, or 500 that the components therein are at 5-, 10-20-, 50-, 100-, 150 1000 ml. 200-fold concentration, as compared to a 1x media formula 0258 Kits and containers may be vacuum sealed or pack tion. Thus, for example, a 5x media formulation, when aged. One example is a “brick-pack” in which the media is diluted to 1x media formulation, can maintain or Sustain cell packaged into a flexible container, such as a bag or a pouch, viability for an amount of time. Exemplary diluted forms of a which is sealed while being evacuated. Such packages may media formulation, media composition or media preparation include one or more access ports (such as valves, luer-locks, are 0.75x. 0.5x, 0.25x, 0.10x or more diluted, which means etc.) to allow introduction of a liquid (e.g., water, embryo that the components therein are at 0.75- 0.5-, 0.25- 0.10-fold tested water, sera, media or other solvent or solution) into the concentration, as compared to a 1x media formulation. Thus, package to facilitate dissolving or resuspending the media. for example, a 0.75x media formulation, when concentrated 0259 Kits and containers may include multiple (two or to a 1x media formulation, can maintain or Sustain cell viabil more) units of a complete media formulation of the invention. ity for an amount of time. Kits and containers may also include an incomplete media 0254 The invention also provides methods of producing formulation lacking one or more components, with the one or media formulations. In one embodiment, a method includes more components packaged separately from the incomplete combining the following components: albumin, an iron car media. Thus, a kit or container may contain 1) an incomplete rier, glutamine, a glycosidase or hydrolase, fibroblast growth media and; 2) components packaged separately, so that when factor (FGF), a salt or mineral, and essential amino acids. In the components are combined with the incomplete media a another embodiment, a method includes combining the fol complete media formulation is produced (e.g., a complete lowing components: albumin, an iron carrier, glutamine, a media formulation having an osmolarity of about 220-330 glycosaminoglycan degradation product, fibroblast growth mOsm/Liter). In a particular non-limiting example, a kit or factor (FGF), a salt or mineral, and essential amino acids. container can include a first and second container, the first Such components can added individually or in combination container including therein a media formulation comprising with each other. For example, albumin can be combined with the following components: an iron carrier, a salt or mineral, an iron carrier, glutamine, a glycosidase or hydrolase, fibro and essential amino acids; and a second container, said the blast growth factor (FGF), a salt or mineral, and essential second container including therein the following compo amino acids; or albumin and glutamine combined together nents: albumin, glutamine, a glycosidase or hydrolase, and can be combined with an iron carrier, a glycosidase or hydro fibroblast growth factor (FGF).). In another particular non lase, fibroblast growth factor (FGF), a salt or mineral, and limiting example, a kit or container can include one more essential amino acids. In particular aspects, the media formu packages that contain albumin, an iron carrier, glutamine; a lation produced is a liquid having an osmolarity of about glycosidase or hydrolase, fibroblast growth factor (FGF), a 220-330 mOsm/Liter. salt or mineral, and essential amino acids, in individual pack 0255 Complete and incomplete media formulations can ages or together in packages (e.g., a salt or mineral and be included or packaged in kits or containers, the kits or essential amino acids combined in an individual package). containers optionally including instructions for maintaining 0260 Complete media formulations include media for survival or proliferation of cells. Specific non-limiting mulations with cells in culture. The invention therefore pro examples of kits and containers include a complete media vides cell cultures that include media formulations of the formulation of the invention, with instructions for maintain invention. In Such embodiments, cultured cells are contacted ing survival or proliferation of stem cells without substantial with a complete media formulation of the invention under differentiation. conditions, such as controlled temperature, humidity, and 0256 A kit or container typically has “packaging mate atmosphere, favoring survival or proliferation of the cells. rial.” which refers to a physical structure housing a compo Contact occurs for a period of time, typically at least 10 or nent(s) of the kit or container. The packaging material can more minutes, or greater than 20 minutes, for example, 30, US 2011/0306129 A1 Dec. 15, 2011
60,90, 120,240 minutes, or more (e.g., 1,2,3,4,5,6,7,8, 20, remain pluripotent) retain pluripotency or proliferate without 23, 26, 30, 34, 36 or 48 hours or more). Substantial differentiation, for one or more passages, e.g., 2. 0261. The term “cell culture” refers to cells sustained, 3, 4, 5 or more passages. maintained, grown or proliferated (expanded) in an in vitro or 0267 Cell cultures that include media formulations of the artificial environment. A "cell culture' is a generic term that invention may include additional Supplements, as set forth can also be used to encompass individual clonal cells, but also herein or that would be known to one skilled in the art. Cell groups of cells, progenitor cells, differentiated and non-dif cultures that include media formulations of the invention may ferentiated cells and mixtures thereof. further include other cells or cell products. A particular non 0262 Cells that are amenable to cell culture include, but limiting example of cells is a feeder cell (e.g., FGF producing are not limited mammalian cells, including somatic, a germ, feeder cells). a normal, diseased, transformed, mutant, animal cells, includ 0268 Reconstituted media may be used to culture cells. In ing primary isolates, secondary or further passaged cells, and Such techniques, cultured cells are contacted with a reconsti immortalized mammalian (e.g., human) cells. Cells may be tuted liquid media formulation under conditions favoring Sur anchorage-dependent or anchorage-independent (i.e., "sus vival or proliferation of the cells. pension’) cells. A non-limiting example of a class of mam 0269 Methods for culturing cells in media formulations malian cells is a stem cell. Such as an embryonic, tissue are therefore provided. In one embodiment, a method specific, germinal or adult stem cell. Stem cells include toti includes growing or incubating the cells in a complete media potent, pluripotent and multipotent stem cells. Another non formulation of the invention that includes the following com limiting example of a class of mammalian cells are tumor or ponents: albumin, an iron carrier, glutamine, a glycosidase or cancer cells, such as breast, prostate, haematopoietic, stom hydrolase, fibroblast growth factor (FGF), a salt or mineral, ach, colon, lung, pancreas, bladder, kidney, brain (e.g., and essential amino acids, at an osmolarity of about 220-330 glioma). Additional particular examples of mammalian cells mOsm/Liter. In another embodiment, a method includes include but are not limited to CHO cells, COS cells, VERO growing or incubating the cells in a complete media formu cells, HEK cells, BHK cells, hybridoma cells, STO cells, lation of the invention that includes the following compo BJ-TERT fibroblasts and HeLa cells. nents: albumin, an iron carrier, glutamine, a glycosaminogly 0263 Totipotent stem cells can give rise to all cell types can degradation product, fibroblast growth factor (FGF), a found in an embryo, fetus, or developed organism, including salt or mineral, and essential amino acids, at an osmolarity of the embryonic components of the trophoblast and placenta about 220-330 mOsm/Liter. In one aspect, the period of time required to Support development and birth. The Zygote and is sufficient to allow cells to proliferate or increase in num the cells at the very early stages following fertilization (i.e., bers, for example, by 25%, 50%, 75%, 100% or more. In the 2-cell stage) are considered totipotent. another aspect, the period of time is for at least about 30, 60. 0264 Pluripotent stem cells are somewhat less plastic in 90, 120, 240 minutes or more, or at least about 1, 2, 3, 4, 5, 6, their differentiative capacity than totipotent stem cells, but 7, 8, 10, 12, 16, 20, 24, 36, or 48 hours or more. In additional can become all cell types that are found in an implanted aspects, cells include stems cells such as embryonic, tissue embryo, fetus, or developed organism. Unlike totipotent stem specific, germinal or adult stem cells; or multipotent, totipo cells, pluripotent stem cells do not form embryonic compo tent or pluripotent stem cells. In a further aspect, the stem nents of the trophoblast or placenta. cells survive or proliferate without substantial differentiation. 0265 A multipotent stem cell is a progeny of a stem cell In yet additional aspects, stem cells, or a majority of stem within a particular tissue, organ, or physiological system that cells in the culture (e.g., 50%, 60%, 70%, 80% or more) is able to divide for many generations (the number of cell remain pluripotent or proliferate without substantial differen divisions may or may not be limited). Under certain condi tiation, for one or more passages, e.g., 2, 3, 4, 5 or more tions, a multipotent stem cell can give rise to daughter cells passages. Methods of culturing cells also include co-cultures, (typically, at least one is an undifferentiated cell) a proportion for example, with feeder cells (e.g. FGF or proteoglycan of which eventually terminally differentiates. producing feeder cells), as set forth herein or would be known 0266 Cultures of stem cells typically include a proportion to the skilled artisan. of stem cells capable of differentiation. A proportion of the 0270. In methods for culturing stem cells in which stem stem cells in a cell culture may therefore undergo spontane cells are passaged, stem cells as a proportion of the overall ous differentiation. In a typical healthy population of stem cell population can increase in numbers, i.e., proliferate or cells, about 80% of the cells are not differentiated and the expand. The invention therefore includes methods of cultur other 20% may be in various stages of differentiation. In cell ing cells, including stem cells, that result in clonal prolifera cultures in which stem cells are passaged, stem cells as a tion or expansion. For example in a starting culture in which proportion of the overall cell population can increase in num stem cells comprise a certain percentage of cells (e.g., bers, i.e., proliferate or expand. For example, a starting cell 10-20% of total cells), a method of the invention increases culture in which stem cells comprise a certain percentage of numbers of stem cells so that the relative proportion of stem total cells (e.g., 10-20% of the total cells in the culture), stem cells in the culture increases over time (e.g., from 10-20% cells increase in numbers so that the proportion of stem cells stem cells to 30, 40, 50, 60, 70.80% or more stem cells). Such in the culture increases (e.g., from 10-20% stem cells to 30, methods optionally retain stem cell pluripotency or stem cells 40, 50, 60, 70, 80% or more stem cells). The invention there proliferate without substantial differentiation for a plurality fore provides cultured cells, including stem cell cultures, that of passages, e.g., 1, 2, 3, 4, 5 or more passages. exhibit proliferation or expansion. In one embodiment, the 0271 Unless otherwise defined, all technical and scien cultured cells include stem cells that retain pluripotency or tific terms used herein have the same meaning as commonly proliferate without substantial differentiation, or a majority of understood by one of ordinary skill in the art to which this stem cells in the culture (e.g., 50%, 60%, 70%, 80% or more invention relates. Although methods and materials similar or US 2011/0306129 A1 Dec. 15, 2011
equivalent to those described herein can be used in the prac mented with MEM-Non Essential Aminoacids and tice or testing of the invention, Suitable methods and materials L-Glutamine from 100x stock solutions (Invitrogen), are described herein. beta mercapto-ethanol, 35 ng/mL (Sigma Aldrich) and 0272 All publications, patents, and other references cited FGF2, 4 ng/ml final concentration (Chemicon-Milli herein are incorporated by reference in their entirety. In case pore). Media was incubated overnight on a confluent of conflict, the present specification, including definitions, culture of inactivated fibroblasts, collected, sterile fil will control. tered and supplemented with 10 ng/ml FGF before stem 0273. As used herein, singular forms “a”, “and” and “the cell culture feeding. include plural referents unless the context clearly indicates 0281 Conditioned media, on inactivated (mitotically otherwise. Thus, for example, reference to “a component' inactive) mouse fibroblasts: Same as Exemplary Media includes a plurality of components or ingredients and refer Formulation indicated above, except incubated for about ence to “a cell' can include a plurality of cells. 24 hours on a culture of inactivated mouse fibroblasts. 0274 As used herein, all numerical values or numerical 0282 Conditioned classic media with hyaluronidase: ranges include whole integers within or encompassing Such 80% Knockout DMEM (Invitrogen) 20% Knockout ranges and fractions of the values or the integers within or Serum Replacement (Invitrogen), Supplemented with encompassing ranges unless the context clearly indicates oth MEM-Non Essential Aminoacids and L-Glutamine erwise. Thus, for example, reference to values such as 25%, from 100x stock solutions (Invitrogen), beta mercapto 50%, 75%, 100%, includes 25% to 50% (i.e.,25,26, 27, 28%, ethanol, 35 ng/mL (Sigma Aldrich) and FGF2, 4 ng/ml etc.), 50 to 75% (50, 51, 52, 53, 54%, etc.), and so forth. In (Chemicon-Millipore). The media was incubated over another example, reference to a concentration or amount night on a confluent culture of inactivated fibroblasts, range of 1 to about 100 g/L includes 1,2,3,4,5,6,7,8,9, 10, collected, sterile filtered and supplemented with 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 g/L, etc., as well as 1.1, ng/ml FGF before stem cell culture feeding. Hyalu 1.2, 1.3, 1.4, 1.5 g/L, etc., 2.1.2.2, 2.3, 2.4, 2.5g/L, etc., and ronidase was added to a final concentration of 1 lug/ml so forth. before use. 0275. The invention is generally disclosed herein using 0283 Human embryonic stem cells derived from human affirmative language to describe the numerous embodiments. blastocysts at California StemCell Inc., were cultured in 75 The invention also specifically includes embodiments in cm2 polystyrene flasks at 4x106 density using the above which particular subject matter is excluded, in full or in part, described conditions. The flasks were coated with Matrigel such as substances or materials, method steps and conditions, 1:30 in base media. Hyaluronidase type 1 purified from protocols, procedures, assays or analysis. Thus, even though bovine testis (Sigma Aldrich) was used. The lyophilized pow the invention is generally not expressed herein in terms of der was dissolved in sterile distilled water at a concentration what the invention does not include, aspects that are not of 1 mg/ml and stored frozen at -20°C. Thawed aliquots were expressly included in the invention are nevertheless dis kept at 4°C. for maximum 1 week. closed. 0284. The human embryonic stem cells growth in above 0276 A number of embodiments of the invention have media formulation was compared to a previously published been described. Nevertheless, it will be understood that vari formulation consisting of KO-DMEM, KO-Serum replace ous modifications may be made without departing from the ment L-Glutamine, NEAA. The media conditioning was per spirit and scope of the invention. Accordingly, the following formed on a mitomycin inactivated mouse embryonic fibro examples are intended to illustrate but not limit the scope of blast layer cultivated at a density of 12x106 cells per 225 invention described in the claims. cm2, overnight at 37° C. The colony sizes were measured daily and compared. The results are shown in FIG. 1. EXAMPLES 0285. The cells grown in the media formulation were fur ther characterized. The stem cell colonies plated after the 3" Example 1 passage in imaging chambers (Nunc) preserved the micro 0277. This example describes studies of 3 passages of scopic morphology with flat colonies, well delimited from the stem cells in a media formulation and the effect of hyalu Surrounding stroma. Labeling for stem cell markers Oct4 and ronidase. The media formulation used: TRA1-81 did show the persistence of the undifferentiated (0278 Exemplary Media Formulation: Probumin state after 3 passages. An in vitro spontaneous differentiation (Chemicon)—1 to 4 mg/ml, Human Globulins (Sigma was studied using a serum containing media and labeling of Aldrich)—0.1 to 0.5 mg/ml. Human Transferrin (Sigma the formed embryoid bodies demonstrated the presence of Aldrich)—20 ug/ml. Sodium Selenite (Sigma Aldrich), cells from all three germinal layers: ectoderm, mesoderm and 1 ng/ml; DMEM-F12 1:1, HEPES free (Invitrogen); endoderm. L-glutamine (or Glutamax) (Invitrogen)—200 uM; 0286 As illustrated in FIG. 2, stem cell colonies devel MEM non essential amino acid solution NEAA (Invit oped as expected in the classic media formulation (FIG. 2A). rogen) 1x; Water for embryo transfer 10 to 20% (Sigma Without conditioning the media based on serum replacement Aldrich); Final osmolarity 250-270 mOsm/L. Media caused massive differentiation (FIG. 2B). The serum free was supplemented before use with FGF2 (Chemicon media formulation developed large homogenous colonies Millipore) to a final concentration of 10 ng/ml, and with with Smooth surfaces and abundant Surrounding Stroma (FIG. hyaluronidase to a final concentration of 1 lug/ml. 2C). Without hyaluronidase the colonies had more fuzzy 0279 Exemplary Media Formulation indicated above, delimitation and tendency to differentiate (FIG. 2D). without hyaluronidase. Example 2 0280 Classic media (conditioned on inactivated mouse fibroblasts): 80% Knockout DMEM (Invitrogen) 20% 0287. The example includes data in which media formu Knockout Serum Replacement (Invitrogen), Supple lations were used to derive new stem cell lines. US 2011/0306129 A1 Dec. 15, 2011
0288 Frozen blastocysts were obtained by consented lagenase IV (approximately 1 mg/ml) for 5-7 minutes fol donation from the West Coast fertility Clinic. After thawing lowed by rinsing with Ca and Mg free phosphate buffer, and maturation in M2 media (Irvine Scientific), the blasto mechanical dissociation with a cell Scraper in media, than the cysts were assisted hatched or spontaneously hatched in the partially dissociated cellular agglomerates were plated in the exemplary Media Formulation in Example 1. The hatched new flasks after 1:3 or 1:4 dilution. blastocysts were than transferred to polystyrene wells coated 0292. In order to assess the potential to differentiate with Matrigel 1:30. After an initial expansion, when the inner toward neural cells, after 5 passages in exemplary invention cell mass growth was limited by the invading trophoblastic media formulation, the stem cells were dissociated and dif cells, the inner cell mass (ICM) was mechanically extracted ferentiated using retinoic acid towards neural lineages. A using fine needles and separately plated on Matrigel in the Smaller culture sample was used to perform a karyotype novel media formulation. After another 7 days the colonies analysis. started to grow with typical morphology (FIG. 3)—Small, 0293 Each day, ten randomly selected colonies in each round, compact immobile cells with a nucleus to cytoplasm flask were subjected to measurement using an microscope proportion of about 80 to 95%, one or multiple prominent ocular inserted micrometric reticule and each corresponding nucleoli are visible, and the colonies are sharply delimited day measurement was averaged over multiple passages. In the from surrounding cells which can be fibroblasts, trophoblasts exemplary invention media formulation stem cell colonies or spontaneously differentiated cells that for stroma. The new grew faster and larger (FIGS. 4 and 5). stem cells were characterized using previously described 0294 The spontaneously differentiating cells that arose methods (Hoffman and Carpenter, Nature Biotechnol. 23:699 from the hESC colonies grown in the exemplary invention (2005); Richards et al., Nature Biotechnol. 20:933 (2002); media formulation, resulted in epithelial morphology, with and Xu et al., Nature Biotechnol. 19:971 (2001)). This study Some tendencies to rosette formations. The exemplary inven demonstrates the efficacy of the proposed media composition tion media formulation manifested a clear tendency towards in deriving new stem cell lines without inactivated feeder ectodermal differentiation of the stem cells, while maintain cells. ing pluripotency (FIG. 6B). The formulation that did not Example 3 cause a clear and immediate neural differentiation, but biased the culture towards the ectodermal lineage. When differenti 0289. This example describes media formulations used to ated into neural cell types, the cultures expressed a high yield expand human embryonic stem cells (hESC) for multiple and purity of neural populations, compared to the cultures passages and maintenance of pluripotency and normal karyo grown in conditioned media (CM). When the embryonic stem type. Identical stem cell flasks were plated with similar den cells are grown in CM, and the differentiation is initiated in a sities and fed with either a) a previously described condi typical differentiation media, there is an initial drop in the tioned media (CM), or b) exemplary invention media number of cells caused by cellular death, which reduces the formulation described below: efficiency of the differentiation. In the exemplary invention
Component Wendor Cat # Vol used Stock Conc. Final Conc. DMEM/F-12 low osmolality Gibco 12660-012 467.5 ml 1x Non essential amino acids Gibco 11140 5 ml 1OOx Glutamax Gibco 3SOSO 5 ml 1OOx Pyruvate Gibco 11360 2.5 ml 1OOx Human Albumin Sigma A1887 12.5 ml 20% Ethanolamine Sigma 411000 5 ul 2 mg/ml Insulin Sigma I8405 500 ul 5 mg/ml Transferrin Sigma TO665 250 pil 20 mg/ml Selenite Sigma S9133 50 ul 10 ug/ml T3 Sigma TO397 50 ill 0.4 mg/ml Beta mercapto ethanol Sigma M7522 35 ul 1.43M
0290 The exemplary invention media formulation was media formulation, at initiation of differentiation, the initial Supplemented at feeding time with 10 ng/ml or 20 ng/ml basic drop in cell counts was absent and resulted in significantly FGF and 0 or 1 lug/ml Hyaluronidase. The CM media was larger numbers of differentiated neural cells (FIG. 10). formulated as previously described: KO-DMEM, 20% KO 0295 The addition of hyaluronidase shifts the differentia Serum replacement, Non essential amino acids 1x. tion tendency toward the endodermal lineage, while the L-Glutamine 2 mM, B-mercaptoethanol 100 uM, bFGF 5 removal hyaluronidase shifts the culture toward ectodermal ng/ml. This composition is incubated overnight on a conflu lineage. Addition of hyaluronidase to the media corrected the ent and mitotically inactivated mouse embryonic fibroblast tendency to ectodermal differentiation resulting in typical culture and 10 ng/mlbFGF is added prior to use for the culture stem cell colonies, with minimal spontaneously differentiat of human embryonic stem cells. ing cells (FIGS. 6C, D, E, F). 0291. The cultures were fed daily and passaged weekly at 0296 Prolonged exposure to hyaluronidase caused differ 1:3 or 1:4 ratios in 75 cm2 plastic flasks coated overnight with entiation toward the endodermal lineage, characterized by 1:30 diluted growth factor reduced Matrigel. The cultures large polygonal cells with abundant cytoplasm and prominent were maintained at 37° C. and in 5% CO2 atmosphere. The nuclei. These cells were alpha-feto protein positive, indica passage was performed by enzymatic dissociation using col tive of endodermal lineages. US 2011/0306129 A1 Dec. 15, 2011
0297. By feeding the stem cell cultures in exemplary was maintained, evidenced by typical colony morphology, invention media formulation with the increase of the bFGF expression of the stemness markers Oct4 and SSEA4, and concentration up to 20 ng/ml and addition of hyaluronidase at pluripotency (ectoderm, mesoderm, endoderm). After 5 pas 1 ug/ml for two days in each passage cycle, undifferentiated sages the cultures manifested a tendency to differentiate pref pluripotent stem cell cultures were grown for many passages erentially towards ectodermal lineages in exemplary inven without signs of extensive differentiation (FIGS. 5 and 6C, D, tion media, if unsupplemented with hyaluronidase. E, F). Stem cell cultures grown in the exemplary invention media formulation maintained pluripotency over more than 5 0301 The yield of neural cells was higher in the stem cell passages, evidenced by expression of the pluripotency mark cultures in exemplary invention media without the addition of ers Octá and SSEA4 (FIG. 7). hyaluronidase compared to cells grown in the CM formula 0298. In vitro differentiation of human embryonic stem tion. This outcome is beneficial when ectodermal lineage cells grown in the exemplary invention media formulation specificity is desired (for example neuro-ectoderm) (FIGS. 9 Supplemented with hyaluronidase resulted in populations and 10). belonging to all germ layers (ectoderm, mesoderm and endo 0302. The ectodermal differentiation tendencies can be derm, FIG. 8). corrected using hyaluronidase for 1-2 days per passage cycle 0299 Karyotype analysis was performed by an indepen and the increasing FGF concentration to 20 g/ml. dent third party service provider (Genzyme Genetics, 0303 Extensive use of hyaluronidase in low concentration Orange, Calif.) and was found to be normal (46, XX) after 7 results in a tendency of endodermal differentiation of the stem passages of growth in the novel media formulation. cell cultures. This outcome is beneficial when endodermal 0300. After extensive growth of hESCs in the exemplary lineage specificity is desired (for example hepatocytes) invention media formulation the typical stem cell phenotype (FIGS. 9 and 10).
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS: 32
SEO ID NO 1 LENGTH: 435 TYPE PRT ORGANISM: Homo sapiens
<4 OOs SEQUENCE: 1
Met Ala Ala His Lieu. Leul Pro Ile Cys Ala Lieu. Phe Lieu. Thir Lieu. Luell 1. 5 15
Asp Met Ala Glin Gly Phe Arg Gly Pro Lell Luell Pro Asn Pro Phe 2O 25 3 O
Thir Thr Wall Trp Asn Ala ASn Thir Glin Trp Luell Glu Arg His Gly 35 4 O 45
Val Asp Wall Asp Wall Ser Wall Phe Asp Wall Wall Ala Asn Pro Gly Glin SO 55 60
Thir Phe Arg Gly Pro Asp Met Thir Ile Phe Tyr Ser Ser Glin Lell Gly 65 70
Thr Tyr Pro Tyr Tyr Thir Pro Thir Gly Glu Pro Wall Phe Gly Gly Lell 85 90 95
Pro Glin Asn Ala Ser Luell Ile Ala His Lell Ala Arg Thir Phe Glin Asp 105 110
Ile Lieu. Ala Ala Ile Pro Ala Pro Asp Phe Ser Gly Luell Ala Wall Ile 115 12O 125
Asp Trp Glu Ala Trp Arg Pro Arg Trp Ala Phe Asn Trp Asp Thir 13 O 135 14 O
Asp Ile Glin Arg Ser Arg Ala Lell Wall Glin Ala Glin His Pro 145 15 O 155 16 O
Asp Trp Pro Ala Pro Glin Wall Glu Ala Wall Ala Glin Asp Glin Phe Glin 1.65 17 O 17s
Gly Ala Ala Arg Ala Trp Met Ala Gly Thir Luell Glin Luell Gly Arg Ala 18O 185 190
Lieu. Arg Pro Arg Gly Luell Trp Gly Phe Gly Phe Pro Asp US 2011/0306129 A1 Dec. 15, 2011 20
- Continued
195 2OO 2O5 Asn Tyr Asp Phe Leu Ser Pro Asn Tyr Thr Gly Glin Cys Pro Ser Gly 21 O 215 22O Ile Arg Ala Glin Asn Asp Gln Lieu. Gly Trp Lieu. Trp Gly Glin Ser Arg 225 23 O 235 24 O Ala Leu Tyr Pro Ser Ile Tyr Met Pro Ala Val Lieu. Glu Gly Thr Gly 245 250 255 Llys Ser Gln Met Tyr Val Glin His Arg Val Ala Glu Ala Phe Arg Val 26 O 265 27 O Ala Val Ala Ala Gly Asp Pro Asn Lieu Pro Val Lieu Pro Tyr Val Glin 27s 28O 285 Ile Phe Tyr Asp Thr Thr Asn His Phe Leu Pro Leu Asp Glu Lieu. Glu 29 O 295 3 OO His Ser Lieu. Gly Glu Ser Ala Ala Glin Gly Ala Ala Gly Val Val Lieu. 3. OS 310 315 32O Trp Val Ser Trp Glu Asn Thr Arg Thr Lys Glu Ser Cys Glin Ala Ile 3.25 330 335 Lys Glu Tyr Met Asp Thir Thr Lieu. Gly Pro Phe Ile Lieu. Asn Val Thr 34 O 345 35. O Ser Gly Ala Lieu. Lieu. Cys Ser Glin Ala Lieu. Cys Ser Gly. His Gly Arg 355 360 365 CyS Val Arg Arg Thr Ser His Pro Lys Ala Lieu. Lieu. Lieu. Lieu. ASn Pro 37 O 375 38O Ala Ser Phe Ser Ile Glin Lieu. Thr Pro Gly Gly Gly Pro Leu Ser Leu 385 390 395 4 OO Arg Gly Ala Lieu. Ser Lieu. Glu Asp Glin Ala Glin Met Ala Val Glu Phe 4 OS 41O 415 Lys Cys Arg Cys Tyr Pro Gly Trp Glin Ala Pro Trp Cys Glu Arg Llys 42O 425 43 O
Ser Met Trp 435
<210s, SEQ ID NO 2 &211s LENGTH: 450 212. TYPE: PRT &213s ORGANISM: Bos taurus
<4 OOs, SEQUENCE: 2 Met Arg Pro Phe Ser Lieu. Glu Val Ser Lieu. His Leu Pro Trp Ala Met 1. 5 1O 15 Ala Ala His Lieu Lleu Pro Val Cys Thir Lieu. Phe Lieu. Asn Lieu. Lieu. Ser 2O 25 3O Met Thr Glin Gly Ser Arg Asp Pro Val Val Pro Asn Gln Pro Phe Thr 35 4 O 45 Thir Ile Trp Asn Ala Asn Thr Glu Trp Cys Met Lys Llys His Gly Val SO 55 6 O Asp Val Asp Ile Ser Ile Phe Asp Val Val Thr Asn Pro Gly Glin Thr 65 70 7s 8O Phe Arg Gly Pro Asn Met Thr Ile Phe Tyr Ser Ser Gln Leu Gly Thr 85 90 95 Tyr Pro Tyr Tyr Thr Ser Ala Gly Glu Pro Val Phe Gly Gly Lieu Pro 1OO 105 11 O US 2011/0306129 A1 Dec. 15, 2011 21
- Continued Glin Asn Ala Ser Lieu. Asn Ala His Lieu Ala Arg Thr Phe Glin Asp Ile 115 12 O 125 Lieu Ala Ala Met Pro Glu Pro Arg Phe Ser Gly Lieu Ala Val Ile Asp 13 O 135 14 O Trp Glu Ala Trp Arg Pro Arg Trp Ala Phe Asn Trp Asp Thir Lys Asp 145 150 155 160 Ile Tyr Arg Glin Arg Ser Arg Ala Lieu Val Glin Lys Glin His Pro Asp 1.65 17O 17s Trp Lieu Ala Pro Arg Val Glu Ala Ala Ala Glin Asp Glin Phe Glu Gly 18O 185 19 O Ala Ala Glu Glu Trp Met Ala Gly. Thir Lieu Lys Lieu. Gly Glin Ala Lieu 195 2OO 2O5 Arg Pro Glin Gly Lieu. Trp Gly Phe Tyr Asn Phe Pro Glu. Cys Tyr Asn 21 O 215 22O Tyr Asp Phe Llys Ser Pro Asn Tyr Thr Gly Arg Cys Pro Leu. Asn Ile 225 23 O 235 24 O Cys Ala Glin Asn Asp Glin Lieu. Gly Trp Lieu. Trp Gly Glin Ser Arg Ala 245 250 255 Lieu. Tyr Pro Ser Ile Tyr Lieu Pro Ala Ala Lieu. Glu Gly. Thir Lys Llys 26 O 265 27 O Thr Gln Met Phe Val Gln His Arg Val Ala Glu Ala Phe Arg Val Ala 27s 28O 285 Ala Gly Ala Gly Asp Pro Llys Lieu Pro Val Lieu Pro Tyr Met Glin Lieu 29 O 295 3 OO Phe Tyr Asp Met Thr Asn His Phe Leu Pro Ala Glu Glu Lieu. Glu. His 3. OS 310 315 32O Ser Lieu. Gly Glu Ser Ala Ala Glin Gly Ala Ala Gly Val Val Lieu. Trp 3.25 330 335 Val Ser Trp Leu Ser Thr Ser Thr Lys Glu Ser Cys Glin Ala Ile Llys 34 O 345 35. O Glu Tyr Val Asp Thr Thr Lieu. Gly Pro Ser Ile Lieu. Asn Val Thr Ser 355 360 365 Gly Ala Arg Lieu. Cys Ser Glin Val Lieu. Cys Ser Gly His Gly Arg Cys 37 O 375 38O Ala Arg Arg Pro Ser Tyr Pro Lys Ala Arg Lieu. Ile Lieu. Asn. Ser Thr 385 390 395 4 OO Ser Phe Ser Ile Llys Pro Thr Pro Gly Gly Gly Pro Leu. Thir Lieu. Glin 4 OS 41O 415 Gly Ala Lieu. Ser Lieu. Glu Asp Arg Lieu. Arg Met Ala Val Glu Phe Glu 42O 425 43 O Cys Arg Cys Tyr Arg Gly Trp Arg Gly Thr Arg Cys Glu Gln Trp Gly 435 44 O 445
Met Trp 450
<210s, SEQ ID NO 3 &211s LENGTH: 462 212. TYPE: PRT <213s ORGANISM: Mus musculus
<4 OOs, SEQUENCE: 3 Met Lieu. Gly Lieu. Thr Gln His Ala Glin Llys Val Trp Arg Met Llys Pro 1. 5 1O 15 US 2011/0306129 A1 Dec. 15, 2011 22
- Continued
Phe Ser Pro Glu Val Ser Pro Gly Ser Ser Pro Ala Thr Ala Gly His 2O 25 3O Lieu. Lieu. Arg Ile Ser Thr Lieu. Phe Lieu. Thir Lieu. Lieu. Glu Lieu Ala Glin 35 4 O 45 Val Cys Arg Gly Ser Val Val Ser Asn Arg Pro Phe Ile Thr Val Trp SO 55 6 O Asn Gly Asp Thr His Trp Cys Lieu. Thr Glu Tyr Gly Val Asp Val Asp 65 70 7s 8O Val Ser Val Phe Asp Val Val Ala Asn Lys Glu Glin Ser Phe Glin Gly 85 90 95 Ser Asn Met Thr Ile Phe Tyr Arg Glu Glu Lieu. Gly Thr Tyr Pro Tyr 1OO 105 11 O Tyr Thr Pro Thr Gly Glu Pro Val Phe Gly Gly Lieu Pro Glin Asn Ala 115 12 O 125 Ser Lieu Val Thir His Lieu Ala His Thr Phe Glin Asp Ile Lys Ala Ala 13 O 135 14 O Met Pro Glu Pro Asp Phe Ser Gly Lieu Ala Val Ile Asp Trp Glu Ala 145 150 155 160 Trp Arg Pro Arg Trp Ala Phe Asn Trp Asp Ser Lys Asp Ile Tyr Arg 1.65 17O 17s Glin Arg Ser Met Glu Lieu Val Glin Ala Glu. His Pro Asp Trp Pro Glu 18O 185 19 O Thir Lieu Val Glu Ala Ala Ala Lys Asn Glin Phe Glin Glu Ala Ala Glu 195 2OO 2O5 Ala Trp Met Ala Gly. Thir Lieu. Glin Lieu. Gly Glin Val Lieu. Arg Pro Arg 21 O 215 22O Gly Leu Trp Gly Tyr Tyr Gly Phe Pro Asp Cys Tyr Asn Asn Asp Phe 225 23 O 235 24 O Lieu. Ser Lieu. Asn Tyr Thr Gly Glin Cys Pro Val Phe Val Arg Asp Glin 245 250 255 Asn Asp Gln Lieu. Gly Trp Lieu. Trp Asn Glin Ser Tyr Ala Lieu. Tyr Pro 26 O 265 27 O Ser Ile Tyr Lieu Pro Ala Ala Leu Met Gly Thr Gly Lys Ser Gln Met 27s 28O 285 Tyr Val Arg His Arg Val Glin Glu Ala Lieu. Arg Val Ala Ile Val Ser 29 O 295 3 OO Arg Asp Pro His Val Pro Val Met Pro Tyr Val Glin Ile Phe Tyr Glu 3. OS 310 315 32O Met Thr Asp Tyr Lieu Lleu Pro Lieu. Glu Glu Lieu. Glu. His Ser Lieu. Gly 3.25 330 335 Glu Ser Ala Ala Glin Gly Val Ala Gly Ala Val Lieu. Trp Lieu. Ser Ser 34 O 345 35. O Asp Llys Thir Ser Thr Lys Glu Ser Cys Glin Ala Ile Lys Ala Tyr Met 355 360 365 Asp Ser Thr Lieu. Gly Pro Phe Ile Val Asn Val Thr Ser Ala Ala Leu 37 O 375 38O Lieu. Cys Ser Glu Ala Lieu. Cys Ser Gly His Gly Arg Cys Val Arg His 385 390 395 4 OO Pro Ser Tyr Pro Glu Ala Lieu. Lieu. Thir Lieu. Asn Pro Ala Ser Phe Ser 4 OS 41O 415 US 2011/0306129 A1 Dec. 15, 2011 23
- Continued Ile Glu Lieu. Thir His Asp Gly Arg Pro Pro Ser Lieu Lys Gly. Thir Lieu. 42O 425 43 O Ser Lieu Lys Asp Arg Ala Gln Met Ala Met Llys Phe Arg Cys Arg Cys 435 44 O 445 Tyr Arg Gly Trp Arg Gly Llys Trp Cys Asp Lys Arg Gly Met 450 45.5 460
<210s, SEQ ID NO 4 &211s LENGTH: 473 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 4 Met Arg Ala Gly Pro Gly Pro Thr Val Thir Lieu Ala Lieu Val Lieu Ala 1. 5 1O 15 Val Ala Trp Ala Met Glu Lieu Lys Pro Thr Ala Pro Pro Ile Phe Thr 2O 25 3O Gly Arg Pro Phe Val Val Ala Trp Asp Val Pro Thr Glin Asp Cys Gly 35 4 O 45 Pro Arg Lieu Lys Val Pro Lieu. Asp Lieu. Asn Ala Phe Asp Val Glin Ala SO 55 6 O Ser Pro Asn Glu Gly Phe Val Asn Glin Asn Ile Thr Ile Phe Tyr Arg 65 70 7s 8O Asp Arg Lieu. Gly Lieu. Tyr Pro Arg Phe Asp Ser Ala Gly Arg Ser Val 85 90 95 His Gly Gly Val Pro Glin Asn Val Ser Lieu. Trp Ala His Arg Llys Met 1OO 105 11 O Lieu. Glin Lys Arg Val Glu. His Tyr Ile Arg Thr Glin Glu Ser Ala Gly 115 12 O 125 Lieu Ala Val Ile Asp Trp Glu Asp Trp Arg Pro Val Trp Val Arg Asn 13 O 135 14 O Trp Glin Asp Lys Asp Val Tyr Arg Arg Lieu. Ser Arg Glin Lieu Val Ala 145 150 155 160 Ser Arg His Pro Asp Trp Pro Pro Asp Arg Ile Val Lys Glin Ala Glin 1.65 17O 17s Tyr Glu Phe Glu Phe Ala Ala Glin Glin Phe Met Lieu. Glu Thir Lieu. Arg 18O 185 19 O Tyr Val Lys Ala Val Arg Pro Arg His Leu Trp Gly Phe Tyr Lieu Phe 195 2OO 2O5 Pro Asp Cys Tyr Asn His Asp Tyr Val Glin Asn Trp Glu Ser Tyr Thr 21 O 215 22O Gly Arg Cys Pro Asp Val Glu Val Ala Arg Asn Asp Gln Lieu Ala Trp 225 23 O 235 24 O Lieu. Trp Ala Glu Ser Thr Ala Lieu. Phe Pro Ser Val Tyr Lieu. Asp Glu 245 250 255 Thir Lieu Ala Ser Ser Arg His Gly Arg Asn. Phe Val Ser Phe Arg Val 26 O 265 27 O Glin Glu Ala Lieu. Arg Val Ala Arg Thr His His Ala Asn His Ala Lieu. 27s 28O 285 Pro Val Tyr Val Phe Thr Arg Pro Thr Tyr Ser Arg Arg Lieu. Thr Gly 29 O 295 3 OO Lieu. Ser Glu Met Asp Lieu. Ile Ser Thir Ile Gly Glu Ser Ala Ala Lieu. 3. OS 310 315 32O US 2011/0306129 A1 Dec. 15, 2011 24
- Continued
Gly Ala Ala Gly Val Ile Lieu. Trp Gly Asp Ala Gly Tyr Thir Thir Ser 3.25 330 335 Thr Glu Thir Cys Glin Tyr Lieu Lys Asp Tyr Lieu. Thir Arg Lieu. Lieu Val 34 O 345 35. O Pro Tyr Val Val Asn Val Ser Trp Ala Thr Glin Tyr Cys Ser Arg Ala 355 360 365 Glin Cys His Gly His Gly Arg Cys Val Arg Arg ASn Pro Ser Ala Ser 37 O 375 38O Thr Phe Lieu. His Leu Ser Thr Asn Ser Phe Arg Lieu Val Pro Gly His 385 390 395 4 OO Ala Pro Gly Glu Pro Gln Lieu. Arg Pro Val Gly Glu Lieu. Ser Trp Ala 4 OS 41O 415 Asp Ile Asp His Lieu. Glin Thr His Phe Arg Cys Glin Cys Tyr Lieu. Gly 42O 425 43 O Trp Ser Gly Glu Glin Cys Gln Trp Asp His Arg Glin Ala Ala Gly Gly 435 44 O 445 Ala Ser Glu Ala Trp Ala Gly Ser His Lieu. Thir Ser Lieu. Lieu Ala Lieu 450 45.5 460 Ala Ala Lieu Ala Phe Thir Trp Thir Lieu. 465 470
<210 SEQ ID NO 5 &211s LENGTH: 473 212. TYPE: PRT <213s ORGANISM: Mus musculus
<4 OOs, SEQUENCE: 5 Met Arg Ala Gly Lieu. Gly Pro Ile Ile Thir Lieu Ala Lieu Val Lieu. Glu 1. 5 1O 15 Val Ala Trp Ala Gly Glu Lieu Lys Pro Thr Ala Pro Pro Ile Phe Thr 2O 25 3O Gly Arg Pro Phe Val Val Ala Trp Asn Val Pro Thr Glin Glu. Cys Ala 35 4 O 45 Pro Arg His Llys Val Pro Lieu. Asp Lieu. Arg Ala Phe Asp Wall Lys Ala SO 55 6 O Thr Pro Asn Glu Gly Phe Phe Asin Glin Asn Ile Thr Thr Phe Tyr Tyr 65 70 7s 8O Asp Arg Lieu. Gly Lieu. Tyr Pro Arg Phe Asp Ala Ala Gly. Thir Ser Val 85 90 95 His Gly Gly Val Pro Glin Asn Gly Ser Lieu. Cys Ala His Leu Pro Met 1OO 105 11 O Lieu Lys Glu Ser Val Glu Arg Tyr Ile Glin Thr Glin Glu Pro Gly Gly 115 12 O 125 Lieu Ala Val Ile Asp Trp Glu Glu Trp Arg Pro Val Trp Val Arg Asn 13 O 135 14 O Trp Glin Glu Lys Asp Val Tyr Arg Glin Ser Ser Arg Glin Lieu Val Ala 145 150 155 160 Ser Arg His Pro Asp Trp Pro Ser Asp Arg Val Met Lys Glin Ala Glin 1.65 17O 17s Tyr Glu Phe Glu Phe Ala Ala Arg Glin Phe Met Lieu. Asn. Thir Lieu. Arg 18O 185 19 O Tyr Val Lys Ala Val Arg Pro Gln His Leu Trp Gly Phe Tyr Lieu Phe US 2011/0306129 A1 Dec. 15, 2011 25
- Continued
195 2OO 2O5 Pro Asp Cys Tyr Asn His Asp Tyr Val Glin Asn Trp Glu Ser Tyr Thr 21 O 215 22O Gly Arg Cys Pro Asp Val Glu Val Ala Arg Asn Asp Gln Lieu Ala Trp 225 23 O 235 24 O Lieu. Trp Ala Glu Ser Thr Ala Lieu. Phe Pro Ser Val Tyr Lieu. Asp Glu 245 250 255 Thr Lieu Ala Ser Ser Val His Ser Arg Asin Phe Val Ser Phe Arg Val 26 O 265 27 O Arg Glu Ala Lieu. Arg Val Ala His Thr His His Ala Asn His Ala Lieu 27s 28O 285 Pro Val Tyr Val Phe Thr Arg Pro Thr Tyr Thr Arg Gly Lieu. Thr Gly 29 O 295 3 OO Lieu. Ser Glin Val Asp Lieu. Ile Ser Thir Ile Gly Glu Ser Ala Ala Lieu. 3. OS 310 315 32O Gly Ser Ala Gly Val Ile Phe Trp Gly Asp Ser Glu Asp Ala Ser Ser 3.25 330 335 Met Glu Thir Cys Glin Tyr Lieu Lys Asn Tyr Lieu. Thr Gln Lieu. Leu Val 34 O 345 35. O Pro Tyr Ile Val Asn Val Ser Trp Ala Thr Glin Tyr Cys Ser Trp Thr 355 360 365 Gln Cys His Gly His Gly Arg CyS Val Arg Arg ASn Pro Ser Ala ASn 37 O 375 38O Thr Phe Lieu. His Lieu. Asn Ala Ser Ser Phe Arg Lieu Val Pro Gly His 385 390 395 4 OO Thr Pro Ser Glu Pro Gln Lieu. Arg Pro Glu Gly Gln Leu Ser Glu Ala 4 OS 41O 415 Asp Lieu. Asn Tyr Lieu. Glin Llys His Phe Arg Cys Glin Cys Tyr Lieu. Gly 42O 425 43 O Trp Gly Gly Glu Glin Cys Glin Arg Asn Tyr Lys Gly Ala Ala Gly Asn 435 44 O 445 Ala Ser Arg Ala Trp Ala Gly Ser His Lieu. Thir Ser Lieu. Lieu. Gly Lieu 450 45.5 460 Val Ala Val Ala Lieu. Thir Trp Thir Lieu. 465 470
<210s, SEQ ID NO 6 &211s LENGTH: 417 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 6 Met Thir Thr Glin Lieu. Gly Pro Ala Lieu Val Lieu. Gly Val Ala Lieu. Cys 1. 5 1O 15 Lieu. Gly Cys Gly Glin Pro Leu Pro Glin Val Pro Glu Arg Pro Phe Ser 2O 25 3O Val Lieu. Trp Asn Val Pro Ser Ala His Cys Glu Ala Arg Phe Gly Val 35 4 O 45 His Lieu Pro Lieu. Asn Ala Lieu. Gly Ile Ile Ala Asn Arg Gly Gln His SO 55 6 O Phe His Gly Glin Asn Met Thr Ile Phe Tyr Lys Asn Gln Leu Gly Lieu. 65 70 7s 8O US 2011/0306129 A1 Dec. 15, 2011 26
- Continued Tyr Pro Tyr Phe Gly Pro Arg Gly Thr Ala His Asn Gly Gly Ile Pro 85 90 95 Glin Ala Lieu Pro Lieu. Asp Arg His Lieu Ala Lieu Ala Ala Tyr Glin Ile 1OO 105 11 O His His Ser Lieu. Arg Pro Gly Phe Ala Gly Pro Ala Val Lieu. Asp Trp 115 12 O 125 Glu Glu Trp Cys Pro Lieu. Trp Ala Gly Asn Trp Gly Arg Arg Arg Ala 13 O 135 14 O Tyr Glin Ala Ala Ser Trp Ala Trp Ala Glin Glin Val Phe Pro Asp Lieu. 145 150 155 160 Asp Pro Glin Glu Gln Lieu. Tyr Lys Ala Tyr Thr Gly Phe Glu Glin Ala 1.65 17O 17s Ala Arg Ala Lieu Met Glu Asp Thir Lieu. Arg Val Ala Glin Ala Lieu. Arg 18O 185 19 O Pro His Gly Leu Trp Gly Phe Tyr His Tyr Pro Ala Cys Gly Asn Gly 195 2OO 2O5 Trp. His Ser Met Ala Ser Asn Tyr Thr Gly Arg Cys His Ala Ala Thr 21 O 215 22O Lieu Ala Arg Asn. Thr Glin Lieu. His Trp Lieu. Trp Ala Ala Ser Ser Ala 225 23 O 235 24 O Lieu. Phe Pro Ser Ile Tyr Lieu Pro Pro Arg Lieu Pro Pro Ala His His 245 250 255 Glin Ala Phe Val Arg His Arg Lieu. Glu Glu Ala Phe Arg Val Ala Lieu. 26 O 265 27 O Val Gly His Arg His Pro Lieu Pro Val Lieu Ala Tyr Val Arg Lieu. Thr 27s 28O 285 His Arg Arg Ser Gly Arg Phe Lieu. Ser Glin Asp Asp Lieu Val Glin Ser 29 O 295 3 OO Ile Gly Val Ser Ala Ala Lieu. Gly Ala Ala Gly Val Val Lieu. Trp Gly 3. OS 310 315 32O Asp Lieu. Ser Lieu. Ser Ser Ser Glu Glu Glu. Cys Trp His Lieu. His Asp 3.25 330 335 Tyr Lieu Val Asp Thr Lieu. Gly Pro Tyr Val Ile Asn Val Thr Arg Ala 34 O 345 35. O Ala Met Ala Cys Ser His Glin Arg Cys His Gly His Gly Arg Cys Ala 355 360 365 Arg Arg Asp Pro Gly Gln Met Glu Ala Phe Lieu. His Lieu. Trp Pro Asp 37 O 375 38O Gly Ser Leu Gly Asp Trp Llys Ser Phe Ser Cys His Cys Tyr Trp Gly 385 390 395 4 OO Trp Ala Gly Pro Thr Cys Glin Glu Pro Arg Pro Gly Pro Lys Glu Ala 4 OS 41O 415
Wall
<210s, SEQ ID NO 7 &211s LENGTH: 481 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 7 Met Llys Val Lieu. Ser Glu Gly Glin Lieu Lys Lieu. Cys Val Val Glin Pro 1. 5 1O 15 US 2011/0306129 A1 Dec. 15, 2011 27
- Continued Val His Lieu. Thir Ser Trp Lieu. Lieu. Ile Phe Phe Ile Leu Lys Ser Ile 2O 25 3O Ser Cys Lieu Lys Pro Ala Arg Lieu Pro Ile Tyr Glin Arg Llys Pro Phe 35 4 O 45 Ile Ala Ala Trp Asn Ala Pro Thir Asp Glin Cys Lieu. Ile Llys Tyr Asn SO 55 6 O Lieu. Arg Lieu. Asn Lieu Lys Met Phe Pro Val Ile Gly Ser Pro Lieu Ala 65 70 7s 8O Lys Ala Arg Gly Glin Asn Val Thir Ile Phe Tyr Val Asn Arg Lieu. Gly 85 90 95 Tyr Tyr Pro Trp Tyr Thr Ser Glin Gly Val Pro Ile Asn Gly Gly Lieu. 1OO 105 11 O Pro Glin Asn. Ile Ser Lieu. Glin Val His Lieu. Glu Lys Ala Asp Glin Asp 115 12 O 125 Ile Asn Tyr Tyr Ile Pro Ala Glu Asp Phe Ser Gly Lieu Ala Val Ile 13 O 135 14 O Asp Trp Glu Tyr Trp Arg Pro Gln Trp Ala Arg Asn Trp Asn. Ser Lys 145 150 155 160 Asp Val Tyr Arg Glin Llys Ser Arg Llys Lieu. Ile Ser Asp Met Gly Lys 1.65 17O 17s Asn Val Ser Ala Thr Asp Ile Glu Tyr Lieu Ala Llys Val Thr Phe Glu 18O 185 19 O Glu Ser Ala Lys Ala Phe Met Lys Glu Thir Ile Llys Lieu. Gly Ile Llys 195 2OO 2O5 Ser Arg Pro Lys Gly Lieu. Trp Gly Tyr Tyr Lieu. Tyr Pro Asp Cys His 21 O 215 22O Asn Tyr Asn Val Tyr Ala Pro Asn Tyr Ser Gly Ser Cys Pro Glu Asp 225 23 O 235 24 O Glu Val Lieu. Arg Asn. Asn. Glu Lieu. Ser Trp Lieu. Trp Asn. Ser Ser Ala 245 250 255 Ala Lieu. Tyr Pro Ser Ile Cys Val Trp Llys Ser Lieu. Gly Asp Ser Glu 26 O 265 27 O Asn. Ile Lieu. Arg Phe Ser Llys Phe Arg Val His Glu Ser Met Arg Ile 27s 28O 285 Ser Thr Met Thr Ser His Asp Tyr Ala Leu Pro Val Phe Val Tyr Thr 29 O 295 3 OO Arg Lieu. Gly Tyr Arg Asp Glu Pro Lieu. Phe Phe Lieu. Ser Lys Glin Asp 3. OS 310 315 32O Lieu Val Ser Thir Ile Gly Glu Ser Ala Ala Lieu. Gly Ala Ala Gly Ile 3.25 330 335 Val Ile Trp Gly Asp Met Asn Lieu. Thir Ala Ser Lys Ala Asn. Cys Thr 34 O 345 35. O Llys Val Lys Glin Phe Val Ser Ser Asp Lieu. Gly Ser Tyr Ile Ala Asn 355 360 365 Val Thr Arg Ala Ala Glu Val Cys Ser Lieu. His Lieu. Cys Arg Asn. Asn 37 O 375 38O Gly Arg Cys Ile Arg Llys Met Trp Asn Ala Pro Ser Tyr Lieu. His Lieu. 385 390 395 4 OO Asn Pro Ala Ser Tyr His Ile Glu Ala Ser Glu Asp Gly Glu Phe Thr 4 OS 41O 415 Val Lys Gly Lys Ala Ser Asp Thr Asp Lieu Ala Wal Met Ala Asp Thr US 2011/0306129 A1 Dec. 15, 2011 28
- Continued
42O 425 43 O Phe Ser Cys His Cys Tyr Glin Gly Tyr Glu Gly Ala Asp Cys Arg Glu 435 44 O 445 Ile Llys Thr Ala Asp Gly Cys Ser Gly Val Ser Pro Ser Pro Gly Ser 450 45.5 460 Lieu Met Thr Lieu. Cys Lieu. Lieu. Lieu. Lieu Ala Ser Tyr Arg Ser Ile Glin 465 470 47s 48O
Lell
<210s, SEQ ID NO 8 &211s LENGTH: 155 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 8 Met Ala Glu Gly Glu Ile Thir Thr Phe Thr Ala Lieu. Thr Glu Lys Phe 1. 5 1O 15 Asn Lieu Pro Pro Gly Asn Tyr Lys Llys Pro Llys Lieu. Lieu. Tyr Cys Ser 2O 25 3O Asn Gly Gly His Phe Lieu. Arg Ile Lieu Pro Asp Gly. Thr Val Asp Gly 35 4 O 45 Thir Arg Asp Arg Ser Asp Gln His Ile Glin Lieu. Glin Lieu. Ser Ala Glu SO 55 6 O Ser Val Gly Glu Val Tyr Ile Llys Ser Thr Glu Thr Gly Glin Tyr Lieu. 65 70 7s 8O Ala Met Asp Thr Asp Gly Lieu. Lieu. Tyr Gly Ser Glin Thr Pro Asn. Glu 85 90 95 Glu Cys Lieu. Phe Lieu. Glu Arg Lieu. Glu Glu Asn His Tyr Asn. Thir Tyr 1OO 105 11 O Ile Ser Lys Llys His Ala Glu Lys Asn Trp Phe Val Gly Lieu Lys Llys 115 12 O 125 Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Glin Lys Ala 13 O 135 14 O Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150 155
<210s, SEQ ID NO 9 &211s LENGTH: 155 212. TYPE: PRT <213s ORGANISM: Mus musculus
<4 OOs, SEQUENCE: 9 Met Ala Glu Gly Glu Ile Thir Thr Phe Ala Ala Lieu. Thr Glu Arg Phe 1. 5 1O 15 Asn Lieu Pro Lieu. Gly Asn Tyr Lys Llys Pro Llys Lieu. Lieu. Tyr Cys Ser 2O 25 3O Asn Gly Gly His Phe Lieu. Arg Ile Lieu Pro Asp Gly. Thr Val Asp Gly 35 4 O 45 Thir Arg Asp Arg Ser Asp Gln His Ile Glin Lieu. Glin Lieu. Ser Ala Glu SO 55 6 O Ser Ala Gly Glu Val Tyr Ile Lys Gly Thr Glu Thr Gly Glin Tyr Lieu. 65 70 7s 8O Ala Met Asp Thr Glu Gly Lieu. Leu Tyr Gly Ser Glin Thr Pro Asn Glu 85 90 95 US 2011/0306129 A1 Dec. 15, 2011 29
- Continued
Glu Cys Lieu. Phe Lieu. Glu Arg Lieu. Glu Glu Asn His Tyr Asn. Thir Tyr 1OO 105 11 O Thir Ser Lys Llys His Ala Glu Lys Asn Trp Phe Val Gly Lieu Lys Llys 115 12 O 125 Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Glin Lys Ala 13 O 135 14 O Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150 155
<210s, SEQ ID NO 10 &211s LENGTH: 155 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 10 Met Ala Ala Gly Ser Ile Thir Thr Lieu Pro Ala Lieu Pro Glu Asp Gly 1. 5 1O 15 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Lieu. 2O 25 3O Tyr Cys Lys Asn Gly Gly Phe Phe Lieu. Arg Ile His Pro Asp Gly Arg 35 4 O 45 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Llys Lieu. Glin Lieu SO 55 6 O Glin Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn 65 70 7s 8O Arg Tyr Lieu Ala Met Lys Glu Asp Gly Arg Lieu. Lieu Ala Ser Lys Cys 85 90 95 Val Thr Asp Glu. Cys Phe Phe Phe Glu Arg Lieu. Glu Ser Asn Asn Tyr 1OO 105 11 O Asn Thr Tyr Arg Ser Arg Llys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115 12 O 125 Arg Thr Gly Glin Tyr Llys Lieu. Gly Ser Lys Thr Gly Pro Gly Glin Lys 13 O 135 14 O Ala Ile Lieu. Phe Lieu Pro Met Ser Ala Lys Ser 145 150 155
<210s, SEQ ID NO 11 &211s LENGTH: 154 212. TYPE: PRT <213s ORGANISM: Mus musculus
<4 OOs, SEQUENCE: 11 Met Ala Ala Ser Gly Ile Thir Ser Lieu Pro Ala Lieu Pro Glu Asp Gly 1. 5 1O 15 Gly Ala Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Lieu. Tyr 2O 25 3O Cys Lys Asn Gly Gly Phe Phe Lieu. Arg Ile His Pro Asp Gly Arg Val 35 4 O 45 Asp Gly Val Arg Glu Lys Ser Asp Pro His Val Llys Lieu. Glin Lieu. Glin SO 55 6 O Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn Arg 65 70 7s 8O Tyr Lieu Ala Met Lys Glu Asp Gly Arg Lieu. Lieu Ala Ser Lys Cys Val 85 90 95 US 2011/0306129 A1 Dec. 15, 2011 30
- Continued
Thr Glu Glu. Cys Phe Phe Phe Glu Arg Lieu. Glu Ser Asn Asn Tyr Asn 1OO 105 11 O Thir Tyr Arg Ser Arg Llys Tyr Ser Ser Trp Tyr Val Ala Lieu Lys Arg 115 12 O 125 Thr Gly Glin Tyr Lys Lieu. Gly Ser Lys Thr Gly Pro Gly Glin Lys Ala 13 O 135 14 O Ile Leu Phe Leu Pro Met Ser Ala Lys Ser 145 150
<210s, SEQ ID NO 12 &211s LENGTH: 238 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 12 Met Gly Lieu. Ile Trp Lieu Lleu Lieu. Lieu. Ser Lieu. Lieu. Glu Pro Gly Trp 1. 5 1O 15 Pro Ala Ala Gly Pro Gly Ala Arg Lieu. Arg Arg Asp Ala Gly Gly Arg 2O 25 3O Gly Gly Val Tyr Glu. His Lieu. Gly Gly Ala Pro Arg Arg Arg Llys Lieu. 35 4 O 45 Tyr Cys Ala Thr Lys Tyr His Leu Gln Lieu. His Pro Ser Gly Arg Val SO 55 6 O Asn Gly Ser Lieu. Glu Asn. Ser Ala Tyr Ser Ile Lieu. Glu Ile Thir Ala 65 70 7s 8O Val Glu Val Gly Ile Val Ala Ile Arg Gly Lieu. Phe Ser Gly Arg Tyr 85 90 95 Lieu Ala Met Asn Lys Arg Gly Arg Lieu. Tyr Ala Ser Glu. His Tyr Ser 1OO 105 11 O Ala Glu. Cys Glu Phe Val Glu Arg Ile His Glu Lieu. Gly Tyr Asn Thr 115 12 O 125 Tyr Ala Ser Arg Lieu. Tyr Arg Thr Val Ser Ser Thr Pro Gly Ala Arg 13 O 135 14 O Arg Glin Pro Ser Ala Glu Arg Lieu. Trp Tyr Val Ser Val Asn Gly Lys 145 150 155 160 Gly Arg Pro Arg Arg Gly Phe Llys Thr Arg Arg Thr Glin Llys Ser Ser 1.65 17O 17s Lieu. Phe Lieu Pro Arg Val Lieu. Asp His Arg Asp His Glu Met Val Arg 18O 185 19 O Gln Leu Glin Ser Gly Lieu Pro Arg Pro Pro Gly Lys Gly Val Glin Pro 195 2OO 2O5 Arg Arg Arg Arg Gln Lys Glin Ser Pro Asp Asn Lieu. Glu Pro Ser His 21 O 215 22O Val Ala Ser Arg Lieu. Gly Ser Glin Lieu. Glu Ala Ser Ala His 225 23 O 235
<210s, SEQ ID NO 13 &211s LENGTH: 2O4 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 13 Met Ser Gly Pro Gly Thr Ala Ala Val Ala Lieu Lleu Pro Ala Val Lieu. 1. 5 1O 15 US 2011/0306129 A1 Dec. 15, 2011 31
- Continued
Lieu Ala Lieu. Lieu Ala Pro Trp Ala Gly Arg Gly Gly Ala Ala Ala Pro 2O 25 3O Thir Ala Pro Asn Gly Thr Lieu. Glu Ala Glu Lieu. Glu Arg Arg Trp Glu 35 4 O 45 Ser Lieu Val Ala Lieu. Ser Lieu Ala Arg Lieu Pro Val Ala Ala Glin Pro SO 55 6 O Lys Glu Ala Ala Val Glin Ser Gly Ala Gly Asp Tyr Lieu. Lieu. Gly Ile 65 70 7s 8O Lys Arg Lieu. Arg Arg Lieu. Tyr Cys Asn Val Gly Ile Gly Phe His Lieu. 85 90 95 Glin Ala Lieu Pro Asp Gly Arg Ile Gly Gly Ala His Ala Asp Thir Ser 1OO 105 11 O Lieu. Lieu. Glu Lieu. Ser Pro Val Glu Arg Gly Val Val Ser Ile Phe Gly 115 12 O 125 Val Ala Ser Arg Phe Phe Val Ala Met Ser Ser Lys Gly Llys Lieu. Tyr 13 O 135 14 O Gly Ser Pro Phe Phe Thr Asp Glu. Cys Thr Phe Lys Glu Ile Leu Lieu. 145 150 155 160 Pro Asn Asn Tyr Asn Ala Tyr Glu Ser Tyr Lys Tyr Pro Gly Met Phe 1.65 17O 17s Ile Ala Lieu. Ser Lys Asn Gly Lys Thir Lys Lys Gly Asn Arg Val Ser 18O 185 19 O Pro Thr Met Llys Val Thr His Phe Leu Pro Arg Lieu. 195 2OO
<210s, SEQ ID NO 14 &211s LENGTH: 268 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 14
Met Ser Lieu. Ser Phe Leu Lleu Lleu Lleu Phe Phe Ser His Lieu. Ile Lieu. 1. 5 1O 15 Ser Ala Trp Ala His Gly Glu Lys Arg Lieu Ala Pro Llys Gly Glin Pro 2O 25 3O Gly Pro Ala Ala Thr Asp Arg Asn Pro Arg Gly Ser Ser Ser Arg Glin 35 4 O 45
Ser Ser Ser Ser Ala Met Ser Ser Ser Ser Ala Ser Ser Ser Pro Ala SO 55 6 O Ala Ser Leu Gly Ser Glin Gly Ser Gly Lieu. Glu Gln Ser Ser Phe Glin 65 70 7s 8O Trp Ser Pro Ser Gly Arg Arg Thr Gly Ser Leu Tyr Cys Arg Val Gly 85 90 95 Ile Gly Phe His Leu Glin Ile Tyr Pro Asp Gly Lys Val Asn Gly Ser 1OO 105 11 O
His Glu Ala Asn Met Lieu Ser Val Lieu. Glu Ile Phe Ala Wal Ser Glin 115 12 O 125 Gly Ile Val Gly Ile Arg Gly Val Phe Ser Asn Llys Phe Lieu Ala Met 13 O 135 14 O Ser Lys Lys Gly Llys Lieu. His Ala Ser Ala Lys Phe Thr Asp Asp Cys 145 150 155 160 Llys Phe Arg Glu Arg Phe Glin Glu Asn Ser Tyr Asn Thr Tyr Ala Ser US 2011/0306129 A1 Dec. 15, 2011 32
- Continued
1.65 17O 17s Ala Ile His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Lieu 18O 185 19 O Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Llys Pro 195 2OO 2O5 Gln His Ile Ser Thr His Phe Leu Pro Arg Phe Lys Glin Ser Glu Gln 21 O 215 22O Pro Glu Lieu Ser Phe Thr Val Thr Val Pro Glu Lys Llys Llys Pro Pro 225 23 O 235 24 O Ser Pro Ile Llys Pro Lys Ile Pro Leu Ser Ala Pro Arg Lys Asn Thr 245 250 255 Asn Ser Val Lys Tyr Arg Lieu Lys Phe Arg Phe Gly 26 O 265
<210s, SEQ ID NO 15 &211s LENGTH: 2O8 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 15 Met Ala Lieu. Gly Glin Llys Lieu. Phe Ile Thir Met Ser Arg Gly Ala Gly 1. 5 1O 15 Arg Lieu. Glin Gly Thr Lieu. Trp Ala Lieu Val Phe Lieu. Gly Ile Lieu Val 2O 25 3O Gly Met Val Val Pro Ser Pro Ala Gly Thr Arg Ala Asn Asn Thr Lieu. 35 4 O 45 Lieu. Asp Ser Arg Gly Trp Gly. Thir Lieu. Lieu. Ser Arg Ser Arg Ala Gly SO 55 6 O Lieu Ala Gly Glu Ile Ala Gly Val Asn Trp Glu Ser Gly Tyr Lieu Val 65 70 7s 8O Gly Ile Lys Arg Glin Arg Arg Lieu. Tyr Cys Asn Val Gly Ile Gly Phe 85 90 95 His Lieu. Glin Val Lieu Pro Asp Gly Arg Ile Ser Gly Thr His Glu Glu 1OO 105 11 O Asn Pro Tyr Ser Lieu. Lieu. Glu Ile Ser Thr Val Glu Arg Gly Val Val 115 12 O 125 Ser Lieu. Phe Gly Val Arg Ser Ala Lieu. Phe Val Ala Met Asn. Ser Lys 13 O 135 14 O Gly Arg Lieu. Tyr Ala Thr Pro Ser Phe Glin Glu Glu. Cys Llys Phe Arg 145 150 155 160 Glu Thir Lieu. Lieu Pro Asn. Asn Tyr Asn Ala Tyr Glu Ser Asp Lieu. Tyr 1.65 17O 17s Glin Gly. Thir Tyr Ile Ala Lieu. Ser Lys Tyr Gly Arg Val Lys Arg Gly 18O 185 19 O Ser Llys Val Ser Pro Ile Met Thr Val Thr His Phe Leu Pro Arg Ile 195 2OO 2O5
<210s, SEQ ID NO 16 &211s LENGTH: 194 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 16 Met His Llys Trp Ile Lieu. Thir Trp Ile Leu Pro Thr Lieu. Leu Tyr Arg US 2011/0306129 A1 Dec. 15, 2011 33
- Continued
1. 5 1O 15 Ser Cys Phe His Ile Ile Cys Lieu Val Gly Thr Ile Ser Leu Ala Cys 2O 25 3O Asn Asp Met Thr Pro Glu Gln Met Ala Thr Asn Val Asn Cys Ser Ser 35 4 O 45 Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly Asp Ile SO 55 6 O Arg Val Arg Arg Lieu. Phe Cys Arg Thr Glin Trp Tyr Lieu. Arg Ile Asp 65 70 7s 8O Lys Arg Gly Llys Wall Lys Gly Thr Glin Glu Met Lys Asn. Asn Tyr Asn 85 90 95 Ile Met Glu Ile Arg Thr Val Ala Val Gly Ile Val Ala Ile Lys Gly 1OO 105 11 O Val Glu Ser Glu Phe Tyr Lieu Ala Met Asn Lys Glu Gly Lys Lieu. Tyr 115 12 O 125 Ala Lys Lys Glu. Cys Asn. Glu Asp Cys Asn. Phe Lys Glu Lieu. Ile Lieu 13 O 135 14 O Glu Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr His Asn Gly 145 150 155 160 Gly Glu Met Phe Val Ala Lieu. Asn Gln Lys Gly Ile Pro Val Arg Gly 1.65 17O 17s Llys Llys Thr Lys Lys Glu Gln Lys Thr Ala His Phe Lieu. Pro Met Ala 18O 185 19 O
Ile Thr
<210s, SEQ ID NO 17 &211s LENGTH: 233 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 17 Met Gly Ser Pro Arg Ser Ala Lieu. Ser Cys Lieu Lleu Lieu. His Lieu. Lieu. 1. 5 1O 15 Val Lieu. Cys Lieu. Glin Ala Glin Glu Gly Pro Gly Arg Gly Pro Ala Lieu 2O 25 3O Gly Arg Glu Lieu Ala Ser Lieu. Phe Arg Ala Gly Arg Glu Pro Glin Gly 35 4 O 45 Val Ser Glin Gln His Val Arg Glu Glin Ser Lieu Val Thr Asp Gln Lieu SO 55 6 O Ser Arg Arg Lieu. Ile Arg Thr Tyr Glin Lieu. Tyr Ser Arg Thir Ser Gly 65 70 7s 8O Llys His Val Glin Val Lieu Ala Asn Lys Arg Ile Asn Ala Met Ala Glu 85 90 95 Asp Gly Asp Pro Phe Ala Lys Lieu. Ile Val Glu Thir Asp Thr Phe Gly 1OO 105 11 O Ser Arg Val Arg Val Arg Gly Ala Glu Thr Gly Lieu. Tyr Ile Cys Met 115 12 O 125 Asn Llys Lys Gly Llys Lieu. Ile Ala Lys Ser Asn Gly Lys Gly Lys Asp 13 O 135 14 O Cys Val Phe Thr Glu Ile Val Lieu. Glu Asn Asn Tyr Thr Ala Leu Gln 145 150 155 160 Asn Ala Lys Tyr Glu Gly Trp Tyr Met Ala Phe Thr Arg Lys Gly Arg US 2011/0306129 A1 Dec. 15, 2011 34
- Continued
1.65 17O 17s Pro Arg Lys Gly Ser Llys Thr Arg Glin His Glin Arg Glu Val His Phe 18O 185 19 O Met Lys Arg Lieu Pro Arg Gly His His Thir Thr Glu Glin Ser Lieu. Arg 195 2OO 2O5 Phe Glu Phe Lieu. Asn Tyr Pro Pro Phe Thr Arg Ser Lieu. Arg Gly Ser 21 O 215 22O Glin Arg Thr Trp Ala Pro Glu Pro Arg 225 23 O
<210s, SEQ ID NO 18 &211s LENGTH: 2O8 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 18 Met Ala Pro Lieu. Gly Glu Val Gly Asn Tyr Phe Gly Val Glin Asp Ala 1. 5 1O 15 Val Pro Phe Gly Asn Val Pro Val Lieu Pro Val Asp Ser Pro Val Lieu. 2O 25 3O Lieu. Ser Asp His Lieu. Gly Glin Ser Glu Ala Gly Gly Lieu Pro Arg Gly 35 4 O 45 Pro Ala Val Thir Asp Lieu. Asp His Lieu Lys Gly Ile Lieu. Arg Arg Arg SO 55 6 O Gln Leu Tyr Cys Arg Thr Gly Phe His Lieu. Glu Ile Phe Pro Asn Gly 65 70 7s 8O Thir Ile Glin Gly Thr Arg Lys Asp His Ser Arg Phe Gly Ile Lieu. Glu 85 90 95 Phe Ile Ser Ile Ala Val Gly Lieu Val Ser Ile Arg Gly Val Asp Ser 1OO 105 11 O Gly Lieu. Tyr Lieu. Gly Met Asn. Glu Lys Gly Glu Lieu. Tyr Gly Ser Glu 115 12 O 125 Lys Lieu. Thr Glin Glu. Cys Val Phe Arg Glu Glin Phe Glu Glu Asn Trp 13 O 135 14 O Tyr Asn Thr Tyr Ser Ser Asn Lieu. Tyr Lys His Val Asp Thr Gly Arg 145 150 155 160 Arg Tyr Tyr Val Ala Lieu. Asn Lys Asp Gly Thr Pro Arg Glu Gly Thr 1.65 17O 17s Arg Thr Lys Arg His Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val 18O 185 19 O Asp Pro Asp Llys Val Pro Glu Lieu. Tyr Lys Asp Ile Lieu. Ser Glin Ser 195 2OO 2O5
<210s, SEQ ID NO 19 &211s LENGTH: 2O8 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 19 Met Trp Llys Trp Ile Lieu. Thir His Cys Ala Ser Ala Phe Pro His Leu 1. 5 1O 15 Pro Gly Cys Cys Cys Cys Cys Phe Leu Lleu Lleu Phe Leu Val Ser Ser 2O 25 3O Val Pro Val Thr Cys Glin Ala Leu Gly Glin Asp Met Val Ser Pro Glu US 2011/0306129 A1 Dec. 15, 2011 35
- Continued
35 4 O 45 Ala Thr Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly SO 55 6 O Arg His Val Arg Ser Tyr Asn His Lieu. Glin Gly Asp Val Arg Trp Arg 65 70 7s 8O Llys Lieu. Phe Ser Phe Thr Llys Tyr Phe Lieu Lys Ile Glu Lys Asn Gly 85 90 95 Llys Val Ser Gly Thr Llys Lys Glu Asn. Cys Pro Tyr Ser Ile Lieu. Glu 1OO 105 11 O Ile Thir Ser Val Glu Ile Gly Val Val Ala Wall Lys Ala Ile Asin Ser 115 12 O 125 Asn Tyr Tyr Lieu Ala Met Asn Llys Lys Gly Lys Lieu. Tyr Gly Ser Lys 13 O 135 14 O Glu Phe Asn. Asn Asp Cys Llys Lieu Lys Glu Arg Ile Glu Glu Asn Gly 145 150 155 160 Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met 1.65 17O 17s Tyr Val Ala Lieu. Asn Gly Lys Gly Ala Pro Arg Arg Gly Glin Llys Thr 18O 185 19 O Arg Arg Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 195 2OO 2O5
<210s, SEQ ID NO 2 O &211s LENGTH: 225 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 2O Met Ala Ala Lieu Ala Ser Ser Lieu. Ile Arg Glin Lys Arg Glu Val Arg 1. 5 1O 15 Glu Pro Gly Gly Ser Arg Pro Val Ser Ala Glin Arg Arg Val Cys Pro 2O 25 3O Arg Gly. Thir Lys Ser Lieu. Cys Gln Lys Glin Lieu. Lieu. Ile Lieu. Lieu. Ser 35 4 O 45 Llys Val Arg Lieu. Cys Gly Gly Arg Pro Ala Arg Pro Asp Arg Gly Pro SO 55 6 O Glu Pro Glin Lieu Lys Gly Ile Val Thir Lys Lieu. Phe Cys Arg Glin Gly 65 70 7s 8O Phe Tyr Lieu. Glin Ala Asn Pro Asp Gly Ser Ile Glin Gly Thr Pro Glu 85 90 95 Asp Thr Ser Ser Phe Thr His Phe Asn Lieu. Ile Pro Val Gly Lieu. Arg 1OO 105 11 O Val Val Thir Ile Glin Ser Ala Lys Lieu. Gly His Tyr Met Ala Met Asn 115 12 O 125 Ala Glu Gly Lieu Lleu Tyr Ser Ser Pro His Phe Thr Ala Glu. Cys Arg 13 O 135 14 O Phe Lys Glu. Cys Val Phe Glu Asn Tyr Tyr Val Lieu. Tyr Ala Ser Ala 145 150 155 160 Lieu. Tyr Arg Glin Arg Arg Ser Gly Arg Ala Trp Tyr Lieu. Gly Lieu. Asp 1.65 17O 17s Lys Glu Gly Glin Val Met Lys Gly Asn Arg Val Llys Llys Thir Lys Ala 18O 185 19 O US 2011/0306129 A1 Dec. 15, 2011 36
- Continued Ala Ala His Phe Lieu Pro Llys Lieu. Lieu. Glu Val Ala Met Tyr Glin Glu 195 2OO 2O5
Pro Ser Lieu. His Ser Wall Pro Glu Ala Ser Pro Ser Ser Pro Pro Ala 21 O 215 22O
Pro 225
<210s, SEQ ID NO 21 &211s LENGTH: 243 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 21 Met Ala Ala Ala Ile Ala Ser Ser Lieu. Ile Arg Glin Lys Arg Glin Ala 1. 5 1O 15 Arg Glu Ser Asn. Ser Asp Arg Val Ser Ala Ser Lys Arg Arg Ser Ser 2O 25 3O Pro Ser Lys Asp Gly Arg Ser Lieu. Cys Glu Arg His Val Lieu. Gly Val 35 4 O 45 Phe Ser Llys Val Arg Phe Cys Ser Gly Arg Lys Arg Pro Val Arg Arg SO 55 6 O Arg Pro Glu Pro Gln Leu Lys Gly Ile Val Thr Arg Lieu Phe Ser Glin 65 70 7s 8O Gln Gly Tyr Phe Leu Gln Met His Pro Asp Gly Thr Ile Asp Gly Thr 85 90 95 Lys Asp Glu Asn. Ser Asp Tyr Thr Lieu. Phe Asn Lieu. Ile Pro Val Gly 1OO 105 11 O Lieu. Arg Val Val Ala Ile Glin Gly Val Lys Ala Ser Lieu. Tyr Val Ala 115 12 O 125 Met Asin Gly Glu Gly Tyr Lieu. Tyr Ser Ser Asp Val Phe Thr Pro Glu 13 O 135 14 O Cys Llys Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val Ile Tyr Ser 145 150 155 160 Ser Thr Lieu. Tyr Arg Glin Glin Glu Ser Gly Arg Ala Trp Phe Lieu. Gly 1.65 17O 17s Lieu. Asn Lys Glu Gly Glin Ile Met Lys Gly Asn Arg Val Lys Llys Thr 18O 185 19 O Llys Pro Ser Ser His Phe Val Pro Llys Pro Ile Glu Val Cys Met Tyr 195 2OO 2O5 Arg Glu Pro Ser Lieu. His Glu Ile Gly Glu Lys Glin Gly Arg Ser Arg 21 O 215 22O Lys Ser Ser Gly Thr Pro Thr Met Asn Gly Gly Lys Val Val Asn Glin 225 23 O 235 24 O Asp Ser Thr
<210s, SEQ ID NO 22 &211s LENGTH: 245 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 22 Met Ala Ala Ala Ile Ala Ser Ser Lieu. Ile Arg Glin Lys Arg Glin Ala 1. 5 1O 15 Arg Glu Arg Glu Lys Ser Asn Ala Cys Lys Cys Val Ser Ser Pro Ser US 2011/0306129 A1 Dec. 15, 2011 37
- Continued
2O 25 3O Lys Gly Llys Thir Ser Cys Asp Lys Asn Llys Lieu. Asn Val Phe Ser Arg 35 4 O 45 Val Llys Lieu. Phe Gly Ser Llys Lys Arg Arg Arg Arg Arg Pro Glu Pro SO 55 6 O Glin Lieu Lys Gly Ile Val Thir Lys Lieu. Tyr Ser Arg Glin Gly Tyr His 65 70 7s 8O Lieu. Glin Lieu. Glin Ala Asp Gly. Thir Ile Asp Gly. Thir Lys Asp Glu Asp 85 90 95 Ser Thr Tyr Thr Lieu. Phe Asn Lieu. Ile Pro Val Gly Lieu. Arg Val Val 1OO 105 11 O Ala Ile Glin Gly Val Glin Thr Lys Lieu. Tyr Lieu Ala Met Asn. Ser Glu 115 12 O 125 Gly Tyr Lieu. Tyr Thr Ser Glu Lieu Phe Thr Pro Glu. Cys Llys Phe Lys 13 O 135 14 O Glu Ser Val Phe Glu Asn Tyr Tyr Val Thr Tyr Ser Ser Met Ile Tyr 145 150 155 160 Arg Glin Glin Glin Ser Gly Arg Gly Trp Tyr Lieu. Gly Lieu. Asn Lys Glu 1.65 17O 17s Gly Glu Ile Met Lys Gly Asn His Val Lys Lys Asn Llys Pro Ala Ala 18O 185 19 O His Phe Leu Pro Llys Pro Leu Lys Val Ala Met Tyr Lys Glu Pro Ser 195 2OO 2O5 Lieu. His Asp Lieu. Thr Glu Phe Ser Arg Ser Gly Ser Gly Thr Pro Thr 21 O 215 22O Llys Ser Arg Ser Val Ser Gly Val Lieu. Asn Gly Gly Llys Ser Met Ser 225 23 O 235 24 O
His ASn Glu Ser Thr 245
<210s, SEQ ID NO 23 &211s LENGTH: 247 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 23 Met Ala Ala Ala Ile Ala Ser Gly Lieu. Ile Arg Glin Lys Arg Glin Ala 1. 5 1O 15 Arg Glu Gln His Trp Asp Arg Pro Ser Ala Ser Arg Arg Arg Ser Ser 2O 25 3O Pro Ser Lys Asn Arg Gly Lieu. Cys Asn Gly Asn Lieu Val Asp Ile Phe 35 4 O 45 Ser Llys Val Arg Ile Phe Gly Lieu Lys Lys Arg Arg Lieu. Arg Arg Glin SO 55 6 O Asp Pro Glin Lieu Lys Gly Ile Val Thr Arg Lieu. Tyr Cys Arg Glin Gly 65 70 7s 8O Tyr Tyr Lieu Gln Met His Pro Asp Gly Ala Lieu. Asp Gly. Thir Lys Asp 85 90 95 Asp Ser Thir Asn. Ser Thr Lieu. Phe Asn Lieu. Ile Pro Val Gly Lieu. Arg 1OO 105 11 O Val Val Ala Ile Glin Gly Val Lys Thr Gly Lieu. Tyr Ile Ala Met Asn 115 12 O 125 US 2011/0306129 A1 Dec. 15, 2011 38
- Continued Gly Glu Gly Tyr Lieu. Tyr Pro Ser Glu Lieu. Phe Thr Pro Glu. Cys Lys 13 O 135 14 O Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val Ile Tyr Ser Ser Met 145 150 155 160 Lieu. Tyr Arg Glin Glin Glu Ser Gly Arg Ala Trp Phe Lieu. Gly Lieu. Asn 1.65 17O 17s Lys Glu Gly Glin Ala Met Lys Gly Asn Arg Val Llys Llys Thir Lys Pro 18O 185 19 O Ala Ala His Phe Lieu Pro Llys Pro Lieu. Glu Val Ala Met Tyr Arg Glu 195 2OO 2O5 Pro Ser Lieu. His Asp Val Gly Glu Thr Val Pro Llys Pro Gly Val Thr 21 O 215 22O Pro Ser Lys Ser Thr Ser Ala Ser Ala Ile Met Asn Gly Gly Llys Pro 225 23 O 235 24 O Val Asn Llys Ser Lys Thr Thr 245
<210s, SEQ ID NO 24 &211s LENGTH: 218 212. TYPE: PRT <213s ORGANISM: Mus musculus
<4 OOs, SEQUENCE: 24 Met Ala Arg Llys Trp Asn Gly Arg Ala Val Ala Arg Ala Lieu Val Lieu. 1. 5 1O 15 Ala Thr Lieu. Trp Lieu Ala Val Ser Gly Arg Pro Lieu Ala Glin Glin Ser 2O 25 3O Glin Ser Val Ser Asp Glu Asp Pro Lieu. Phe Lieu. Tyr Gly Trp Gly Lys 35 4 O 45 Ile Thr Arg Lieu. Glin Tyr Lieu. Tyr Ser Ala Gly Pro Tyr Val Ser Asn SO 55 6 O Cys Phe Lieu. Arg Ile Arg Ser Asp Gly Ser Val Asp Cys Glu Glu Asp 65 70 7s 8O Glin Asn. Glu Arg Asn Lieu. Lieu. Glu Phe Arg Ala Val Ala Lieu Lys Thr 85 90 95 Ile Ala Ile Lys Asp Val Ser Ser Val Arg Tyr Lieu. Cys Met Ser Ala 1OO 105 11 O Asp Gly Lys Ile Tyr Gly Lieu. Ile Arg Tyr Ser Glu Glu Asp Cys Thr 115 12 O 125 Phe Arg Glu Glu Met Asp Cys Lieu. Gly Tyr Asn Glin Tyr Arg Ser Met 13 O 135 14 O Llys His His Lieu. His Ile Ile Phe Ile Glin Ala Lys Pro Arg Glu Glin 145 150 155 160 Leu Glin Asp Glin Llys Pro Ser Asin Phe Ile Pro Val Phe His Arg Ser 1.65 17O 17s Phe Phe Glu Thr Gly Asp Gln Leu Arg Ser Lys Met Phe Ser Leu Pro 18O 185 19 O Lieu. Glu Ser Asp Ser Met Asp Pro Phe Arg Met Val Glu Asp Wall Asp 195 2OO 2O5 His Leu Val Lys Ser Pro Ser Phe Gln Lys 21 O 215
<210s, SEQ ID NO 25 US 2011/0306129 A1 Dec. 15, 2011 39
- Continued
&211s LENGTH: 2O7 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 25 Met Ala Glu Val Gly Gly Val Phe Ala Ser Lieu. Asp Trp Asp Lieu. His 1. 5 1O 15 Gly Phe Ser Ser Ser Lieu. Gly Asn Val Pro Leu Ala Asp Ser Pro Gly 2O 25 3O Phe Lieu. Asn. Glu Arg Lieu. Gly Glin Ile Glu Gly Llys Lieu. Glin Arg Gly 35 4 O 45 Ser Pro Thr Asp Phe Ala His Lieu Lys Gly Ile Lieu. Arg Arg Arg Glin SO 55 6 O Lieu. Tyr Cys Arg Thr Gly Phe His Leu Glu Ile Phe Pro Asn Gly Thr 65 70 7s 8O Val His Gly Thr Arg His Asp His Ser Arg Phe Gly Ile Leu Glu Phe 85 90 95 Ile Ser Lieu Ala Val Gly Lieu. Ile Ser Ile Arg Gly Val Asp Ser Gly 1OO 105 11 O Lieu. Tyr Lieu. Gly Met Asn. Glu Arg Gly Glu Lieu. Tyr Gly Ser Lys Llys 115 12 O 125 Lieu. Thir Arg Glu. Cys Val Phe Arg Glu Glin Phe Glu Glu Asn Trp Tyr 13 O 135 14 O Asn Thr Tyr Ala Ser Thr Lieu. Tyr Lys His Ser Asp Ser Glu Arg Glin 145 150 55 160 Tyr Tyr Val Ala Lieu. Asn Lys Asp Gly Ser Pro Arg Glu Gly Tyr Arg 1.65 17O 17s Thr Lys Arg His Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val Asp 18O 185 19 O Pro Ser Lys Lieu Pro Ser Met Ser Arg Asp Leu Phe His Tyr Arg 195 2OO 2O5
<210s, SEQ ID NO 26 &211s LENGTH: 216 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 26 Met Gly Ala Ala Arg Lieu Lleu Pro Asn Lieu. Thir Lieu. Cys Lieu. Glin Lieu. 1. 5 1O 15 Lieu. Ile Lieu. Cys Cys Glin Thr Glin Gly Glu Asn His Pro Ser Pro Asn 2O 25 3O Phe Asin Glin Tyr Val Arg Asp Glin Gly Ala Met Thr Asp Gln Lieu. Ser 35 4 O 45 Arg Arg Glin Ile Arg Glu Tyr Glin Lieu. Tyr Ser Arg Thir Ser Gly Lys SO 55 6 O His Val Glin Val Thr Gly Arg Arg Ile Ser Ala Thr Ala Glu Asp Gly 65 70 7s 8O Asn Llys Phe Ala Lys Lieu. Ile Val Glu Thir Asp Thr Phe Gly Ser Arg 85 90 95 Val Arg Ile Lys Gly Ala Glu Ser Glu Lys Tyr Ile Cys Met Asn Lys 1OO 105 11 O Arg Gly Lys Lieu. Ile Gly Llys Pro Ser Gly Lys Ser Lys Asp Cys Val 115 12 O 125 US 2011/0306129 A1 Dec. 15, 2011 40
- Continued
Phe Thr Glu Ile Val Lieu. Glu Asn Asn Tyr Thr Ala Phe Glin Asn Ala 13 O 135 14 O Arg His Glu Gly Trp Phe Met Ala Phe Thr Arg Glin Gly Arg Pro Arg 145 150 155 160 Glin Ala Ser Arg Ser Arg Glin Asn Glin Arg Glu Ala His Phe Ile Llys 1.65 17O 17s Arg Lieu. Tyr Glin Gly Glin Lieu Pro Phe Pro Asn His Ala Glu Lys Glin 18O 185 19 O Lys Glin Phe Glu Phe Val Gly Ser Ala Pro Thr Arg Arg Thr Lys Arg 195 2OO 2O5 Thr Arg Arg Pro Gln Pro Leu. Thr 21 O 215
<210s, SEQ ID NO 27 &211s LENGTH: 2O7 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 27 Met Tyr Ser Ala Pro Ser Ala Cys Thr Cys Lieu. Cys Lieu. His Phe Leu 1. 5 1O 15 Lieu. Lieu. Cys Phe Glin Val Glin Val Lieu Val Ala Glu Glu Asn Val Asp 2O 25 3O Phe Arg Ile His Val Glu Asn Glin Thr Arg Ala Arg Asp Asp Val Ser 35 4 O 45 Arg Lys Glin Lieu. Arg Lieu. Tyr Glin Lieu. Tyr Ser Arg Thir Ser Gly Lys SO 55 6 O His Ile Glin Val Lieu. Gly Arg Arg Ile Ser Ala Arg Gly Glu Asp Gly 65 70 7s 8O Asp Llys Tyr Ala Glin Lieu. Lieu Val Glu Thir Asp Thr Phe Gly Ser Glin 85 90 95 Val Arg Ile Lys Gly Lys Glu Thr Glu Phe Tyr Lieu. Cys Met Asn Arg 1OO 105 11 O Lys Gly Llys Lieu Val Gly Llys Pro Asp Gly. Thir Ser Lys Glu. Cys Val 115 12 O 125 Phe Ile Glu Lys Val Lieu. Glu Asn. Asn Tyr Thr Ala Lieu Met Ser Ala 13 O 135 14 O Lys Tyr Ser Gly Trp Tyr Val Gly Phe Thr Lys Lys Gly Arg Pro Arg 145 150 155 160 Lys Gly Pro Llys Thr Arg Glu Asin Glin Glin Asp Wal His Phe Met Lys 1.65 17O 17s Arg Tyr Pro Lys Gly Glin Pro Glu Lieu Gln Llys Pro Phe Llys Tyr Thr 18O 185 19 O Thr Val Thr Lys Arg Ser Arg Arg Ile Arg Pro Thr His Pro Ala 195 2OO 2O5
<210s, SEQ ID NO 28 &211s LENGTH: 216 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 28 Met Arg Ser Gly Cys Val Val Val His Val Trp Ile Lieu Ala Gly Lieu. 1. 5 1O 15 US 2011/0306129 A1 Dec. 15, 2011 41
- Continued
Trp Lieu Ala Val Ala Gly Arg Pro Lieu Ala Phe Ser Asp Ala Gly Pro 2O 25 3O His Val His Tyr Gly Trp Gly Asp Pro Ile Arg Lieu. Arg His Lieu. Tyr 35 4 O 45 Thir Ser Gly Pro His Gly Lieu Ser Ser Cys Phe Lieu. Arg Ile Arg Ala SO 55 6 O Asp Gly Val Val Asp Cys Ala Arg Gly Glin Ser Ala His Ser Lieu. Lieu 65 70 7s 8O Glu Ile Lys Ala Val Ala Lieu. Arg Thr Val Ala Ile Lys Gly Val His 85 90 95 Ser Val Arg Tyr Lieu. Cys Met Gly Ala Asp Gly Llys Met Glin Gly Lieu. 1OO 105 11 O Lieu. Glin Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro 115 12 O 125 Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Lieu Pro Val Ser 13 O 135 14 O Lieu. Ser Ser Ala Lys Glin Arg Glin Lieu. Tyr Lys Asn Arg Gly Phe Lieu. 145 150 155 160
Pro Leu Ser His Phe Lieu. Pro Met Leu Pro Met Wall Pro Glu Glu Pro 1.65 17O 17s Glu Asp Lieu. Arg Gly His Lieu. Glu Ser Asp Met Phe Ser Ser Pro Lieu. 18O 185 19 O Glu Thir Asp Ser Met Asp Pro Phe Gly Lieu Val Thr Gly Lieu. Glu Ala 195 2OO 2O5 Val Arg Ser Pro Ser Phe Glu Lys 21 O 215
<210s, SEQ ID NO 29 &211s LENGTH: 211 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 29 Met Ala Pro Lieu Ala Glu Val Gly Gly Phe Lieu. Gly Gly Lieu. Glu Gly 1. 5 1O 15 Lieu. Gly Glin Glin Val Gly Ser His Phe Leu Lleu Pro Pro Ala Gly Glu 2O 25 3O Arg Pro Pro Lieu. Lieu. Gly Glu Arg Arg Ser Ala Ala Glu Arg Ser Ala 35 4 O 45 Arg Gly Gly Pro Gly Ala Ala Glin Lieu Ala His Lieu. His Gly Ile Lieu SO 55 6 O Arg Arg Arg Glin Lieu. Tyr Cys Arg Thr Gly Phe His Lieu. Glin Ile Lieu 65 70 7s 8O Pro Asp Gly Ser Val Glin Gly Thr Arg Glin Asp His Ser Leu Phe Gly 85 90 95 Ile Lieu. Glu Phe Ile Ser Val Ala Val Gly Lieu Val Ser Ile Arg Gly 1OO 105 11 O Val Asp Ser Gly Lieu. Tyr Lieu. Gly Met Asn Asp Llys Gly Glu Lieu. Tyr 115 12 O 125 Gly Ser Glu Lys Lieu. Thir Ser Glu. Cys Ile Phe Arg Glu Glin Phe Glu 13 O 135 14 O Glu Asn Trp Tyr Asn Thr Tyr Ser Ser Asn Ile Tyr Lys His Gly Asp US 2011/0306129 A1 Dec. 15, 2011 42
- Continued
145 150 155 160 Thr Gly Arg Arg Tyr Phe Val Ala Lieu. Asn Lys Asp Gly. Thr Pro Arg 1.65 17O 17s Asp Gly Ala Arg Ser Lys Arg His Glin Llys Phe Thr His Phe Lieu Pro 18O 185 19 O Arg Pro Val Asp Pro Glu Arg Val Pro Glu Lieu. Tyr Lys Asp Lieu. Lieu 195 2OO 2O5 Met Tyr Thr 21 O
<210s, SEQ ID NO 3 O &211s LENGTH: 209 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 30 Met Asp Ser Asp Glu Thr Gly Phe Glu. His Ser Gly Lieu. Trp Val Ser 1. 5 1O 15 Val Lieu Ala Gly Lieu. Lieu. Lieu. Gly Ala Cys Glin Ala His Pro Ile Pro 2O 25 3O Asp Ser Ser Pro Lieu. Lieu. Glin Phe Gly Gly Glin Val Arg Glin Arg Tyr 35 4 O 45 Lieu. Tyr Thr Asp Asp Ala Glin Glin Thr Glu Ala His Lieu. Glu Ile Arg SO 55 6 O Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Glin Ser Pro Glu Ser Lieu. 65 70 7s 8O Lieu. Glin Lieu Lys Ala Lieu Lys Pro Gly Val Ile Glin Ile Lieu. Gly Val 85 90 95 Llys Thir Ser Arg Phe Lieu. Cys Glin Arg Pro Asp Gly Ala Lieu. Tyr Gly 1OO 105 11 O Ser Lieu. His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Lieu. Lieu. Lieu. 115 12 O 125 Glu Asp Gly Tyr Asn Val Tyr Glin Ser Glu Ala His Gly Lieu Pro Lieu. 13 O 135 14 O His Lieu Pro Gly Asn Llys Ser Pro His Arg Asp Pro Ala Pro Arg Gly 145 150 155 160 Pro Ala Arg Phe Lieu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu 1.65 17O 17s Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp 18O 185 19 O Pro Leu Ser Met Val Gly Pro Ser Glin Gly Arg Ser Pro Ser Tyr Ala 195 2OO 2O5
Ser
<210s, SEQ ID NO 31 &211s LENGTH: 170 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 31 Met Arg Arg Arg Lieu. Trp Lieu. Gly Lieu Ala Trp Lieu. Lieu. Lieu Ala Arg 1. 5 1O 15 Ala Pro Asp Ala Ala Gly Thr Pro Ser Ala Ser Arg Gly Pro Arg Ser 2O 25 3O US 2011/0306129 A1 Dec. 15, 2011 43
- Continued
Tyr Pro His Lieu. Glu Gly Asp Val Arg Trp Arg Arg Lieu. Phe Ser Ser 35 4 O 45 Thr His Phe Phe Lieu. Arg Val Asp Pro Gly Gly Arg Val Glin Gly Thr SO 55 6 O Arg Trp Arg His Gly Glin Asp Ser Ile Lieu. Glu Ile Arg Ser Val His 65 70 7s 8O Val Gly Val Val Val Ile Lys Ala Val Ser Ser Gly Phe Tyr Val Ala 85 90 95 Met Asn Arg Arg Gly Arg Lieu. Tyr Gly Ser Arg Lieu. Tyr Thr Val Asp 1OO 105 11 O Cys Arg Phe Arg Glu Arg Ile Glu Glu Asn Gly His Asn. Thir Tyr Ala 115 12 O 125 Ser Glin Arg Trp Arg Arg Arg Gly Glin Pro Met Phe Lieu Ala Lieu. Asp 13 O 135 14 O Arg Arg Gly Gly Pro Arg Pro Gly Gly Arg Thr Arg Arg Tyr His Lieu 145 150 155 160
Ser Ala His Phe Lieu. Pro Wall Lieu. Wal Ser 1.65 17O
<210s, SEQ ID NO 32 &211s LENGTH: 251 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 32 Met Lieu. Gly Ala Arg Lieu. Arg Lieu. Trp Val Cys Ala Lieu. Cys Ser Val 1. 5 1O 15 Cys Ser Met Ser Val Lieu. Arg Ala Tyr Pro Asn Ala Ser Pro Lieu. Lieu. 2O 25 3O Gly Ser Ser Trp Gly Gly Lieu. Ile His Leu Tyr Thr Ala Thr Ala Arg 35 4 O 45 Asn Ser Tyr His Lieu. Glin Ile His Lys Asn Gly His Val Asp Gly Ala SO 55 6 O Pro His Glin Thr Ile Tyr Ser Ala Leu Met Ile Arg Ser Glu Asp Ala 65 70 7s 8O Gly Phe Val Val Ile Thr Gly Val Met Ser Arg Arg Tyr Lieu. Cys Met 85 90 95 Asp Phe Arg Gly Asn Ile Phe Gly Ser His Tyr Phe Asp Pro Glu Asn 1OO 105 11 O Cys Arg Phe Gln His Glin Thr Lieu. Glu Asin Gly Tyr Asp Val Tyr His 115 12 O 125 Ser Pro Glin Tyr His Phe Lieu Val Ser Lieu. Gly Arg Ala Lys Arg Ala 13 O 135 14 O Phe Leu Pro Gly Met Asn Pro Pro Pro Tyr Ser Glin Phe Leu Ser Arg 145 150 155 160 Arg Asn Glu Ile Pro Lieu. Ile His Phe Asn Thr Pro Ile Pro Arg Arg 1.65 17O 17s His Thr Arg Ser Ala Glu Asp Asp Ser Glu Arg Asp Pro Lieu. Asn. Wall 18O 185 19 O Lieu Lys Pro Arg Ala Arg Met Thr Pro Ala Pro Ala Ser Cys Ser Glin 195 2OO 2O5 Glu Lieu Pro Ser Ala Glu Asp Asn. Ser Pro Met Ala Ser Asp Pro Lieu. US 2011/0306129 A1 Dec. 15, 2011 44
- Continued
21 O 215 22O Gly Val Val Arg Gly Gly Arg Val Asn. Thir His Ala Gly Gly Thr Gly 225 23 O 235 24 O Pro Glu Gly Cys Arg Pro Phe Ala Lys Phe Ile 245 250
1. A method of biasing hESCs without substantial differ culturing hESCs in a complete media formulation contain entiation the method comprising: ing: albumin, transferrin, glutamine, hyaluronidase, culturing hESCs in selectively varied concentrations of between about 10 and about 20 ng/ml bFGF, a salt or hyaluronidase added to a complete media formulation mineral, and essential amino acids, at an osmolarity of containing: albumin, transferrin, glutamine, bFGF, a salt about 220-330 mOsm/Liter; and, or mineral, and essential amino acids at an osmolarity of selectively varying the concentration of hyaluronidase about 220-330 mOsm/Liter whereby increasing or from about 1 lug/ml to about Oug/ml. decreasing the concentration of hyaluronidase selec 17. The method of claim 16, the media further comprising tively biases the differential tendency of the hES towards an extracellular matrix of laminin or fibronectin. one of ectodermal or endodermal lineage 18. The method of claim 17 the method further comprising 2. The method of biasing of claim 1, wherein the albumin culturing the hESCs in the complete media formulation on the is at a concentration of about 5 to 100 grams/Liter. extracellular matrix for a period of time allowing the hESCs 3. The method of biasing of claim 1, wherein the transferrin to increase in number by 25%, 50%, 75%, 100% or more. is at a concentration of about 5 to 100 g/ml. 19. A method of biasing hESCs towards endodermal lin 4. The method of biasing of claim 1, wherein the glutamine eages the method comprising: is at a concentration of about 10 to 40 mg/Liter. culturing hESCs in a complete media formulation contain 5. The method of biasing of claim 1, wherein the glutamine ing: albumin, transferrin, glutamine, hyaluronidase, comprises a dipeptide. bFGF, a salt or mineral, and essential amino acids at an 6. The method of biasing of claim 5, wherein the dipeptide osmolarity of about 220-330 mOsm/Liter; and, comprises glutamine-alanine. increasing the concentration of bFGF from between about 7. The method of biasing of claim 1, wherein the salt or 10 and about 20 ng/ml; and, mineral is one or more of Sodium, potassium, calcium, mag maintaining the concentration of hyaluronidase at about 1 nesium, copper, manganese, molybdenum, selenium, iron, or ug/ml for 1-2 days in each passage cycle. Z10. 20. A method of biasing hESCs towards neuro-ectodermal 8. The method of biasing of claim 1, wherein the essential lineages the method comprising: amino acids comprise arginine; cystine; histidine; isoleucine; culturing hESCs in a complete media formulation contain leucine; methionine; phenylalanine; threonine; tryptophan; ing: albumin, hyaluronidase, transferrin, glutamine, tyrosine; and valine. between about 10 and about 20 ng/ml bFGF, a salt or mineral, essential amino acids at an osmolarity of about 9. The method of biasing of claim 1, the media further 220-330 mOsm/Liter, and, varying the concentration of comprising an extracellular matrix of laminin or fibronectin. hyaluronidase from about 0 to about 1 lug/ml thereinfor 10. The method of biasing of claim 9 the method further a period of time the concentration is about 0. comprising culturing the hESCs in the complete media for 21. The method of claim 20, wherein the albumin is at a mulation on the extracellular matrix for a period of time concentration of about 5 to 100 grams/Liter. allowing the hESCs to increase in number by 25%, 50%, 22. The method media formulation of claim 20, wherein 75%, 100% or more. the transferrin is at a concentration of about 5 to 100 ug/ml. 11. The method of biasing of claim 9 the method further 23. The method of claim 20, wherein the glutamine is at a comprising culturing the hESCs in the complete media for concentration of about 10 to 40 mg/Liter. mulation on the extracellular matrix for at least 30 minutes to 24. The method of claim 20, wherein the glutamine com 48 hours. prises a glutamine-alanine dipeptide. 12. The method of biasing of claim 1 wherein the concen 25. The method of claim 20, wherein the salt or mineral is tration of hyaluronidase is between about 0 to about 1 g/ml. one or more of sodium, potassium, calcium, magnesium, 13. The method of biasing of claim 1 wherein the concen copper, manganese, molybdenum, Selenium, iron, or zinc. tration of hyaluronidase is between about 1 to about 10 g/ml. 26. The method of claim 20, wherein the essential amino 14. The method of biasing of claim 1 wherein the concen acids comprise arginine; cystine; histidine; isoleucine; leu tration of hyaluronidase is between about 1 to about 25 g/ml. cine; methionine; phenylalanine; threonine; tryptophan; 14. The method of biasing of claim 1 wherein the concen tyrosine; and valine. tration of hyaluronidase is between about 1 to about 50 g/ml. 27. The method of claim 20, the media further comprising 15. The method of biasing of claim 1 wherein the concen an extracellular matrix of laminin or fibronectin. tration of hyaluronidase is between about 1 to about 100 28. The method of claim 20, wherein the period of time is g/ml. between 1 hour and 48 hours. 16. A method of biasing hESCs towards ectodermal lin eages the method comprising: c c c c c