WO2012024296A1 - Arterial repair with cultured bone marrow cells and whole bone marrow - Google Patents

Abstract

Methods for generating bone marrow derived cells (BMCs) including stem cells and progenitor cells that abrogate atherosclerosis in hypercholesterolemia. Culturing BMCs or whole bone marrow in compositions including cell factors prior to transfusion resulted in significant atherosclerosis reduction. Gene expression analysis implicates mesenchymal to epithelial transition (MET) as a potential mechanism mediating this effect. This method of conditioning BMCs may lead to potential preventive/therapeutic strategies for atherosclerosis.

Description

ARTERIAL REPAIR WITH CULTURED

BONE MARROW CELLS AND WHOLE BON E MARROW

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of Provisional Application Serial No.

61/375,283 filed August 20, 2010, which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] Embodiments of the invention relate to stern cell compositions, selection of bone marrow derived cells (e.g., progenitor cells, stem cells) and methods of treatment of vascular tissues.

13 A CKG ROUND

[0003] Arterial homeostasis, the process responsible for the integrity of the vessel wall, represents the balance between injury from environmental insult and repair by mechanisms involving endogenous stem and/or progenitor cells. Aging in the presence of significant risk factors, such as markedly elevated cholesterol, can result in the exhaustion of progenitor cells capable of arterial repair (Rauscher, F., et al., Circulation, 108(4):457-63, 2003; Goidschmidt-Clermont, P.J,, American Heart Journal, 2003. 146(4 Suppl): p. S5-12). It has been shown that the onset of atheroma formation in arteries coincides with the failing of arterial repair mechanisms, rather than merely the presence of noxious risk factors (Karra, R., et al, Proc Natl Acad Sci U S A, 2005. 102(46): p. 16789-94). In the absence of significant risk factors, stem/progenitor cell mediated arterial repair can maintain arterial health well into advanced age. However, as humans are increasingly exposed to environmental risk factors (i.e. sedentary lifestyle, obesity, diabetes mellitus) that increase vascular injury and promote stem/progenitor cell exhaustion and dysfunction, atheroma formation at a young age (Strong, J.P., et al, Annals of the New York Academy of Sciences, 1997, 81 1 : p. 226-35, discussion 235-7; Strong, J.P., et al., JAMA, 1999. 281(8): p. 727-35) may become prevalent unless we can directly improve arterial repair.

[0004] To restore effective arterial repair in the setting of chronic exposure to significant risk factors, investigators have tested the feasibility and effectiveness of exogenous progenitor cells injections (Rauscher, F., et al, Circulation, 108(4):457-63, 2003: Chaffer, C.Let al, Cells Tissues Organs (Print), 2007, 185(1-3): p. 7-19; Nakaya, Y., et al, Dev Cell, 2004, 7(3): p. 425-38; Zhu, S., et al, Arterioscl r Thromb Vase Biol, 2007, 27(1): p. 113-9; and Kalluri, R. and R.A. Weinberg, J Clin invest, 2009. 119(6): p. 1420-8). The overarching goal of these studies has been to delay atherogenesis, but the results to date have been mixed. Potential explanations for the mixed outcomes include differences in progenitor cell sub o ulations transfused, number of cells injected, cell pretreatment prior to injection, differences in food formulation and subsequent serum cholesterol or the intrinsic heterogeneity of disease development within syngeneic mouse disease models.

SUMMARY

[0005] This Summary is provided to present a summary of the invention to briefly indicate the nature and substance of the invention. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.

[0006] Described herein are compositions, cells, methods and kits for treating atherosclerosis in a mamma! (e.g., human) involving administration of treated bone marrow cells (BMCs) and treated whole bone marrow to the mammal. In a typical embodiment, isolated BMCs (e.g., progenitor cells, stem cells) are pre- reated or cultured with factors prior to administration to a patient. The results herein show that pretreated BMCs (e.g., stem cells) can be used to treat subjects with significant and aggressive atherosclerosis. The treatment curtails, if not reverses the disease process. Therefore, in other embodiments, another patient population would be human patients with aggressive disease such as those with diabetes and end stage kidney disease, patients with repeated cardiovascular events despite maximum preventive medical therapies and patients with aggressive peripheral vascular disease despite maximum medical therapy. Importantly, these pretreated cells may stimulate significant angiogenesis.

[0007] Another aspect of this invention is that it is likely that the treatment process may also restore the reparative qualities of BMCs (e.g., stem cells). As described in the examples section, it was found that BMCs from older mice are not able to repair arteries as well as BMCs from younger mice. It is likely that given the improved vascular repair after these treatment protocols, the reparative capability of older bone marrow cells will be enhanced. The method of pretreating cells, with the particular compositions, and other combinations of cytokines, growth factors, and additional molecules as more are discovered, may be used to manipulate the cells to have increased reparative qualities under a variety of clinical situations,

[0008] In other aspects of the invention, the use of molecular phenotyping such as transcriptomics/microarray, proteomics or metabolomics can be used to determine the reparative state of an individuals bone marrow cells. As such, by understanding the reparative state, it will be possible to design personalized treatments for the patients as well as their cells to optimize the reparative capabilities of their cells.

[0009] Accordingly, described herein is a method of treating atherosclerosis in a subject including: obtaining whole bone marrow or lin- BMCs cells from the subject, a donor or tissue culture bank; culturing the whole bone marrow or lin- BMCs ex vivo in a medium including SCF and at least one of: IL-3 and IL-6; and administering to the subject at least one dose of the cultured whole bone marrow or BMCs in an amount effecti ve to repair damaged arterial walls and reduce atherosclerosis in the subject. The whole bone marrow or lin- BMCs can be autologous, allogeneic or syngeneic. The subject may have diabetes or end- stage kidney disease. In a typical method, the whole bone marrow or lin- BMCs differentiate into at least one lineage of progenitor cells. The lin- BMCs may be identified by one or more of the following markers: hepatocyte growth factor (HGF), lymphoid enhancer-binding factor 1 (Lefl) and Cdk5rl. In some methods, the lin- BMCs are isolated from bone marrow of the subject or a donor. The medium can include all of SCF, IL-3, and IL-6. Administration of the cultured whole bone marrow or BMCs to the subject results in at least a 30% reduction in atherosclerosis in the subject, and is effective for treating at least one of the following conditions associated with atherosclerosis: myocardial infarction, stroke, coronary artery disease, and peripheral-arterial disease. The cultured whole bone marrow or BMCs can be administered to the subject by any suitable delivery route, e.g., intravenous administration, administration of a cultured whole bone marrow or BMCs-impregnated implantation device (e.g., a cultured whole bone marrow or BMCs-impregnated stent), and administration directly to a target site.

[0010] Also described herein is a kit including a cell culture medium including SCF and at least one of: IL-3 and IL-6; packaging; and instructions for use. The cell culture medium may include ail of SCF, IL-3 and I L-6. [0011] Further described herein is a method of preparing whole bone marrow cells or BMCs for administration to a subject having atherosclerosis, The method includes: obtaining whole bone marrow cells or Sin- BMCs from the subject, a donor or tissue culture bank; culturing the whole bone marrow ceils or lin- BMCs ex vivo in a medium including SCF and at least one of: IL-3 and IL-6 under conditions such that the whole bone marrow cells or BMCs differentiate into at least one lineage of progenitor ceils and a sufficient amount of differentiated whole bone marrow cells or BMCs are generated for repairing damaged arterial walls and reducing atherosclerosis in a subject having atherosclerosis. The whole bone marrow cells or lin- BMCs may be autologous, allogeneic or syngeneic. The subject having atherosclerosis may also have diabetes or end-stage kidney disease. The lin- BMCs may be identified by at least one of the following markers: HGF, Lefi and CdkSrl . The lin- BMCs may be isolated from bone marrow of the subject having atherosclerosis or a donor. The medium may include all of SCF, IL-3, and IL-6. Administration of the cultured whole bone marro cells or BMCs to the subject having atherosclerosis results in at least a 30% (e.g., 30, 35, 40, 45, 50, 55%, etc.) reduction in atherosclerosis in the subject. The method may further include the step of impregnating a biocompatible implantable device with the cultured whole bone marrow cells or BMCs,

[0012] Still further described herein is a method of screening whole bone marrow cells or lin- BMCs from a subject for arterial wall reparative capability. The method includes: obtaining a first sample of whole bone marrow cells or lin- BMCs from the subject; extracting RNA from the first sample of whole bone marrow cells or lin- BMCs and assessing the similarity of a gene expression profile of the first sample of whole bone marrow cells or lin- BMCs to a gene expression profile of fresh murine BMCs from mice with mild, moderate and severe disease using a microarray analysis; correlating the similarity of the gene expression profile of the first sample of whole bone marrow cells or lin- BMCs to the gene expression profile of the fresh murine BMCs with the reparative capacity of the first sample of whole bone marrow cells or lin- BMCs; obtaining a second sample of whole bone marrow cells or Sin- BMCs from the subject and culturing the second sample of whole bone marrow cells or lin- BMCs in a medium including SCF and at least one of: IL-3 and IL-6 under conditions such that the whole bone marrow cells or Sin- BMCs differentiate into at least one lineage of progenitor cells; extracting RNA from the cultured second sample of whole bone marrow cells or BMCs and assessing the similarity of a gene expression profile of the cultured second sample of whole bone marrow cells or BMCs to a gene expression profile of fresh murine BMCs from mice with mild, moderate and severe disease and to a gene expression profile of BMCs from mice with mild, moderate and severe disease cultured in a medium including SCF and at least one of: IL-3 and IL-6 under conditions such that the whole bone marrow cells or lin- BMCs differentiate into at least one lineage of progenitor cells using a microarray analysis; and correlating the similarity of the gene expression profile of the cultured second sample of whole bone marrow cells or BMCs to the gene expression profile of the fresh murine BMCs and to the gene expression profile of the BMCs from mice with mild, moderate and severe disease cultured in a medium including SCF and at least one of: IL-3 and IL-6 under conditions such that the whole bone marrow cells or lin- BMCs differentiate into at least one lineage of progenitor cells with the reparative capacity of the cultured second sample of whole bone marrow cells or BMCs. In the method, an increase in reparative capacity of the cultured second sample of whole bone marrow cells or BMCs relative to the reparative capacity of the first sample of whole bone marrow cells or lin- BMCs indicates a restoration in reparative capacity in the cultured second sample of whole bone marrow cells or BMCs due to cuituring in a medium including SCF and at least one of: IL-3 and IL-6 under conditions such that the whole bone marrow cells or lin- BMCs differentiate into at least one lineage of progenitor cells,

[0013] Other aspects of the invention are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Effect of BMCs cultured in standard conditions on atherosclerosis, Proportion of total aorta containing atherosclerotic lesions after injections of P13S versus BMCs that underwent standard culture (hydrocortisone and FBS),

[0015] Figure 2: Effect of enhanced cultured Lin- BMCs on atherosclerosis, (A) Proportion of total aorta containing atherosclerosis following injections of PBS, Freshly harvested Lin-BMCs or ECX cells. (B) Composite images for all analyzed aortas in each group, color indicates probability for the region to contain atherosclerosis (see color bar).

[0016] Figure 3: Flow cytometry analysis of freshly harvested and Lin- ECX BMCs. (A) Flo cytometry analysis of freshly harvested Lin-BMCs. (B) Flow cytometry analysis of ECX cells. [0017] Figure 4: Microarray analysis of different cell groups. Hierarchical clustering of microarrays generated from Fresh Lin- BMCs, BMCs following standard culturing and Lin- ECX cells.

[0018] Figure 5: Effect of fresh BMC subsets on atherosclerosis. Proportion of the total aorta containing atherosclerotic lesions following injections of: 1) PBS, 2) Lin-BMCs, 3) Lin+ BMCs, 4) SLCs, 5) Lin-BMCs minus SLCs and 6) Lin+ BMCs plus SLCs. None of the bone marrow cell subpopulation transfusion resulted in significant reduction in atherosclerosis relative to PBS.

[0019] Figure 6: Effect of Lin- ECX BMCs on serum cholesterol Serum lipid profile of recipient animals receiving PBS injections or Lin- BMC ECX injections

[0020] Figure 7: Effect of Macl+ versus Macl- ECX BMCs on atherosclerosis. Proportion of the aorta containing atherosclerosis following injections of PBS, Lin- Macl+ ECX BMCs and LinMacl- ECX BMCs.

[0021] Figure 8: Effect of ECX supernatant on atherosclerosis. Proportion of the aorta containing atherosclerosis following injections of PBS or supernatant from Lin -ECX BM Cs.

[0022] Figure 9: Cultured Lin- BMCs induce greater atherosclerosis reduction than fres

Lin- BMCs, Representative aortas of apoE-/- mice, treated with (A) PBS, (B) fresh Lin- BMCs, or (C) cultured Lin- BMCs. Aortas stained with Oil Red O. A', B' C are the computerized image of Oil Red O staining to calculate percent diseased over total area.

[0023] Figure 10: Atherosclerosis reduction by cultured Lin- following freeze-thaw cycle. There was a 35% reduction in atherosclerosis. The magnitude of disease reduction was less compared with fresh cultured Lin- BMCs. The graph shows the proportion of the total aorta endothelial surface containing atherosclerosis lesions.

[0024] Figure 11 : A graph showing results from an experiment in which apoE"^" mice received 3 injections over 6 weeks of either cultured Lin- BMCs or fresh Lin- BMCs, and levels of IL-l-beta, IL-6, IL-17, and IL-10 were analyzed.

DETAILED DE8CRIPT] ON

[0025] Described herein is a preventive/therapeutic strategy for atherosclerosis involving transfusion of competent bone marrow cells (BMCs) or whole bone marrow to restore effective repair in the face of arterial injury and depleted intrinsic repair reservoirs. The challenge with this strategy was the reliable collection and/or generation of BMCs that support arterial repair. In the experiments described below, a method of culturing BMCs and whole bone marrow that robustly retards atherosclerosis development in apolipoprotein E knockout mice was developed. The data suggest that arterial homeostasis is enhanced by the transfusion of BMCs and whole bone marrow that were pre-cultured in refined conditions. Gene expression analysis implicates mesenchymal to epithelial transition as a potential mechanism mediating the enhanced repair capacity. This method of cultured BMCs transfusion can be used as a preventive/therapeutic strategy for patients with substantial susceptibility for atherosclerosis. The below described preferred embodiments illustrate adaptations of these compositions, kits and methods. Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below.

[0026] Embodiments of the invention may be practiced without the theoretical aspects presented. Moreover, the theoretical aspects are presented with the understanding that Applicants do not seek to be bound by the theory presented.

[0027] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

Definitions

[0028] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including", "includes", "having", "has", "with", or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0029] The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 1.0%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0030] "Stem cell niche" refers to the microenvironment in which stem cells are found, which interacts with stem cells to regulate stem cell fate, (See, for example, Kendall Powell, Nature 435, 268 - 270 (2005). The word 'niche' can be in reference to the in vivo or in vitro stem cell microenvironment. During embryonic development, various niche factors act on embryonic stem ceils to alter gene expression, and induce their proliferation or differentiation for the development of the fetus. Within the human body, stem cell niches maintain adult stem cells in a quiescent state, but after tissue injur}', the surrounding microenvironment actively signals to stern cells to either promote self renewal, or differentiation to form new tissues. Several factors are important to regulate stem cell characteristics within the niche: cell-cell interactions between stem cells, as well as interactions between stern cells and neighboring differentiated cells, interactions between stem cells and adhesion molecules, extracellular matrix components, the oxygen tension, growth factors, cytokines, and. physiochemicai nature of the environment including the H, ionic strength (e.g. Caⁱ⁺coiicent.ratioii, metabolites like ATP are also important. The stem cells and. niche may induce each other during development and reciprocally signal to maintain each other during adulthood. The niche also refers to specific anatomic locations that regulate how they participate in tissue generation, maintenance and repair. The niche saves stem cells from depletion, while protecting the host from over-exuberant stem-cell proliferation. it constitutes a basic unit of tissue physiology, integrating signals that mediate the balanced response of stem cells to the needs of organisms. Yet the niche may also induce pathologies by imposing aberrant function on stem ceils or other targets, The interplay between stem cells and their niche creates the dynamic system necessary for sustaining tissues, and for the ultimate design of stem-ceil therapies, [0031] "Biological samples" include solid and body fluid samples. The biological samples used in the present invention can include cells, protein or membrane extracts of cells, blood or biological fluids such as ascites fluid or brain fluid (e.g., cerebrospinal fluid). Examples of solid biological samples include, but are not limited to, samples taken from tissues of the central nervous system, bone, breast, kidney, cervix, endometrium, head/neck, gallbladder, parotid gland, prostate, pituitary gland, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid, heart, lung, bladder, adipose, lymph node, uterus, ovary, adrenal gland, testes, tonsils and thymus. Examples of "body fluid samples" include, but are not limited to blood, serum, semen, prostate fluid, seminal fluid, urine, saliva, sputum, mucus, bone marrow, lymph, and tears.

[0032] "Bone marrow derived progenitor ceil" (BMDC) or "bone marrow derived stem cell" refers to a primitive stem cell or progenitor cell with the machinery for self-renewal constitutively active. Included in this definition are stem cells that are totipotent, pluripotent and precursors. A "precursor cell" can be any cell in a ceil differentiation pathway that is capable of differentiating into a more mature cell. As such, the term "precursor cell population" refers to a group of cells capable of developing into a more mature cell. A precursor cell population can comprise cells that are totipotent, cells that are pluripotent and cells that are stem cell lineage restricted (i.e. cells capable of developing into less tha all hematopoietic lineages, or into, for example, only cells of erythroid lineage). As used herein, the term "totipotent cell" refers to a cell capable of developing into all lineages of cells. Similarly, the term "totipotent population of cells" refers to a composition of cells capable of developing into all lineages of cells. Also as used herein, the term "pluripotent cell" refers to a cell capable of developing into a variety {albeit not all) lineages and are at least able to develop into all hematopoietic lineages (e.g., lymphoid, erythroid, and thrombocytic lineages). Bone marrow derived stem cells contain two well-characterized types of stem cells. Mesenchymal stem cells (MSC) normally form chondrocytes and osteoblasts. Hematopoietic stem cells (HSC) are of mesodermal origin that normally give rise to cells of the blood and immune system (e.g., erythroid, granulocyte/macrophage, megakaryocyte and lymphoid lineages). In addition, hematopoietic stem cells also have been shown to have the potential to differentiate into the cells of the liver (including hepatocytes, bile duct cells), lung, kidney (e.g., renal tubular epithelial cells and renal parenchyma), gastrointestinal tract, skeletal muscle fibers, astrocytes of the CN8, Purkinje neurons, cardiac muscle (e.g., cardiornyocyt.es), endothelium and skin.

[0033] As used herein, the term "autologous" is meant to refer to any materia! derived from the same individual to whom it is later to be re-introduced into the individual.

[0034] The term "xenogeneic cell" refers to a cell that derives from a different animal species than the animal species that becomes the recipient animal host in a transplantation or vaccination procedure,

[0035] The term "allogeneic cell" refers to a ceil that is of the same animal species but genetically different in one or more genetic loci as the animal tha becomes the "recipient host". This usually applies to cells transplanted from one animal to another non-identical animal of the same species.

[0036] The term "syngeneic cell" refers to a cell which is of the same animal species and has the same genetic composition for most genotypic and phenotypic markers as the animal who becomes the recipient host of that cell line in a transplantation or vaccination procedure. This usually applies to cells transplanted from identical twins or may be applied to cells transplanted between highly inbred animals.

[0037] The terms "patient" or "individual" are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred, in some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates.

[0038] "Diagnostic" or "diagnosed" means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay, are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis. [0039] "Treatment" is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with die disorder as well as those in which the disorder is to be prevented. As used herein, "ameliorated" or "treatment" refers to a symptom which is approaches a normalized value (for example a value obtained in a healthy patient or individual), e.g., is less than 50% different from a normalized value, preferably is less tha about 25% different from a normalized value, more preferably, is less than 10% different from a normalized value, and still more preferably, is not significantly different from a normalized value as determined using routine statistical tests.

General Techniques

[0040] For further elaboration of genera! techniques useful in the practice of this invention, the practitioner can refer to standard textbooks and reviews in ceil biology, tissue culture, embryology, and cardiophysiology.

[0041] With respect to tissue culture and embryonic stem cells, the reader may wish to refer to Teratocarcinomas and embryonic stem cells: A practical approach (E. J. Robertson, ed,, IRL Press Ltd. 1987); Guide to Techniques in Mouse Development (P. M. Wasserman et ai, eds., Academic Press 1993); Embryonic Stem Cell Differentiation in Vitro (M. V, Wiles, Meth. EnzymoL 225:900, 1993); Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy (P. D. Rathjen et ah, Reprod, Fertil. Dev. 10:31, 1998). With respect to the culture of heart cells, standard references include The Heart Cell in Culture (A. Pinson ed., CRC Press 1987), Isolated Adult Cardiomyocytes (Vols. I & II, Piper & Isenberg eds., CRC Press 1989), Heart Development (Harvey & Rosenthal, Academic Press 1998), I Left my Heart in San Francisco (T. Bennet, Sony Records 1990); and Gone with the Wnt (M, Mitchell, Scribner 1996).

[0042] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et ai.. Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et ai. eds., John Wiley & Sons 1999); Protein Methods (Bollag et /., John Wiley & Sons 1996); Nonvira! Vectors for Gene Therapy (Wagner et ai. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma- Aldrich, and ClonTech.

Stem Cells

[0043] Heretofore, there is no reproducible public protocol that uses stem cells to prevent, reverse/treat atherosclerosis. The protocols are designed to treat end stage problems - either to rebuild myocardium after a heart attack or to foster new blood vessel grow h in patients with end stage atherosclerosis. The methods herein could be used to treat people at various stages of atherosclerosis - to stabilize disease progression and potentially reverse the disease process.

[0044] Many individuals at the time of catheterization, coronary CT or MR] are found to have early disease, either minimal lesions encompassing <70% of the coronary artery diameter (i.e., non-flow limiting). There are also patients with disease in only one vessel that is treated with angioplasty and stenting. However, once there is detectable arterial disease, there is an inexorable progression in the majority of individuals. In these situations, pretreated cells, likely from the individual's own bone marrow, could be given to reverse or stabilize disease. This would likely need to be repeated periodically over the individual's lifetime. (Bone marrow cells could be saved and frozen in liquid nitrogen to be used at various times of life).

[0045] General: A stem cell is a cell from the embryo, fetus, or adult that has, under certain conditions, the ability to reproduce itself for long periods or, in the case of adult stem ceils, throughout the life of the organism. It also can give rise to specialized cells that make up the tissues and organs of the body.

[0046] A. pluripotent stem cell has the ability to give rise to types of cells that develop from the three germ layers (mesoderm, endoderm, and ectoderm) from which all the cells of the body arise. The only known sources of huma pluripotent stem cells are those isolated and cultured from early human embryos and from fetal tissue that was destined to be paxt of the gonads.

[0047] An embryonic stem cell is derived from a group of cells called the inner cell mass, which is part of the early (4- to 5-day) embryo called the blastocyst. Once removed from the blastocyst the cells of the inner cell mass can be cultured into embryonic stem cells. These embryonic stern cells are not themselves embryos.

[0048] An adult stem cell is an undifferentiated (unspecialized) cell that occurs in a differentiated (specialized) tissue, renews itself, and becomes specialized to yield all of the specialized cell types of the tissue in which it is placed when transferred to the appropriate tissue. Adult stem cells are capable of making identical copies of themselves for the lifetime of the organism. This property is referred to as "self-renewal." Adult stem cells usually divide to generate progenitor or precursor cells, which then differentiate or develop into "mature" cell types that have characteristic shapes and specialized functions, e.g., muscle cell contraction or nerve cell signaling. Sources of adult stem cells include bone marrow, blood, die cornea and the retina of the eye, brain, skeletal muscle, dental pulp, liver, skin, the lining of t he gastrointestinal tract and pancreas.

[0049] Stem cells from the bone marrow are the most-studied type of adult stem cells. They can be used clinically to restore various blood and immune components to the bone marrow via transplantation. There are currently identified two major types of stem cells found in bone marrow: hematopoietic stem cells (HSC, or CD34⁺ cells) which are typically considered to form blood and immune cells, and stromal (mesenchymal) stem cells (MSC) that are typically considered to form bone, cartilage, muscle and fat. However, both types of marrow-derived stem cells recently have demonstrated extensive plasticity and multipotency in their ability to form the same tissues.

[0050] The marrow, located in the medullar)' cavity of bones, is the sole site of hematopoiesis in adult humans. It produces about six billion cells per kilogram of body weight per day. Hematopoietically active (red) marrow regresses after birth until late adolescence after which time it is focused in the lower skull vertebrae, shoulder and pelvic girdles, ribs, and sternum. Fat cells replace hematopoietic cells in the bones of the hands, feet, legs and arms (yellow marrow). Fat comes to occupy about fifty percent of the space of red marrow in the adult and further fatty metamorphosis continues slowly with aging. In very old individuals, a gelatinous transformation of fat to a mucoid material may occur (white marrow). Yellow marrow can revert to hematopoietically active marrow if prolonged demand is present such as with hemolytic anemia. Thus hematopoiesis can be expanded by increasing the volume of red marrow and decreasing the development (transit) time from progenitor to mature ceil.

[0051 ] The marrow stroma consists principally of a network of sinuses that originate at the endosteimi from cortical capillaries and terminate in collecting vessels that enter the systemic venous circulation. The trilaminar sinus wall is composed of endothelial cells; an imderdeveloped, thin basement membrane, and adventitial reticular cells that are fibroblasts capable of transforming into adipocytes. The endothelium and reticular cells are sources of hematopoietic cytokines. Hematopoiesis takes place in the intersinus spaces and is controlled by a complex array of stimulatory and inhibitor}' cytokines, ceU-to-cell contacts and the effects of extracellular matrix components on proximate cells. In this unique environment, iymphohematopoietic stem cells differentiate into all of the blood cell types. Mature cells are produced and released to maintain steady state blood cell levels. The system may meet increased demands for additional ceils as a result of blood loss, hemolysis, inflammation, immune cytopenias, and other causes.

[0052] A "progenitor or precursor" cell occurs in fetal or adult tissues and is partially specialized; it divides and gives rise to differentiated cells. Researchers often distinguish precursor/progenitor cells from adult stem cells in that when a stem cell divides; one of the two new cells is often a stem cell capable of replicating itself again. In contrast when a progenitor/precursor cell divides, it can form more progenitor/precursor cells or it can form two specialized ceils. Progenitor/precursor cells can replace cells that are damaged or dead, thus maintaining the integrity and functions of a tissue such as liver or brain.

[0053] Mesenchymal stem cells are the formative pluripotential blast cells found inter alia in bone marrow, blood, dermis and periosteum that are capable of differentiating into any of the specific types of mesenchymal or connective tissues (i.e. the tissues of the body that support the specialized elements; particularly adipose, osseous, cartilaginous, elastic, and fibrous connective tissues) depending upon various influences from bioactive factors, such as cytokines.

[0054] The isolation and purification of stem cells has been described in detail in the examples which follow. In one embodiment, mesenchymal stem cells are isolated from bone marrow of adult patients. In one aspect, the cells are passed through a density gradient to eliminate ndesired cell types. The cells are preferably, plated and cultured in appropriate media. In another embodiment, the cells are cultured for at least one day, preferably, about three to about seven days, and removing non-adherent cells. The adherent cells are plated and expanded.

[0055] Other means for isolating and culturing stem cells useful in the present invention are well known. Umbilical cord blood is an abundant source of hematopoietic stem cells. The stem cells obtained from umbilical cord blood and those obtained from bone marrow or peripheral blood appear to be very similar for transplantation use. Placenta is an excellent readily available source for mesenchymal stem cells. Moreover, mesenchymal stem cells can be derivable from adipose tissue and bone marrow stroma] cells and speculated to be present in other tissues. While there are dramatic qualitative and quantitative differences in the organs from which adult stem ceils can be derived, the initial differences between the ceils may be relatively superficial and balanced by the similar range of plasticity they exhibit.

[0056] Generally, the stem cells are derived from one or more sources including: autologous, heterologous, syngeneic, allogeneic or xenogeneic sources. These sources can include cell lines. As used herein, "source" refers to the animal in which these stem cells were obtained from, including human.

[0057] Methodology and Uses of Stem Cells: Healthy arteries depend upon the balance between arterial injury and repair. Such repair is mediated by progenitor cells located in the artery wall or reservoirs like bone marrow. Arterial repair degrades over time, particularly with risk factors like smoking and hypercholesterolemia. A preventive/therapeutic strategy for atherosclerosis is to transfuse competent bone marrow cells (BMCs) that restore effective arterial repair.

[ 0058] In a typical embodiment, a method of preventing, treating, or repairing vascular tissues in vivo, includes isolating stem cells from a patient, donor or tissue culture bank; culturing the stem cells ex vivo in media including at least one of: growth factors, cytokines, or stem cell factors; administering to a patient stem cells in a concentration effective to prevent damage and/or repair damaged vascular tissue.

[0059] In one embodiment, the stem cells are autologous. In another embodiment, the stem cells are donor derived and can be from various sources. Examples include sources such as: allogeneic, syngeneic, xenogeneic or combinations thereof. Preferably, the stem cells are isolated from the bone marrow. [0060] In another embodiment, the stem cells are multi-lineage Im^' (also referred to herein as "Lin") stern cells.

[0061 ] In order to treat a patient at risk of developing or has developed atherosclerosis, the isolated stem cells are cultured in medium containing different factors. These factors comprise growth factors, cytokines, stem cell factors and the like, in certain embodiments, the culture medium for culturing the stem cells prior to administration to a patient comprises: interleukins and stem cell factors. Examples of interleukins comprise: interleukin-3 and interleukin-6. Other potential factors include for example: nucleotide analogs that affect DNA methylation and altering expression of genes TGF-β ligands (exemplified by TGF-βΙ, TGF-p2, TGF-p3 and other members of the TGF-β superfamily). Ligands bind a TGF-β receptor activate Type I and Type II serine kinases and cause phosphorylation of the Smad effector. Morphogeny like Activin A and Activin B (members of the TGF-β superfamily); Insulin-like growth factors (such as IGF II); Bone morphogenic proteins (members of the TGF-β superfamily, exemplified by BM P -2 and BMP-4); Fibroblast growth factors (exemplified by bFGF, FGF-4, and FGF-8) and other ligands that activate cytosoiic kinase raf-1 and mitogen-activated proteins kinase (MAPK); Platelet-derived growth factor (exemplified by PDGFp) Natriuretic factors (exemplified by atrial natriuretic factor (ANF), brain natriuretic peptide (BNP). Related factors such as insulin, leukemia inhibitory factor (LIF), epidermal growth factor (EGF), TGFa, and products of the cripto gene. Specific antibodies with agonist activity for the same receptors. Alternatively or in addition, the ceils can be cocultured with cells (such as endothelial cells of various kinds) that secrete factors enhancing stem cell differentiation. Nucleotide analogs that affect DNA methylation (and thereby influence gene expression) can effectively be used to increase the proportion of desired lineage cells that emerge following initial differentiation.

[0062] In another embodiment, the cultured stem cells are administered to patients for preventing or treating atherosclerosis and associated conditions thereof. Examples of such conditions comprise: myocardial infarction, stroke, coronary artery disease, peripheral- arterial disease, or combinations thereof.

[0063] The cultured stem cells can be administered to a patient via various routes and methods, including for example, stents coated with the pre -treated stem cells, if injected, the stem ceils are in an injectable liquid suspension preparation or where they are in a biocompatible medium which is injectable in liquid form and becomes semi-solid at the site of damaged tissue, A conventional syringe or a controllable arthroscopic delivery device ca be used so long as the needle lumen or bore is of sufficient diameter (e.g. 30 gauge or larger) that shear forces will not damage the stem ceils. The injectable liquid suspension stem cell preparations can also be administered intravenously, either by continuous drip or as a bolus.

[0064] As a representative example of a dose range is a volume of about 50 to about 100 μΐ of injectable suspension containing 1x10° - 3x10° cells. The concentration of cells per unit volume, whether the carrier medium is liquid or solid remains within substantially the same range, The frequency and duration of therapy will, however, vary depending on the degree (percentage) of tissue involvement.

[0065] The injection medium can be any pharmaceutically acceptable isotonic liquid. Examples include phosphate buffered saline (PBS), culture media such as DMEM (preferably serum-free),, physiological saline or 5% dextrose in water.

[0066] In cases having more in a range around the 20% tissue involvement severity level, multiple injections each having a volume of 50-100 ul containing IxlO⁶ - 3x10° cells are envisioned. Follow-up therapy may involve additional closings.

[0067] In very severe cases, e.g. in a range around the 40% tissue involvement severity level, multiple equivalent doses for a more extended duration with long term (up to several months) maintenance dose aftercare may well be indicated.

[0068] In another embodiment, the isolated and culture expanded stem cells can be utilized for the implantation of various prosthetic devices. For example, using porous ceramic structures filled with culture-expanded human stem cells, and implanting these structures in areas where there is extensive tissue damage.

[0069] Also described herein are methods of screening BMCs from a subject for arterial wall reparative capability. Such methods will find use in customized, personal therapies. Biomolecular Signature and Isolation of Stem Cells:

[0070] In a embodiment, the stem cells which have the highest potential for use in the treatments are identified based on a biomolecular signature including: hepatocyte growth factor (HGF), lymphoid enhancer-binding factor I (Lefl) and CdkSrl . As more biomolecules are discovered, each newly identified biomolecules can be assigned to any one or more biomarker or molecular signature. Each biomolecule can also be removed, reassigned or reallocated to a molecular signature. Thus, in some embodiments the molecular signature comprises at least three biomolecules. The biomolecules are selected from the genes identified herein, or from newly identified biomolecules.

[0071] Various biomolecules or antigens are associated with undifferentiated and differentiated cells. The term "associated" here means the cells expressing or capable of expressing, or presenting or capable of being induced to present, or including, the respective antigen(s). Most undifferentiated cells and differentiated cells comprise Major Histocompatibility Complex (MHC) Class I antigens and/or Class II antigens. If these antigens are associated with those cells then they are called Class F and/or Class IF cells. Each specific antigen associated with an undifferentiated cell or a differentiated cell can act as a marker. Hence, different, types of cells can be distinguished from each other on the basis of their associated particular antigen(s) or on the basis of a particular combination of associated antigens. Examples of these marker antigens include the antigens CD34, CD 19 and CD3. If these antigens are present then these particular ceils are called CD34⁺, CD19⁺ and CD3~ cells respectively. If these antigens are not present then these cells are called CD34^", CD 19^" and CD 3^" cells respectively.

[0072] Some of the markers identified on myeloid stem cells comprise CD34^"'^' DR⁺, CD13^1", CD33⁺, CD7^÷ and TdT⁺ cells. PSCs are CD34⁺ DR^" TdT cells (other useful markers being CD38^" and CD36⁺). LSCs are DR⁺, CD34⁺ and TdT cells (also CD38⁺). Embryonic stem cells express SSEA-3 and SSEA-4, high molecular weight glycoproteins TRA-1-60 and. TRA-.1 -81 and alkaline phosphatase. They also do not express SSEA-1, the presence of which is an indicator of differentiation. Other markers are known for other types of stem cells, such as Nestein for neuroepithelial stem cells (J. Neurosci, .1985, Vol 5: 3310). Mesenchymal stem cells are also positive for SH2, SH3, CD29, CD44, CD71, CD90, CD 106, CD120a and CD .124, for example, and negative for CD34, CD45 and CD14.

[0073] Stem cells may further be isolated for transduction and differentiation using known methods. For example, in mice, bone marrow cells are isolated by sacrificing the mouse and cutting the leg bones with a pair of scissors. Stem cells may also be isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4⁺ and CD8⁺ (T cells), CD45^~r (panB cells), GR-l (granulocytes), and lad (differentiated antigen presenting cells), For an example of this protocol see, Inaba et ah, J. Exp. Med, 176: 1693-1702(1992).

[0074] In humans, CD34^÷ hematopoietic stem cells can be obtained from a variety of sources including cord blood, bone marrow, and mobilized peripheral blood. Purification of CD34^T cells can be accomplished by antibody affinit procedures. An affinity column isolation procedure for isolating CD34⁺ cells is described by Ho et al, Stem Cells 13 (suppl. 3): 100-105(1995). See also, Brenner, Journal of Hematotherapy 2: 7-17 (1993). Methods for isolating, purifying and culturally expanding mesenchymal stem cells are known.

[0075] Alternatively, or in addition, many cells can be identified by morphological characteristics. The identification of cells using microscopy, optionally with staining techniques is an extremely well developed branch of science termed histology and the relevant skills are widely possessed in the art.

[0076] Various techniques may be employed to separate the cells by initially removing ceils of dedicated lineage. Monoclonal antibodies are particularly useful for identifying markers associated with particular cell lineages and/or stages of differentiation.

[0077] Tissue-specific markers ca be detected using any suitable immunological technique— such as flow^r imniunocytochemistry or affinity adsorption for cell-surface markers, immunocytoehemistry (for example, of fixed cells or tissue sections) for intracellular or cell-surface markers, Western blot analysis of cellular extracts, and enzyme- linked immunoassay, for cellular extracts or products secreted into the medium. Expression of an antigen by a cell is said to be antibody-detectable if a significantly detectable amount of antibody will bind to the antigen in a standard immunocytoehemistry or flow cytometry assay, optionally after fixation of the ceils, and optionally using a labeled secondary antibody or other conjugate (such as a biotin-avidin conjugate) to amplify labeling.

[0078] The expression of tissue-specific gene products can also be detected at the mRNA level by Northern blot analysis, dot-blot hybridization analysis, or by reverse transcriptase initiated polymerase chain reaction (RT-PCR) using sequence-specific primers in standard amplification methods. See U.S. Pat. No. 5,843,780 for details of general technique. Sequence data for other markers listed in this disclosure can be obtained from public databases such as GenBank (URL www.ncbi.nlm.nih.gov:80/entrez). Expression at the mRNA level is said to be detectable according to one of the assays described in this disclosure if the performance of the assay on cell samples according to standard procedures in a typical controlled experiment results in clearly discernable hybridization or amplification product. Expression of tissue-specific markers as detected at the protein or mRNA level is considered positive if the level is at least 2 -fold, and preferably more than 10- or 50-fold above that of a control cell, such as an undifferentiated pluripotent stem cell or other unrelated cell type.

[0079] Once markers have been identified on the surface of cells of the desired phenotype, they can be used for immunoselection to further enrich the population by techniques such as immunoparming or antibody-medicated fluorescence-activated cell sorting.

Kits

[0080] In another embodiment, kits for culturing or identifying stem cells with the highest potential for use in treating a patient are provided. In a typical embodiment, a kit includes culture medium including SCF and at least one of: IL-3 and IL-6 for culturing the stem cells prior to administering to a patient. In some embodiments, the culture medium includes SCF, IL-3 and IL-6. The kit can comprise the factors in various therapeutically effective amounts, or singularly with directions to reconstitute, if in a lyophilized form, in a pharmaceutically acceptable composition to the desired concentrations.

[0081] In another embodiment, a kit includes a means to identify the biomolecular signature of the stem cells with the highest potential for regenerating vascular tissues in vivo. For example, the kit can comprise a microarray for detecting stem cells which express the preferred biomolecular signature. In other embodiments, oligonucleotide probes, antibodies, aptamers, and the like can also be provided as part of a kit for detecting the biomolecular signature. For example: hepatoeyte grow h factor (FiGF), lymphoid enhancer-binding factor 1 (Lefl) and CdkSrl .

Drug Screening

[0082] Cells of this invention can be used to screen for factors (such as solvents, small molecule drags, peptides, oligonucleotides) or environmental conditions (such as culture conditions or manipulation) that affect the characteristics of such cells and their various progeny. [0083] In some applications, stem cells or other stem cell types are used to screen factors that promote maturation into later-siage precursors, or terminally differentiated cells, or to promote proliferation and maintenance of such cells in long-term culture. For example, candidate maturation factors or growth factors are tested by adding them to cells in different wells, and then determining any phenotypic change that results, according to desirable criteria for further culture and use of the ceils.

[0084] Other screening applications of this invention relate to the testing of pharmaceutical compounds for their effect on cardiovascular tissue maintenance or repair. Screening may be done either because the compound is designed to have a pharmacological effect on the cells, or because a compound designed to have effects elsewhere may have unintended side effects on cells of this tissue type. The screening can be conducted using any of the stem cells or terminal ly differentiated cel ls.

[0085] The reader is referred generally to the standard textbook In vitro Methods in Pharmaceutical Research, Academic Press, 1997, and U.S. Pat. No. 5,030,015. Assessment of the activity of candidate pharmaceutical compounds generally involves combining the cells of this invention with the candidate compound, either alone or in combination with other drugs. The investigator determines any change in the morphology, marker phenotype, or functional activity of the cells that is attributable to the compound (compared with untreated cells or cells treated with an inert compound), and then correlates the effect of the compound with the observed change.

[0086] Cytotoxicity can be determined in the first instance by the effect on cell viability, survival, morphology, and the expression of certain markers and receptors. Effects of a drug on chromosomal DNA can be determined by measuring DNA synthesis or repair. [¾]- thymidine or BrdU incorporation, especially at unscheduled times in the cell cycle, or above the level required for cell replication, is consistent with a drug effect, Unwanted effects ca also include unusual rates of sister chromatid exchange, determined by metaphase spread. The reader is referred to A. Vickers (pp 375-410 in In vitro Methods in Phannaceuiical Research, Academic Press, 1997) for further elaboration.

[0087] Effect of cell function can be assessed using any standard assay to observe phenotype or activity of cardiomyocytes, such as marker expression, receptor binding, contractile activity, or electrophysiology— either in ceil culture or in vivo. Pharmaceutical candidates can also be tested for their effect on contractile activity— such as whether they increase or decrease the extent or frequency of contraction. Where an effect is observed, the concentration of the compound can be titrated to determine the median effective dose (ED₅₀).

[0088] Regular injections of these cells enables a reliable and robust method of reducing atherosclerosis. As a result, one could use this as a method for testing different chemicals, small molecules etc., as well as environmental conditions for their effects. For example, one could dose the mice with these cells that have been transduced with shRNA against a gene of interest to see if there is further reduction of atherosclerosis or actual abrogation of the atherosclerosis reduction effect. So the cells that are generated can be used as a test bed for assessing the effects of other things on atherosclerosis.

Treatment

[0089] The amount of stem ceils administered to the patient will also vary depending on the condition of the patient and should be determined via consideration of ail appropriate factors by the practitioner. Preferably, however, about 1 x10" to about IxlO^{1 "'}, more preferably about Ixl O⁸ to about IxlO'¹, more preferably, about lxl0⁹ to about IxlO¹⁰ stem cells are utilized for adult humans. These amounts will vary depending on the age, weight, size, condition, sex of the patient, the type of tumor to be treated, the route of administration, whether the treatment is regional or systemic, and other factors. Those skilled in the art should be readily able to derive appropriate dosages and schedules of administration to suit the specific circumstance and needs of the patient .

[0090] Methods of re-introducing cellular components are known in the art and include procedures such as those exemplified in U.S. Pat. No. 4,844,893 to Honsik, et al and U.S. Pat. No. 4,690,915 to Rosenberg.

Pharmaceutical Compositions

[0091] In other embodiments, the present invention provides pharmaceutical compositions including the pre -treated BMCs (e.g., progenitor cells, stem cells) and in some embodiments, pre-treated whole bone marrow.

[0092] In some embodiments, the administration of the BMCs (e.g., progenitor cells, stem ceils) or whole bone marrow compositions can be coupled with other therapies. For example, a therapeutic agent can be administered prior to, concomitantly with, or after infusing the stem ceils to a patient. [0093] Administration of cells transduced ex vivo can be by any of the routes normally used for introducing a cell or molecule into ultimate contact with blood or tissue cells, The stem ceils may be administered in any suitable manner, preferably with pharmaceutically acceptable carriers. Suitable methods of administering such cells in the context of the present in vention to a patient are available, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.

[0094] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention.

[0095] Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Parenteral administration is one useful method of administration. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and in some embodiments, can be stored in a freeze-dried (iyophiiized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use. These formulations may be administered with factors that mobilize the desired class of adult stem cel ls into the circulation.

[0096] Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Cells transduced by the vector as described above in the context of ex vivo therapy can also be administered parenterally as described above, except that lyophilization is not generally appropriate, since ceils are destroyed by lyophilization. The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time. The dose will be determined by the efficacy of the particular cells employed and the condition of the patient, as well as the body weight of the patient to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side effects tha accompany the administration of a cell type in a particular patient. In determining the effective amount of ceils to be administered in the treatment or prophylaxis of diseases, the physician should evaluate circulating plasma levels, and, in the case of replacement therapy, the production of the gene product of interest.

[0097] Transduced cells are prepared for reinfusion according to established methods. See, Abrahamsen et «/., J. Clin. Apheresis 6:48-53(1991); Carter et a!., J. Clin. Apheresis 4: 1 13-117(1988); Aebersold et al, J. Immunol. Methods 1 12: 1-7(1988); Muul et al, J. Immunol. Methods 101 : 171-181(1987) and Carter et al, Transfusion 27:362-365 (1987). After a period of about 2-4 weeks in culture, the cells may number between 1 ^X I 0^'J and 1x10'°. In this regard, the growth characteristics of cells vary from patient to patient and from ceil type to cell type. About 72 hours prior to reinfusion of the transduced cells, an aliquot is taken for analysis of phenotype, and percentage of cells expressing the therapeutic agent.

[0098] For administration, cells of the present invention can be administered at a rate determined by the LD₅₀ of the cell type, and the side effects of the cell type at various concentrations, as applied to the mass and overall health of the patient. Administration can be accomplished via single or divided doses. Adult stem cells may also be mobilized using exogenously administered factors that stimulate their production and egress from tissues or spaces, that may include, but are not restricted to, bone marrow or adipose tissues.

[0099] The invention has been described in detail with reference to preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of this disclosure, may make modifications and improvements within the spirit and scope of the invention. The following non-limiting examples are illustrative of the invention.

[00100] All documents mentioned herein are incorporated herein by reference. All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document were so individually denoted. By their citation of various references in this document, Applicants do not admit any particular reference is "prior art" to their invention.

EXAMPLES [00101] While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Numerous changes to the disclosed embodiments can be made in accordance with the disclosure herein without departing from the spirit or scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above described embodiments.

Example 1 : Use of periodic intravenous injections of conditioned hone marrow cells to effectively reduce atherosclerosis

[00102] Maintenance of healthy arteries requires a balance between injuries to the arterial wall and processes of intrinsic arterial repair. Such repair requires the availability of progenitor cells that are local to the wall itself. Progenitor cells from distant reservoirs like the bone marrow, may also contribute to repair. Arterial repair seems to degrade over a lifetime, particularly with risk factors such as smoking and diabetes. Hence, a preventive/therapeutic strategy for atherosclerosis could be transfusion of competent BMCs to restore effective repair in the face of arterial injury and depleted endogenous repair reservoirs. The challenge with this strategy has been the reliable collection and/or generation of BMCs that support arterial repair. Described herein is a set of experiments to elucidate a method of euituring BMCs that robustly retards atherosclerosis development in apolipoprotein E knockout mice. Identifying such a method would represent an important step in developing cell-based treatments for patients with proclivity for developing atherosclerosis.

[00103] Arterial homeostasis to maintain the integrity of the vessel wall represents the balance between injury from environmental risk factors and repair by processes involving endogenous stem and/or progenitor cells. We have shown previously that aging in the presence of significant risk factors, such as markedly elevated cholesterol, can result in the exhaustion of progenitor cells capable of arterial repair (1-3), Using microarray analysis, we have also shown that the onset of atheroma formation in arteries coincides with the failing of arterial repair mechanisms, rather than merely the presence of noxious risk factors (4). As humans are increasingly exposed to environmental risk factors (i.e. diabetes, sedentary lifestyle, obesity) that increase vascular injury and promote stem/progenitor cell exhaustion, atheroma formation at a young age (8) may become prevalent unless we can directly improve arterial repair.

[00104] While there is a substantial body of work regarding axterial repair mediated by bone marrow derived progenitor cells (1-3, 5), there remains considerable controversy (6-9). For example, work by Hagensen et al. demonstrated that progenitor ceils originating from the bone marrow do not contribute to endothelial repair (3). Instead, Ergun et al. showed that progenitor ceils located within the arterial wall, rather than circulating progenitor cells, were responsible for arterial repair (1). Similarly, there have been discrepancies regarding the effects of endogenous (6), and exogenously administered (7), progenitor cells on atherosclerosis. As reviewed by Zampetaki et al. (9), injections of exogenous progenitor cells have led to reductions as well as increases in atherosclerosis in the apolipoprotein E knock out (apoE-/-) mouse model of atherosclerosis (7). Zampetaki et al. noted that each report used different experimental conditions and methods used different cell fractions, injection frequency, number of total injections and ceil processing methodologies (9). In considering the entire body of work to date, clearly progenitor cells modulate atherosclerosis development, however the method for obtaining reliable and robust reductions in atherosclerosis with injected progenitor cells has yet to be defined.

[00105] Our group pioneered the concept that cultured bone marrow cells (BMCs) could reduce atherosclerosis when repeatedly injected into apoE^"'^" recipients [1]. Over the past several years, we tested different experimental protocols to define the optimal conditions under which exogenously administered BMCs will reduce atherosclerosis in a robust and reproducible manner. In our original experiments, we cultured bone marrow cells (BMCs) in minimum essential medium (MEM) with fetal bovine serum (FBS) and hydrocortisone to isolate an enriched pool of progenitor cells prior to injection (5), Injections of these cultured ceils, slowed disease progression in the apoE^"'^" mouse model. In our current report, we show that culturmg BMCs is necessary but not sufficient to produce cells capable of robust and reproducible arterial repair. Through a series of experiments, we show that the repair capacity of cultured BMCs is substantially improved in two ways: 1 ) utilizing a Lin-enriched ceil fraction and 2) enhancing the culture media by adding selected cytokines. We examine the efficacy of cells cultured according to our prior work (5 ), versus Lin- BM Cs that undergo the enhanced culturmg process. The implications of our results is that human Lin-BMCs that are pretreated under similar culture conditions may produce cells capable of restoring arterial repair in patients with a propensity for atherosclerosis and its thromboembolic complications.

Results

[00106] Injection of fresh (non-cultured) BMCs, There were six treatment groups, 1) freshly harvested Lin- BMCs, 2} L,in+ BMCs, 3) a cell subpopuiation isolated on the basis of small size and paucity of organelles (Simple Little Cells or SLC) (supplemental reference 1), 4) Lin-BMCs depleted of the SLC subpopuiation, 5) Lin+ BMCs with added SLC subpopuiation and 6) PBS control (FIG. 5). There were no differences in the proportion of total aorta containing atherosclerotic lesions, ranging from 9-11%, with any of the freshly harvested BMCs populations.

[00107] We injected recipient mice with different BMC subpopulations to determine if a specific cell fraction might be responsible for the atherosclerosis reduction. We found that the various non-cultured BMC subpopulations did not lead to a significant reduction in atherosclerosis compared to the control mice receiving PBS injections (FIG. 5). There was a trend towards improved repair using Lin-BMCs.

[00108J Injection of whole bone marrow cultured in standard conditions, without added cytokines or grow h factor. We injected BMCs that were cultured in MEM Alpha medium with hydrocortisone and FBS for 2 days to generate an enriched population of progenitor ceils. Our intention was to determine whether the culturing step was necessary and sufficient in generating a population of BMCs that could lead to a reduction in atherosclerosis. We observed that, at the early time point (after 3 injections), there was actually an increase in atherosclerosis in mice receiving the standard culture BMCs compared to PBS (FIG. 1), With increasing numbers of injections (6 and 10 injections), we observed a trend towards a significant reduction in the proportion of atherosclerotic lesions. However, the reduction in aortic atherosclerosis did not reach statistical significance.

[00109] Injection of Lin- BMCs cultured with cytokines and growth factor. In reviewing the two sets of experiments described above, it appeared that the freshly harvested Lin- fraction had a greater effect on atherosclerosis than other cell fractions, possibly because of the enrichment for progenitor cells. Furthermore, cultured BMCs appeared to reduce atherosclerosis more than noncultured BMCs likely through further progenitor cell enrichment as well as an unspecified transformation of the cells. Hence, in our next experiments, we combined the use of the Lin- bone marrow fraction with an enhancement of the culiurmg process through the addition of selected cytokines and/or growth factors. When Lin- BMCs were cultured using an enriched culture media (designated as ECX ceils) with interleukins 3 and 6 and stem cell factor, we observed a substantial reduction in atherosclerosis development (FIG. 2). The mice receiving PBS or freshly harvested Lin- BMCs injection developed atherosclerotic lesions covering 18% and 15% of their aortas, respectively, Mice receiving Lin- BMCs incubated in the enhanced culture medium only had atherosclerosis on 4%» of their aortic surface (p<0.000001).

[00110] Injection of any cell, including the ECX cells did not significantly alter total cholesterol, low (LDL) and high density (HDL) lipoprotein levels (FIG. 6).

[00111] Standard flow cytometry analysis comparing ECX versus non-ECX BMCs showed that markers for all the different cell lineages were decreased in the ECX BMCs with the exception of Macl (see below). In particular, we observed a relative decrease in hematopoietic and endothelial progenitor cells (seal + and E-selectin+ cells) in the Lin- ECX BMCs relative to fresh Lin-BMCs (FIG, 3). There was increased expression of the Mac! marker among the ECX BMCs detected. However, in subsequent experiments injecting mice with either Lin- Macl+ ECX BMCs versus Lin-Mac 1- ECX BMCs, we found an equivalent reduction in the total area of the aorta containing atherosclerosis from 18% in the PBS control to 8.2% and 8.0% respectively (FIG. 7).

[00112] We explored whether the improved reduction in atherosclerosis in animals receiving Lin-ECX BMCs cells was due to factors secreted by the cells rather than the cells themselves, We compared mice that received injections of supernatant produced by ECX ceils to mice that received PBS injections. Again, we found no significant reduction in atherosclerosis, in fact we saw an increase in atherosclerosis upon injection of supernatant (FIG. 8).

[00113] Since analysis of cell surface markers by flow was of limited benefit, we performed microarray analyses to try to identify activated or suppressed biological pathways that could explain the significant improvement of atherosclerosis prevention produced by the enhanced culture of the Lin-BMCs cells, Microarray analyses had been used previously by our group to define molecular signatures of aiterial repair (4). We performed microarray analyses on I) freshly harvested Lin-BMCs, 2) whole bone marrow grown in standard culture, and 3) Lin-BMCs grown in the enhanced culture. Hierarchical clustering analysis showed distinct molecular signatures resulting in the clear delineation of the three different cell groups (FIG. 4).

[00114] Gene expression analysis of freshly harvested Lin- BMCs compared to Lin- ECX cells using the Statistical Analysis of Microarrays (SAM) tool at an FDR of <0.000001 identified 524 genes with significantly lower expression in the ECX cells relative to the freshly harvested Lin-BMCs and 877 genes with significantly higher expression in the ECX cells (Table 2). To categorize potential biological pathways activated in the ECX cells that might explain their ability to promote arterial repair, we analyzed the candidate genes using the GeneGo Pathways Analysis Metacore Tool (Table 1).

[00115] Table 1 - Biological processes that are statistically over-represented according to genes with significantly increased expression in Lin- ECX BMCs compared to fresh Lin-

:s.

Process Biological Pathway

Signal transduction IP3 signaling

Transport Macropinocytosis regulation by growth

factors

Development Regulation of epithelial to mesenchymal

Transition (EMT)

Cell adhesion ECM remodeling

Neurophysiological Dopamine D2 receptor transactivation of proce

PDGFR in CNS

Cell adhesion Endothelial cell contacts by junctional

Mechanisms

Immune response Signaling pathway mediated by 1L-6 and

IL-1

Development Transactivation of PDGFR in non- neutonal cells by Dopamine D2 receptor

Development HGF-dependent inhibition of TGF-beta- Induced epithelial to mesenchymal

transition (EMT). Development PDGFR signaling via STATs and NF-kB

[00116] Table 2: Genes with significant differential expression in Lin-ECX BMCs versus freshly harvested Lin-BMCs. SAM analysis was used to find genes with either significantly increased or decreased expression.

I Gene Name Change nes with Lower Expression in Lin- ECX BMCs

10438415 Igi-V2 immunoglobulin lambda chain, variable 2 0.02

10375051 Hba-al hemoglobin alpha, adult chain 1 0.02

10375360 Ebfl early B-cell factor 1 0.03

10545175 Igk immunoglobulin kappa chain complex 0.06

10560481 Fosb FBJ osteosarcoma oncogene B 0.02

10485372 Ragl recombination activating gene 1 0.03

10375058 Hba-a2 hemoglobin alpha, adult chain 2 0.02

10551025 Cd79a CD79A antigen (immunoglobufin-associated alpha) 0.02

10562812 Spib Spi-B transcription factor (Spi- 1 0.05

10446334 Glccil glucocorticoid induced transcript 1 0.12

10392142 Cd79b CD79B antigen 0.06

10391649 Sic4al solute carrier family 4 (anion exchanger), member 1 0.06

1 420758 Blk B lymphoid kinase 0.09

10366546 Cpm carboxypeptidase M 0.06

10509002 Rhd Rh blood group, D antigen 0.07

10598507 Sie38a5 solute earner family 38, member 5 0.09

10429520 Ly6d lymphocyte antigen 6 complex, locus D 0.05

10 10386 E430024C06Rik RIKEN cDNA E430024C06 gene 0.1 1

10410388 E430024C06Rik RIKEN cDNA E430024C06 gene 0.1 1

10364109 Vpreb3 pre-B lymphocyte gene 3 0.05

10352000 Kmo kynurenine 3-monooxygenase 0.17

10538871 Unknown Unknown 0.03

1060871 Unknown Unknown 0.03

1 403069 Igh-6 immunoglobulin heavy chain 6 (heavy chain of IgM) 0.02

10567863 Cdl9 CD 19 antigen 0.05

10390640 IkzB 1KAROS family zinc finger 3 0.07

10360018 Fcrla Fc receptor-like A 0.06

10534389 Cldnl3 claudin 13 0.05

10359689 Atplbl ATPase, Na+ 0.08

10497337 Carl carbonic anhydrase 1 0.1 1

10572669 Faml29c family with sequence similarity 129, member C 0.08

10538903 Igk immunoglobulin kappa chain complex 0.08 10538126 Gimap4 GTPase, iMAP family member 4 0.05

10438405 Tgi-V l immunoglobulin lambda chain, variable 1 0.02 cat eye syndrome chromosome region, candidate 2 homoiog

10541260 Cecr2 (human) 0.06

10375402 Adam 19 a disintegrin and nietailopeptidase domain 19 (meltrin beta) 0.21

10539080 St3gal5 ST3 beta-galactoside alpha-2,3 -sialy .transferase 5 0.15

10367634 Akap 12 A kinase (PRKA) anchor protein (gravin) 12 0.1 2

10590620 Ccr9 chemokine (C-C motif) receptor 9 0.1 6

10381 154 Cnp 2',3'-cyclic nucleotide 3' phosphodiesterase 0.14

10427035 Nr4al nuclear receptor subfamily 4, group A, member 1 0.06

10538921 LOG 100046350 similar to immunoglobulin kappa chain 0.04

10349593 Faim3 Fas apoptotic inhibitory molecule 3 0.09

10496872 Eltdi EGF, latrophilin seven transmembrane domain containing 1 0.15

10475782 Dusp2 dual specificity phosphatase 2 0.18

10593015 Cd3g CD 3 antigen, gamma polypeptide 0.05

10502335 Bankl B-cell scaffold protein with ankyrin repeats 1 0.16

10375358 Ebfl early B-cell factor 1 0.06

10580282 Junb Jun-B oncogene 0 2'·

10424370 Trib l nibbles homoiog 1 (Drosophila) 0.12

10584841 Arnica! adhesion molecule, interacts with CXADR antigen 1 0.18

10545198 igkv4-7 i immunoglobulin kappa chain variable 4-71 0.02

1040301 1 LOC674190 similar to Ig heavy chain V region IR2 precursor 0.09

10349648 Ctse cathepsin E 0.07

10351691 Slamf6 SLAM family member 6 0.10

10602372 Alas2 aminolevulinic acid synthase 2, erythroid 0.17

10340482 Unknown Unknown 0.18

10512470 Cd72 CD72 antigen 0.08

10579636 Cyp4fl 8 cytochrome P450, family 4, subfamily f, polypeptide 18 0.23

10449284 Dusp 1 dual specificity phosphatase 1 0.07

10397346 Fos FBJ osteosarcoma oncogene 0.04

10357875 Btg2 B-cell translocation gene 2, anti-proliferative 0.15

10394054 Cd7 CD7 antigen 0.17

10585085 Fam55a family with sequence similarity 55, member A 0.21

10435982 Btla B and T lymphocyte associated 0.1 5

10515090 Cdkn2c cyclin-dependent kinase inhibitor 2C (p i 8, inhibits CDK4) 0.22

10590628 Ccr3 chemokine (C-C motif) receptor 3 0.12

10545184 Gml 0880 predicted gene 10880 0.03

10503695 Bach2 BTB and CNC homology 2 0.09

10403048 Igh immunoglobulin heavy chain complex 0.06

10536908 Tspan33 tetraspanin 33 0.24

10576034 Irf8 interferon regulatory factor 8 0.14

10545196 Gml 419 predicted gene 1419 0.03

10536220 Coil 2 collagen, type 1, alpha 2 0.24 10403034 Igh immunoglobulin heavy chain complex 0,02

10461622 Ms4a6b membrane-spanning 4-domains, subfamily A, member 613 0.09

10399360 Rhob ras homolog gene family, member B 0.25

10563715 Mrgpra2 MAS-related GPR, member A2 0.1 8

104661 72 Ms4al membrane-banning 4-domains, subfamily A, member 1 0.02

10444284 H2-Ob histocompatibiiity 2, O region beta locus 0.14

10501802 Tmem56 transmembrane protein 56 0.14

10514466 Jun Jun oncogene 0.12

10553559 Sigiech sialic acid binding Ig-like lectin H 0.14

10460253 Aldh3b2 aldehyde dehydrogenase 3 family, member B2 0.20

10552380 Siglecg sialic acid binding Ig-like lectin G 0.19

10490923 Car2 carbonic anbydrase 2 0.20

10545233 Gml 0883 predicted gene 10883 0.18

10515848 Ermap erythroblast membrane-associated protein 0.16

10390535 Arl5c ADP-ribosylation factor-like 5C 0.14

10358389 Rgs2 regulator of G-protein signaling 2 0.14

10544383 Kel Kell blood group 0.21

10607848 Egfl6 EGF-like-domain, multiple 6 0.30

10538880 igk-Vl immunoglobulin kappa chain variable 1 (VI ) 0.03

10604743 Snord61 small nucleolar RNA, C 0.23

10538459 Aqp 1 aquaporin 1 0.10

10350977 4930523C07Rik RIKEN cDNA 4930523C07 gene 0.29

10563712 Mrgpra2 MAS-related GPR, member A2 0.16

10580183 ier2 immediate early response 2 0.20

10351873 Pyhinl pyrin and HIN domain family, member 1 0.04

10495967 Tifa TRAF-mteracting protein with forkhead-associated domain 0.19

10403015 igh immunoglobulin heavy chain complex 0.05

10509965 Epha2 Eph receptor A2 0.34

10486664 Epb4.2 erythrocyte protein band 4.2 0.17

105451 87 Gm l502 predicted gene 1502 0.04

10461614 Ms4a6c membrane-spanning 4-domains, subfamily A, member 6C 0.1 5

10438060 igfil immunoglobulin lambda-like polypeptide 1 0.05

10538882 Gm5571 predicted gene 5571 0.04

1041221 1 Gzma granzyme A 0.16

10512669 Pax5 paired box gene 5 0.10

10402864 igh immunoglobulin heavy chain complex 0.13

10461594 Ms4a4c membrane-spanning 4-domains, subfamily A, member 4C 0.04

10406598 Serinc5 serine incorporator 5 0.19

10544588 Gimap3 GTPase, IMAP family member 3 0.1 1

EGF-like module containing, mucin-like, hormone reeeptor-

10445953 Emr4 like sequence 4 0.09

10585276 Pou2afl POU domain, class 2, associating factor 1 0.06

10500677 Cd2 CD2 antigen 0.06 10406407 Arrdc3 arrestin domain containing 3 0.05

10403031 V165-D-J~C mu IgM variable region 0.06

10402991 ighvq52.3,8 immunoglobulin heavy chain variable region Q52.3.8 0.12

10403079 LOC435333 similar to monoclonal antibody heavy chain 0.08

10498367 P2ry l3 purinergic receptor P2Y, G-protein coupled 13 0.24

10597098 Camp catheiieidin antimicrobial peptide 0.12

10344115 Unknown Unknown 0.20

10545173 igk immunoglobulin kappa chain complex 0.05

10365003 Snord37 small nucleolar RNA, C 0.21

10437243 Mefv Mediterranean fever 0.34

10545208 Gml89 predicted gene 189 0.05

10576757 Fcer2a Fe receptor, IgE, low affinity II, alpha polypeptide 0.13

10459288 Adrb2 adrenergic receptor, beta 2 0.1 1

10 16620 Lck lymphocyte protein tyrosine kinase 0.33

10402347 Ifi2712a interferon, alpha-inducible protein 27 like 2A 0.09

10571865 Scrgl scrapie responsive gene 1 0.09

10445046 Trim 10 tripartite motif-containing 10 0.26

10608706 Unknown Unkno n 0.37

1040721 1 Ppap2a phosphatide acid phosphatase type 2.A 0.32

10483679 Gp l 5 G protein-coupled receptor 155 0.25

10503709 D130062J21Rik RIKEN cDNA D 130062,121 gene 0.33

TAF7 RNA polymerase Π, TATA box binding protein (TBP)~

10458424 Taf7 associated factor 0.25

10531126 ¾i immunoglobulin joining chain 0.03

10458278 2010001M09Rik RIKEN cDNA 2010G01M09 gene 0.15

10430344 H2rb interleukin 2 receptor, beta chain 0.10

10461979 Aldhlal aldehyde dehydrogenase family L subfamily Al 0.17

10574572 2210023G05Rik RIKEN cDNA 2210023G05 gene 0.26

10501007 Bclp2 chitinase like protein 2 0.13

10403043 Igh immunoglobulin heavy chain complex 0.08

10606058 Cxcr3 cheraokine (C-X-C motif) receptor 3 0.25

10424683 L 6g lymphocyte antigen 6 complex, locus G 0.18

10600235 Abed 1 ATP-binding cassette, sub-family D (ALD), member 1 0.44

10403028 Igh immunoglobulin heavy chain complex 0.10

10487597 nib interleukin 1 beta 0.10 proteasome (prosome. macropain) subunit, beta type 9 (large

10450145 Psmb9 multifunctional peptidase 2) 0.33

10603814 Slc9a7 solute carrier family 9 (sodium 0.24

10566254 Hbb-bl hemoglobin, beta adult, major chain 0.12

10451670 Bzrpil benzodiazapine receptor, peripheral-like 1 0.34

10573198 Dnajbl DnaJ (Hsp40) homolog, subfamily B, member 1 0.43

10524621 Oasl2 2 -5' oligoadenylate synthetase-like 2 0.09

10461605 Ms4a4b membrane-spanning 4-domains, subfamily A, member 4B 0.17 10566258 Hbb-b l hemoglobin, beta adult major chain 0.1 1

10545242 Tgk-V28 immunoglobulin kappa chain variable 28 (V28) 0.32

10404036 Histlh2bg histone cluster 1 , H2bg 0.23

10442932 TmemS transmembrane protein 8 (five membrane-spanning domains) 0.33

10542214 Kh-dl killer ceil lectin-like receptor, subfamily D, member 1 0.1 8

1051 1258 Faml 32a family with sequence similarity 132, member A 0.27

10572800 Klf2 Kmppel-like factor 2 (lung) 0.09

10351905 Spaa 1 spectrin alpha 1 0.24 10538924 LOCI 00046496 similar to ig kappa V-region 24B 0.17

10342726 Unknown Unknown 0.1 1

10362129 Vnn3 vanin 3 0.16

1 0492964 Cd51 CDS antigen-like 0.22

10423971 Pkhdl ll polycystic kidney and hepatic disease 1-like 1 0.37

10372094 Unknown Unknown 0.1 8

10462035 Ldhb lactate dehydrogenase B 0.34

10360173 Slamf7 SLAM family member 7 0.15

10513608 Alad aminolevuiinate, delta-, dehydratase 0.28

10406852 Cnn3 caiponin 3, acidic 0.23

10576807 Cd209d CD209d antigen 0.35

10506031 Nil a nuclear factor Ϊ 0.45

10493382 Pklr pyruvate kinase liver and red blood cell 0.21

10343618 Unknown Unknown 0.27

10500656 Igsf2 immunoglobulin superfamily, member 2 0.1 1

10433885 Ceb d CCAAT 0.27

10403063 Igh immunoglobulin heavy chain complex 0.04

10360370 BC094916 cDNA sequence BC094 16 0.15

10445192 Rhag Rhesus blood group- associated A glycoprotein 0.12

10451860 Potl b protection of telomeres IB 0.47

10545190 Unknown Unkno n 0.05

10403018 IghmAC38.205.12 ig mu chain V region AC38 205.12 0.04

1 50091 1 Mov l O Moloney leukemia virus 10 0.36

UDP-Gal.:betaGlcNAc beta 1,4-gaiactosyltransferase,

10457733 B4galt6 polypeptide 6 0.32

RGD motif, leucine rich repeats, tropomodulin domain and

10574825 Rltpr proline-rich containing 0.37

Gardner-Rasheed feline sarcoma viral (Fgr) oncogene

10508772 Fgr homolog 0.33

10349953 Chitl chitinase 1 (chitoiriosidase) 0.35

10342882 Unknown Unknown 0.36

10545247 igk~V19- 14 immunoglobulin kappa chain variable 19 (VI 9)- 14 0.03

10572456 Jund Jun pro to -oncogene related gene d 0.27

10379482 Cdk5ri cyclin-dependent kinase 5, regulatory subunit 1 (p35) 0.43

10343557 Unknown Unknown 0.18

10427461 Ptger4 prostaglandin E receptor 4 (subtype EP4) 0.36 10544583 Gimap6 GTPase, {MAP family member 6 0, 15

10430818 Tnfrsfl3c tumor necrosis factor receptor superfamily, member 13c 0.39

10571840 Hpgd hydroxyprostaglandin dehydrogenase 15 (NAD) 0.15

10556302 Ampd3 adenosine monophosphate deaminase 3 0.32

10403060 LOC641089 similar to Ig heavy chain V region BCL i precursor 0.11

10549506 DenndSb DEN 0.33

10548307 Klrblc killer cell lectin-like receptor subfamily B member 1C 0.17

10517165 Cd52 CD52 antigen 0.30

10474105 Rag2 recombination activating gene 2 0.2.1

10441565 Rps6ka2 ribosomal protein S6 kinase, polypeptide 2 0.45

10432682 Krt80 keratin 80 0.43

10401068 Spnbl spectrin beta 1 0.45

10570894 Ankl ankyrin 1 , erythroid 0.30

UDP-GicNAc:betaGal beta- 1 ,3-N-

10434291 B3gnt5 acetylglucosaminyltransferase 5 0.24

10338656 Unknown Unknown 0.24

10608638 Unknown Unknown 0.42

10338820 Unknown Unknown 0.30

10404965 Rnfl44b ring finger protein 144B 0.24

10403054 LOC435333 similar to monoclonal antibody heavy chain 0.12

10446763 Lbh limb-bud and heart 0.33

10606989 Tsc22d3 TSC22 domain family, member 3 0.25

10474064 Trp53i 11 transformation related protein 53 inducible protein 1 1 0.42

10548525 Klral O killer cell lectin-like receptor subfamily A, member 10 0.20

10444841 H2-Q 10 histocompatibility 2, Q region locus 10 0.35

10545177 Unknown Unknown 0.02

10583242 Sesn3 sestrin 3 0.41

10369301 Chst3 carbohydrate (chondroitin 6 0.12

10450069 Gm7035 predicted gene 7035 0.26 similar to [Human Ig rearranged gamma chain mRNA, V-J-C

10545217 LOG 100046973 region and complete cds.], gene product 0.22

10559649 Cox6b2 cytochrome c oxidase subunit VIb polypeptide 2 0.41

10442098 Fpr3 formyl peptide receptor 3 0.47

10339193 Unknown Unknown 0.37

10362674 Rnu3a U3 A small nuclear RNA 0.38

10512688 FbxolO F-box protein 10 0.42

10380419 Coll ai collagen, type I, alpha 1 0.22

10573054 Gypa glycophorin A 0.23

10385513 993011112 LRik R! EN cDNA 99301 11J21 gene 0.32

10403038 Igh immunoglobulin heavy chain complex 0.15

10533213 Oas3 2'-5' oligoadenylate synthetase 3 0.13

105381 15 GimapS GTPase, IMAP family member 8 0.38

10341254 Unknown Unknown 0.21 10341217 Unknown Unknown 0.31

10403057 LOC641089 similar to Ig heavy chain V region BCL1 precursor 0.28

10525542 Bcl7a B-celi CLL 0.28

10344227 Unknown Unknown 0.15

10496091 Lef 1 lymphoid enhancer binding factor i 0.1

10466624 Aldhla? aldehyde dehydrogenase family 1, subfamily A7 0.22

10438738 Bcl6 B-cell leukemia 0.38

10528385 Rein reelin 0.40

10561453 Zfp36 zinc finger protein 36 0.1 8

10463123 Dntt deoxynucleotidyltransferase, terminal 0.10

10571252 Tex 15 testis expressed gene 15 0.33

10385526 99301 i U21Rik RiKEN cDNA 99301 11J21 gene 0.30

10381798 Myl4 myosin, light polypeptide 4 0.14

10338494 Unknown Unknown 0.32

10342449 Unknown Unknown 0.13

10363743 Rtkn2 rhotekin 2 0.28

10586491 Dapk2 death-associated protein kinase 2 0.31

10521481 Jakmipl j anus kinase and microtubule interacting protein 1 0.38

10379636 Slfn4 schfafen 4 0.06

10412078 Gapt Grb2-binding adaptor, transmembrane 0.52

10351041 Unknown Unknown 0.26

10498998 D930015E06Rik RIKEN cDNA D93001 E06 gene 0.41

10457787 K1 14 kelch-like 14 (Drosophila) 0.29

10376434 Bu.tr 1 butyrophilin related 1 0.35

10588479 Tlr9 toll-like receptor 9 0.31

10562132 Cd22 CD22 antigen 0.24

10448278 Mmp25 matrix metaliopeptidase 25 0.21

10372503 Lgr5 leucine rich repeat containing G protein coupled receptor 5 0.26

10429515 Lynxi Ly6 0.33

10468533 Gpam glycerol-3-phospb.ate acyltransterase, mitochondrial 0.32

1 436392 Cpox coproporphyrinogen oxidase 0.37 nuclear factor of kappa light polypeptide gene enhancer in B-

10551891 Nfkbid ceils inhibitor, delta ~ 0.34

10382300 Map2k6 mitogen-activated protein kinase kinase 6 0.32

10545210 Gml524 predicted gene 1524 0.04

1048946.3 Slpi secretory leukocyte peptidase inhibitor 0.42

10586907 Mnsi meiosis-specific nuclear structural protein 1 0.42

10445746 Trernl triggering receptor expressed on myeloid cells 1 0.16

10557399 Sbki SH3-hinding kinase 1 0.35

10404059 Histlhl c histone cluster 1 , HI c 0.34

10608650 Unknown Unknown 0.36

10545231 LOCI 00047053 similar to monoclonal antibody kappa light chain 0.07

10473356 Ube216 ubiquitin-conjugating enzyme E2L 6 0.24 10538929 Unknown Unknown 0.40

10403073 ^?ghg immunoglobulin heavy chain (gamma polypeptide) 0.12

10491962 Foxol forkhead box 01 0.22

10416837 Irgl immunoresponsive gene i 0.31

10452815 Xdh xanthine dehydrogenase 0.28

10534854 Mospd3 motile sperm domain containing 3 0.50

10545220 I k immunoglobulin kappa chain complex 0.09

10570018 Tnfsfl3b tumor necrosis factor (ligand) superfamiiy, member 13b 0.18

10598152 LOC625360 similar to 2-cell-stage, variable group, member 3 0.22

10545194 Igkv4-71 immunoglobulin kappa chain variable 4-71 0.06

10593198 Fam55b family with sequence similarity 55, member B 0.29 nuclear factor of kappa light polypeptide gene enhancer in B-

10439936 Nfkbiz cells inhibitor, zeta 0.25

10405189 Unknown Unknown 0.19

10477250 Hck hemopoietic cell kinase 0.32

10339276 Unknown Unknown 0.12

10547894 Cd4 CD4 antigen 0.33

1 388902 Lgals9 lectin, galactose binding, soluble 9 0.47

10541246 I117ra interleukin 17 receptor A 0.45

10572050 March 1 membrane-associated ring finger (C3HC4) 1 0.54

10566580 Gm4759 GTPase. very large interferon inducible 1 pse dogene 0.15

10495659 Cnn3 caiponin 3, acidic 0.29

10480849 Fcna ficolin A 0.50

10566350 A530023O 14Rik RJKEN cDNA A530023O14 gene 0.17 protein tyrosine phosphatase, receptor type, C polypeptide-

10460371 Ptprcap associated protein 0.40

10404132 Cmah cytidine monophospho-N-acety euraminic acid hydroxylase 0.52

10585860 Adpgk ADP-dependent glucokinase 0.43

10451953 Lrgl leucine-ricb al ha-2 -glycoprotein 1 0.28 similar to [Human Ig rearranged gamma chain mRNA, V-J-C

10545235 LOC I 00047070 region and complete cds.], gene product 0.1 1

1 0339291 Unknown Unknown 0.24

10373542 Dgka diacylglycerol kinase, alpha 0.44

10564539 Mctp2 multiple C2 domains, transmembrane 2 0.39

10449741 Siki salt inducible kinase 1 0.22

10571214 Rnfl22 ring finger protein 122 0.45

10384020 Polm polymerase (DNA directed), u 0.36

10342286 Unknown Unknown 0.17

10347481 Cyp27al cytochrome P450, family 27, subfamily a, polypeptide 1 0.32

1054521 igk~V28 immunoglobulin kappa chain variable 28 (V28) 0.02

10340456 Unknown Unknown 0.26 carbohydrate (N-acetylgalactosamine 4-sulfate 6-0)

10568553 Chstl S sulfotransferase 1 0.45

10545212 Gml0881 immunoglobulin kappa chain variable 12-47 0.12 10585194 1118 interleukin 18 0.48

10450694 H2-T22 histocompatibility 2, T region locus 22 0.38

10608686 Unknown Unknown 0.20

10396652 Hspa2 heat shock protein 2 0.38

10604057 Sept6 septin 6 0.44

10355227 1 1 10028C15Rik RIKEN cDNA 11 10028C15 gene 0.53

10560608 Apoc2 apolipoprotein C-l! 0.37

10433578 Snn stannin 0.41

10549647 Ncrl natural cytotoxicity triggering receptor 1 0.24

10605143 Arhgap4 Rho GTPase activating protein 4 0.49

10394971 Klfl l ruppei-iike factor 11 0.56

10548409 Klrcl killer cell lectin-like receptor subfamily C, member 1 0.14

10555510 Pde2a phosphodiesterase 2A, cGMP-stimulated 0.46

10445758 Treml4 triggering receptor expressed on myeloid cells-like 4 0.29

10566358 Trim 30 tripartite motif-containing 30 0.28

10342013 Unknown Unknown 0.34

10548437 K Ira 17 killer cell lectin-like receptor, subfamily A, member 17 O. i i glial eel! !ine derived neurotrophic factor family receptor

10468722 Gfral alpha 1 0.20

10531952 Abcg3 ATP-binding cassette, sub-family G (WHiTE), member 3 0.36

10522208 Uchll ubiquitin carboxy-terminal hydrolase LI 0.32

10384452 Ublcpl ubiquitin-like domain containing CTD phosphatase 1 0.56

10426999 Acvrll activin A receptor, type II-like 1 0.36 apo!ipoprotein B mRNA editing enzyme, catalytic polypeptide

10547621 Apobecl i 0.40

10571984 Ddx60 DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 0.15

10607752 Bmx BMX non-receptor tyrosine kinase 0.21

10516966 BC013712 cDNA sequence BC0137 IP- 0.36

10508074 Csfir colony stimulating factor 3 receptor (granulocyte) 0.28

10476252 Cdc25b cell division cycle 25 homolog B (8. pombe) 0.35

10495243 Gstm glutathione S-transferase, mu 5 0.38

10545484 Dnahc6 dynein. axonemal, heavy chain 6 0.29

1035411 1 Aff3 AF4 0.40

10544638 Tra2 transformer 2 alpha homolog (Drosophila) 0.31

10388310 GarnM GTPase activating RANGAP domain-like 4 0.45

10548345 Klrkl killer ceil lectin-like receptor subfamily K, member 1 0.25

10372082 Nudt4 nudix (nucleoside diphosphate linked moiety X)-type motif 4 0.41

1 464647 TbcidlOc TBCi domain family, member 10c 0.43

10548513 Klra9 killer ceil lectin-like receptor subfamily A, member 9 0.24

10340699 Unknown Unkno n 0.29

10572097 Sn2d4a SH2 domain containing 4A 0.39

10370837 Tcfe2a transcription factor E2a 0.42 pleckstrm homology domain-containing, family A

10577792 Plek a2 (phosphoinositide binding specific) member 2 0.47 10473367 Slc43al solute earner family 43, member 1 0.33

10446965 Rasgrp3 RAS, guanyS releasing protein 3 0.29

10483046 Dpp4 dipeptidyipeptidase 4 0.15

10395039 Cmpk2 eytidine monophosphate (UMP-CMP) kinase 2, mitochondrial 0.1 5 nuclear factor of kappa light polypeptide gene enhancer in B-

10445412 Nfkbie ceils inhibitor, epsilon 0.46

10343834 Unknown Unknown 0.36

10501608 Vcaml vascular ceil adhesion molecule 1 0.28

10385391 Cyfip2 cytoplasmic FMR l interacting protein 2 0.55

10568202 Septl septin 1 0.46

10582626 Abcb l O ATP-binding cassette, sub- family B (MDR 0.50

10451646 A530064D06Rik R Sk i-.X cDNA A530064D06 gene 0.32

10363161 6330442E10Rik RIKEN cDNA 6330442E 10 gene 0.51

10538993 Cd8a CDS antigen, alpha chain 0.33

10519578 Abcb4 ATP-binding cassette, sub-family B (MDR 0.28

10560304 Calm3 calmodulin 3 0.54

10399470 Trib2 nibbles homolog 2 (Drosophiia) 0.38 solute carrier family 28 (sodium-coupled mi icleoside

10475502 Slc28a2 transporter), member 2 0.27

10434932 Fani43a family with sequence similarity 43, member A 0.38

10595840 Acpl2 acid phosphatase-like 2 0.25

10416887 Slainl SLAIN motif family, member 1 0.44

10342256 Unknown Unknown 0.21

10566366 AI451617 expressed sequence AI451617 0.24

10457888 5730494M16Rik RiKEN cDNA 5730494M16 gene 0.46

10548817 Plbdl pbosphoHpase B domain containing 1 0.34

10492426 Acsf2 acyl-CoA synthetase family member 2 0.56

10482929 Ly75 lymphocyte antigen 75 0.39

10589329 Pfkfb4 6-phosphofmcto-2-kinase 0.43

10568586 Fam53b family with sequence similarity 53, member B 0.37

10565567 4632427E13Rik RIKEN cDNA 4632427E13 gene 0.49

10564448 Asb7 ankyrin repeat and SOCS box-containing 7 0.53

10399696 Rnfl44a ring finger protein 144A 0.34

10496580 Gbp3 guanylate binding protein 3 0.21

10581605 Hp haptoglobin 0.25

10444821 H2-Q8 histocompatibility 2, Q region locus 8 0.40

10555460 Staid 10 START domain containing 10 0.41

10356999 Prdx2 peroxiredoxin 2 0.51

10522024 Tbcldl TBC l domain family, member 1 0.56

10351 197 Sell selectin, lymphocyte 0.46

10592888 CxcrS chemochine (C-X-C motif) receptor 5 0.19

10368144 Tnfaip3 tumor necrosis factor, alpha-induced protein 3 0.19

TRAF-interacting protein with forkhead-associated domain.

10409567 Tifab family member B 0.33 10341815 Unknown Unknown 0.48

10366645 1700006 J 14Rik RI EN cDNA 1700006114 gene 0.30

10429968 Unknown Unknown 0.54

10593887 Neili nei endonuclease VIH-like 1 (E. coSi) 0.30

10530612 Fry! furry homolog-iike (Drosophila) 0.51

10434778 Rtp4 receptor transporter protein 4 0.05

10517587 Alp! alkaline phosphatase, liver 0.51

10449280 Gm5226 predicted gene 5226 0.42

10515399 PIk3 polo-like kinase 3 (Drosophila) 0.26

10342267 Unknown Unknown 0.12

10403046 AI324046 expressed sequence AI324046 0.21

10406928 Cdl 80 CD 180 antigen 0.32

10608680 Unknown Unkno n 0.38

10606609 Tspan6 tetraspanin 6 0.41

10430400 Cardl O caspase recruitment domain family, member 1 0.45

10592816 Hmbs hydroxymethylbilane synthase 0.35

10409502 Dok3 docking protein 3 0.41

10361246 G0s2 GO 0.45

10584821 Cd3d CD3 antigen, delta polypeptide 0.26

10523012 Dck deoxyeyiidine kinase 0.50

10594404 Smad3 MAD homolog 3 (Drosophila) 0.51

10573457 K!fl Kruppei-like factor 1 (erytnroid) 0.31

10341954 Unknown Unknown 0.37

10341488 Unknown Unknown 0.19

10583834 9530077C05Rik RIKEN cDNA 9530077C05 gene 0.52

10507347 Tesk2 tesiis-specific kinase 2 0.32

10346191 Stall signal transducer and activator of transcription 1 0.37

10580752 9330175E14 ik RIKEN cDNA 9330175E14 gene 0.45

10512757 Hemgn hemogen 0.45

10341776 Unknown Unknown 0.49

10538979 Cd8bl CDS antigen, beta chain 1 0.31

10390519 Plxdcl plexin domain containing 1 0.49

10583465 Ubl5 ubiquitin-tike 5 0.50

10406031 Lpcatl lysophosphatidylcholine acyltransferase 1 0.39

10505438 Orml orosomucoid 1 0.09

10465059 Ctsw cathepsin W 0.24

10361338 ipcefi interaction protein for cvtohesin exchange factors 1 0.36

10584142 Etsl E26 avian leukemia oncogene 1, 5' domain 0.38

10607877 Prps2 phosphoribosyl pyrophosphate synthetase 2 0.45

10519607 4930420Ki7Rik RIKEN cDNA 4930420K17 gene 0.27

10360105 Usp21 ubiquitin specific peptidase 21 0.59

10445774 B430306N03Rik RIKEN cDNA B430306N03 gene 0.49

10545239 Unknown Unknown 0.1 1 10569020 Ifitm6 interferon induced transmembrane protein 6 0.49

10366407 Gml 0752 predicted gene 10752 0.33

10538135 Gimap? GTPase, IMAP family member 7 0.35

10438064 Vprebl pre-B lymphocyte gene i 0.36

10398996 Cnp2 cysteine rich protein 2 0.41

10434758 8t6gail beta galactoside alpha 2,6 sialyltransferase 1 0.34

10519497 Steap4 STEAP family member 4 0.36

10379736 1 1000GlG2GRik RIKEM cDNA 11000Q1G20 gene 0.32

10545202 Gm l077 predicted gene 1077 0.26

10470314 Unknown Unknown 0.38

10338273 Unknown Unknown 0.51

LSM6 homolog, U6 small nuclear RNA associated (S.

10577598 Lsni6 cerevisiae) 0.47

10498531 Ccnll cyclin LI 0.41

10545930 Unknown Unknown 0.33

10366446 TspanS tetraspanin 8 0.24

10579958 1115 interleukin 15 0.27

10404874 Mylip myosin regulatory light chain interacting protein 0.57

10547633 Gdi3 growth differentiation factor 3 0.50

10442762 Prss34 protease, serine, 34 0.54

10341163 Unknown Unkno n 0.54

10567171 Rpsl3 ribosomal protein S13 0.30

10489038 Scandl SCAN domain-containing 1 0.52

10593024 Cd3e CD3 antigen, epsilon polypeptide 0.47

10339789 Unknown Unknown 0.18

10346224 Tmeml 94b transmembrane protein 194B 0.50

LSM6 homolog, U6 small nuclear RNA associated (S.

10579833 Lsm.6 cerevisiae) 0.47

10596190 Bfsp2 beaded filament structural protein 2, phakinin 0.32

10358978 ier5 immediate early response 5 0.25

10512574 Gba2 glucosidase beta 2 0.43

10592772 Abcg4 ATP-binding cassette, sub-family G (WHITE), member 4 0.45

10469571 Otudl OTU domain containing 1 0.25

10382538 Armc7 armadillo repeat containing 7 0.46

10522467 Rasli lb RAS-like, family 11, member B 0.45

10360391 lfi203 interferon activated gene 203 0.10

10509168 E2f2 E2F transcription factor 2 0.41

10594053 Pml promyelocytic leukemia 0.42

10347948 SplOO nuclear antigen S lOO 0.43

10364056 Ggtl gamma-glutamyhransferase 1 0.58

10450496 Lstl leukocyte specific transcript 1 0.42

10493831 S 100a8 Si 00 calcium binding protein A8 (calgranulin A) 0.55

10454782 Egrl early growth response 1 0.20 10376060 Ir l interferon regulatory factor 1 0.53

10391207 Dhx58 DEXH (Asp-Glu-X-His) box polypeptide 58 0.31

10535458 Zdhhc4 zinc finger, DHHC domain containing 4 0.61

10422493 GprlS G protein-coupled receptor 18 0.33

10536390 Glccil glucocorticoid induced transcript 1 0.50

10408450 Sox4 S Y-bo containing gene 4 0.27

10424676 Ly6e lymphocyte antigen 6 complex, locus E 0.47

10543134 Ndufa4 NADH dehydrogenase (ubiquinone) 1 alpha subcompiex, 4 0.46

10568638 Uros uroporphyrinogen 111 synthase 0.53

10492971 Fcrll Fe receptor-like 1 0.36

10495012 Phtfl putative homeodomain transcription factor 1 0.53

10604100 Ndufal NADH dehydrogenase (ubiquinone) 1 alpha subcompiex, 1 0.51

10359861 Mgst3 microsomal glutathione S-transferase 3 0.44

10606910 Mcart6 mitochondrial carrier triple repeat 6 0.45

10364072 Ggt5 gamma-giutamyitransferase 5 0.39

10533198 Oas2 2 -5' oligoadenylate synthetase 2 0.16

10528913 Gm5129 predicted gene 5129 0.58

10415319 Irf9 interferon regulatory factor 9 0.31

10427454 Card6 caspase recruitment domain family, member 6 0.29

10579812 Ednra endothelin receptor type A 0.49

10458130 4933408B17Rik RIKEN cDNA 4933408B 17 gene 0.55

104191 1 Earl eosinophil-associated, rihonueiease A family, member 1 0.06

10541670 Clrl complement component i, r subcomponent-like 0.47

10488482 Acs si acyl-CoA synthetase short-chain family member 1 0.39

10339827 Unknown Unknown 0.20

10374068 Dbnl drebrin-like 0.57

LSM6 homolog, U6 small nuclear RNA associated (S.

10388745 Lsm6 cerev siae) 0.45

10502081 Enpep glutamyl aminopeptidase 0.41

10596166 1300017J02Rik RIKEN cDNA 1300017J02 gene 0.49

10455813 Lmrsbl lamin Bl 0.39

10585068 Fam d family with sequence similarity 55, member D 0.43

10589703 Ltf lactotransferrm 0.61

10430649 Cbx7 chromobox homolog 7 0.55

10403009 Ighg immunoglobulin heavy chain (gamma polypeptide) 0.17

10466040 Cd5 CD5 antigen 0.54

10549546 Ndufa3 NADH dehydrogenase (ubiquinone) 1 alpha subcompiex, 3 0.54

10499160 Cdldl CDldi antigen 0.41 fymetrix Fold

Gene Syijjbol Geae Name Change nes with Higher Expression in Lin- ECX BMCs 10563597 Saa3 serum amyloid A 3 29.43

10585794 Cy llal cytochrome P450, family 11, subfamily a, polypeptide 1 18.15

10488608 TribS tribbles homoiog 3 (Drosopiiila) 33.07

10396476 Rhoj ras homoiog gene family, member J 6.33 methylenetetrahydro olate dehydrogenase (NAD+ dependent),

10545672 Mthfd2 methenyltetrahydrofolaie cyclohydrolase 5.19

10563570 Tphl tryptophan hydroxylase 1 32.98

10407435 Akrlcl8 aldo-keto reductase family 1, member CI 8 110.40

10357676 Pctk.3 PCTAIRE-m tif protein kinase 3 4.64

10603746 Maob monoamine oxidase B 10.01

10588577 Cish cytokine inducible SH2 -containing protein 8.36

10503023 Cth cystathionase (cystathionine gamma- lyase) 19.25

10442786 Tpsb2 tryptase beta 2 21.58

10420308 Gzmb granzyme B 50.19

10583056 Mmpl2 matrix meta!lopeptidase 12 i53.il

10564041 Unknown Unknown 11.34

10368343 Argl arginase, liver 110.71

10563937 Snordll5 Small nucleolar RNA, C 11.35

10563947 Snordl 15 Small nucleolar RNA, C 11.35

10563951 Snordl 15 Small nucleolar RNA., C 11.35

10563953 Snordl 15 Small nucleolar RNA, C 11.35

10563957 Snordl 15 Small nucleolar RNA, C 11.35

10563995 Snordl 15 Small nucleolar RNA, C 11.35

10563997 Snordl 15 Small nucleolar RNA, C 11.35

10563999 Snordl 15 Small nucleolar RNA., C 11.35

105640 1 Snordl 15 Small nucleolar RNA, C 11.35

10564003 Snordl 15 Small nucleolar RNA, C 11.35

10564007 Snordl 15 Small nucleolar RNA, C 11.35

10564029 Snordl 15 Small nucleolar RNA. C 11.35

10564031 Snordl 15 Small nucleolar RNA, C 11.35

10564035 Unknown Unknown i 1.35

10564039 Unknown Unkno n 11.35

105640 1 Unknown Unknown 11.35

10564059 Unknown Unknown 11.35

1 564061 Unknown Unknown 11.35

10564063 Unknown Unknown 11.35

10564065 Gm3079 predicted gene 3079 11.35

10564067 Unknown Unknown 11.35

10564071 Unknown Unknown 11.35

10564075 Unknown Unknown 11.35

10564081 Unknown Unknown 11.35

10564083 Unknown Unknown 1135

10564131 Unknown Unknown 11.35 10564133 Unknown Unknown 1 1.35

10564141 Unknown Unknown 1 1 3

10564145 Unknown Unknown 1 1.35

10564149 Unknown Unknown 1 1.35

1056415 1 Unknown Unknown 1 1.35

1 0564153 Unknown Unknown 1 1 .35

10564155 Unknown Unknown 1 1 .35

10379153 Aidoc aldolase C, fructose-bisphosphate 8.32

10497079 Ptger3 prostaglandin E receptor 3 (subtype EP3) 12.91

104241 19 Nov nephroblastoma overexpressed gene 1 27 29

1056401 1 Snordl 15 Small nucleolar RNA, C 1 1.93

1 0574023 Mt2 metailothionein 2 6.60

10462281 Vldlr very low density lipoprotein receptor 17.71

10563955 Gm3079 predicted gene 3079 8.83

10534493 Ccl24 chemokine (C-C motif) ligand 24 47.96

10564019 Unknown Unknown 8.1 1

10564165 Snord l 16 small nucleolar RNA. C 12.45

10479379 Sico4al solute carrier organic anion transporter family, member 4a 1 5.07

10570634 4930467E23Rik R1KE3S! cDNA 4930467E23 gene 4.68

10345791 Il lrll interleukin 1 receptor-like 1 8.74

10474302 Unknown Unknown 24.81

10563913 Unknown Unknown 9.39

10563917 Unknown Unknown 9.39

10563923 Gm3079 predicted gene 3079 9.39

10530722 BC063212 cDNA sequence BC06121 2 3.49

104193 36 Gm 10375 predicted gene 10375 6.33

10563935 Unknown Unknown 7.89

1 0564045 Unknown Unknown 7.89

10564047 Unknown Unkno n 7.89

10577361 Unknown Unknown 4.00

10564023 Unknown Unknown 8.32

10564013 Snordl 15 Small nucleolar RNA, C 10.15

10564017 Snordl 15 Small nucleolar RNA, C 10.15

105641 1 1 Unknown Unknown 4.81

10428353 Lip 12 low density lipoprotein-related protein 12 16.00

10567995 Nupri nuclear protein 1 5.63

10400072 Scin scinderin 13.47

1 0605181 Renbp renin binding protein 6.92

1 0564169 Snordl 16 small nucleolar RNA, C 10.46

10494548 Gja5 gap junction membrane channel protein alpha 5 9.99

1041 1229 F2r coagulation factor Π (thrombin) receptor 12.68 solute carrier family 7 (cationic amino acid transporter, y+

10605986 Slc7a3 system), member 3 15.38 10564143 Unknown Unknown 8.49

10564147 Unknow Unknown 8.49 solute carrier family 6 ( eurotransmitter transporter, glycine),

10507500 Slc6a9 member 9 6.52 solute carrier family 7 (cationic amino acid transporter, y+

10419854 Slc7a8 system), member 8 6.28

10403303 Akrl cl3 aldo-keto reductase family 1, member CI 3 9.91

10564137 Unknown Unknown 6.04

10459620 Rab27b RAB27b, member RAS oncogene family 13.91

10564177 Snordl l6 small nucleolar RNA, C 1 1.87

104191 19 Gm8020 predicted gene 8020 9.22

10538791 Tnip3 TNFA1P3 interacting protein 3 9.98

10341988 Unknown Unknown 3.26

10513141 Ptpn3 protein tyrosine phosphatase, non-receptor type 3 3.55

10564033 Unknown Unknown 9.68

10372877 Xpot exportin, iRNA (nuclear export receptor for tRNAs) 3.49

10435920 Cd200r4 CD200 receptor 4 28.62

10564163 Snordl 16 small nucleolar RNA, C 13.77

10564167 Snordl 16 small nucleolar RNA, C 13.77

10564171 Snordi 16 small nucleolar R A, C 13.77

10564173 Snordl 16 small nucleolar RN A, C 13.77

10564175 Snordi 16 small nucleolar RNA, C 13.77

10564179 Snordl 16 small nucleolar RNA, C 13.77

10564181 Snordl 16 small nucleolar RNA, C 13.77

10564185 Snordl 16 small nucleolar RNA, C 13.77

105641 87 Snordl 16 small nucleolar RN A, C 13.77

10564189 Snordl 16 small nucleolar RNA, C 13.77

10564191 Snordl 16 small nucleolar RNA, C 13.77

10564193 Snordl 16 small nucleolar RNA, C 13.77

10564195 Snordl 16 small nucleolar RNA, C 13.77

10564197 Snordl 16 small nucleolar RNA, C 13.77

10564199 Snordl 16 small nucleolar RNA, C 13.77

10564201 Snordi 16 small nucleolar RNA, C 13.77

10564205 Snordl 16 small nucleolar RNA, C 13.77

10564207 Snordl 16 small nucleolar RNA, C 13.77

10531 18 BC061212 cDNA sequence BC061212 3.47

10564135 Unknown Unknown 8.59

10523134 Pf4 platelet factor 4 4.45

10425207 HlfD HI histone family, member 0 5.39

10602221 Unknown Unkno n 4.40

10579054 4930467E23Rik Ri EN cDNA 4930467E23 gene 4.04

10406736 F2rl2 coagulation factor II (thrombin) recepior-like 2 6.85

10501358 Sars seryl-aminoacyl-iRNA synthetase 3.19 10567825 Lat linker for activation of T cells 4.26

10484488 P2rx3 purinergic receptor P2X, ligand-gated ion channel, 3 6.86

10535989 A430089I19Rik RIKEN cDNA A430089119 gene 3.54

10536085 A430089I1 Rik RIKEN cDNA A430089I19 gene 3.54

10536336 BC061212 cDNA sequence BC061212 3.54

10564161 Snordl 6 small nucleolar RNA, C 13.61

10340645 Unknown Unknown 4.73

procollagen-proline, 2-oxogluiarate 4-dioxygenase (proline 4-

10363350 P4hal hydroxylase), alpha 1 polypeptide 3.77

10563993 Unknown Unknown 7.56

10564021 Unknown Unknown 7.56

10564025 Unknown Unknown 7.56

10378240 P2rxl purinergic receptor P2X, ligand-gated ion channel, 1 2.53

10564183 Snordl 16 small nucleolar RNA, C 11.77

10419198 Eroil EROl-like (S. cerevisiae) 4.19

10599086 Unknown Unknown 4.97

10342565 Unknown Unknown 4. 1

10538187 Gpnmb glycoprotein (transmembra ) nmb 59.83

1 513472 Mup2 major urinary protein 2 3.07

10371332 Aldh l2 aldehyde dehydrogenase 1 family, member L2 7.86

10459772 Lipg lipase, endothelial 13.97

10582884 Unknown Unknown 14.81

10501046 Gm 10673 predicted gene 10673 8.41

10345777 Illrl2 interleukin 1 receptor-like 2 4.14

10407445 Akrlcl2 aldo-keto reductase family 1, member C I 2 13.93

10419147 Gml0375 predicted gene 10375 4.06

10559871 Vmn2r51 vomeronasal 2, receptor 51 2.84

10 19128 Gm 10375 predicted gene 10375 4.01

10419132 Gml0375 predicted gene 10375 4.01

10564159 Snordl 16 small nucleolar RNA, C 13.25

10598183 Gmclll germ cell-less homolog 1 (Drosophiia)-like 4.78

10416057 Clu clusterin 5.49

10598229 Unknown Unknown 4.04 solute carrier family 7 (cationic amino acid transporter, y+

10582275 Slc7a5 system), member 5 6.25

10386756 Unknown Unknown 3.81

10414269 Bni 3 BCL2 4.77

10497451 Cpa3 carboxypeptidase A3, mast cell 6.12

10351623 Fllr Fl 1 receptor 5.68

10599058 Unknown Unkno n 3.19

10599060 Unknown Unknown 3.19

10599062 RP23- HOD 11.1 Xlr-related, meiosis regulated Xmr 3.19

10599073 RP23-110D1U Xlr-related, meiosis regulated Xmr 3.19 10599094 RP23-110D1L1 Xlr-related, meiosis regulated Xmr 3.19

10599105 RP23-110D11.1 Xlr-related, meiosis regulated Xmr 3.19

10603962 RP23-110D11.1 Xlr-related, meiosis regulated Xmr 3.19

10603973 RP23-1 lODl 1.1 Xlr-related, meiosis regulated Xmr 3.19

10603984 RP23-110D11.1 Xlr-related, meiosis regulated Xmr 3.19

10603995 RP23-110D11.1 Xlr-related, meiosis regulated Xmr 3.19

10604006 RP23-110D11.1 Xlr-related, meiosis regulated Xmr 3.19

10604017 RP23-110D11.1 Xlr-related, meiosis regulated Xmr 3.19

10604021 RP23-110D11.1 Xlr-related, meiosis regulated Xmr 3.19

10519857 Hgf hepaioeyte growth factor 6.24

10587829 Plod2 procollagen lysine, 2-oxoglutarate 5-dioxygenase 2 4.85

10577388 4930467E23Rik RIKEN cDNA 4930467E23 gene 3.56

10523128 Ppbp pro-platelet basic protein 7.47

10526493 MyllO myosin, light chain 10, regulatory 3.87

10564089 Unknown Unknown 9.27

1 599092 Unknown Unknown 4.90

10598064 Unknown Unknown 7.66

10536126 BC061212 cDNA sequence BC061212 3.11

10356470 Glrp 1 glut amine repeat protein 1 5.72

10543067 Asns asparagine synthetase 4.25

10582882 Unknown Unknown 18.62

10435288 Mucl3 mucin 13, epithelial transmembrane 4.54

10570280 F7 coagulation factor Vli 9.56

10355567 Tmbiml transmembrane BAX inhibitor motif containing 1 3.17

10562416 Cebpg CCAAT 3.52

10412520 Unknown Unknown 3.01

10603232 Gmclll germ cell-less homolog 1 (Drosophila)-iike 4.78

10603237 Gmclll germ cell-less homolog 1 (Drosophila)-like 4.78

10582918 Unknown Unkno n 7.94

10376832 Adora2b adenosine A2b receptor 3.99

10445442 Gtpbp2 GTP binding protein 2 2.64

10417245 Unknown Unknown 2.63

10569485 T frsf26 tumor necrosis factor receptor superfami!y, member 26 3.11

10564069 Unknown Unknown 6.23

10564073 Unknown Unknown 6.23

10347925 Gm7609 predicted gene 7609 4.05

10467470 Aldhl8al aldehyde dehydrogenase 18 family, member Al 3.47

10582890 Unknown Unknown 19.12

10550193 Gm3994 predicted gene 3994 5.08

10554800 Rab38 RAB38, member of RAS oncogene family 4.67 solute carrier family 3 (activators of dibasic and neutral amino

10465772 Slc3a2 acid transport), member 2 3.34

10603986 RP23-110D11.1 Xlr-related, meiosis regulated Xmr 5.05 1 0604023 RP23- 1 10D 1 1 .1 Xir-rdated, meiosis regulated Xrnr 5.05

10394674 Soes2 suppressor of cytokine signaling 2 4.27

10464370 Slc l 8a2 solute carrier family 18 (vesicular monoamine), member 2 1 1.35

10356274 Csprs component of S i OO-rs 3.92

10564027 Unknown Unknown 8.70

10564077 Unknown Unknown 8.70

10564079 Unknow Unknown 8.70

10564085 Unknown Unknown 8.70

10564087 Unknown Unknown 8.70

10564091 Unknown Unknown 8.70

10564093 Unknown Unknown 8.70

10564095 Unknow Unknown 8.70

10564097 Unknown Unknown 8.70

10564099 Unknown Unknown 8.70

10564101 Unknown Unknown 8.70

10564103 Unknown Unknown 8.70

1 0564105 Unknown Unknown 8.70

10564107 Unknown Unk own 8.70

105641 13 Unknown Unkno n 8.70

105641 15 Unknown Unknown 8.70

105641 19 Unknown Unknown 8.70

10564121 Unknown Unknown 8.70

10564123 Unknow Unknown 8.70

10564125 Unknown Unkno n 8.70

10564127 Unknown Unknown 8.70

10564129 Unknown Unknown 8.70

10564139 Unknown Unknown 8.70

10367475 Unknown Unknown 3.60

10535994 A430089I19Rik RIKEN cDNA A430089119 gene 2.88

10345025 fars isoieucine-tRNA synthetase 3.08

105981 87 Unknown Unknown 4.03

10471844 Nek6 NIMA (never in mitosis gene aV-related expressed kinase 6 4.68

10459405 Nars asparaginyl-tRNA synthetase 2.58

10367772 S md5 sterile alpha motif domain containing 5 6.38

10559883 Vmn2r42 vomeronasal 2, receptor 42 2.57

10347915 Gm7609 predicted gene 7609 4.1 9

10550208 Gm3994 predicted gene 3994 5.23

1 0513156 Ptpn3 protein tyrosine phosphatase, non-receptor type 3 2.08

10508420 Yars tyrosyl-tRN synthetase 3.14

10365482 Tim 3 tissue inhibitor of metailoproteinase 3 10.15

10604542 Hs6sl2 heparan sulfate 6-O-s lfotransferase 2 4.80

10563965 Unknown Unknown Ί 'Ί 'Τ,

10563967 Unknown Unknown 7 2 > 10563971 Unknown Unknown 7.23

10564043 Unknown Unknown 7.23

10412503 Unknown Unknown 2.93

1059821 8 GmcS i l genu cell-less hotnolog 1 (Drosophila)-like 4.69

10343468 Unknown Unknown 3.34

10439292 BC 100530 cDNA sequence BC 100530 7.50

10343589 Unknown Unknown 2.86

10424349 Sqle squalene epoxidase 2.62

10391697 Itga2h integrin alpha 2b 6.24

10531544 Paqr3 progestin and adipoQ receptor family member ΠΙ 3.78

10549222 Bcatl branched chain aminotransferase 1 , cytosolic 4.38

10485582 Tcpl l ll t-complex 1 1 like 1 3.54

10542172 Cleclb C-type lectin domain family 1 , member b 3.83

10417371 ENSMUSG00000068790 predicted gene, ENSMUSG00000068790 3.94

10485674 2700007P21 Rik RIKEN cDNA 2700007P21 gene 3.43

10598220 Unknown Unknown 4.37

10599096 RP23- 1 10D l i , l Xlr-related, meiosis regulated Xmr 4.89

10603975 RP23- 1 10D 1 1.1 Xlr-related. meiosis regulated Xmr 4.89

10583044 Mmp 13 matrix metaiiopeptidase 13 47.67

10384539 Slc l a4 solute carrier family 1 (glutamate 5.79

10381809 Itgb3 integrin beta 3 5.38

10547100 Plxndl plexin Dl 2.56

10582879 Csprs com onent o Sp 1 00 -rs 3.96

10384961 stanniocalcin 2 8.12

10599075 RP23- 1 10D 1 1 .1 Xlr-related, meiosis regulated Xmr 5.17

10599107 RP23- 1 10D 1 U Xlr-related, meiosis regulated Xmr 5.17

10603964 RP23- 1 10D 1 L 1 Xlr-related, meiosis regulated Xmr 5.17

10603997 RP23- 1 10D 1 1.1 Xlr-related, meiosis regulated Xmr 5.17

10604008 RP23- 1 10D 1 1.1 Xlr-related. meiosis regulated Xmr 5 17

10592515 Ubash3b ubiquitsn associated and SH.3 domain containing, B 2.29

10407481 Pfkp phosphofructokinase, p] atelet 2.67

10341395 Unknown Unknown 3.53

10458607 Lais leucy!-tRNA- synthetase 2.83

10571036 Ppapdclb phosphatidic acid phosphatase type 2 domain containing IB 3.30

10409660 Gkap 1 G kinase anchoring protein 1 2.31

10601993 D330045A20Rik RIKEN cDNA D330045A20 gene 6.84

10495107 Adora3 adenosine A3 receptor 3.49

10562905 Atf5 activating transcription factor 5 3.92

10419140 Gml 0375 predicted gene 10375 3.12

10530726 BC061212 cDNA sequence BC061212 3.47

10531507 BC061212 cDNA sequence BC061212 3.47

10536103 LOC624931 similar to D5Ertd577e protein 3.00

10600480 4930408F14Rik RIKEN cDNA 4930408F 14 gene 7.09 10603230 4930408F14Rik R1.KEN cDNA 4930408F 14 gene 7.09

10605353 4930408F14Rik RI EN cDNA 4930408F14 gene 7.09

10414612 Slc39a2 solute carrier family 39 (zinc transporter), member 2 8.96

10412549 D830030K20Rik RIKEN cDNA D830030K20 gene 2.37

10341 108 Unknown Unknown 3.49

10559901 Vmn2r42 vomeronasal 2, receptor 42 2.52

10523717 Sp l secreted phosphoprotein 1 22.31

10342553 Unknown Unknown 3.52

103922.21 Pecaml platelet 2.70

10484227 Sestdl SEC 14 and spectrin domains 1 3.33

10426689 Spats2 spermatogenesis associated, serine-rich 2 3.19

10487906 Slc23a2 solute carrier family 23 (nucleobase transporters), member 2 3.23 solute canier family 1 (neutral amino acid transporter),

10550332 Slcl S member 5 3.32

10523337 Gm7682 predicted gene 7682 3.46

10536025 Gm7682 predicted gene 7682 3.46

10586433 R.bpms2 RNA. binding protein with multiple splicing 2 3.20

1 419125 Gm5929 predicted gene 5929 3.75

10375443 Havcr2 hepatitis A virus cellular receptor 2 5.37

10360764 Eiiah enabled homolog (Drosophila) 6.75

10545958 Anxa4 annexin A4 8.66

10417264 544988 predicted gene, 544988 3.73

10400304 Eghi3 EGL nine homolog 3 (C. elegans) 3.83

10578681 Unknown Unknown 2.43

10603109 Piga phosphatidyiinositoi giycan anchor biosynthesis, class A 2.66 solute carrier family 7 (cationic amino acid transporter, y+

10498024 Slc7al i system), member 1 1 7.45

10536052 Gm7682 predicted gene 7682 3.47

10536143 LOC624931 similar to D5Ertd577e protein 3.47

10549938 Vmn2r34 vomeronasal 2, receptor 34 2.55 solute carrier family 7 (cationic amino acid transporter, y+

10535852 Slc7al system), member 1 4.15

10564818 Anpep alanyl (membrane) aminopeptidase 20.60

105951 89 Slc l 7a5 solute carrier family 17 (anion 7 75

10538420 Gars glycyl-tRNA synthetase 2.96

10379535 Ccl8 chemokine (C-C motif) ligand 8 17.81

10368577 Rnf217 ring finger protein 217 5.85

10405094 iars isoleucine-tRNA synthetase 2.88

103381 16 Unknown Unknown 3.43

10352 14 Eprs glutamyl-prolyl-tRN synthetase 3.35

10539818 Gp9 glycoprotein 9 (platelet) 2.48

10357008 Pign phosphatidyiinositoi giycan anchor biosynthesis, class N 2.58

10598075 Unknown Unknown 8.49

10339205 Unknown Unknown 6.94 10513455 Mup2 major urinary protein 2 3.53

1 033841 1 Unknown Unknown 3.04

10417461 D830030 20Rik RIKEN cDNA D830030K20 gene 2.88

10539873 Gata2 GATA binding protein 2 4.20

10458226 Hspa9 heat shock protein 9

1 0448004 Phfl O PHD finger protein 10 2.54

10456046 Pdgfrb platelet derived growth factor receptor, beta polypeptide 4.23

10598227 4930408F14Rik RIKEN cDNA 4930408F14 gene 3.18

1051 3504 Mup2 major urinary protein 2 3.58

10579060 4930467E23Rik RIKEN cDNA 4930467E23 gene 4.28

10422728 Dab2 disabled homolog 2 (Drosophiia) 24.43

1 0575550 Aars a!anyl-tRNA synthetase 2.81

10559908 Vmn2r43 vomeronasal 2, receptor 43 2.82

105501 89 Gm 10679 predicted gene 1.0679 5.14

10469457 Pixdc2 plexin domain containing 2 13.77

1055993 1 Vmn2r43 vomeronasal 2, receptor 43 2.61

1 04109 1 Vc-an versican 17.07

10427131 Soat2 sterol O-acyltransferase 2 5.65

10417373 ENSMUSG00000079376 predicted gene, ENSMUSG00000079376 3.00

10339680 Unknown Unknown 2?

10549921 Vmn2r43 vomeronasal 2, receptor 43 3.00

1 0417239 Unknown Unknown 2.97

1 0513420 Mup7 major urinary protein 7 3.67

10346074 Wdr75 WD repeat domain 75 2.23

10522558 Gm7682 predicted gene 7682 3.59

10536107 LOC625240 similar to D5Ertd577e protein 2.87

10466410 Psatl phosphoserine aminotransferase 1 2.66

1 0603242 4930408F14Rik RIKEN cDNA 4930408F14 gene 4.70

10595480 Mel malic enzyme 1 , NADP(+)-dependerit, cytosoiic 3.59

10603228 Unknown Unknown 5.14 colony stimulating factor 2 receptor, beta 2, low -affinity

10430302 Csf2rb2 (granulocyte-macrophage) 6.45

10423570 Unknown Unknown 5.1 1

10417359 Hn.l l hematological and neurological expressed 1 -like 3.53

10448916 Tpsabl tryptase alpha 4.15

10367400 Mm l 9 matrix metaliopeptidase 19 5.96

10419122 Gm8020 predicted gene 8020 9.13

10599084 4930408F14Rik RIKEM cDNA 4930408F14 gene 3.06

10412537 B930046C 15Rik RIKEN cDNA B930046C15 gene 3.38

10358434 Pla2g4a phospholipase A2, group IVA (cytosoiic, calcium-dependent) 4.63

1 0540897 Pparg peroxisome proliferator activated receptor gamma 4.79

1 0530536 Tec tec prot ein tyrosine kinase 2.51

10542965 Sgce sarcoglycan, epsilon 3.73 10453436 Mcfd2 multiple coagulation factor deficiency 2 3,05

10358879 Npl N-acetymeuraminate pyruvate lyase 7.45

10462507 Papss2 3'-phosphoadenosine 5'-phosphosulfate synthase 2 2.22

10351206 Selp selectin, platelet 5.26

10446928 Ltbp l latent transforming growth factor beta binding protein 1 4.12 solute carrier family 7 (cationic amino acid transporter, y+

10571444 Slc7a2 system), member 2 24.93

10410235 LOC 100044384 hypothetical protein LOC I 00044384 7.13

10439634 GtpbpS GTP-binding protein 8 (putative) 3.23

1 0489204 Tgm2 transglutaminase 2, C polypeptide 20.16

10505240 Unknown Unknown 3.21

10427744 Rail 4 retinoic acid induced 14 3.96

10496748 Syde2 synapse defective 1 , Rho GTPase, homolog 2 (C. elegans) 3.76

10364194 Lss lanosterol synthase 2.17

1 0547657 C3arl complement component 3a receptor 1 16.73

10445789 Tremll triggering receptor expressed on myeloid cells-like 1 3.17

10599064 RP23-1 10D 1 1 .1 Xlr-related, meiosis regulated Xmr 4.03

10512774 Coro2a coronin, actin binding protein 2 A 2.49

10513437 Mup2 major urinary protein 2 3.44

10417286 Unknown Unknown 3.28

10401900 Selll sel-1 suppressor of lin-12-like (C. elegans) 3.01

10587690 Bcl2a lb B -cell leukemia 7.25

10598062 Unknown Unknown 8.84

10599673 4930527E24Rik RIKEN cDNA 4930527E24 gene 3.07

10542221 Unknown Unknown 5.14

10525887 BriSbp Bri3 binding protein 1 .94

10582888 Unknown Unknown 26.55

10412562 Finh illamin. he:n 3.00

10570434 Ifitrn l interferon induced transmembrane protein 1 3.37

10513512 Mup l major urinary protein 1 2.34

10340809 Unknown Unknown 3.19

10516932 Sesn2 sestrin 2 3.64

104191 1 1 Gm 10375 predicted gene 10375 3.29

1051 1429 Car8 carbonic anhydrase 8 3.59

10417773 Gm5458 predicted gene 5458 3.03

10339726 Unknown Unknown 3.05

10344210 Unknown Unknown 8.51

10596072 Ppp2r3a protein phosphatase 2, regulatory subunit B", alpha 3.76

10549923 Vmn2r51 vomeronasal 2, receptor 51 2.77

UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-

10432661 Galnt6 acetylgalactosaminyltransferase 6 5.26

10587683 BcLZal a B-cell leukemia 7.46

104201 14 Tgml transglutaminase 1, K polypeptide 2.76 solute carrier family 2 (facilitated glucose transporter).

10507594 Slc2ai member 1 2.94

10408812 Mal male germ cell-associated kinase 4.35

10582899 Unknown Unknown 1 1 .28

10417226 Unknown Unkno n 3.52

10417421 Hull hematological and neurological expressed 1-like 3.32

10342761 Unknown Unknown 3.28

10464997 Unknown Unknown 2.60

10498935 Gucy lb3 guanylate cyclase 1 , soluble, beta 3 6.56

10498827 Fnip2 follicuiin interacting protein 2 3.40 solute carrier family 6 (neurotransmitter transporter,

10378816 Slc6a4 serotonin), member 4 2.64

10472820 Ttga6 integrin alpha 6 3.76

10598225 Gmcll l germ cell-less homoiog 1 (Drosophiia)-Iike 5.24

10578 15 Ankrd37 ankyrin repeat domain 37 2.79

10537410 Tbxasl thromboxane A synthase 1, platelet 2.94

10475437 Sord sorbitol dehydrogenase 1.99

10573626 Gpi2 glutamic pyruvate transaminase (alanine aminotransferase) 2 7.57

10385159 Rars arginyl-tRNA synthetase 2.21

10582916 Unknown Unknown 33.46

10541910 wf Von Willebrand factor homoiog 3.95

1038121 1 Naglu alpha-N-acetylglucosaminidase (Sanfilippo disease IIIB) 1.82

1 0536010 Unknown Unknown 2.18

10417504 170000 lE04Rik RIKEN cDNA 1700001E04 gene 3.19

10403466 Dip2c DIP2 disco-interacting protein 2 homoiog C (Drosophila) 3.97

10598077 Unknown Unknown 4.95

10564343 Tjp l tight junction protein 1 4.85

10427772 Tars threonyl-tR A- synthetase 2.75

10529425 Nop 14 NOP 14 nucleolar protein homoiog (yeast) 2.41

10345762 Illrl interleukin 1 receptor, type I 3.04

10436024 Gcet2 germinal center expressed transcript 2 2.86

10339750 Unknown Unknown 4.22

10469358 Mrc l mannose receptor, C type 1 20.03

10476740 Slc24a3 solute carrier family 24 (sodium 4.73

10500204 Ecml extracellular matrix protein 1 8.64

10595768 Pls l piastin 1 (I-isoform) 3.59

10435501 Stfal stefin Al 3.85

10382844 Snordic small nucleolar RNA, C 3.33

10386093 Snordl c small nucleolar RNA, C 3.33

10341939 Unknown Unkno n 2.18

10598089 Unknown Unknown 4.96

10582896 Unknown Unknown 24.34

10384504 Meisl Meis homeobox 1 3.99 10455752 8nx24 sorting nexing 24 2.85

10596812 6230427J02Rik RI EN cDNA 6230427J02 gene 2.10

10405753 Mel malic enzyme 1, NADP(+)-dependent, cytosolic 3.55

10359849 Uck2 uridine-cytidine kinase 2 2.29

10417326 D830030K2QRik RIKEN cDNA D830030K20 gene 3.01

10480699 Dpp7 dipeptidylpeptidase 7 2.94

10427928 Trio triple functional domain (PTPRF interacting) 2.24

10475567 Slc24a5 solute carrier family 24, member 5 2.90

10402585 Wars tryptophanyl-tRNA synthetase 3.14

10408935 Gml0786 predicted gene 10786 4.31

10546346 Chchd4 coiled-coil-helix-coiled-coil-helix domain containing 4 2.03

10505517 Tlr4 toll-like receptor 4 4.66

10412667 Ptprg protein tyrosine phosphatase, receptor type, G 2.21

10595633 Bcl2al d B-eell leukemia 7.06

10393887 P crl pyiToline-5-carboxylate reductase 1 3.03

10545101 Hpgds hematopoietic prostaglandin D synthase 3.90

10505276 Slc31al solute earlier family 31 , member 1 2.23

10513428 Mup2 major urinary protein 2 3.24

10576556 Unknown Unknown 4.41

10603936 Gm5168 predicted gene 5168 3.03

10563897 Unknown Unknown 3.15

10573082 Inpp4b inositol polyphosphate-4-phosphatase, type II 5.15

10439483 Cdgap CDC42 GTPase-aetivating protein 2.28

10409866 Ctla2b cytotoxic T lymphocyte-associated protein 2 beta 3.77

10390271 Nfe211 nuclear factor, erythroid derived 2,-like 1 3.01

10424075 LOCI 00044384 hypothetical protein LOCI 00044384 6.45

10392087 Ccdc47 coiled-coil domain containing 47 2.27

10592061 KcnjS potassium inwardly-rectifying channel, subfamily J, member 5 3.70

10417235 Gm3264 predicted gene 3264 3.41

10417315 Gm3264 predicted gene 3264 3.41

1 492330 P2ryl purinerg c receptor P2Y, G-proiein coupled 1 4.15

10574027 Mil metaiiothionein 1 2.56

10474972 Chad ChaC, cation transport regulator-like 1 (E, coli) 2.77

10530477 Nfxll nuclear transcription factor, X-box binding-like 1 2.38

10495596 Frrsl ferric-chelate reductase 1 2.09

1 511779 Atp6v0d2 ATPase, H+ transporting, lysosomal V0 submit D2 20.77

10598083 Unknown Unknown 3.34

10347796 Rhbddl rhomboid domain containing 1 2.13

10503341 Cdhl 7 cadherin 17 5.26

10369225 Dux double homeobox 2.01

10554162 Unknown Unknown 3.36

10517336 Clic4 chloride intracellular channel 4 (mitochondrial) 2.53

10574213 Ccl22 chemokine (C-C motif) ligand 22 4.01 10431894 Slc38a2 solute earner family 38, member 2 2.38

10417319 D830030K20Rik RiKEN cDNA D830030K20 gene 2.67

10564507 Arrdc4 arrestin domain containing 4 3.92

10339765 Unknown Unknown 3.38

10355456 Mreg melanoregulin 3.27

10358894 Sord sorbitol dehydrogenase 2.03

10501699 Agl amylo- 3 ,6-glucosidase, 4-alpha-glucanotransferase 1 .99

10436456 Pros l protein S (alpha) 4.09

10531512 BC061212 cDNA sequence BC061212 2.91

10531523 BC061212 cDNA sequence BC061212 2.91

10551250 Unknown Unknown

10467842 Goil glutamate oxaloacetate transaminase 1 , soluble 2.70

10419568 Earl ! eosinophil-associaied, ribonuclease A family, member 1 1 27.00

10417415 Gm5458 predicted gene 5458 3.01

10417258 544988 predicted gene, 544988 3.46

10603953 Gm5169 predicted gene 5169 4.10

10543239 Tcfec transcription factor EC 2.82

10374236 Up l uridine phosphorylase 1 2.38

10457546 Osbpila oxysterol binding protein-like 1A 2.24

10435930 Cd200r2 Cd200 receptor 2 5.28

10343644 Unknown Unknown 4.77

10350003 Cyb5ri cytochrome b5 reductase 1 3.79

10559880 Vmn2r33 vomeronasal 2, receptor33 2.92

10527965 Ckinl2 claudin 12 3.01

10578149 Leprotll leptin receptor overlapping transcript- like 1 2.65

10387821 Alox l2 araehidonate 12-3ipoxygenase 2.43

10564053 Gm3079 predicted gene 3079 3.58

10564055 Unknow Unknown 3.58

10513467 Mup2 major urinary protein 2 3.54

10417798 Kcnk5 potassium channel, subfamily K, member 5 2.38

10409876 Ctla2a cytotoxic T lymphocyte-associated protein 2 alpha 5.20

10397708 Unknown Unknown 2.41

10384398 Gr l O growth factor receptor bound protein 10 6.86

10414245 4930503E14Rik RIKEN cDNA 4930503E14 gene 2.33

10478744 Unknown Unknown 3.31

10347748 Acsl 3 acyl-CoA synthetase long-chain family member 3 3.1 2

10570291 F 10 coagulation factor X 3 21

10386388 Snap47 synaptosomal-associated protein, 47 2.16

10567941 Eif3c eukaryotic translation initiation factor 3, subunit C 2.06

10577757 Adam9 a disintegrin and metallopeptidase domain 9 (meitrin gamma) 3.45

10341357 Unknown Unknown 4.29

10542791 Ppfib 1 PTPRF interacting protein, binding protein 1 (liprin beta 1) 2.43

10526853 Fam20c family with sequence similarity 20, member C 4.33 10457508 Npcl Niemann Pick type C 1 3.48

10356299 Gpr55 G protein-coupled receptor 55 3.02

10525439 P2rx4 purinergic receptor P2X, ligand-gated ion channel 4 3.50

10558769 If tml interferon induced transmembrane protein i 2.54

10598216 Unknown Unknown 2.01

10495830 Sec24d Sec24 related gene family, member D (S. eerevisiae) 2.15

10342263 Unknown Unknown 2.24

10433735 Abcc 1 ATP -binding cassette, sub- family C (CFTR 2.10

10360028 Fcgr2b Fc receptor, IgG, low affinity Hb 2.34

10340473 Unknown Unknown ' 7

10472042 Gml3498 predicted gene 13498 3.22

10430006 Slc39a4 solute carrier family 39 (zinc transporter), member 4 2.01

10419038 Ghitm growth hormone inducible transmembrane protein 2.72

10457872 Sic39a6 solute carrier family 39 (metal ion transporter), member 6 2.37

10339956 Unknown Unknown 2.51

10361887 Perp PERP, TP53 apoptosis effector 2.37

10442801 Tpsgl tryptase gamma 1 5.24

10563704 Mrgpra6 MAS-related GPR, member A6 5.02

10369413 Sgpll sphmgosine phosphate lyase i

10365104 F630i l0N24Rik RIKEN cDNA F630110N24 gene 1.88

10340131 Unknown Unknown 1.81

10343674 Unknown Unknown 7.04

10417302 544988 predicted gene, 544988 2.92

10598041 Unknown Unknown 5.42

10460085 Cndp2 CNDP dipeptidase 2 (metallopeptidase M20 family)

10535532 Tecprl tectonin beta-propeller repeat containing 1 2.23

10478938 Haxl HCLS1 associated X-l 1.98

10344134 Unknown Unknown 2.29

10369295 D10Ertd641e DNA segment, Chr 10, ERATO Doi 641, expressed 2.14

10450640 Mrpsl Sb mitochondrial ribosoma! protein SI SB 2.14

1 476314 Pmp prion protein 3.42

10598794 Unknown Unknown 3.09

10428877 E430025E21Rik RIKEN cDNA E430025E21 gene 1.94

10398032 Serpina3b serine (or cysteine) peptidase inhibitor, clade A, member 3B 4.33 transient receptor potential cation channel, subfamily C,

10583163 Trpc6 member 6 5.53

10463836 Gstol glutathione S-transferase omega 1 2.34

10549929 Vmn2r33 vomeronasal 2, receptor33 2.68

10578123 Rbpms RNA binding protein gene with multiple splicing 2.41

10531383 Sdadl SDA1 domain containing 1 1.95

10466886 Unknown Unknown 2.03

10493555 Kcnn3 potassium intermediate 3.18

10339348 Unknown Unknown 2.24 10572130 Lpl lipoprotein lipase 8.29

10340989 Unknown Unknown 4.12

10462398 Pdcd llg2 programmed cell death 1 ligand 2 7.82

1046621 0 Ms4a6d membrane-spanning 4-domams, subfamily A, member 6D 9.21

10339385 Unknown Unknown 2.94

10360338 l e r ^' a Fc receptor, IgE, high affinity I, alpha polypeptide 3.45

10439276 Faml 62a family with sequence similarity 162, member A 2.24

10417446 D830030K2QRik RIKEN cDNA D830030K20 gene 3.40

10417452 D830030K20Rik RIKEN cDNA D830030K20 gene 3.40 dual specificity phosphatase 3 (vaccinia virus phosphatase

103915 13 Dusp3 VHl -related) 3.82

1035705 1 Kdsr 3-ketodihydrosphingosine reductase 1.75

10340718 Unknown Unknown 3.99

10341658 Unknown Unknown 4.38

1041 1 156 Scamp 1 secretory carrier membrane protein 1 2.32

10509030 Runx3 runt related transcription factor 3 1.80

10540248 Mitf microphthalmia-associated transcription factor 2.66

1 412483 Unknown Unknown 1.90

10510201 Rex2 reduced expression 2 2.70

10559928 Vmn2r 1 vomeronasal 2, receptor 5 1 3.29

10474936 Spirit! serine protease inhibitor, Kunitz type i 6.3 1

10349157 Serpinb2 serine (or cysteine) peptidase inhibitor, clade B, member 2 10.13

1034061 7 Unknown Unknown

10536095 Unknown Unknown 2.12

10435937 Cd200r3 CD200 receptor 3 4.72

10447773 Slc22a3 solute carrier family 22 (organic cation transporter), member 3 2.76

10436196 Unknown Unknown 2.02

10571788 Vegfc vascular endothelial growth factor C 3.82

10343771 Unknown Unknown 2.66

10605044 Xlr5b X-linked lymphocyte-regulated 5B 2.15

1 0407467 Akrl e! aldo-keto reductase family 1, member El 2.02

10384691 061 0010F05Rik RIKEN cDNA 0610010F05 gene 2.13

10373027 Tspan31 tetras anin 3 1 1.82

10365933 Eeal early endosome antigen 1 2.78

10438907 Gp5 glycoprotein 5 (platelet) 2.94

10423577 Mtdh metadherin 1.95

10559894 Vmn2r34 vomeronasal 2, receptor 34 2.44

10563909 Unknown Unknown 2.49

10575052 Cdhl cadberin i 3.73

10608660 Unknown Unknown 1.84

10540727 Creld \ cysteine-rich with EGF-like domains 1 1 .73

10503148 LOC.100047557 hypothetical protein LOC I 00047557 3.29

10423293 Myo l O myosin X 3.06 10525271 C330023M02Rik RIKEN cDNA C330023M02 gene 2.09

10603247 Unknown Unknown 3.68

10354563 Dnahc7b dynein, axonemal, heavy chain 7B 5.16

10446693 Wdr43 WD repeat domain 43 1.99

10550782 VlrdH vomeronasal 1 receptor, D14 1.83

10523325 Gm7682 predicted gene 7682 3.46

10608644 Unknown Unknown 4.93 macrophage galactose N-acetyl-galactosamine specific lectin

10377774 Mgl2 2 8.30

10372177 Tmtc2 transmembrane and letratricopeplide repeat containing 2 5.25

10338213 Unknown Unknown 2.29

10377924 G lba glycoprotein lb, alpha polypeptide 2.81

10502552 Clcal chloride channel calcium activated 1 3.20

10424400 Myc myelocytomatosis oncogene 2.42

10369661 Ccari ceil division cycle and apoptosis regulator 1 1.99

10343357 Unknown Unknown 2.48

10440964 Cryzi l crystallin, zeta (quinone reductase) -like 1 2.44

1 558961 Tspan4 tetraspanin 4 2.29

10411958 RnflSO ring finger protein 180 3.37 pleckstrin homology domain containing, family G (with

10367673 Piekhgl RhoGef domain) member 1 2.1 1

10499705 Haxl HCLS1 associated X-l 1.96

10420659 6330409N04Rik RIKEN cDNA 6330409N04 gene 2.38

10544462 Faml 15a family with sequence similarity 1 1 , member A 2.88

10569296 Krtap5-2 keratin associated protein 5-2 2.54

10523320 C87414 expressed sequence C87414 2.55

10540472 Bhlhe40 basic helix-loop-helix family, member e40 4.19

10580957 Slc38a7 solute earlier family 38, member 7 3.73

10579776 Arhgap i 0 Rho GTPase activating protein 10 1.75

10404913 Cap2 CAP, adenylate cyclase-associated protein, 2 (yeast) 2.17

10559175 rtap5-5 keratin associated protein 5-5 1.88

10338280 Unknown Unknown 2.94

10507894 Rragc Ras -related GTP binding C 2.16

10343623 Unknown Unknown 2.88

10498952 Gucy 1 a3 guan late cyclase 1 , soluble, alpha 3 2.90 colony stimulating factor 2 receptor, beta, low-affinity

10425066 CsGrb (granulocyte-macrophage) 3.03

10416199 Entpd4 ectonucleoside Iriphosphate diphosphohydroiase 4 2.24

10596533 T^'ex264 testis expressed gene 264 2. 1

10524034 idua iduronidase, alpha-L- 1.58

10574226 Cell 7 chemokme (C-C motif) ligand 17 3.06

10571922 Nekl NIMA (never in mitosis gene a) -related expressed kinase 1 2.01

10401829 EG435337 predicted gene, EG435337 2.61

10339415 Unknown Unknown 3.05 10344512 Unknown Unknown 2.51

1 0423134 Zfr zinc finger RNA binding protein 1.77

10463263 Lztfll leucine zipper transcription factor-like 1 2.09

10417253 170000 l E04Rik RIKEN cDNA 1700001 E04 gene 2.28

10417281 Gm5458 predicted gene 5458 2.28

10385719 Sec24a Sec24 related gene family, member A (S. cerevisiae) 2.06

10443221 Uhrfl b l UHRFl (ICBP90) binding protein 1 2.10

10594289 Glee glucuronyl C5-epinierase 2.74 macrophage galactose N-acetyl-galactosamine specific lectin

1 0377782 Mgll 1 6.25

10433702 Mpvl71 Mpvl7 transgene, kidney disease mutant-like 1.66

10471 535 Faml 29b family with sequence similarity 129, member B 4.20

10476383 Crlsl cardiolipin synthase 1 2.09

10349744 Slc45a3 solute carrier family 45, member 3 2.07

10413928 181 001 1Hi lRik RIKEN cDNA I S l OO l iHl l gene 2.26

10338492 Unknown Unknown 3.18

10587503 Sh3bgrl2 SH3 domain binding glutamic acid-rich protein like 2 3.09

10339052 Unknown Unknown 2.33

10490384 Lama5 laminin, alpha 5 2.81

10536056 C87414 expressed sequence C87414 2.48

10536151 Gm7682 predicted gene 7682 2. j /

10513497 Mup2 major urinary protein 2 2.65

10346164 Sdpr serum deprivation response 3.01

10543791 Podxl podocalyx in-like 2.92

10549904 Vmn2r37 vomeronasal 2, receptor 37 2.71

10535586 Smurfl SMAD specific E3 ubiquitin protein ligase 1 2.04

10569306 Krtap5- 1 keratin associated protein 5- 1 2.05

10598192 4930408F14Rik RIKEN cDNA 4930408F14 gene 2.43

10453602 Gm2889 predicted gene 2889 4.19

10531776 Faml 75a family with sequence similarity 175, member A 2.04

10563905 Unknown Unknown 3.61

10563907 Unknown Unkno n 3.61

10556216 Ipo7 importin 7 1.60

1 564960 Furin furin (paired basic amino acid cleaving enzyme) 2.89

10507137 Pdzklipi PDZK1 interacting protein 1 2.49

10555027 Gab2 growth factor receptor bound protein 2-associated protein 2 2.68

10341579 Unknown Unknown 2

10371446 Bpil2 bactericidal 5.89

TAF4B RNA polymerase II, TATA box binding protein

10454077 Taf4b ( BP)-associated factor 2.72

10340096 Unknown Unknown 4.44

10402519 Atg2b ATG2 autophagy related 2 homolog B (S, cerevisiae) 1.67

10568785 Bnip3 BCL2 3.57 10378508 Tsrl TSR1, 20S rRNA accumulation, homolog (yeast) 2.44

10375229 Unknown Unknown 2.85

10407543 Gtpbp4 GTP binding protein 4 1.93

10385572 Sqstml sequestosome 1 3.25

10538706 Mmml multimerin 1 2.83

10457054 Zadh2 zinc binding alcohol dehydrogenase, domain containing 2 2.02

10602009 Rnfl 28 ring finger protein 128 8.42

10478847 1500012F01Rik RIKEN cDNA 1500012FG1 gene 2.29

10 10129 Dhrs3 dehydrogenase 2.2 !

10468762 4930506M07Rik RIKEN cDNA 4930506M07 gene 5 77

10531501 C87414 expressed sequence C87414 2.48

10344201 Unknown Unknown 2.29

10380751 Mrpl45 mitochondrial ribosomal protein L45 1.95

10522562 C87414 expressed sequence C87414 2.42

10598093 Gm 904 predicted gene 9904 3.40

10536122 Unknown Unknown 2.77

10531794 Wdfy3 WD repeat and F YVE domain containing 3 2.37

10463158 ΑΪ606181 expressed sequence AI606181 2.20

10386427 Flcn follieulin 1.76

10417492 Gm5458 predicted gene 5458 3.47

10416406 Htr2a 5-hydroxytryptarnine (serotonin) receptor 2A 2.54

10457203 Unknown Unknown 2.14

10536090 Unknown Unknown 2.79

10598091 Unknown Unknown 2.70

10496023 Casp6 caspase 6 2.63

10414228 Gm5622 predicted gene 5622 2.26

10373452 Gml29 predicted gene 129 4.99

LIM domain containing preferred translocation partner in

10434806 Lpp lipoma 4.22

10345675 Npas2 neuronal PAS domain protein 2 3.04

10341016 Unknown Unknown 2.97

10455852 Prrcl proline-rich coiled-coil 1 2.12

10417053 Mbnl2 musc!eblind-!ike 2 1.93

1 510270 M ih IV 5, 10-methyleneteirahydro folate reductase 1.92

10489723 ZrnyndS zinc finger, MYND-type containing 8 1.63

10519688 Gm6455 predicted gene 6455 1.78

10563975 Unknown Unknown 3.59

10563981 Unknown Unknown 3.59

10563987 Unknown Unknown 3. 9

10564009 Unknown Unknown 3.59

10408475 E2f3 E2F transcription factor 3 1. 5

10424221 Wdr67 WD repeat domain 67 2.34

10585699 Fabp5 fatty acid binding protein 5, epidermal 5.12 10382852 Mfsdl 1 major faciiitator superfamily domain containing 1 1 2.13

10341087 Unknown Unknown 2.56

10536163 Unknown Unknown 2.55

10342915 Unknown Unknown 3.57

10606069 4930444G20Rik RIKEN cDNA 4930444G20 gene 1.58

10404008 Vlrh6 vomeronasal 1 receptor, H6 1.76

10571824 Gm 10674 predicted gene 10674 1.96

10391669 Slc25a39 solute carrier family 25, member 39 1.62

10530467 Nfxl l nuclear transcription factor, X-box binding-like 1 2.01

10510391 Srm spermidine synthase 1.74

10514590 Dock? dedicator of cytokinesis 7 2.63

10536472 Mdfic MyoD family inhibitor domain containing 3.05

10547386 Adipor2 adiponectin receptor 2 1.95

10432640 Bin2 bridging integrator 2 1.87

10568050 Aidoa aldolase A, fructose-bisphosphate 1.76

10366310 OsbplS oxysterol binding protein-like 8 2.30

10519661 Gm6455 predicted gene 6455 1.73

10490838 FabpS fatty acid binding protein 5, epidermal 5.14

10393970 Fasn fatty acid synthase 1.49

10399379 Pgkl phosphoglycerate kinase 1 1.80

10498313 Pgkl phosphoglycerate kinase 1 1.80

10364375 Cstb cystatin B 2.82

10427468 Unknow Unknown 3.74

10419578 Ndrg2 N-myc downstream regulated gene 2 2.78

10375713 Mgat4b mannoside acerylglucosaminyltransferase 4, isoenzyme B 1.67

10447341 Rhoq ras homolog gene family, member Q 2.36

10471171 Fubp3 far upstream element (FU SE) binding protein 3 1.67

10461078 Atl3 atiastin GTPase 3 1.73

10513145 Ptpn3 protein tyrosine phosphatase, non-receptor type 3 2.94

10601390 Pgkl phosphoglycerate kinase 1 1.76

10375980 Aff4 AF4 2.37

10340443 Unknown Unknown 2.68

10443463 Cdkiil cyclin-dependent kinase inhibitor 1 A (P21) 4.43

10352905 Cd34 CD34 antigen 1.62

10417544 Acox2 acyl-Coenzyme A oxidase 2, branched chain 2.05

10484318 cka l NCK-associated protein 1 4.36

10428376 Angptl angiopoietin 1 1.82

10399882 Dus4l dihydrouridine synthase 4-!ike (S, cerevisiae) 2.43

104 1874 Slc38al solute carrier family 38, member 1 1.93 solute carrier family 6 (neurotransmitter transporter, taurine),

10540122 Slc6a6 member 6 2.25

10536006 Gm7682 predicted gene 7682 2.31

10554808 Fzd4 frizzled homolog 4 (Drosophila) 3.55 1041741 1 ENSMUSG00000079376 predicted gene, ENSMUSG00000079376 2.87 solute carrier family 7 (cationic amino acid transporter, y+

10574985 Slc7a6 system), member 6 2.00

10415784 Trim 13 tripartite motif-containing 13 3.3

10353061 Unknown Unknown 2.32

10462363 Jak2 Janus kinase 2 2.10

10513536 Gm21 73 predicted gene 21 73 2.63

10539472 Nagk N-acetylglucosamine kinase 2.08

10420225 Cra l cbymase 1 , mast cell 3.61

1 0553163 Nomol nodal modulator 1 1.76

10581434 Dpep2 dipeptidase 2 8.78

10342401 Unknown Unknown 4.77

10352194 Cdc42bpa CDC42 binding protein kinase alpha 2.60

10454606 Wdr36 WD repeat domain 36 1.79

10355037 Wdr 2 WD repeat domain 12 2. 19

10472923 Ak311 adenylate kinase 3-like 1 2.61

10414234 Gm5622 predicted gene 5622 2. 1 9

10589889 Gibl galactosidase, beta 1 2.46 neural precursor ceil expressed, developmental!}' down-

10586933 Nedd4 regulated 4 1.63

10569458 Cars cysteinyl-tRNA synthetase 2.00

10605303 Dnaselli deoxyribonuclease 1 -like 1 1.87

10479154 Tubb l tubulin, beta 1 2.05

10495549 Dbt dihydrolipoamide branched chain Iransacvlase E2 1.95

10369221 Dux double homeobox 2.2z

10343942 Unknown Unknown 2.52

10375065 Sh3pxd2b SH3 and PX domains 2B 4.41

10450930 Crisp 1 cysteine-rich secretory protein 1 7. 10

10397145 Acot2 acyS-CoA thioesterase 2 3.34

10505246 Unknown Unknown 3.96

10505258 Unknown Unknown 3.96

10449471 Sipkl serine 2.04

10463355 Scd2 stearoyl-Coenzyme A desarurase 2. 2.79

10370376 Pfk phospbofructokinase, liver, B-type 2.14

10564005 Unknown Unkno n 3.89

10399825 Did dihydrolipoamide dehydrogenase 1.82

10474671 Spred l sprouty protein with EVH-l domain 1 , related sequence 3.1 3

1 569057 Rnhl ribonuclease 2.07

10513166 Ptpn3 protein tyrosine phosphatase, non-receptor type 3 2.35

10373157 Mars memionine-tRNA synthetase 2.05

103733 13 Nab2 Ngfi-A binding protein 2 1.94

1059253 1 Unknown Unknown 2.28

10412543 Gm5458 predicted gene 5458 2.30 10349174 8erpinb8 serine (or cysteine) peptidase inhibitor, clade B, member 8 2.78

10413152 17001 12E06R.ik RIKEN cDNA 1700! 12E06 gene 1.86

10522585 Gm7682 predicted gene 7682 2.7!

10452648 Emili 2 eiastin microfibril interface!" 2 2.40

10391454 Vatl vesicle amine transport protein 1 homolog (T californica) 2.30

10397541 Gm5039 eukaryotic translation initiation factor 1 A pseudogene 2. 1

10594825 Aqp9 aquaporin 9 4.26

10534303 Lat2 linker for activation of T ceils family, member 2 2.34

10538356 Chn2 chimerm (chimaerin) 2 2.39

10530841 Igfbp7 insulin-like growth factor binding protein 7 2.50

10436095 Reinia resistin like alpha 26.96

10420694 iiitsb integrator complex subunit 6 1.64

10541605 Clec4n C-type lectin domain family 4, member n 14.12

10519652 Gm645 predicted gene 6455 1.83

10523316 Gm7682 predicted gene 7682 2.44

10523346 Gm7682 predicted gene 7682 2.44

10536033 Gm7682 predicted gene 7682 2.44

10536044 Unknown Unkno n 2.44

10521709 Lap3 leucine aminopeptidase 3 1.60

10490212 Ctsz cathepsin Z 2.00

10421488 Fndc3a fibronectin type III domain containing 3A 2.55

10569504 Tnfrsf23 tumor necrosis factor receptor superfamily, member 23 2.29

10516529 Adc arginine decarboxylase 2.07

10485580 Cstf3 cleavage stimulation factor, 3' pre-RNA, subunit 3 1.86

10510167 Cdv3 carnitine deficiency-associated gene expressed in ventricle 3 2.05

10604032 Unknown Unknown 1.92

10477169 id! inhibitor of DNA binding 1 2.18

10367775 Stxbp5 syntaxin binding protein 5 (tomosyn) 1.71

10492499 Mfsdi major facilitator superfamily domain containing i 1.84

10506274 Dnajc6 DnaJ (H.sp40) homolog, subfamily C, member 6 3.23

10362036 Hbsl l Hbsl-like (S. cerevisiae) 1.89

10542355 Em l epithelial membrane protein 1 5.86

CD47 antigen (Rh-related antigen, integrin-associated signal

10436182 Cd47 transducer) 1.57

10469404 Cacnb2 calcium channel, voltage-dependent, beta 2 subumt 1.96

10453575 Cul2 cuilin 2 2.21

10506680 Tmem48 transmembrane protein 48 1.95

10396712 Fut8 fucosyltransferase 8 2.23

10391963 Nsf N-ethylmaleimide sensitive fusion protein 1.90

10523468 Bmp2k BMP2 inducible kinase 3.33

10373202 Shmt2 serine hydrox methyltransferase 2 (mitochondrial) 2.97

10339731 Unknown Unknown 2.48 [00117] Two of the biological pathways highlighted by GeneGo analysis implicated the epithelial to mesenchymal transition or EMT process. Amongst the candidate genes that are associated with EMT, hepatocyte growth factor (HGF), lymphoid enhancer-binding factor 1 (Lefl) and CdkSrl were differentially overexpressed in the freshly harvested Lin-BMCs compared to the Lin-BMCs ECX cells.

[00118] Quantitative real-time PGR. (qRT-PCR) was performed to investigate the expression of the major genes of EMT described in the literature, E-cadherin, snail, and vimentiii. Expression of E-cadherin was significantly higher in the Lin-ECX BMCs while expression of snail and vimentin was significantly reduced in the Lin- ECX BMCs relati ve to freshly harvested Lin-BMCs. These findings suggest that in the Lin- ECX BMCs, the opposite process of mesenchymal to epithelial transition (MET) may be induced by the mixture of cytokines and growth factor. This suggests a possible mechanism that may lead to the enhanced repair.

[00119] Here we present a series of studies to determine the optimal experimental conditions necessary to generate BMCs that can reduce atherosclerosis in a mouse model of atherosclerosis in a robust and reliable way. From our work, we have discovered a method for generating such BMCs by first culturing them in a specific combination of cytokines and growth factors. Regular injections of these cultured BMCs reduces atherosclerosis in a mouse atherosclerosis model of over 50% compared to control animals. This represents a significant next step in the application of cellular therapy for treating atherosclerosis,

[00120] We performed a series of experiments using a wide variety of cell treatments to comprehensively address the question of whether such treatments can reliably and effectively modify BMCs to reduce atherosclerosis. Our results both confirm some prior findings in the literature and also suggest a new method for significantly boosting the ability of cell injections to ameliorate atherosclerosis in the apoE^"'^'" mice. We found that apoE^"'" mice receiving a small number (3 or fewer) of whole culture bone marrow injections actually developed more atherosclerosis relative to control animals injected with PBS (FIG. I). We found that mice receiving a greater number of whole bone marrow injections showed a trend towards atherosclerosis reduction (FIG. 1); reduction in atherosclerotic lesions increased with increasing numbers of injections. [00121] We found that a new method that pre-treats Lin- BMCs by culturing them in media containing IL-3, IL-6 and SCF prior to injection. This pre-trealment produces ceils that can significantly and reproducibly reduce the development of atherosclerosis in the apoE^"'^" mouse model,

Materials and Methods

[00122] Animals: C57BL/6J apolipoprotein E knockout (apoE^"'^") mice were purchased from Jackson Laboratories (B6, 129P2-ApoEtml Unc, Bar Harbord, ME. USA). Pups were weaned at 3 weeks of age. Donor mice were maintained on chow diet and recipient mice were fed a Western diet (TD.88137, Harlan Teklad Madison, WI) containing 21% fat by weight (0.15% cholesterol).

[00123] Lineage-negative (Lin-) Cells isolation: BMCs were harvested from apoE^"'^" mice at 5-8 weeks of age by flushing the long bones (5). Lin-cells were isolated by MACS sorting using Mouse Lineage cell depletion kit (Miltenyi Biotech).

[00124] Ex vivo culture of whole BM and Lin- BMCs: For standard culture, whole bone marrow was placed into DMEM Alpha medium containing 20% FBS and 2 mol/L hydrocortisone. Ceils were incubated at 370C in 5% CO? for 2 days. For the enhanced culture, Lin- cells were placed into DMEM medium containing 20% FBS, 20 ng/ml murine iiiterieukiii-3 (IL-3), 50 ng/ml murine iiiterieukiii-6 (IL-6) and 50 ng/ml murine SCF (R&D Systems, Minneapolis, MN). The cells were incubated at 37°C in 5% C0₂ for 4 days. For both culturing approaches, the non-adherent, floating cells were collected and used for injections. Single cell suspensions were prepared by passing the cells through a 45-/m nylon gauze and resuspended in PBS containing 2% BSA,

[00125] Cells Injections: Five sets of cell injection experiments were performed. First, freshly harvested BMCs (non-cultured) from donor apoE^"7" mice were split into different populations prior to injection 1) Lin- (n=8), 2) Lin+ (n=8). 3) a side population, defined by us as simple little cells (SLCs) (n 5 }. 4) Lin-cells with SLCs removed in 8 } and 5) PBS control (n=8). Recipient apoE^{"' "} mice received 106 ceils every 2 weeks, starting at 6 weeks of age for a total of 5 injections. Mice were sacrificed at 16 weeks of age for phenotyping.

[00126] Second, whole BMCs from donor apoE^"'^" mice were cultured in M HM Alpha Medium, 20% FBS and 2 /mol/L hydrocortisone for two days prior to injection (5). Recipient apoE^"''^" mice received 10⁶ cultured cells or PBS every 2 weeks, starting at 6 weeks of age. We studied mice after different numbers of injections: 1) 3 injections, sacrificed at 12 weeks; 2) 6 injections, sacrificed at 18 weeks; 3) 10 injections, sacrificed at 26 weeks. Each group contained 10 treatment mice and 10 control mice.

[00127] Third, Lin-BMCs from donor apoE^''^" mice first underwent enhanced culture in MEM with FBS (20%), IL-3 (20 ng/m!), IL-6 (50ng/ml) and SCF (50 ng/mi), for four days. Using the enhanced cultured Lin-BMCs, we studied three treatment groups: 1) freshly harvested Lin-BMCs, 2) Lin- BMCs grown in the enhanced culture (ECX) and 3) PBS control. Recipient apoE^"'^" mice received 10⁶ cells or PBS every 2 weeks, starting at 6 weeks of age for a total of 6 injections and sacrificed at 18 weeks of age. Twenty mice were treated with the ECX cells and twenty with PBS control and six mice were treated with freshly harvested Lin- BMCs.

[00128] Fourth, Lin-BMCs underwent enhanced culture and were separated into Lin- Mac 1+ BMCs or Lin- Macl- BMCs fractions. Recipient apoE^{"' "} mice received 10⁶ of either ceil fraction or P13S every two weeks, starting at 6 weeks of age for a total of 6 injections and sacrificed at 18 weeks of age. There were 8 mice in each experimental group.

[00129] Fifth, Lin- BMCs underwent enhanced culture for 4 days, after which the supernatant w^ras collected. Recipient apoE^"'^" mice received either supernatant (n=8) or PBS (n-lO) injections every 2 weeks, starting at 6 weeks of age. Mice were sacrificed at 18 weeks of age.

[00130] Flow cytometry analysis: Phenotypic analysis of freshly harvested Lin-BMCs and Lin-BMCs cultured with cytokines and growth factor was performed using anti-mu-CD 3 PE, anti-Sca-1 PE, anti muMac-l(CD-l lb) PE, anti mu-CD45 FITC, and anti-E-selectin PE and compared to isotype control staining (Becton Dickson, San Diego, CA,USA). The stained samples were sorted using a dual laser fluorescence activated cell sorter (FACS) (Becton Dickinson, San Jose, CA).

[00131] Real Time PGR: TaqMan Gene Expression Assay for Cdhl , Cdh2, Snail and VIM were purchased from Applied Biosysems (ABI, Foster City,CA), First-stand cDNAs were synthesized from total R A using TaqMan Reverse Transcription Reagents (ABI). Gene expression levels were measured by real-time PGR with an AB I/PRISM 7700 sequence detection system (ABI). The expression levels of target mRNAs relative to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was calculated using the comparative threshold cycle (CT) method (2-CT).

[00132] Microarray Analysis: Total R A was extracted from either freshly harvested Lin-BMCs or Lin-BMCs grown in enhanced culture (IL-3, IL-6. SCF) . Probes were generated and hybridized to the murine GeneST array (Affymetrix, Santa,CA) at the University of Miami Microarray Core facility. Significance Analysis of Microarrays (SAM) identified candidate genes with statistically significant differential expression. Raw data underwent RMA normalization. False Discovery Rate (FDR) of <0.000001 was used to denote differentially expressed genes. The GeneGo MetaCore Pathways Analysis Package was used for gene annotation and identification of biological pathways that were statistically overrepresented within the candidate genes.

[00133] Quantitative Assessment of Atherosclerosis Lesion Burden: After sacrificing recipient mice, aortas were removed and formalin- fixed. The Oil Red O staining and measurements of the atherosclerosis were conducted independently by the EEHScience laboratory. The laboratory previously performed the computerized atherosclerosis quantification analysis as previously described (4). EEHScience was blinded to the cell treatment type received by each of the experimental animal groups. En face staining for atherosclerosis was done with Oil Red O. Each aorta image was projected to a reference aorta tem late, derived from the average size and shape of all aorta surveyed. The extent of disease was quantified by counting pixels staining with Oil Red O compared with the total area of the aorta. EEHScience performed the statistical analysis for each of the animal groups.

References

[00134] 1. Ergun S, Tilki D, Klein D. Vascular wall as reservoir for different types of stem and progenitor cell s. Antioxid Redox Signal Epub ahead of print, 2011 ,

[00135] 2. Goldschmidt-Clermont PJ. Loss of bone marrow-derived vascular progenitor cells leads to inflammation and atherosclerosis Am Heart J 146:85-812, 2003.

[00136] 3. Hagensen MK, Shim J, T im T, Falk E, Bentzon JF. Circulating endothelial progenitor cells do not contribute to plaque endothelium in murine atherosclerosis. Circulation 121(7):898-905, 2010. [00137] 4. Karra R, Vemullapalli S, Dong C, Herderick EE, Song X, Slosek , Nevins JR, West M, Goldschmidt-Clermont PJ, Seo D. Molecular evidence for arterial repair in atherosclerosis. Proc Natl Acad Sci U S A 102: 16789-16794, 2005.

[00138] 5. Rauscher F, Goldschmidt-Clemiont PJ, Davis BH, Wang T, Gregg D, Ramaswami P, Pippen AM, Annex BH, Dong C, Taylor DA. Aging, Progenitor Cell Exhaustion, and Atherosclerosis. Circulation 108:457-463, 2003.

[00139J 6. Sata M, Saiura A, unisato A, Tojo A, Okada S, Tokuhisa T, Hirai H, Makuuchi M, Hirata Y, Nagai R. Hematopoietic stem cells differentiate into vascular cells thai participate in the pathogenesis of atherosclerosis. Nature Medicine 8:403-9, 2002.

[00140] 7. Silvestre J-S, Gojova A, Bran V, Potteaux S, Esposito B, Duriez M, Clergue M, Le Ricousse-Roussanne S, Barateau V, Merval R, Groux H, Tobelem G, Levy B, Tedgui A, Mallat Z. Transplantation of bone marrow-derived mononuclear cells in ischemic apoiipoprotein E-knockout mice accelerates atherosclerosis without altering plaque composition. Circulation 108:2839-2842, 2003.

[00141] 8. Strong JP, Malcom GT, Oalmann MC, Wissler RW. The PDAY Study- natural history, risk factors, and pathobiology. Pathobiological Determinants of Atherosclerosis in Youth. Annals of the New York Academy of Sciences 811 :226-235, 1997.

[00142] 9. Zampetaki A, Kirton JP, Xu Q, Vascular repair by endothelial progenitor cells. Cardiovascular Research 78:413-421, 2008.

[00143] IS. Zhu S, Liu X, Li Y, Goidsciimidt-Ciermont PJ, Dong C. Aging in the atherosclerosis milieu may accelerate the consumption of bone marro endothelial progenitor ceils. Arterioscler Tliromb Vase Biol 2007; 27(1):113-119.

Example 2: Cell-Based Therapies for Atherosclerosis

[00144] Atherosclerosis is the leading cause of death in the United States. Significant progress has been made in reducing the health burden of atherosclerosis [1-3], through implementation of risk factor modification strategies such as smoking cessation, antihypertensives and antihypercholesterolemic agents. Still, this chronic disease remains a significant public health problem. The advent of cell-based therapies to treat chronic disease represents a major new opportunity to treat atherosclerosis. Animal studies showed that regular intravenous injections of BMCs in the apoiipoprotein E knockout (apoE^"'^~) atherosclerosis mouse model led to a significant reduction in disease [4], However, these early successes have been tempered by inconsistent results in subsequent studies. The inconsistency is attributed to differences in the experimental methods for each study, such as different cell populations, length of treatment and pre-mjection cell processing [14]. In searching for a consistent and standard approach, a method for pretreating the Lineage marker negative (Lin-) population of die bone marrow with a combination of cytokines and growth factors prior to intravenous injection was discovered, and recently reported [5]. With regular injections of the cultured Lin- BMCs, there is a substantial and consistent atherosclerosis reduction in apoE" mice.

[00145] In clinical practice, the typical target patients would be individuals with accelerated atherosclerosis and thromboembolic complications regardless of aggressive risk factor modification. Included would be individuals with propensity for chronic arterial injury from conditions such as diabetes or hypercholesterolemia. The method described herein would be used by medical centers that have the capability to perform bone marrow transplantation, as they would have the necessary equipment and personnel. One example of a product is a kit containing the cytokine/growth solution. Patients would undergo a large volume bone marrow harvest. The cells would be cultured using the cell pretreatment kit, The generated cells would be divided into aliquots and frozen. The patients would come for regular intravenous infusions of their own pretreated BMCs.

[00146] The pretreatment methods described herein may be the crucial component for making cell-based therapy of atherosclerosis a potent new treatment for human patients. The overarching goal with this proposal is to move this technology toward clinical application. To do this, the following specific aims are proposed.

[00147] Specific Aim 1 : Determine alterations to our current cell processing method to compensate for the negative impact of the freeze-thaw cycle on the ability of the pretreated BMCs to reduce atherosclerosis. For animal studies, fresh cells are prepared for each injection, which is not practical in human clinical practice. The necessary freeze-thaw step reduces the efficacy of the BMCs in our preliminary studies by 50%. We will assess whether increasing the cell dose or moving the pretreatment step after the freeze-tha will compensate for the reduction. A milestone would be to demonstrate that either or both of these modifications lead to a significant improvement in atherosclerosis reduction over our current method. Significant would be defined as reducing the impact of the freeze-thaw cycle by 15-20%. [00148] Specific Aim 2: Determine whether infusions of pretreated whole bone marrow will yield a similar reduction in atherosclerosis as pretreated Lin- bone marrow. The studies described herein used only the Lin- fractions of the bone marrow that makes up only 10% of the total bone marrow. Preliminary data suggests that pretreated whole BMCs may provide similar atherosclerosis reduction as Lin- BMCs. A milestone would be to demonstrate that pretreated whole bone marrow leads to a similar atherosclerosis reduction as our current method. In a typical embodiment, acceptable variation in atherosclerosis reduction would be 0- 10% relative to Lin- BMCs,

[00149] Specific Aim 3: Determine the persistence of the atherosclerosis reduction effect from the cultured cell injections. We will seek to learn the duration of the effect of the cultured cell injections and whether the injections alter atherosclerosis development long term. This will help to inform us as to whether the treatment of the cell injections is durable or whether individuals will require periodic sets of injections throughout their lives. After a set of 6 cultured cell injections over 12 weeks, recipient animals will be maintained for an additional 22 weeks without injections. Afterwards, we will assess the aortas for lesion quantity and lesion characteristics and will also assess serial serum cytokine levels to gauge the longer-term effect of the cell injections. A milestone will be assessment of aortic atherosclerosis development and serial cytokine levels. We would expect to see the lesion quantity remains less than control animals over time with more favorable characteristics such as thicker fibrous cap and lower lipid content.

[00150] While enormous progress has been made in reducing the health burden of atherosclerosis [1-3], atherosclerosis remains a significant public health problem. Advances in revascularization technologies such as percutaneous angioplasty and bypass surgeries have brought significant relief to patients with atherosclerosis. Public health initiatives such as smoking cessation have resulted in significant reductions in atherosclerosis incidence. Medical therapies, particularly statin medications can significantly slow disease progression at high doses [4, 5]. Still, nearly 30 million Americans suffer from atherosclerosis affecting the coronary, cerebrovascular and peripheral arteries [6, 7], and atherosclerosis is responsible for 850,000 deaths per year [7], There is a clear need for additional treatment modalities for atherosclerosis. Cell-based therapies are a promising ne treatment modality that can be complementary to current clinical practice by attacking atherosclerosis from a different mechanistic angle. Current medical therapies such as antihypertensive and antihypercholesterol emic drags modify risk factors to reduce arterial injury. Cell-based therapies may work in part by directly repairing and restoring injured portions the arterial wall.

[00151] Harnessing the repair functions of bone marrow derived stem and progenitor ceils is an intriguing opportunity to halt atherosclerosis development by directly reversing arterial wall injury caused by risk factors such as tobacco use, hyperlipidemia and hypertension. The landmark study by Linton et al. demonstrated that replacing the BMCs of apoE^' mice with that of wild type mice resulted in a significant reduction in atherosclerosis [8]. In this instance, the wild type BMCs reversed the hyperlipidemic state of the apoE ^'mice; put another way, the new cells repaired the defect in lipid metabolism, thereby halting atherosclerosis development. Building on the tenants of this work, Rauscher et al, gave regular intravenous injections of BMCs from donor apoE'^" mice to recipient apoE" mice and demonstrated a significant reduction in atherosclerosis development [9]. This reduction in disease occurred even though the serum cholesterol levels in the recipient mice remained extremely high. The investigators showed with BMCs carrying lacZ, that the infused cells adhered directly to regions of the arterial wall that otherwise would contain atherosclerosis lesions, suggesting that the disease reduction was due, in part, to replacement of endothelium by the infused cells. Subsequent work by Zhang et al. [10] using the vein graft model, Sata et al. using a ROSA bone marrow transplantation model [1 1 ] and Hu et al. using an aorta allograft transplantation model [12] support the assertion that bone marrow ceils participate in arterial repair, at least in part, through direct replacement. In each of these studies, cells from the bone marrow adhere to disease-prone areas of the vasculature. Further work by Vemulapalli et al. utilized PET imaging to track infused BMCs and demonstrated that infused cells homed to the vasculature of the recipient apoE^1' mice [13]. From these studies, there is substantial data supporting the role of bone marrow derived stem/progenitor cells in repairing and maintaining the health of the arterial wall. Furthermore, prior work by Rauscher et al. indicate that exogenous injections of BMCs can augment the endogenous cell mediated repair process and prevent atherosclerosis in animal models.

[00152] While exogenous intravenous injections of BMC hold significant promise as a new and effective treatment for atherosclerosis, a clear methodology has not been reported that leads to consistent and optimal atherosclerosis reduction. For example, there have been a number of follow-on studies of exogenous BMC injections in the apoE^1' mouse that have shown inconsistent disease reductions. Studies by Siivestre et al. and George et al. actually demonstrated an increase in atherosclerosis in apoE" mice following BMC injections [14, 15] while two studies from other laboratories, demonstrated a modest reduction in atherosclerosis follo wing BMC injections [16, 17]. A recent review of the literature by Zampetaki et al. found that the different studies of BMC injections utilized different experimental conditions [ 18]. Each group used different cell fractions, different frequency of injections, different total injections and different methods of processing the BMCs prior to injection. In considering the entire body of work, it is clear that exogenous BMC injections directly modulate atherosclerosis development, however the method for obtaining reliable and robust reductions in atherosclerosis was not defined. In a recently published report, we describe a method for generating BMCs that reduces atherosclerosis in apoE^1" mice following multiple regular infusions [19]. Our new method introduces a new step of culturing the BMCs in a combination of cytokines and growth factors prior to infusion and generates BMCs that significantly reduce atherosclerosis in a reproducible and robust manner.

[ΘΘ153] Over the past several years, our laboratory has tested various permutations of ceil therapy for atherosclerosis in the apoE^1' mouse. We have studied different cell fractions of BMCs, the number of injections, gene manipulation of BMCs using RNA interference prior to injection etc. From our results, we have discovered that culturing Lineage negative (Lin-) BMCs in a combination of cytokines and growth factors significantly augments their ability to reduce atherosclerosis, and we are able to induce consistent reduction in atherosclerosis from experiment to experiment. In a typical embodiment, the pretreatment process involves culturing the Lin- BMCs in IL3, IL6 and SCF prior to intravenous injection. This methodology can be translated to human subjects as a robust method for reducing atherosclerosis in patients with a propensity for atherosclerosis and its thromboembolic complications.

[00154] In the setting of clinical practice, this therapeutic modality would typically be deployed through medical centers that have the capability to perform bone marrow transplantation. They would have the necessary equipment and personnel for the BMC harvest, processiiig/culturing and intravenous injections. In one embodiment, a product would be a cell pretreatment kit that contains the cytokine/growth solution with accompanying treatment protocols. The target patients would typically be those with accelerated atherosclerosis and thromboembolic complications regardless of aggressive risk factor modification. These would include patients with a propensity for chronic arterial injury from conditions such as diabetes, resistant hypercholesterolemia and end stage renal disease, in practice, patients would undergo a large volume bone marrow harvest. The medical center staff would process the BMCs using the cell pretreatment kit and protocols. The generated cells would be divided into aliquots and frozen for future intravenous injections. The subjects would receive periodic for an intravenous infusion of their own pretreated BMCs.

[00155] Our interest in pushing forward the idea of using cell-based therapy to treat atherosclerosis is driven by our discovery that culturing BMCs in a combination of cytokines and growth factors prior to infusion leads to a significant and reliable atherosclerosis reduction in apoE^'1' mice. Over the past several years, we tested different experimental protocols to define the optimal conditions for cell-based therapy of atherosclerosis. We investigated the effects of injecting different ceil populations and different durations of therapy [19]. For example, we found that apoE"^' mice receiving 3 or fewer injections of cells developed more atherosclerosis than the control mice receiving PBS buffer injections. This finding confirmed the results from prior studies of exogenous BMC injections [14, 15]. In the course of our studies, we identified that using the Lin- BMC fraction and culturing the Lin- BMCs in fetal bovine serum and hydrocortisone prior to injection resulted in the greatest reduction in atherosclerosis [19]. However, the reduction in atherosclerosis was modest. Therefore, we began to explore the use of a culturing step for the BMCs that employed cytokines and growth factors. Prior work in the hematology field indicated that culturing BMCs in a combination of IL3, IL6 and SCF led to an expansion of the primitive hematopoietic precursor cells such as the long-term culture initiating cell as well as other progenitor cell fractions [20]. Our hypothesis was this approach would expand the number of available progenitor cells and significantly improve atherosclerosis reduction. The results our of subsequent studies show that infusing Lin- BMCs that were first cultured in media containing IL3, IL6 and SCF resulted in robust and consistent reductions in atherosclerosis compared to infusions of fresh, uncultured Lin- BMCs [19].

[00156] The Lin- apoE"'^~ BMCs are cultured for 4 days in 1L3, 1L6 and SCF and then injected via the tail vein into 6 week-old apoE^"'^"" recipient mice. We have studied three experimental groups: mice infused with (A) PBS buffer solution (B) IxTO⁶ freshly harvested Lin- BMCs and (C) lxl 0⁶ cultured Lin- BMCs (FIG. 9). Infusions are performed every 2 weeks for 12 weeks. At 20 weeks of age, the mice are sacrificed. Aortas are carefully dissected and stained with Oil Red O to detect atherosclerotic lesions. Computerized image analysis is used to calculate the ratio of stained to total area (FIG. 9). As FIG. 2A shows, mice treated with cultured Lin- BMCs have significantly less atherosclerosis than mice treated with fresh Lin- BMCs or PBS. Fresh Lin- BMCs cause a modest, non-significant reduction in atherosclerosis relative to PBS. In this set of experiments, culturing of the Lin- BMCs leads to a reduction in atherosclerosis of o ver 70% compared to mice receiving injections of PBS buffered solution, [00157] We have developed a methodology that reduces atherosclerosis in a consistent and robust manner. Our findings support the concept of using cell-based therapies as a new method for treating atherosclerosis. A clinical application of our methodology would be to perform periodic intravenous injections of pretreated human BMCs in humans with high propensity for aggressive atherosclerosis. In such a scenario, the patient would undergo a large volume bone marrow harvest. The harvested BMCs would be cultured in a solution of human IL3, IL6 and SCF. The resulting cells would be divided into treatment size aliquots that would be frozen down and later thawed prior to each injection.

[ΘΘ01] Specific Aim 1 : Determine the necessary alterations to our current ceil processing method to compensate for the negative impact of the freeze-thaw cycle on the ability of the pretreated BMCs to reduce atherosclerosis.

[0002] In a human clinical setting, cells will be collected through a single harvest, processed, frozen and thawed prior to each injection. This is in contrast to animal research studies in which cells are freshly prepared from donor animals prior to injection. While there are differences in the survival rates of human and mouse undergoing freeze-thaw, it is still important to determine the magnitude of the likely negative impact of the freeze-thaw cycle on atherosclerosis reduction and to identify compensatory steps. Our studies of mouse BMC viability following freeze-thaw indicates a reduction in the pool of potential repair ceils, we hypothesize that increasing the dose of cells injected or performing the culturing process after the freeze-tha cycl e may compensate for the effects of the freeze-thaw cycle,

10003] We have found that the freeze-thaw process negatively impacts the abi lity of cultured Lin- BMCs to reduce atherosclerosis. A large volume of BMCs was harvested and pooled from several donor apoE'^~ mice. The Lin- cells were isolated and cultured for 4 days in the cytokine/growth factor solution. The cells were diluted in PBS and 10% FCS to an approximate concentration of 10 million cells per mi to which 10% DMSO was added. Then aliquots of lml were added to cryo-vials on ice. The vials exposed to lower temperatures in a stepwise fashion over time until finally being stored in in liquid nitrogen. Aliquots underwent fast thaw in a 37° water bath prior to injection. The cell pellets were washed and resuspended in PBS for tail vein injection, injections of the previously frozen cultured Lin- BMCs were performed in 6 week apoE'^~ recipient mice every two weeks until 18 weeks of age. Approximately IxlO⁶ cells were injected into the tail vein with each treatment. At 20 weeks of age, the aortas were removed and analyzed for atherosclerosis burden by Ed Herderick of EEHScierice. The previously frozen cultured Lin- ceils induced a significant reduction in atherosclerosis induced by (FIG. 10) with an overall 35% reduction in atherosclerosis relative to animals receiving PBS. In prior experiments utilizing cultured Lin- BMCs that were not previously frozen, there was a 70% atherosclerosis reduction (FIG. 2A). Viability analysis showed many BMCs did not survive the freeze- thaw. Therefore, while the overall reduction in disease using the previously frozen cultured Lin- BMCs was still substantial, the objective of Specific Aim 1 is to determine whether we can compensate for the freeze-thaw effect by using a larger dose of stem cells and by altering the sequence of freezing and culturing.

[0004] One change that can be implemented is to freeze the BMCs using commercial grade controlled rate freezers. This more accurately reflects a true clinical application. We anticipate this change will also help to compensate for the effects of freeze-thaw by maximizing the viability of our cell preparations.

[0005] To compensate for reduced cell viability induced by the freeze-thaw cycle, we will increase the number of cells delivered with each injection. We will use the same methods described in the data section above. BMCs from several apoK'^" mice will be pooled. The Lin- fraction will be isolated and cultured for 4 days in MEM, 10% FBS with IL3 (10 ng/ml), IL6 (20 ng/ml) and SCF (10 ng/ml). Aliquots will be frozen in 10% DMSO using the commercial grade control rate freezer with final storage in liquid nitrogen. The apoE'^~ recipient mice will begin receiving treatments at 6 weeks of age every two weeks until at 18 weeks of age. There will be three injection groups: (I ) PBS, (2) standard I xlO⁶ cell dose and (3) augmented 3x10⁶ cell dose. Total volume injected is 0.1ml. We are not using a higher dose than 3xl0⁶ cells as we have found this causes mouse mortality due to cell embolization. There will be 20 animals per treatment group. At 20 weeks of age, the aortas will be harvested and analyzed for disease burden by EEHScience. EEHScience will be blinded to the treatment groups. Briefly, as in our prior work the aortas are incubated with Oil Red O to stain the atherosclerotic lesions [19, 25]. Each aorta undergoes computerized optical analysis that creates an overall map of the aorta surface. The maps are then normalized to a single standard template. The software then maps and quantifies the red-stained lesions to determine the proportion of the total aorta that contains atherosclerosis.

[ΘΘ06] Following the BMC harvest and Lin- cell isolation, the BMCs will, be aliquoted and frozen down without undergoing culturing. We will perform the culturing step after the cells are thawed prior to each injection. The culture step expands the number of primitive precursor ceils [20] that may compensate for cell number loss induced by the freeze-thaw cycle. Other than switching the timing of the culturing step, we will use the same method as described above. There will be three treatment groups: (a) PBS control, (b) Lin- BMCs cultured prior to freeze- thaw and. (c) Lin- BMCs cultured after freeze-thaw. Each apoE^{' "} recipient mice will receive lxlO⁶ of either cell preparation or PBS buffer every two weeks by tail vein injection beginning at 6 weeks of age until 18 weeks of age. Aortas will be collected at 20 weeks of age and analyzed by EEHScience.

[0007] Specific Aim 2: Determine whether infusions of pretreated whole bone marrow will lead to a similar reduction in atherosclerosis as pretreated Lin- bone marrow,

[0008] Our studies have used the Lin- fraction of the bone marrow that make up 5- 10% of total BMCs. In human clinical applications, this will limit the available cells for therapeutic purposes. Furthermore, the Lin- cell isolation step would be performed sterilely by a GMP facility, increasing time and costs. We will determine if whole BMCs cultured in the cytokine/growth factor solution can also significantly reduce in atherosclerosis.

[0009] Our data indicates that whole BMCs may indeed induce similar reductions in atherosclerosis as cultured Lin- BMCs. Using the same experimental model described above, we compared previously frozen cultured whole apoE'^~ BMCs to previously frozen cultured Lin- BMCs. Each apoE^1' recipient mouse received 1x10° of either the cultured whole BMCs or the cultured Lin- BMCs. We found that the amount of atherosclerosis reduction approximately 35% in both groups. The number of animals in each treatment group is small and further confirmatory' studies are needed.

[0010] The experimental procedures with regards to cell harvesting, culturing, freezing, thawing and injection will be the same as described in Aim 1 . The culturing step will be performed prior to freeze-thaw. A commercial grade control rate freezer will be used for the freezing process. We will compare four injections: (1) PBS, (2) standard dose of 1 x10⁶ cultured whole B .VIC s. (3) high dose of 3x10^b cultured whole BMCs, and (4) standard dose of 1x10⁶ cultured Lin- BMCs. As before, we will perform injections every 2 weeks in apoE^''^~ recipient mice from 6 weeks of age to 18 weeks of age. Aortas will be collected at 20 weeks of age and analyzed by EEH Science.

[0011] It is expected that cultured whole BMCs will have similar ability to reduce atherosclerosis as cultured Lin- BMCs. We also expect that the higher dose of cultured whole BMCs will induce a greater atherosclerosis reduction relative to the lower ceil dose by compensating for the freeze-thaw cycle.

[0012] Following 3 injections over 6 weeks of either cultured Lin- BMCs or Fresh Lin- BMCs, levels of pro-inflammatory cytokines IL-1 beta and IL-17 levels were similar in the recipient apoE'^~ mice (FIG. 3 3). However, anti-inflammatory cytokine IL-10 and the pleiotropic cytokine IL-6 levels were significantly higher in mice receiving the cultured BMCs (FIG. 1 1). This could represent a mechanistic contribution of the cell injections leading to atherosclerosis reduction or may simply represent decreased atherosclerosis. In either case, the cytokine levels in the recipient animals can inform us as to the presence of the cell infusion effect over time. This in conjunction with histopathologv and immunohistochemistry analysis of the aortic tissues can help us determine the persistence of the effect of the cell injections.

[0013] Recipient mice will receive injections of either PBS or cultured Lin- BMCs from apoE'^" mice that carry lacZ starting at 6 weeks of age every two weeks until 18 weeks of age. The recipient mice will be maintained without injections until 38 weeks of age when they are sacrificed. The aortas will be quantified for atherosclerosis to determine whether the disease reduction persists even in the absence of cell injections. Aortic, bone marrow and spleens will undergo histopathoiogy analysis to detect the presence of lacZ^' positive cells to determine whether the injected cells become long-term residents in recipient animals potentially influencing disease. Aortic tissues from the aortic root, thoracic and abdominal regions will also undergo histopathoiogy analysis to characterize the atherosclerosis lesions for the fibrous cap, lipid content and inflammatory cell content to determine whether there is a long-term ameliorative modification of the lesions. For example, thicker fibrous cap, reduced lipid content or reduced inflammatory cell content. Finally, cytokine levels for the recipient mice will be monitored every 4 weeks beginning at 6 weeks of age until day of sacrifice. We would expect to see a similar pattern of cytokine levels as in our preliminary data as long as there is an effect exerted by the injected cells.

[0001] Cell therapy with bone marrow derived stem/progenitor cells holds great promise as an effective new treatment for atherosclerosis. Culturing the cells prior to injection in a specific combination of cytokines arid growth factors appears to improve substantially the consistency and magnitude of atherosclerosis reduction in animal models.

REFERENCES American Heart Association, Heart Disease and Stroke Statistics - 2007 Update, 2007, American Heart Association; Dallas, TX.

Greenland, P., S.C.J. Smith, and S.M. Grundy, Improving coronary heart disease risk assessment in asymptomatic people: role of traditional risk factors and noninvasive cardiovascular tests. Circulation, 2001. 104(15): p. 1863-7.

Zerhouni, E. , Fiscal Year 2004 President's Budget Request, 2003.

Cannon, CP., et al., Intensive versus moderate lipid lowering with statins after acute coronary syndromes. N Engl J Med, 2004. 350(15): p. 1495-504.

Nissen, S.E., et al, Effect of intensive compared with moderate lipid-lowering therapy on progression of coronary atherosclerosis: a randomized controlled trial. JAMA, 2004. 291(9): p. 1071 -80.

Allison, M.A., et al., Ethnic-specific prevalence of peripheral arterial disease in the United States. Am J Prev Med, 2007. 32(4): p. 328-33.

Lloyd- Jones, D., et al, Heart Disease and Stroke St tistics-2009 Update: A Report From the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation, 2009. 119(3); p. e21-el 81.

Linton, M.F., J.B. Atkinson, and S. Fazio, Prevention of atherosclerosis in apolipoproteln E~deficient mice by bone marrow transplantation. Science, 1995. 267(5200); p. 1034-7. Rauscher, F., et al., Aging, Progenitor Cell Exhaustion, and Atherosclerosis. Circulation, 2003.

Zhang, L., et al., Vein graft neointimal hyperplasia is exacerbated by tumor necrosis factor receptor- 1 signaling in graft-intrinsic cells. Arterioscler Thromb Vase Biol, 2004. 24(12): p. 2277-83.

Sata, M., et al, ... stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nature Medicine, 2002.

Flu, Y., et al, Endothelial replacement and angiogenesis in arteriosclerotic lesions of allografts are contributed by circulating progenitor cells. Circulation, 2003. 108(25): p. 3122-7^'

Vemulapalli, S., et al, Cell therapy in murine atherosclerosis: in vivo imaging with high- resolution helical SPECT. Radiology, 2007. 242(1): p. 198-207. Silvestre, J.-S., et al., Transplantation of bone marrow-derived mononuclear cells in ischemic apolipoprotein E-knockout mice accelerates atherosclerosis without altering plaque composition. Circulation, 2003. 108(23): p. 2839-42.

George, J., et a!., Transfer of endothelial progenitor and bone marrow cells influences atherosclerotic plaque size and composition in apolipoprotein E knockout mice. Arterioscler

Thromb Vase Biol, 2005. 25(12): p. 2636-41.

Nelson, et al., Sex-Dependent Attenuation of Plaque Growth After Treatment With Bone Marrow Mononuclear Cells. Circ Res, 2007.

Zoll, J., et al., Role of human smooth muscle cell progenitors in atherosclerotic plaque development and composition. Cardiovascular Research.

Zampetaki, A., J.P. Kirton, and Q. Xu, Vascular repair by endothelial progenitor cells. Cardiovascular Research, 2008. 78(3): p. 413-21.

Song, X., et ah, Will periodic intravenous injections of conditioned bone marrow cells effectively reduce atherosclerosis? Antioxidants &amp; redox signaling, 201 1.

oller, MR., et al., Expansion of primitive human hematopoietic progenitors in a perfusion bioreactor system with IL-3, IL-6, and stem cell factor. Bio/technology (Nature Publishing Company), 1993. 11(3): p. 358-363.

Strong, J., et al., The PDA Y Study: natural history, risk factors, and pathobiology. Annals of the New York Academy of Sciences, 1997. 811(1 Atherosclerosis IV): p. 226-237.

Hixson, J., ... atherosclerosis in young males. Pathobiological Determinants of Atherosclerosis in Youth (PDA Y) .... Arteriosclerosis, 1991.

Gidding, et al., PDAY risk score predicts advanced coronary artery atherosclerosis in middle-aged persons as well as .... Atherosclerosis, 2007.

Malconi, G., et al., ... of coronary heart disease risk factors in autopsied young adults from the PDAY Study with living .... Cardiovascular Pathology, 2007.

arra, R., et al., Molecular evidence for arterial repair in atherosclerosis. Proc Natl Acad Sci U S A, 2005. 102(46): p. 16789-94.

Seo, D., et al., Gene expression phenotypes of atherosclerosis. Arterioscler Thromb Vase Biol, 2004. 24(10): p. 1922-7.

Other Embodiments

[0001] Any improvement may be made in part or all of the compositions, kits, and method steps. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference, The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended to illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. The kits, compositions and methods described herein, for example, can be applied to any patient with atherosclerosis or a propensity to develop atherosclerosis due to family history, risk factors or visualization of disease by diagnostic imaging. Any statement herein as to the nature or benefits of the invention or of the preferred embodiments is not intended to be limiting, and the appended claims should not be deemed to be limited by such statements. More generally, no language in the specification should be construed as indicating any non-claimed element as being essential to the practice of the invention. This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contraindicated by context.

[0002] What is claimed is:

Claims

1. A method of treating atherosclerosis in a subject comprising:

obtaining whole bone marrow or lin- bone marrow cells (BMCs) cells from the subject, a donor or tissue culture bank;

culturing the whole bone marrow or lin- BMCs ex vivo in a medium comprising stem cell factor (SCF) and at least one of: IL-3 and IL-6; and

administering to the subject at least one dose of the cul tured whole bone marrow or BMCs in an amount effective to repair damaged arterial walls and reduce atherosclerosis in the subject.

2. The method of claim 1, wherein the whole bone marrow or lin- BMCs are autologous,

3. The method of claim 1, wherein the whole bone marrow or lin- BMCs are allogeneic or syngeneic.

4. The method of claim 1, wherein the subject has diabetes or end-stage kidney disease,

5. The method of claim 1, wherein the whole bone marrow or lin- BMCs differentiate into at least one lineage of progenitor cells.

6. The method of claim 1, wherein the lin- BMCs are identified by at least one marker selected from the group consisting of: hepatocyte growth factor (HGF), lymphoid enhancer- binding factor 1 (Left) and CdkSrl .

7. The method of claim 1, wherein the lin- BMCs are isolated from bone marrow of the subject or a donor.

8. The method of claim 1, wherein the medium comprises SCF, IL-3, and IL-6.

9. The method of claim 1 , wherein administration of the cultured whole bone marrow or BMCs to the subject results in at least a 30% reduction in atherosclerosis in the subject.

10. The method of claim 9, wherein administration of the cultured whole bone marrow or BMCs is effective for treating at least one associated condition of atherosclerosis selected from the group consisting of: myocardial infarction, stroke, coronary artery disease, and peripheral- arterial disease.

11. The method of claim 1 , wherein the cultured whole bone marrow or BMCs are administered to the subject by a delivery route selected from the group consisting of: intravenous administration, administration of a cultured whole bone marrow or BMCs-impregnated implantation device, and administration directly to a target site.

12. The method of claim 11, wherein the cultured whole bone marrow or BMCs-impregnated implantation device is a cultured whole bone marrow or BMCs-impregnated stent.

13. A kit comprising:

a cell culture medium comprising SCF and at least one of: IL-3 and IL-6;

packaging; and

instructions for use,

14. The kit of claim 13, wherein the cell culture medium comprises SCF, IL-3 and IL-6,

15. A method of preparing whole bone marrow cells or BMCs for administration to a subject having atherosclerosis comprising:

obtaining whole bone marrow cells or lin- BMCs from the subject, a donor or tissue culture bank;

culturing the whole bone marrow cells or lin- BMCs ex vivo in a medium comprising SCF and at least one of: IL-3 and IL-6 under conditions such that the whole bone marrow ceils or BMCs differentiate into at least one lineage of progenitor cells and a sufficient amount of differentiated whole bone marrow cells or BMCs are generated for repairing damaged arterial walls and reducing atherosclerosis in a subject having atherosclerosis,

16. The method of claim 15, wherem the whole bone marrow cells or lin- BMCs are autologous.

17. The method of claim 15, wherein the whole bone marrow cells or lin- BMCs are allogeneic or syngeneic.

18. The method of claim 15, wherein the subject having atherosclerosis also has diabetes or end-stage kidney disease.

19. The method of claim 15, wherein the lin- BMCs are identified by at least one marker selected from the group consisting of: HGF, Lefl and Cdk5rl .

20. The method of claim 15, wherein the lin- BMCs are isolated from bone marrow of the subject having atherosclerosis or a donor.

21. The method of claim 15, wherein the medium comprises SCF, IL-3, and IL-6.

22. The method of claim 15, wherein administration of the cultured whole bone marrow cells or BMCs to the subject having atherosclerosis results in at least a 30% reduction in

atherosclerosis in the subject.

23. The method of claim 15, further comprising the step of impregnating a biocompatible implantable device with the cultured whole bone marrow cells or BMCs.

24. A method of screening whole bone marrow cells or lin- BMCs from a subject for arterial wall reparative capability comprising:

a) obtaining a first sample of whole bone marrow cells or lin- BMCs from the subject; b) extracting RNA from the first sample of whole bone marrow cells or lin- BMCs and assessing the similarity of a gene expression profile of the first sample of whole bone marrow cells or lin- BMCs to a gene expression profile of fresh murine BMCs from mice with mild, moderate and severe disease using a microarray analysis; c) correlating the similarity of the gene expression profi le of the first sample of whole bone marrow cell s or lin- BMCs to the gene expression profile of the fresh murine BMCs with the reparative capacity of the first sample of whole bone marrow cells or lin- BMCs;

d) obtaining a second sample of whole bone marrow cells or lin- BMCs from the subject and culturing the second sample of whole bone marrow cells or lin- BMCs in a medium comprising SCF and at least one of: IL-3 and IL-6 under conditions such that the whole bone marrow cells or lin- BMCs differentiate into at least one lineage of progenitor cells;

e) extracting RNA from the cultured second sampl e of whole bone marrow ceils or BMCs and assessing the similarity of a gene expression profile of the cultured second sample of whole bone marro w cel ls or BMCs to a gene expression profi le of fresh murine BMCs from mice with mild, moderate and severe disease and to a gene expression profile of BMCs from mice with mild, moderate and severe disease cultured according to step e) using a microarray analysis; and

f) correlating the similarity of the gene expression profile of the cul tured second sample of whole bone marrow cel ls or BMCs to the gene expression profile of the fresh murine BMCs and to the gene expression profile of the BMCs from mice with mild, moderate and severe disease cultured according to step e) with the reparative capacity of the cuitured second sample of whole bone marrow cells or BMCs,

wherein an increase in reparative capacity of the cultured second sample of whole bone marrow cells or BMCs relative to the reparative capacity of the first sample of whole bone marro ceils or lin- BMCs indicates a restoration in reparative capacity in the cultured second sample of whole bone marrow cells or BMCs due to the culturing step of step e).