Methods and compositions for modulating stem cells

FIELD OF THE INVENTION

[0002] The present invention generally relates to methods for enriching stem cell population and for modulating stem cell differentiation, as well as to therapeutic applications of such methods. More particularly, the invention pertains to genes differentially expressed in hematopoietic stem cells and to methods of using these genes to modulate stem cell differentiation.

BACKGROUND OF THE INVENTION

[0003] Hematopoiesis (hemopoiesis) is a process whereby multi-potent stem cells give rise to lineage-restricted progeny. The molecular basis of hematopoiesis remains poorly understood. Hematopoietic stem cells (HSCs) are the only cells in the hematopoietic system that produce other stem cells and give rise to the entire range of blood and immune system cells. These cells are able to self-proliferate, so as to maintain a continuous source of regenerative cells. When subject to particular environments and/or factors, they can differentiate to dedicated progenitor cells, where the dedicated progenitor cells may serve as the ancestor cell to a limited number of blood cell types.

[0004] HSCs and their progenies at the various development stages all play an important role in the normal function of the mammalian immune system. HSCs are of prominent therapeutic importance in many circumstances. In many diseased states, the disease is a result of some defect in the maturation process. In other situations, such as transplantation, there is a need to prevent the immune system from rejecting the transplant by irradiating the host. In neoplasia, a patient may be irradiated and/or treated with chemotherapeutic agents to destroy the neoplastic tissue, which often also damage or destroy the host immune system. Further, other situations such as a severe insult to the immune system also result in a substantial reduction in stem cells and injury to the immune system. In all these situations, it will frequently be desirable to restore stem cells to the host. For example, HSCs are the active component in bone marrow transplantation (BMT), and transplant of highly purified HSC will completely restore the hematopoietic system in a manner indistinguishable from unfractioned bone marrow.

[0005] Despite decades of research, there are currently no satisfactory methods to expand the numbers of HSCs or accurately enumerate the numbers of expanded and engraftable HSCs cells following in vitro culture. There is a need in the art for better methods for isolating, enriching, and enumerating transplantable HSCs. The instant invention fulfills this and other needs.

SUMMARY OF THE INVENTION

[0006] In one aspect, the invention provides methods for inhibiting differentiation of mammalian stem cells. The methods entail (a) providing a population of stem cells, (b) introducing a vector comprising an HSC differentiation-inhibiting polynucleotide of the present invention into the stem cells, and (c) expressing a polypeptide encoded by the polynucleotide by culturing the modified stem cells, thereby inhibiting differentiation of the stem cells. In some of the methods, the stem cells are isolated from bone marrow. In some preferred methods, the stem cells are human hematopoietic stem cells. The human stem cells can be first selected for expression of CD38 and Thy prior to introduction of the vector. In some of the methods, the HSC differentiation-inhibiting polynucleotide encodes GATA-binding protein 3 or ID3.

[0007] In a related aspect, the invention provides methods for increasing the effective dose of hematopoietic stem cells in a mammalian subject. The methods require (a) providing a population of hematopoietic stem cells, (b) introducing into the cells an HSC differentiation-inhibiting polynucleotide of the present invention, and c) administering the genetically modified cells that express an HSC differentiation-inhibiting polypeptide to a mammalian subject; thereby increasing the effective dose of hematopoietic stem cells in the subject. In some of these methods, the administered stem cells are a subpopulation of the modified cells that are selected for expression of the polypeptide prior to administering to the subject. In some preferred methods, the subject is human, and the hematopoietic stem cells are human hematopoietic stem cells. In these methods, the hematopoietic stem cells can be selected for expression of CD34 and Thy prior to introducing into the cells the HSC differentiation-inhibiting polynucleotide.

[0008] In another related aspect, the present invention provides methods for inhibiting hematopoietic stem cell differentiation using an HSC differentiation-inhibiting polypeptide identified by the present inventor. The methods entail contacting a population of HSCs with an effective amount of the HSC differentiation-inhibiting polypeptide which inhibits differentiation of the HSCs. In some of the methods, the HSCs are present in an in vitro cell culture. In some other methods, the HSCs are present in a subject grafted with the HSCs. In some preferred methods, the subject is human.

[0009] In another aspect, the invention provides methods for isolating a population of cells that are enriched for hematopoietic stem cells (HSCs). These methods comprise (a) obtaining a sample of cells containing hematopoietic stem cells, (b) selecting cells from the sample based on expression or lack of expression of at least one known HSC surface marker, and at least one novel HSC molecule marker identified in the present invention, and (c) separating cells with the known HSC marker and at least one of the novel molecule markers; thereby isolating a population of human cells enriched for hematopoietic stem cells.

[0010] Preferably, the hematopoietic stem cells enriched with these methods are human HSCs. In some methods, the known human HSC marker is CD34+ and Thy+. In some of the methods, the at least one novel HSC marker is a human HSC surface molecule identified in the present invention.

[0011] In another aspect, the invention provides methods for enumerating hematopoietic stem cells in a population of cells. The methods entail (a) contacting the population of cells with an antibody that specifically binds to one novel HSC surface marker identified in the present invention under conditions that allow the antibody to specifically bind to the HSC surface marker, and (b) quantifying the cells recognized by the antibody; thereby enumerating hematopoietic stem cells in the population of cells. In some of these methods, the hematopoietic stem cells are human HSCs, and the population of cells are first selected for expression of CD34 and Thy prior to the contacting.

[0012] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]
FIG. 1 shows schematic structure of expression vectors for overexpressing various HSC differentiation-inhibiting genes.

[0014]
FIG. 2 shows that ID3 over-expression increases the number of colony forming cells in CFC assay.

[0015]
FIG. 3 shows upregulated expression of various transcription factors in mouse HSCs.

DETAILED DESCRIPTION

[0016] I. Overview The present invention is predicated in part on the discovery by the present inventor that a number of genes are differentially expressed in hematopoietic stem cell populations (see Examples below). It was also found that some of these HSC genes slow down HSC differentiation or enhance HSC activities when they are overexpressed in HSCs. These genes are therefore termed HSC differentiation-inhibiting genes.

[0017] Using HSCs enriched from blood of normal human donors, it was found that sequences upregulated in the human HSCs include genes encoding hormones, enzymes, histone, transcription factors, secreted proteins, surface markers, and other molecules. Table 1 lists examples of these genes that are upregulated in human HSCs (CD4+Thy+) as compared to non stem cells (CD4+Thy−). Further, using HSCs isolated from two different sources, bone marrow and peripheral blood, the present inventor identified a set of genes that are differentially expressed in HSCs from both sources. Some of these genes are shown in Table 2.

[0018] Similarly, in a mouse HSC population (CD34−CD38+), a number of genes encoding proteins with diverse biochemical and cellular functions were also upregulated, including genes encoding surface antigens, transcription factors or growth factors (see Tables 3 and 4). These novel HSC genes are enriched in HSCs compared to their differentiated progeny (e.g., CD34+ CD38+ progenitor cells) or CD34+CD38− facilitator cells.

[0019] Without being bound in theory, the molecules upregulated in HSCs could play various functions in modulating HSC growth and differentiation, as well as regulating activities and functions of progenitor cells that differentiated from the HSCs. For example, increased levels of some of the surface receptors, growth factors, and secreted proteins shown in Table 2 could act in synergy in inhibiting HSC differentiation and promoting their expansion.

[0020] In accordance with these discoveries, the present invention provides methods for modulating HSC differentiation. Inhibition of HSC differentiation allows continued growth and expansion of the HSC population, and therefore provide engraftable HSCs with increased dosage and higher potency. A number of the upregulated HSC genes identified herein (e.g., shown in Tables 1, 3, and 4) can potentially function as HSC differentiation-inhibitors. For example, polypeptides encoded by the novel HSC genes disclosed herein (e.g., the growth factors or hormones shown in Table 2) can be used to inhibit HSC differentiation in vitro (e.g., by applying to an HSC cell culture) and in vivo (e.g., by administering to a subject engrafted with bone marrow or HSCs). Differentiation inhibiting activities of these molecules were exemplified by GATA3 and ID3 as shown in the Examples below.

[0021] As indicated by the GenBank accession numbers or other identification numbers or descriptions in Tables 1, 3, and 4, sequences of the upregulated human and mouse HSC genes disclosed herein are all known in the art. Thus, as detailed below, the HSC differentiation-inhibiting polynucleotide sequences can be easily obtained commercially, from the sources disclosed in the public databases, or isolated using routine techniques of molecular biology. The encoded polypeptides can also be obtained commercially or easily produced with standard procedures of recombinant techniques.

[0022] The invention also provides methods for isolating and enriching HSCs. The currently known HSC markers are not satisfactory because they cannot accurately predict homogeneity and hematopoiesis activities of cells bearing the markers. The discovery of genes differentially expressed in HSCs provides novel molecular markers for selecting and enriching HSCs. For example, antibodies against novel surface markers disclosed in the present invention (e.g., those in Tables 2, 3, 4 and 5) can be used to isolate human and mouse HSCs from a crude population of cells (e.g., bone marrow or peripheral blood). The methods can also be directed to cell populations already enriched for one or more of the known HSCs makers (e.g., CD34+, Thy+ in human, and CD38+, c-kit+, Sca1+ in mice). Further enrichment using these novel markers can lead to more homogeneous HSCs with more potent hematopoiesis activities.

[0023] In both the autologous and allogeneic setting, the time to recover from BMT is directly related to the dose of HSCs transplanted. Even a modest 2 to 3-fold expansion of engraftable HSC would afford great benefit to patients by minimizing the duration of cytopenia when patients are most susceptible to infection. Thus, isolation and expansion of more homogeneous HSCs in vitro in accordance with the present invention would make autologous and allogeneic HSC transplantation safer and more effective.

[0024] The practice of the present invention will employ, unless otherwise indicated conventional techniques of cell biology, molecular biology, cell culture, immunology and the like which are in the skill of one in the art. These techniques are fully disclosed in the art, e.g., in Sambrook et al., “Molecular Cloning A Laboratory Manual,” Cold Springs Harbor Laboratory Press (3rd ed. 2001); Carter and Sweet, “Methods of Enzymology,” Academic Press (1997); and Harlow and Lane, “Antibodies, A Laboratory Manual,” Cold Spring Harbor Press (1998).

[0025] The following sections provide more specific guidance for making and using the compositions of the invention, and for carrying out the methods of the invention.

1TABLE 1Genes upregulated in human CD34+Thy+ HSCs from peripheral bloodClassificationNameDescriptionHistoneH2BFLHomo sapiens H2B histone family, memberAHistoneH2AFAHuman histone genesHistoneH2A/lHomo sapiens H2A histone family, member LHistoneH1F2Histone 2A-like protein geneHistoneH2B/hHomo sapiens H2B histone family, member HHistoneHH2A/cHuman histone H2AFC geneHistoneH2AFQHomo sapiens H2A histone family, member QHLAHLA-DPB1Human MHC class II lymphocyte antigen beta chainHLAHLA-DQB1Human MHC class II HLA-DR2-Dw12 mRNA DQw1-betaHLAHLA-EHomo sapiens HLA-E geneSecreted-complementPTSHomo sapiens 6-pyruvoyltetrahydroprotein synthaseSecreted-complementHFL1Human factor H homologue mRNA complete cdsSecreted-growthMDKHomo sapiens midkine (neurite growth-promoting factor 2)factorSecreted-hormoneOXTHomo sapiens oxytocin, prepro-(neurophysin 1) mRNASecreted-hormoneAVPHomo sapiens arginine vasopressin mRNASignaling-GTPR-RasHuman R-rasSignaling-GTPGCHFRHomo sapiens GTP cyclohydrolase I feedback regulatory proteinSignaling-GTPGUCY1A3Homo sapiens guanylate cyclase 1, soluble, alpha 3Signaling-KinaseWAF1Human DNA sequence from PAC 431A14WAF1Signaling-KinaseITPKBHomo sapiens inositol 1,4,5-triphosphate 3-kinase BSignaling-KinasePPKCLHomo sapiens protein kinase C, etaSignaling-KinasePPKCZHomo sapiens protein kinase C, zetaSignaling-SH3SKAP55Homo sapiens src kinase-associated phosphoprotein of 55 kDaStressPTGS2Homo sapiens prostaglandin-endoperoxide synthase 2StressCYP2A13Human cytochrome P450StressCYP2D6Human mRNA for cytochrome P450 dbl variant bStress-apoptosisBCL2A1Homo sapiens BCL-2-related protein 1StructuralCALB1Homo sapiens calbindin 1StructuralElastinHuman elastin geneStructuralKRT18Human mRNA fragment for cytokeratin 18Surface-IgIGMHuman gene for immunoglobulin muSurface-IgVH4Human IgM heavy chain variable V-D-J region (VH4) geneSurface-otherAPPHomo sapiens APP complete sequenceSurface-receptorBDKRB1Human bradykinin B1 receptorSurface-receptorTLR1Human mRNA for KIAA0012 geneSurface-receptor5T4Homo sapiens 5T4 oncofetal trophoblast glycoproteinSurface-receptorEFL-2Homo sapiens EHK1 receptor tyrosine kinase ligandSurface-receptorEV12AHomo sapiens ecotropic viral integration site 2ASurface-receptorFLT3Homo sapiens fms-related tyrosine kinase 3Surface-receptorTNFSF10Human tumor necrosis factor (ligand) superfamily, member 10Surface-receptorLTBHuman lymphotoxin betaSurface-receptorCDW52Homo sapiens mRNA for CAMPATH-1Surface-receptorCLECSF2Homo sapiens C-type lectin (activation-induced)Surface-unknownGliPRHuman glioma pathogenesis-related proteinTransportLRPHomo sapiens Irp mRNATranscription-RUNTAML1Human AML1 proteinTranscription-PAR-bZIPTEFHuman hepatic leukemia factorTranscription-FKHFKHRHomo sapiens forkhead proteinTranscription-MN1Homo sapiens chromosome 22q11.2 MDR regionsuppressorTranscription-bHLHID1Homo sapiens inhibitor of DNA binding 1Transcription-bHLHID3Homo sapiens HLH 1R21 mRNA for helix-loop-helix proteinTranscription-bHLHEPAS1Homo sapiens endothelial PAS domain protein 1Transcription-bHLHID2Homo sapiens inhibitor of DNA binding 2Transcription-GATAHGATA3Homo sapiens GATA-binding protein 3Transcription-HMGhTcf-4Homo sapiens mRNA for hTCF-4Transcription-HOXPHOX1Human homeobox proteinTranscription-HOXMEIS1Homo sapiens MEIS proteinTranscription-RBP-MSHomo sapiens RNA-binding protein gene with multiple slicingslicingTranscription-TCEA2Homo sapiens transcription elongation factor ATranslationUnknownDIF2IEX-1 = radiation-inducible immediate-early geneUnknownHomo sapiens chromosome 17clone hRPC.906_A_24UnknownHomo sapiens chromosome 22q13 BAC clone CIT987SK-384D8UnknownA-362G6.1Human chromosome 16 BAC clone CIT987SK-A-362G6UnknownLST1Homo sapiens LST1 mRNAUnknownKIAA0125Homo sapiens KIAA0125 gene product

[0026]

2

TABLE 2

Genes Upregulated in Human HSCs from both Bone Marrow and Peripheral Blood

Classification
Name
Description

Hormone
AVP

Homo sapiens
arginine vasopressin mRNA

Hormone

Corticotropin releasing hormone-binding protein

Enzyme
GUCY1A3

Homo sapiens
guanylate cyclase 1, soluble, alpha 3

Enzyme
PPKCZ

Homo sapiens
protein kinase C, zeta

Enzyme

Iduronate 2-sulfatase (Hunter syndrome)

Transcription factor
HLF
Human hepatic leukemia factor

Transcription factor
GATA3

Homo sapiens
GATA-binding protein 3

Transcription
Evil

Homo sapiens
ecotropic viral integration site 1

Transcription
PMX1
Paired mesoderm homeo box 1

Transcription
MN1
Meningioma (disrupted in balanced translocation)

Secreted protein

Tetranectin (plasminogen-binding protein)

Secreted protein

H factor (complement)-like 1

Surface molecule

Transient receptor potential channel 1

Surface molecule
DLK1
Delta-like homolog (Drosophila)

Surface molecule
EphA3
Ephrin-A3

Surface molecule
TNFSF10
Human tumor necrosis factor (ligand) superfamily, member 10

Surface molecule

Interferon induced transmembrane protein

Surface molecule

Ecotropic viral integration site 2A

Surface molecule

Sortilin-related receptor, L(DLR class) A rep

Surface molecule

Major histocompatibility complex, class I, E

Surface molecule

KIAA0125 gene product

[0027] II. Definition

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991). In addition, the following definitions are provided to assist the reader in the practice of the invention.

[0029] The term “analog” is used herein to refer to a molecule that structurally resembles a reference molecule but which has been modified in a targeted and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the reference molecule, an analog would be expected, by one skilled in the art, to exhibit the same, similar, or improved utility. Synthesis and screening of analogs, to identify variants of known compounds having improved traits (such as higher binding affinity for a target molecule) is an approach that is well known in pharmaceutical chemistry.

[0030] As used herein, “contacting” has its normal meaning and refers to combining two or more agents (e.g., polypeptides or small molecule compounds) or combining agents and cells (e.g., a polypeptide and a cell). Contacting can occur in vitro, e.g., combining two or more agents or combining a test agent and a cell or a cell lysate in a test tube or other container. Contacting can also occur in a cell or in situ, e.g., contacting two polypeptides in a cell by coexpression in the cell of recombinant polynucleotides encoding the two polypeptides, or in a cell lysate.

[0031] An “effective amount or dose” is an amount sufficient to effect beneficial or desired results. An effective amount may be administrated in one or more administrations. Determination of an effective amount is within the capability of those skilled in the art. Particularly preferred subjects of the invention in general include living mammals such as human, mice and rabbit, most preferred are humans. The administration of an HSC differentiation-inhibiting polypeptide, or a genetically modified cell comprising a polynucleotide sequence of the invention, may be by conventional means, for example, injection, oral administration, inhalation and others. Appropriate carries and diluents may be included in the administration of the polypeptide or the modified cells. Samples including the modified cells and progeny thereof may be taken and tested to determine transduction efficiency.

[0032] The term “fragment” when used in connection with an amino acid sequence means a part of a reference sequence and having at least 10 amino acid residues, preferably 50 amino acids residues, even more preferably 100 amino acid residues and most preferably 200 amino acid residues which are substantially identical to the reference amino acid sequences. Where referring to a nucleotide sequence, the term means a nucleotide sequence including part of the reference sequence and comprising as few as at least 30, 50, 75, 80, 100 or more contiguous nucleotides, preferably at least 200, 300, 400, 500, 600, or more contiguous nucleotides, even more preferably at least 800, 1000, 1500, 2000 or more contiguous nucleotides that are identical to the reference sequence.

[0033] The term “functional equivalent” when referring to a polypeptide means a protein having a like function and like or improved specific activity, and a similar amino acid sequence. In some embodiments, a functionally equivalent is a variant in which one or more amino acid residues are substituted with conserved or non-conserved amino acid residues, or one in which one or more amino acid residues includes a substituent group. Conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and Ile; interchange of the hydroxl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements among aromatic residues Phe and Tyr.

[0034] A “heterologous sequence” or a “heterologous nucleic acid,” as used herein, is one that originates from a source foreign to the particular host cell, or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that, although being endogenous to the particular host cell, has been modified. Modification of the heterologous sequence can occur, e.g., by treating the DNA with a restriction enzyme to generate a DNA fragment that is capable of being operably linked to the promoter. Techniques such as site-directed mutagenesis are also useful for modifying a heterologous nucleic acid.

[0035] The term “homologous” when referring to proteins and/or protein sequences indicates that they are derived, naturally or artificially, from a common ancestral protein or protein sequence. Similarly, nucleic acids and/or nucleic acid sequences are homologous when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Homology is generally inferred from sequence similarity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of similarity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence similarity is routinely used to establish homology. Higher levels of sequence similarity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more can also be used to establish homology. Methods for determining sequence similarity percentages, e.g., BLASTP and BLASTN using default parameters, are well known and described in the art.

[0036] The terms “identical sequence” and “sequence identity” in the context of two nucleic acid sequences or amino acid sequences refer to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. A “comparison window”, as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482; by the alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443; by the search for similarity method of Pearson and Lipman (1988) Proc. Nat. Acad. Sci U.S.A. 85:2444; by computerized implementations of these algorithms (including, but not limited to CLUSTAL in the PC/Gene program by Intelligentics, Mountain View, Calif.; and GAP, BESTFIT, BLAST, FASTA, or TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., U.S.A.). The CLUSTAL program is well described by Higgins and Sharp (1988) Gene 73:237-244; Higgins and Sharp (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-10890; Huang et al (1992) Computer Applications in the Biosciences 8:155-165; and Pearson et al. (1994) Methods in Molecular Biology 24:307-331. Alignment is also often performed by inspection and manual alignment.

[0037] The term “isolated” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring nucleic acid, polypeptide, or cell present in a living animal is not isolated, but the same polynucleotide, polypeptide, or cell separated from some or all of the coexisting materials in the natural system, is isolated, even if subsequently reintroduced into the natural system. Such nucleic acids can be part of a vector and/or such nucleic acids or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. When referring to a cell population, it means that homogeneous cells expressing a given set of molecular markers constitute at least 60%, preferably 75%, more preferably 90%, and most preferably 95% of the total number of cells in the population.

[0038] The terms “substantially identical” nucleic acid or amino acid sequences means that a nucleic acid or amino acid sequence comprises a sequence that has at least 90% sequence identity or more, preferably at least 95%, more preferably at least 98% and most preferably at least 99%, compared to a reference sequence using the programs described above (preferably BLAST) using standard parameters. For example, the BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)). Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues. In a most preferred embodiment, the sequences are substantially identical over the entire length of the coding regions.

[0039] The terms “nucleic acid” and “polynucleotide” refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in manner similar to naturally occurring nucleotides. A “polynucleotide sequence” is a nucleic acid (which is a polymer of nucleotides (A,C,T,U,G, etc. or naturally occurring or artificial nucleotide analogues) or a character string representing a nucleic acid, depending on context. Either the given nucleic acid or the complementary nucleic acid can be determined from any specified polynucleotide sequence.

[0040] The term “operably linked” refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance. A polylinker provides a convenient location for inserting coding sequences so the genes are operably linked to the promoter. Polylinkers are polynucleotide sequences that comprise a series of three or more closely spaced restriction endonuclease recognition sequences.

[0041] As used herein the term “overexpression” refers to expression of a polypeptide brought about by genetic modification of a host cell with a nucleic acid sequence encoding the polypeptide. Overexpression may take place in cells normally lacking expression of the polypeptide (e.g., an HSC differentiation-inhibiting polypeptide). It can also occur in cells with endogenous expression of the polypeptide. While overexpression may take place in any cell type, preferred host cells for overexpressing an HSC differentiation-inhibiting polypeptide are hematopoietic stem cells.

[0042] The terms “polypeptide” and “protein” are used interchangeably herein, and refer to a polymer of amino acid residues, e.g., as typically found in proteins in nature. A “mature protein” is a protein which is full-length and which, optionally, includes glycosylation or other modifications typical for the protein in a given cell membrane.

[0043] A “variant” of a molecule such as an HSC differentiation-inhibiting polypeptide is meant to refer to a molecule substantially similar in structure and biological activity to either the entire molecule, or to a fragment thereof. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the sequence of amino acid residues is not identical. In some embodiments, a variant differs in amino acid sequence from a reference polypeptide by one or more substitutions, additions, deletions, truncations which may be present in any combination. Among preferred variants are those that vary from a reference polypeptide by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like characters. The following non-limiting list of amino acids are considered conservative replacements: a) alanine, serine, and threonine; b) glutamic acid and asparatic acid; c) asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalaine, tyrosine and tryptophan. Most highly preferred are variants that retain the same biological function and activity as the reference polypeptide from which it varies.

[0044] III. Promoting HSC Expansion by Inhibiting Differentiation

[0045] In addition to novel markers and methods for isolating HSCs, the invention also provides methods for inhibiting or blocking differentiation of mammalian hematopoietic stem cells, thereby promoting expansion of the stem cells. A number of the novel HSC marker genes identified in the present invention can inhibit or block HSC differentiation. Examples of such differentiation-inhibiting genes are shown in Tables 1 and 2 (for human HSC) and Tables 3 and 4 (for mouse HSC). For example, as described in the Examples below, human stem cells overexpressing GATA-binding protein 3 slows differentiation of the cells. HSCs overexpressing ID3 increased colony forming cells, indicating enhanced HSC activity as compared to a control. These differentiation-inhibiting molecules can be used in the present invention to inhibit HSC differentiation and thereby promoting expansion in vitro. They can also be used in vivo to increase the effective dose of engrafted HSCs in a subject.

[0046] The term HSC differentiation-inhibiting molecules (polynucleotides and the encoded polypeptides) include the molecules shown in Tables 1-4 that inhibit or slow HSC differentiation. Polynucleotides with substantial sequence identity are also encompassed. In addition, they also include variants, analogs, fragments, or functional derivatives of the HSC differentiation-inhibiting molecules shown in Tables 1-4. These differentiation-inhibiting molecules can be obtained from any species. Preferably, they are from mammalian species including human, mouse, and chicken. The HSC differentiation-inhibiting molecules can also be from any source whether natural, synthetic or recombinant.

[0047] Differentiation is defined as the restriction of the potential of a cell to self-renew and is normally associated with a change in the functional capacity of the cell. The term “inhibiting” or “blocking” differentiation is used broadly in the context of this invention and includes not only the prevention of differentiation but also encompasses altering or slowing differentiation process of a cell. Differentiation of a stem cell can be determined by methods well known in the art and these include analysis for surface markers associated with cells of a defined differentiated state.

[0048] An HSC differentiation-inhibiting polypeptide of the present invention encodes an HSC differentiation-inhibiting polypeptide that blocks or slows down differentiation of the HSC cells (e.g., as listed in Tables 1-4). As shown in the Tables, these molecules include hormones, secreted proteins, or growth factors. These molecules also include transcription factors. One or more of these HSC differentiation-inhibiting polypeptides, or fragments thereof, can be applied to HSC cells in vitro, e.g., in a cell culture. These cells can be cultured and grown as described herein or other methods well known in the art. The appropriate amount of these differentiation-inhibiting polypeptides to be used in the cultures can be easily determined in accordance with stem cell culturing procedures described herein or knowledge well known in the art. By culturing the HSC in the presence of these molecules, differentiation of the cells can be inhibited or slowed, resulting in enhanced growth of engraftable HSCs.

[0049] In addition to promoting HSC expansion in vitro, the HSC differentiation-inhibiting polypeptides of the invention can also be administered directly to a subject to promote in vivo growth of HSCs. For example, a subject engrafted with bone marrow or a population of HSCs can also be administered an effective amount of an HSC differentiation-inhibiting polypeptide or fragment thereof (e.g., the secreted proteins or growth factors shown in Table 1 and Tables 3-4). The polypeptide can be administered to the subject prior to, concurrently with, or subsequent to transplantation of the bone marrow or HSCs. Preferably, the polypeptide and the HSCs are administered to the subject simultaneously.

[0050] Other than using a differentiation-inhibiting polypeptide, inhibition of HSC differentiation can also be achieved using an HSC differentiation-inhibiting polynucleotide to genetically modify HSCs. HSC differentiation-inhibiting polynucleotides suitable for these methods include some of the genes upregulated in HSCs (as shown in Tables 1 and 3). They encode HSC differentiation-inhibiting polypeptides that block or slow down differentiation of the HSC cells. Some of these methods require first isolation of a population of hematopoietic cells, e.g., a population of CD34+Thy+ human cells or CD34−CD38+ mouse cells as described above, from a source of such cells. An HSC differentiation-inhibiting polynucleotide of the invention can then be introduced into the cells whereby the cells are genetically modified.

[0051] Once the cells are genetically modified, they are cultured in the presence of at least one cytokine in an amount sufficient to support growth of the modified cells. The modified cells are then selected wherein the encoded polypeptide is overexpressed and differentiation is blocked. The genetically modified cells thus obtained may be used immediately (e.g., in transplant), cultured and expanded in vitro, or stored for later uses. The modified HSCs may be stored by methods well known in the art, e.g., frozen in liquid nitrogen.

[0052] Genetic modification as used herein encompasses any genetic modification method of introduction of an exogenous or foreign gene into mammalian cells (particularly human stem cell and hematopoietic cells). The term includes but is not limited to transduction (viral mediated transfer of host DNA from a host or donor to a recipient, either in vitro or in vivo), transfection (transformation of cells with isolated viral DNA genomes), liposome mediated transfer, electroporation, calcium phosphate transfection or coprecipitation and others. Methods of transduction include direct co-culture of cells with producer cells (Bregni et al., Blood 80:1418-1422, 1992) or culturing with viral supernatant alone with or without appropriate growth factors and polycations (Xu et al., Exp. Hemat. 22:223-230, 1994).

[0053] Various in vitro and in vivo assays are well known in the art for the measurement of the functional compositions of hematopoietic cell populations. See, e.g., Quesenberry et al. eds., Stem Cell Biology and Gene Therapy, Wiley-Liss Inc. 1998—Chapter 5, Hematopoietic Stem cells: Proliferation, Purification and Clinical Applications, pgs 133-160. Other examples of suitable assays are also known in the art. For example, the long term culture-initiating cell (LTCIC) assay involves culturing a cell population on stromal cell monolayers for approximately 5 weeks and then testing in a 2 week semisolid media culture for the frequency of clonogenic cells retained (Sutherland et al., Blood 74:1563 (1989)). The Colony Forming Cells (CFC) assay or Colony-Forming Unit Culture (CFUC) assay involves use of cell count as the number of colony-forming units per unit volume or area of a sample. The assay is used to measure clonal growth of quickly maturing progenitors in semi-solid media supplemented with serum and growth factors. Depending on the growth factors used to stimulate growth mature and/or primitive progenitors may be determined. Cobblestone area forming colony (CAFC) assays measure clonal proliferation of long-lived progenitors supported by stromal cell monolayers and growth factor/serum supplemented media. On the appropriate stromal monolayers, cells pluripotent for myeloid and lymphoid lineages may be determined. (Young et al., Blood 88:1619, 1996). SCID-hu bone assays measure the proliferation and multilineage differentiation of cells with bone marrow repopulating activity. These cells are likely to contribute to durable engraftment in clinical transplantation. SCID-hu thymus assays measure the proliferation and differentiation in thymocytes. Both bone marrow repopulating and more mature T-lineage progenitors may be measured.

[0054] A polynucleotide encoding an HSC differentiation-inhibiting molecule is typically introduced to a host cell in a vector. The vector typically includes the necessary elements for the transcription and translation of the inserted coding sequence. Methods used to construct such vectors are well known in the art. For example, techniques for constructing suitable expression vectors are described in detail in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y. (3rd Ed., 2000); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1999).

[0055] Vectors may include but are not limited to viral vectors, such as baculovirus, retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses; bacteriophages; cosmids; plasmid vectors; synthetic vectors; and other recombination vehicles typically used in the art. Vectors containing both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.). Specific examples include, pSG, pSV2CAT, pXtl from Stratagene; and pMSG, pSVL, pBPV and pSVK3 from Pharmacia.

[0056] Preferred vectors include retroviral vectors (see, Coffin et al., “Retroviruses”, Chapter 9 pp; 437-473, Cold Springs Harbor Laboratory Press, 1997). Vectors useful in the invention can be produced recombinantly by procedures well known in the art. For example, WO94/29438, WO97/21824 and WO97/21825 describe the construction of retroviral packaging plasmids and packing cell lines. Exemplary vectors include the pCMV mammalian expression vectors, such as pCMV6b and pCMV6c (Chiron Corp.), pSFFV-Neo, and pBluescript-Sk+. Non-limiting examples of useful retroviral vectors are those derived from murine, avian or primate retroviruses. Common retroviral vectors include those based on the Moloney murine leukemia virus (MoMLV-vector). Other MoMLV derived vectors include, Lmily, LINGFER, MINGFR and MINT (Chang et al., Blood 92:1-11, 1998). Additional vectors include those based on Gibbon ape leukemia virus (GALV) and Moloney murine sacroma virus (MOMSV) and spleen focus forming virus (SFFV). Vectors derived from the murine stem cell virus (MESV) include MESV-MiLy (Agarwal et al., J. of Virology, 72:3720-3728, 1998). Retroviral vectors also include vectors based on lentiviruses, and non-limiting examples include vectors based on human immunodeficiency virus (HIV-1 and HIV-2).

[0057] In producing retroviral vector constructs, the viral gag, pol and env sequences can be removed from the virus, creating room for insertion of foreign DNA sequences. Genes encoded by foreign DNA are usually expressed under the control a strong viral promoter in the long terminal repeat (LTR). Selection of appropriate control regulatory sequences is dependent on the host cell used and selection is within the skill of one in the art. Numerous promoters are known in addition to the promoter of the LTR. Non-limiting examples include the phage lambda PL promoter, the human cytomegalovirus (CMV) immediate early promoter; the U3 region promoter of the Moloney Murine Sarcoma Virus (MMSV), Rous Sacroma Virus (RSV), or Spleen Focus Forming Virus (SFFV); Granzyme A promoter; Granzyme B promoter, CD34 promoter; and the CD8 promoter. Additionally inducible or multiple control elements may be used.

[0058] Such a construct can be packed into viral particles efficiently if the gag, pol and env functions are provided in trans by a packing cell line. Therefore, when the vector construct is introduced into the packaging cell, the gag-pol and env proteins produced by the cell, assemble with the vector RNA to produce infectious virons that are secreted into the culture medium. The virus thus produced can infect and integrate into the DNA of the target cell, but does not produce infectious viral particles since it is lacking essential packaging sequences. Most of the packing cell lines currently in use have been transfected with separate plasmids, each containing one of the necessary coding sequences, so that multiple recombination events are necessary before a replication competent virus can be produced. Alternatively the packaging cell line harbors a provirus. The provirus has been crippled so that although it may produce all the proteins required to assemble infectious viruses, its own RNA cannot be packaged into virus. RNA produced from the recombinant virus is packaged instead. Therefore, the virus stock released from the packaging cells contains only recombinant virus. Non-limiting examples of retroviral packaging lines include PA12, PA317, PE501, PG13, PSI.CRIP, RDI 14, GP7C-tTA-G10, ProPak-A (PPA-6), and PT67. Reference is made to Miller et al., Mol. Cell Biol. 6:2895, 1986; Miller et al., Biotechniques 7:980, 1989; Danos et al., Proc. Natl. Acad. Sci. USA 85:6460, 1988; Pear et al., Proc. Natl. Acad. Sci. USA 90:8392-8396, 1993; and Finer et al., Blood 83:43-50, 1994.

[0059] Other suitable vectors include adenoviral vectors (see, Frey et al., Blood 91:2781, 1998; and WO 95/27071) and adeno-associated viral vectors. These vectors are all well know in the art, e.g., as described in Chatterjee et al., Current Topics in Microbiol. And Immunol., 218:61-73, 1996; Stem cell Biology and Gene Therapy, eds. Quesenberry et al., John Wiley & Sons, 1998; and U.S. Pat. Nos. 5,693,531 and 5,691,176. The use of adenovirus-derived vectors may be advantageous under certain situation because they are not capable of infecting non-dividing cells. Unlike retroviral DNA, the adenoviral DNA is not integrated into the genome of the target cell. Further, the capacity to carry foreign DNA is much larger in adenoviral vectors than retroviral vectors. The adeno-associated viral vectors are another useful delivery system. The DNA of this virus may be integrated into non-dividing cells, and a number of polynucleotides have been successful introduced into different cell types using adeno-associated viral vectors.

[0060] In some embodiments, the construct or vector will include two or more heterologous polynucleotide sequences; a) the nucleic acid sequence encoding an HSC differentiation-inhibiting polypeptide of the invention, and b) one or more additional nucleic acid sequence. Preferably the additional nucleic acid sequence is a polynucleotide which encodes a selective marker, a structural gene, a therapeutic gene, a ribozyme, or an antisense sequence.

[0061] A selective marker may be included in the construct or vector for the purposes of monitoring successful genetic modification and for selection of cells into which DNA has been integrated. Non-limiting examples include drug resistance markers, such as G148 or hygromycin. Additionally negative selection may be used, for example wherein the marker is the HSV-tk gene. This gene will make the cells sensitive to agents such as acyclovir and gancyclovir. Selection may also be made by using a cell surface marker, for example, to select overexpression of an HSC differentiation-inhibiting polypeptide by fluorescence activated cell sorting (FACS). The NeOR (neomycin/G148 resistance) gene is commonly used but any convenient marker gene may be used whose gene sequences are not already present in the target cell can be used. Further non-limiting examples include low-affinity Nerve Growth Factor (NGFR), enhanced fluorescent green protein (EFGP), dihydrofolate reductase gene (DHFR) the bacterial hisD gene, murine CD24 (HSA), murine CD8a(lyt), bacterial genes which confer resistance to puromycin or phleomycin, and beta.-glactosidase.

[0062] The additional polynucleotide sequence(s) may be introduced into the host cell on the same vector as the polynucleotide sequence encoding the polypeptides of the invention or the additional polynucleotide sequence may be introduced into the host cells on a second vector. In a preferred embodiment, a selective marker will be included on the same vector as the HSC differentiation-inhibiting polynucleotide.

[0063] Typically, the host cells for expressing the HSC differentiation-inhibiting polynucleotide are mammalian stem cells, e.g., HSCs from humans, mice, monkeys, farm animals, sport animals, pets, and other laboratory rodents and animals. These cells can be obtained, cultured, and manipulated as described above and in Potten C. S. ed., Stem Cells, Academic Press, 1997; Stem Cell Biology and Gene Therapy, eds. Quesenberry et al., John Wiley & Sons Inc., 1998; and Gage et al., Ann. Rev. Neurosci. 18:159-192, 1995.

[0064] IV. Novel Molecular Markers for Isolating and Enriching HSCs

[0065] As detailed in the Examples below, the present inventor identified a number of genes that are differentially expressed in human and mouse HSCs. These genes, which can play a role in regulating hematopoiesis as well as activities of HSCs and progenitor cells, are suitable as markers for selecting and enriching HSCs from diverse populations of cells. As exemplified in Tables 1-4, these HSC markers include transmembrane proteins (e.g., receptors), growth factor, transcription factors, as well as other proteins with diverse cellular and biochemical functions.

[0066] Employing these novel HSC markers, the present invention provides methods for isolating stem cells from any vertebrate, particularly mammalian, species. In general, one or more of the novel markers can be targeted in the methods. Selection with these markers can be performed alone with a crude population of cells (e.g., bone marrow). The selection scheme can also be used in combination with other selection and purification procedures, e.g., to further select HSCs from cells already enriched for other known HSC surface markers.

[0067] In some embodiments, the novel markers for selecting and enriching HSCs are cell surface markers. As described in the Examples, a number of the genes upregulated in the human and mouse HSCs encode transmembrane proteins (see also Tables 2 and 7). These proteins provide novel surface markers for isolating HSCs from or enumerating HSCs in a population of diverse cells (e.g., bone marrow). These methods are useful for isolating stem cells from primates, e.g. human, monkeys, gorillas, domestic animals, bovine, equine, ovine, porcine, and etc. Isolation of HSCs bearing these novel markers can be performed with the same procedures disclosed herein for the other phenotypic markers.

[0068] In some embodiments, selection of the novel HSC markers utilizes antibodies that recognize the novel HSC markers. This includes preparing an antibody to a novel HSC marker (e.g., a surface marker) of the invention and purifying the antibody. By exposing a population of hematopoietic cells or crude cells to the antibody and allowing the exposed cells to bind with the antibody, cells bearing the novel HSC marker can be isolated. Techniques including antibody preparation and purification are well known and routinely practiced in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1998). Such antibodies encompass any antibody or fragment thereof either native or recombinant, synthetic or naturally derived, which retains sufficient specificity to bind specifically to an HSC marker. They may be monoclonal or polyclonal, and can be produced using the novel HSC marker protein or a fragment or variant thereof. In addition, antibodies that recognize some of these marker proteins may also be obtained commercially.

[0069] When combined with other selection procedures, the particular order by which hematopoietic cells are separated from other cells is not critical to this invention. When a genetically modified HSC cell is to be selected (as detailed above), the specific cell types may be separated either prior to genetic modification or after genetic modification. In some methods, crude cell samples are initially separated by markers indicating unwanted cells, then with a negative selection, followed by separations for markers or marker levels indicating that the cells belong to the stem cell population, and finally positive selection with novel markers of the present invention. In some other methods, following the initial crude separation, the cells can be directly subject to enrichment for at least one of the novel HSC markers.

[0070] For example, an initial crude cell population can be first purified to remove major cell families from the bone marrow or other hematopoietic cell source. A negative selection can then be carried out by targeting some of the cell surface antigens (e.g., Lin, CD34 for mouse HSCs). A further positive selection can be performed to isolate a cell population with specific stem cell markers (e.g., CD34 and Thy for human HSC, and c-kit, Sca-1, or CD38 for mouse HSC). Thereafter, additional selections can be carried out using one or more of the novel HSC surface markers disclosed herein.

[0071] The starting cell populations for selecting and enriching HSC can be obtained from bone marrow or other hematopoietic source. Stem cells and progenitor cells from bone marrow constitute only a small percentage (e.g., about 0.01 to about 0.1%) of the bone marrow cells. Bone marrow cells may be obtained from a source of bone marrow, e.g. tibiae, femora, spine, fetal liver, and other bone cavities. Other sources of hematopoietic stem cells include embryonic yolk sac, fetal live, fetal and adult spleen, and blood including adult peripheral blood and umbilical cord blood (To et al., Blood 89:2233-2258, 1997).

[0072] Procedures for isolation of bone marrow are well known in the art. For example, an appropriate solution may be used to flush the bone. For example, the solution can be a balanced salt solution conveniently supplemented with fetal calf serum or other naturally occurring factors. These components can be present in conjunction with an acceptable buffer at low concentration, generally from about 5 to 25 mM. Convenient buffers include but are not limited to HEPES, phosphate and lactate buffers. Bone marrow can also be aspirated from the bone in accordance with other conventional techniques well known in the art.

[0073] As indicated above, to isolate the HSC cells, a relatively crude separation can be initially used to remove major cell families from the bone marrow or other hematopoietic cell source. Various techniques may be employed to separate the cells to initially remove cells of dedicated lineage. These include physical separation, magnetic separation using antibody-coated magnetic beads, affinity chromatography, and cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody. Also included is the use of fluorescence activated cell sorters (FACS) wherein the cells can be separated on the basis of the level of staining of the particular antigens. These techniques are well known to those of ordinary skill in the art and are described in various references including U.S. Pat. Nos. 5,061,620; 5,409,8213; 5,677,136; and 5,750,397; and Yau et al., Exp. Hematol. 18:219-222, 1990).

[0074] Monoclonal antibodies are particularly useful for this initial separation procedure. The antibodies may be attached to a solid support to allow for separation. In some methods, magnetic bead separations are used to attach the antibodies. Conjugating the antibodies with markers such as magnetic beads, e.g., using biotin-avidin link, allows for direct separation of bound cells from the unbound cells. Antibodies (e.g., monoclonal antibodies) directed to the various surface markers of these differentiated cells can be obtained commercially or prepared using methods routinely practiced in the art.

[0075] To select HSCs, this initial separation allows removal of large numbers of cells of the hematopoietic system of various lineages, such as thymocytes, T-cells, pre-B cells, B-cells, granulocytes, myelomonocytic cells, and platelets. Cells that can be separated in this stage also include other minor cell populations, e.g., megakaryocytes, mast cells, eosinophils and basophils. Generally, at least about 70%, usually 80% or more of the total hematopoietic cells will be removed. Since there will be positive selection at the later selection steps, it is not essential to remove at the initial stage every dedicated cell class, such as the minor population members, the platelets, and erythrocytes. However, it is preferable that there be positive selection for all of the cell lineages, so that in the final positive selection the number of dedicated cells present is minimized.

[0076] Phenotypes of surface antigen of the dedicated lineage cells are known in the art. For example, CD34 is expressed on most immature T-cells also called thymocytes, and these cells lack cell surface expression of CD1, CD2, CD3, CD4, and CD8 antigens. CD45RA is a useful T-cell marker. The best known T-cell marker is the T-cell receptor (TCR). There are presently two defined types of TCRs, TCR-2 (consisting of α and β polypeptides) and TCR-1 (consisting of δ and γ polypeptides). B cells may be selected, for example, by expression of CD19 and CD20. Myeloid cells may be selected, for example, by expression of CD14, CD15, and CD16. NK cells may be selected based on expression of CD56 and CD16. Erythrocytes may be identified by expression of glycophorin A. Compositions enriched for progenitor cells capable of differentiation into myeloid cells, dendritic cells, or lymphoid cells also include the phenotypes CD45RA+ CD34+ Thy1+ and CD45RA+ CD10+ Lin−CD34+. Other useful markers for various cell types are also known in the art.

[0077] The separation techniques employed should maximize the retention of viability of the fraction to be collected. For the initial separations, various techniques of differing efficacy may be employed. The particular technique employed will depend upon efficiency of separation, cytotoxicity of the methodology, ease and speed of performance, and necessity for sophisticated equipment and/or technical skill. Procedures for separation may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, e.g. complement and cytotoxins, and “panning” with antibody attached to a solid matrix, e.g. plate. Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, e.g. a plurality of color channels, low angle and obtuse light scattering detecting channels, and impedance channels.

[0078] Following the initial coarse selection, positive and/or negative selection using various other known stem cell markers as well as the novel HSC markers disclosed herein can be followed. In some methods, human HSCs are isolated using markers such as CD34+ and Thy+ as discussed in the Examples below. In some methods, human HSCs are selected for a phenotype of CD34+ Thy1+ Lin−. Other examples of enriched phenotypes include: CD2−, CD3−, CD4−, CD8−, CD10−, CD14−, CD15−, CD19−, CD20−, CD33−, CD34−, CD38lo/−, CD45RA−, CD 59+/−, CD71−, CDW109+, glycophorin−, AC133+, HLA−DR+/−, c-kit+, and EM+. Lin− refers to a cell population selected on the basis of lack of expression of at least one lineage specific marker, for example CD2, CD3, CD14, and CD56. The combination of expression markers used to isolate and define an enriched HSC population may vary depending on various factors and may vary as other expression markers become available.

[0079] Similarly, mouse HSCs can be selected for one or more of the known markers such as Lin−, c-kit+, Sca-1+, CD38+, and CD34− (see Example 3). In other methods, murine HSCs with similar properties to the human CD34+ Thy-1+ Lin− may be identified by kit+Thy-1.1lo Lin−/lo Sca-1+ (KTLS). Other phenotypes are well known, e.g., as described in U.S. Pat. No. 6,451,558. When CD34 expression is combined with selection for Thy-1, a composition comprising approximately fewer than 5% lineage committed cells can be isolated (U.S. Pat. No. 5,061,620).

[0080] Once the cells are harvested and optionally separated, the cells are cultured in a suitable medium comprising a combination of growth factors that are sufficient to maintain growth. The term culturing refers to the propagation of cells on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (either morphologically, genetically or phenotypically) to the parent cell. Methods for culturing stem cells and hematopoietic cells are well known to those skilled in the art. Any suitable culture container may be used, and these are readily available from commercial vendors. The seeding level is not critical, and it will depend on the type of cells used. In general, the seeding level will be at least 10 cells per ml, more usually at least about 100 cells per ml and generally not more than 106 cells per ml.

[0081] Various culture media can be used and non-limiting examples include Iscove's modified Dulbecco's medium (IMDM), X-vivo 15 and RPMI-1640. These are commercially available from various vendors. The formulations may be supplemented with a variety of different nutrients, growth factors, such as cytokines and the like. In general, the term cytokine refers to any one of the numerous factors that exert a variety of effects on cells, such as inducing growth and proliferation. The cytokines may be human in origin or may be derived from other species when active on the cells of interest. Included within the scope of the definition are molecules having similar biological activity to wild type or purified cytokines, for example produced by recombinant means, and molecules which bind to a cytokine factor receptor and which elicit a similar cellular response as the native cytokine factor.

[0082] The medium can be serum free or supplemented with suitable amounts of serum such as fetal calf serum, autologous serum or plasma. If cells or cellular products are to be used in humans, the medium will preferably be serum free or supplemented with autologous serum or plasma (see, e.g., Lansdorp et al., J. Exp. Med. 175:1501, 1992; and Petzer et al., PNAS 93:1470, 1996).

[0083] Examples of compounds that can be used to supplement the culture medium are thrombopoietin (TPO), Flt3 ligand (FL), c-kit ligand (KL, also known as stem cell factor, SCF, or Stl), Interleukin (e.g., IL-1, IL-2, IL-3, IL-6, soluble IL-6 receptor, IL-11, and IL-12), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), MIP-1:c, and erythropoietin (EPO). These compounds may be used alone or in any combination. When murine stem cells are cultured, a preferred non-limiting medium includes mIL-3, mIL-6 and mSCF.

[0084] Concentration range of these compounds to be used in cultures can be determined according to knowledge well known in the art. For example, a general preferred range of TPO is from about 0.1 ng/mL to about 5000 μg/mL, more preferred is from about 1.0 ng/mL to about 1000 ng/mL, even more preferred from about 5.0 ng/mL to about 300 ng/mL. A preferred concentration range for each of FL and KL is from about 0.1 ng/mL to about 1000 ng/mL, more preferred is from about 1.0 ng/mL to about 500 ng/mL. IL-6 is a preferred factor to be included in the culture, and a preferred concentration range is from about 0.1 ng/mL to about 500 ng/mL, and more preferred from about 1.0 ng/mL to about 100 ng/mL. Hyper IL-6, a covalent complex of IL-6 and IL-6 receptor may also be used in the culture.

[0085] Other molecules can also be added to the culture media, for instance, adhesion molecules, such as fibronection or RetroNectin™ (commercially produced by Takara Shuzo Co., Otsu Shigi, Japan). Fibronectin is a glycoprotein that is found throughout the body, and its concentration is particularly high in connective tissues where it forms a complex with collagen.

[0086] V. Therapeutic Applications

[0087] HSC's are the active component in bone marrow transplantation (BMT). The use of purified HSCs transplant as opposed to bone marrow provides the advantage that transplant of harmful non-HSC cells in the bone marrow is avoided. In the autologous cancer or autoimmune setting, the use of purified HSCs minimizes the possibility of giving tumor or diseased cells back to the patient along with the bone marrow. In allogenic transplantion, using high doses of HSCs overcomes rejection by the recipient immune system. Thus, expansion of HSCs would make autologous and allogeneic HSC transplantation safer and more effective.

[0088] The present invention provides methods for inhibiting HSC differentiation and promoting HSC expansion in vivo in a subject, e.g., a human subject engrafted with HSCs. Using HSC differentiation-inhibiting molecules identified in the present invention, these methods allow expansion of non-differentiated stem cells and increase the dose of HSCs either ex vivo or in vivo, thereby potentially allowing more rapid engraftment. The HSC differentiation-inhibiting molecules can be expressed in the engrafted HSCs. It can also be separately provided to the subject receiving the HSC graft, e.g., expressed from a vector introduced into the subject. In addition, the HSC differentiation-inhibiting molecules can also be administered to the subject as an expressed polypeptide, e.g., a growth factor. As a result, differentiation of the cells is blocked or slowed down, resulting in expansion of non-differentiated stem cells.

[0089] Some methods of the invention provide ex vivo gene therapy for transplanting genetically modified HSCs cells into a subject. For example, vectors expressing an HSC differentiation-inhibiting polypeptide can be delivered to HSCs explanted from an individual subject, followed by reimplantation of the cells into a subject, usually after selection for cells that have incorporated the vector. Procedures for modifying host cells with an HSC differentiation-inhibiting polynucleotide (e.g., GATA3) are described above. In addition, ex vivo cell transfection for diagnostics, research, or for gene therapy (e.g., via re-infusion of the transfected cells into the host organism) is well known in the art. For a review of gene therapy procedures, see Anderson, Science 256: 808-813, 1992; Nabel & Felgner, TIBTECH 11: 211-217, 1993; Mitani & Caskey, TIBTECH 11: 162-166, 1993; Mulligan, Science 260: 926-932, 1993; Dillon, TIBTECH 11: 167-175, 1993; Miller, Nature 357: 455-460, 1992; Van Brunt, Biotechnology 6: 1149-1154, 1998; Vigne, Restorative Neurology and Neuroscience 8: 35-36, 1995; Kremer & Perricaudet, British Medical Bulletin 51: 31-44, 1995; Haddada et al., in Current Topics in Microbiology and Immunology (Doerfler & Böhm eds., 1995); and Yu et al., Gene Therapy 1: 13-26, 1994).

[0090] For therapeutic applications, the genetically modified HSC cells are maintained for a period of time sufficient for overexpression of HSC differentiation-inhibiting polypeptide. A suitable time period will depend inter alia upon cell type used and is readily determined by one skilled in the art. In general, genetically modified cells of the invention may overexpress HSC differentiation-inhibiting polypeptide for the lifetime of the host cell. Preferably, for hematopoietic cells the time period will be in the range of 1 to 45 days, more preferably in the range of 1 to 30 days, even more preferably in the range of 1 to 20 days, still more preferably in the range of 1 to 10 days, and most preferably in the range of 1 to 5 days.

[0091] Other than ex vivo gene therapy, vectors expressing an HSC differentiation-inhibiting polypeptide can also be delivered in vivo. This is carried out by administering to an individual subject the expression vector, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application. Methods for in vivo gene therapy are also well known in the art, e.g., as described in the literatures noted above.

[0092] As described above, other than gene therapy, therapeutic expansion of HSCs in a subject can also be achieved by directly applying an HSC differentiation-inhibiting polypeptide (or its fragment or functional derivative) to a subject. The subject can be simultaneously engrafted with HSCs. The subject can also be one that has not been subject to HSC transplant. Typically, in such applications, the HSC differentiation-inhibiting polypeptide (e.g., GATA3) is administered to the subject in a pharmaceutical composition. The pharmaceutical compositions typically comprise at least one active ingredient together with one or more acceptable carriers thereof. Suitable carriers for preparing the pharmaceutical compositions, appropriate dosages, and suitable routes of administration of the compositions can all be readily determined by following methods well known in the art. See, e.g., Gilman et al., eds., Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; Avis et al., eds., Pharmaceutical Dosage Forms: Parenteral Medications, published by Marcel Dekker, Inc., N.Y., 1993; and Lieberman et al., eds., Pharmaceutical Dosage Forms: Tablets, published by Marcel Dekker, Inc., N.Y., 1990.

EXAMPLES

[0093] The following examples are provided to illustrate, but not to limit the present invention.

Example 1

Genes Upregulated in Human HSCs

[0094] This Example describes RNA profiling of human hematopoietic stem cells and characterization of genes upregulated in the HSCs. All procedures and assays employed herein to study the human HSCs have been described in the art, e.g., as noted above.

[0095] CD34+ cells were first isolated from blood of six normal human donors using magnetic beads. Flow activated cell sorting (FACS) was then used to purify CD34+Thy+ (stem enriched) and CD34+Thy− (stem depleted) cell populations. The two populations of cells (total 12 samples, 6 CD34+Thy+ and 6 CD34+Thy−) were assayed for bioactivity with the CFC assay. RNA profiling (Thy+ vs Thy−) was then carried out to identify genes differentially expressed in stem cells. Results of the profiling are shown in Table 1. The data indicate that the upregulated genes encode proteins with diverse biochemical and cellular functions.

[0096] In addition, genes upregulated in CD34+Thy+ HSCs from two different sources, bone marrow and peripheral blood, were compared for overlapping sequences that are enriched in HSCs from both sources. A total of 30 genes were found to have been upregulated in HSCs from both sources. An exemplary list of these genes is shown in Table 2. Both HSC types contain transcription factors some of which are known proto-oncogenes (e.g., GATA3, HLF, Evi1, PMX1, MN1, ATF3).

[0097] Further, the results indicate that HSCs from peripheral blood, but not HSCs from bone marrow, are enriched in histones and inhibitory HLH transcription factors (ID1, ID2, and ID3). The data also suggest new cell surface markers for HSCs. Examples include 5T4, EphA3, TNFSF3, EVI2b, DLK1. Several potential neuropeptides are also upregulated, including Vasopression (AVP), Oxytocin (OXT), and Vasodilators.

Example 2

Inhition of HSC Differentiation by Overexpressing an HSC Differentiation-Inhibiting Polypeptide

[0098] The Example describes effects on HSC differentiation by constitutive expression of an HSC differentiation-inhibiting gene in CD34+Thy+ cells using retroviral vectors. First, effect of overexpressing ID3 was analyzed with colony-forming cell (CFC) assay. Other assays such as cobblestone area forming cell (CAFC) assay and NOD/SCID (nonobese diabetic mice with severe combined immunodeficiency disease) repopulating cell assay can also be used in these analyses. These assays can be performed as described as described above and are well known in the art (e.g., Kusadasi et al., Leukemia 14: 1944-53, 2000; and Larochelle et al., Nature Medicine, 2: 1329-1337, 1996).

[0099]
FIG. 1 illustrates the schematic structure of the retroviral vectors used in the study. Gene X in the figure denotes any of these HSC genes (e.g., ID3) to be examined. The vectors also express the green-fluorescence protein (GFP). When the GFP gene is transfected into or infected cells, the encoded GFP shines green under ultraviolet light and thus enables the detection of the transfected or infected cell in a simple manner.

[0100] A vector harboring the HSC gene (e.g., ID3 or GATA3) was transfected into the CD34+ cells. Cells expressing the gene were sorted and assayed with the CFC assay. As shown in FIG. 2, ID3 over-expression increased the number of colony forming cells (e.g., primitive BFU-E colonies). This suggests enhanced HSC activity, indicating that differentiation of the stem cells has been slowed down.

[0101] The HSC differentiation-inhibiting genes were also examined for their effects on HSC growth in liquid culture. The effect of GATA3 over-expression on human HSC differentiation was examined in liquid culture. Here, stem cells were transfected with the same vectors described above (which harbor the ID1 gene, GATA3 gene, or no HSC gene), and grown in liquid culture. CD34+ and GFP+ cells were sorted. Expression of CD34 was monitored during the culture. Cells without transfection were used in a control analysis. The results indicate that, as compared to the control, ID1 had no effect on differentiation of the CD34+ cells. However, expression of GATA3 significantly slowed the differentiation process as indicated by the rate of reduction of CD4+ cells.

Example 3

Novel Molecular Markers Expressed in Mouse HSCs

[0102] This Example describes use of RNA expression profiling to characterize purified mouse HSCs. Mouse HSCs were purified using a combination of antibodies to cell-surface markers. The following three cell populations were purified from murine bone marrow as described in Zhao et al., Blood 96: 3016-22, 2000; and Zhong et al., Blood 100: 3521-6, 2002.

3Cell typeImmunophenotypeHSC activityLT-HSCLin−, c-kit+, Sca-1+, CD38+, CD34−1×FacilitatorLin−, c-kit+, Sca-1+, CD38−, CD34+0.1×CellsProgenitorLin−, c-kit+, Sca-1+, CD38+, CD34+0.1×Cells

[0103] Cells were purified from normal BL6 mice using flow cytometry. Three different preparations of sorted cells for each population were prepared and combined prior to the isolation of total RNA. The RNA was quantified using the Ribogreen fluorescence-based solution assay (e.g., as described in Jones et al., Anal Biochem 265: 368-74, 1998). 10 ng of each pooled RNA preparation was labeled in duplicates using the triple labeling procedure (as described, e.g., in Hrabovszky et al., J. Histochem. Cytochem. 43: 363-370, 1995) and hybridized to affymetrix U74A gene chips according to the manufacturer's instructions. Intensity values were obtained for each gene and sample using GeneChip software. These Average difference (AD) values were exported to a spreadsheet program and analyzed by first filtering for genes which are expressed above a threshold criteria (50 in at least two samples), and whose average for each population was expressed >2× or <2× between any two cell populations and where ANOVA analysis showed a significant difference (P<0.01) between any two populations.

[0104] Examples of genes upregulated in HSCs are shown in Table 3. The genes were analyzed for patterns using Genespring software and arranged by functional gene classification using GO ontogeny. Accession numbers or identification numbers from other public databases of these genes, as well as levels of up-regulation of these genes in HSCs as compared to non-HSCs, are also shown in the table.

Example 4

Characterization of Genes Differentially Expressed in Mouse HSCs

[0105] To correlate stem cell activity of the three subsets with gene expression, a hypothetical stem cell activity pattern corresponding to the in vivo repopulating activity of the three subsets was generated and used for comparison of the normalized expression levels of each differentially expressed gene identified above. Principle Component Analysis (PCA) on the stem cell expression data was performed to identify gene expression patterns. This is an unsupervised computational method used to identify major patterns in diverse data types including gene expression data (Alter et al., Proc Natl Acad Sci USA 97:10101-10106, 2000; and Holter et al., Proc Natl Acad Sci USA 97:8409-8414, 2000). The correlation analysis of the gene expression patterns of the differentially expressed genes with stem cell activity identified genes with highly significant (Pearson R>0.95) correlations. These genes are shown in Table 4. In addition to genes upregulated in HSCs, the analysis also identified genes whose expression negatively correlated with LTR HSCs (i.e., down-regulated expression). Examples of these genes are shown in Table 5.

[0106] Some of the differentially expressed genes were further analyzed and classified according to their biological functions. The results are shown in Table 6. As shown in Tables 3, 4, and 6, the upregulated genes in mouse HSCs also encode proteins of diverse biological properties, similar to genes upregulated in the human HSCs. For example, a number of transmembrane proteins were enriched in the mouse HSCs, as exemplified in Table 7. These molecules can be useful as novel surface markers for isolating HSCs. Some of transcription factors that are upregulated in the mouse HSCs are shown in Table 8. Their upregulated expression levels in the CD34−CD38+ HSCs relative to that in the facilitator cells (CD38−CD34+) and progenitor cells (CD34+CD38+) are shown in FIG. 3.

[0107] The expression of several known transcription regulation factors was found to correlate positively with LTR HSC activity. These include Cited2, GATA3, Hdac3, Irf6, Jun B, Nmyc1, Rnps1, Xbp1, and Zfp292. Little is known regarding the role of these specific transcription factors in the control of HSC biology. These essential transcription factors could play an important role in regulating HSC development and differentiation.

[0108] To determine if any of the differentially expressed transcription factors are themselves regulating transcription in LTR HSCs, we performed a search of putative upstream regulatory regions (10 kb upstream of start codons) of the interrogated genes for binding sites of the nine transcription factors. Statistical analysis of these results revealed that only the binding sites of GATA were significantly enriched (P<0.05) within the differentially expressed genes. Interestingly, this list contains a large fraction (20 of 52) of the genes whose expression positively correlated with HSC activity, suggesting the possibility that Gata may play an important role in the control of LTR HSC biology. A small number of gene (3 of 20) whose expression is negatively correlated with HSC activity also contained Gata binding sites, suggesting the possibility that low levels of Gata expressed in STR HSC may influence gene expression at later stages.

[0109] To confirm the data from expression profiling, we performed semi-quantitative RT-PCR on total RNA extracted from the three BM subsets for three of the LTR HSC genes identified. These included the transcription factors Gata 3, Jun B, and the thrombopoietin receptor c-Mpl. The results demonstrated that all three mRNAs are expressed at significantly higher levels in CD38+CD34− cells compared to the other two subsets.

4TABLE 3Genes Upregulated in Mouse HSCs HSC/SymbolDescriptionRefSeqSwiss Prot Keywordsnon-HSCAU044919expressed sequence AU044919AU044919Glycoprotein79.7Immunoglobulin C regionImmunoglobulin domainKlf2Kruppel-like factor 2 (lung)NM 008452Activator DNA-binding44.9Metal-binding Nuclearprotein RepeatTranscription regulationZinc-fingerCar1carbonic anhydrase 1NM 009799Lyase Zinc36.8Mm.220154Mus musculus anti-HIV-1 reverseNANone30.1transcriptase single-chain variablefragment mRNA, complete cds2010309G21RikRIKEN cDNA 2010309G21 genenoneImmunoglobulin C region28.8Immunoglobulin domainNAM80423:Mus castaneus IgK chainM80423None20.9gene, C-region, 3 end/cds = (0,322)/gb = M80423/gi = 196865/ug = Mm.46804/len = 323 mRNAFragilisFragilisNM 025378None17.1Smoc1SPARC related modular calciumNM 022316None15.8binding 15830413E08RikRIKEN cDNA 5830413E08 geneNM 029083None14.95830431A10RikRIKEN cDNA 5830431A10 genenoneNone14.4AI325941expressed sequence AI325941AI325941None14.2Cdkn1ccyclin-dependent kinase inhibitor 1C (P57)NM 009876Alternative splicing14.1Cell cycleLisch7liver-specific bHLH-Zip transcriptionnoneNone13.9factorAW108012expressed sequence AW108012AW108012None13.8Akr1c13aldo-keto reductase family 1, member C13NM 013778None13.30910001L24RikRIKEN cDNA 0910001L24 geneNM 022419None12.7AI842353expressed sequence AI842353AI842353None11.7Tgm2transglutaminase 2, C polypeptideNM 009373Acyltransferase Calcium-11.4binding TransferaseNckap1NCK-associated protein 1noneTransmembrane11.3Serpina3gserine (or cysteine) proteinasenoneNone11.3inhibitor, clade A, member 3G1700008C22RikRIKEN cDNA 1700008C22 genenoneNone10.4Nmyc1neuroblastoma myc-related oncogene 1NM 008709DNA-binding Nuclear10.4protein PhosphorylationProto-oncogeneZfhx1azinc finger homeobox 1aNM 011546Activator DNA-binding10.4Homeobox Metal-bindingNuclear protein RepeatRepressor Transcriptionregulation Zinc-fingerH2-Eb1histocompatibility 2, class II antigen E betaNM 010382Glycoprotein MHC II10.0Signal TransmembraneAU044919expressed sequence AU044919AU044919Glycoprotein9.9Immunoglobulin C regionImmunoglobulin domainGbp2guanylate nucleotide binding protein 2NM 010260None9.5Gabbr1gamma-aminobutyric acid (GABA-B)NM 019439Alternative splicing Coiled9.5receptor, 1coil G-protein coupledreceptor GlycoproteinPostsynaptic membraneRepeat SignalTransmembraneD8Ertd69eDNA segment, Chr 8, ERATO Doi 69,noneNone9.2expressedGata3GATA binding protein 3NM 008091Activator DNA-binding9.1Nuclear protein T-cellTranscription regulationZinc-fingerC130052I12RikRIKEN cDNA C130052I12 geneNM 146047None8.70610025I19RikRIKEN cDNA 0610025I19 geneNM 029555None8.6Tcf15transcription factor 15NM 009328None8.6H2-Aahistocompatibility 2, class II antigen A, alphaNM 0103783D-structure Glycoprotein8.5MHC II SignalTransmembraneTal1T-cell acute lymphocytic leukemia 1NM 011527Chromosomal8.3translocationDifferentiation DNA-binding PhosphorylationProto-oncogeneTranscription regulationMyoz1myozenin 1NM 021508None7.94930421J07RikRIKEN cDNA 4930421J07 genenoneNone7.4Igh-6immunoglobulin heavy chain 6noneAlternative splicing7.3(heavy chain of IgM)GlycoproteinImmunoglobulin C regionImmunoglobulin domainTransmembraneHoxb5Homeo box B5NM 008268Developmental protein7.3DNA-binding HomeoboxNuclear proteinTranscription regulationCol9a1procollagen, type IX, alpha 1NM 007740Alternative splicing7.2Cartilage CollagenConnective tissueExtracellular matrixGlycoproteinHydroxylation RepeatSignalMeis1myeloid ecotropic viral integrationNM 010789None7.1site 1Ela1elastase 1, pancreaticnoneNone7.0Hiat1hippocampus abundant gene transcript 1NM 008246None7.0Fahfumarylacetoacetate hydrolaseNM 010176Hydrolase Phenylalanine6.9catabolism TyrosinecatabolismCypf13cytochrome P450 CYP4F13NM 130882None6.7NA:Mus musculus transcription factorAF020200None6.5PBX3b (PBX3b) mRNA, completecds/cds = (118, 1173)/gb =AF020200/gi = 2432016/ug =Mm.7331/len = 2467 mRNAIgjimmunoglobulin joining chainNM 152839Glycoprotein Signal6.3NA:AV336991 Mus musculus cDNA, 3AV336991None6.2end/clone = 6332407A01/clone_end = 3/gb = AV336991/gi = 6377043/ug = Mm.99212/len = 201/NOTE = replacement forprobe set(s) 100264_f_at on MG-U74A mRNACtla2bcytotoxic T lymphocyte-associatednoneRepeat Signal T-cell6.1protein 2 betaSerpinb6serine (or cysteine) proteinaseNM 009254Serine protease inhibitor5.8inhibitor, clade B, member 6SerpinMm.29940ESTsNANone5.8AU043625expressed sequence AU043625NM 133910None5.8Col4a1procollagen, type IV, alpha 1noneBasement membrane5.6Collagen Connective tissueExtracellular matrixGlycoproteinHydroxylation RepeatSignalIgh-4immunoglobulin heavy chain 4noneAlternative splicing5.5(serum IgG1)GlycoproteinImmunoglobulin C regionImmunoglobulin domainSiat6sialyltransferase 6 (N-acetyllacosaminideNM 009176Glycoprotein5.4alpha 2,3-sialyltransferase)Glycosyltransferase Golgistack Signal-anchorTransferaseTransmembraneIgk-Cimmunoglobulin kappa chain, constant regionnoneNone5.4SdprSerum deprivation responseNM 138741None5.4Dusp1dual specificity phosphatase 1NM 013642Cell cycle Hydrolase5.3Cited2Cbp/p300-interacting transactivator,NM 010828Alternative splicing5.2with Glu/Asp-rich carboxy-terminalNuclear proteindomain, 2Eporerythropoietin receptorNM 010149Glycoprotein Receptor5.1Signal TransmembraneMm.200980Mus musculus, Similar toNANone5.0translocation protein 1, cloneIMAGE: 5347105, mRNA, partial cdsAtf2activating transcription factor 2noneActivator Alternative5.0splicing DNA-bindingMetal-binding Nuclearprotein PhosphorylationTranscription regulationZinc-fingerCcne1cyclin E1NM 007633Cell cycle Cell division5.0Cyclin Nuclear proteinPhosphorylationMllt3myeloid/lymphoid or mixed lineage-NM 027326None4.9leukemia translocation to 3 homolog(Drosophila)D5Ertd40eDNA segment, Chr 5, ERATO Doi 40, expressednoneNone4.9Zfp216zinc finger protein 216NM 009551None4.8SypsynaptophysinNM 009305Calcium-binding4.8Glycoprotein NervePhosphorylation RepeatSynapse SynaptosomeTransmembraneNedd4neural precursor cell expressed,NM 010890Ligase Repeat Ubiquitin4.7developmentally down-regulted gene 4conjugationPbx1pre B-cell leukemia transcription factor 1NM 008783None4.76330407G11RikRIKEN cDNA 6330407G11 geneNM 023423None4.6Ash1absent, small, or homeotic discs 1NM 138679None4.5(Drosophila)Lrmplymphoid-restricted membrane proteinNM 008511None4.5Casp8ap2caspase 8 associated protein 2NM 011997None4.5Mm.30163Mus musculus, clone IMAGE: 4952607, mRNANANone4.5Ctslcathepsin LNM 009984Glycoprotein Hydrolase4.5Lysosome Signal Thiolprotease ZymogenSfpqsplicing factor proline/glutamine richNM 023603None4.4(polypyrimidine tract binding proteinassociated)2010004A03RikRIKEN cDNA 2010004A03 genenoneNone4.3Car2carbonic anhydrase 2NM 009801Lyase Zinc4.2Mm.22896ESTsNANone4.1AI573938expressed sequence AI573938noneNone3.9Vaspvasodilator-stimulated phosphoproteinnoneActin-binding3.9PhosphorylationAA408451expressed sequence AA408451AA408451None3.7Pftk1PFTAIRE protein kinase 1NM 011074None3.6TiegTGFB inducible early growth responseNM 013692DNA-binding Metal-binding3.6Nuclear proteinRepeat RepressorTranscription regulationZinc-fingerIgk-V28immunoglobulin kappa chain variable 28 (V28)noneImmunoglobulin C region3.6Immunoglobulin domainMm.1806Mus musculus, Similar to KIAA1404 protein,NANone3.5clone IMAGE: 5252426, mRNA, partial cdsMm.25115ESTsNANone3.5Ccrn4lCCR4 carbon catabolite repression 4-likenoneBiological rhythms3.5(S. cerevisiae)Cpocoproporphyrinogen oxidaseNM 007757Heine biosynthesis Iron3.5MitochondrionOxidoreductase Porphyrinbiosynthesis Transit peptideNupr1nuclear protein 1NM 019738None3.5Mm.5510similar to gene overexpressed in astrocytomaNANone3.4[Homo sapiens]Rab33bRAB33B, member of RAS oncogene familyNM 016858Golgi stack GTP-binding3.4Lipoprotein PrenylationProtein transport9430065L19RikRIKEN cDNA 9430065L19 geneNM 146083None3.4Pgrprogesterone receptorNM 008829DNA-binding Nuclear3.4protein Receptor Steroid-binding Transcriptionregulation Zinc-fingerLOC218490similar to Transcription factor BTF3 (RNANM 145455Alternative splicing3.4polymerase B transcription factor 3)Nuclear proteinTranscription regulation4930434H03RikRIKEN cDNA 4930434H03 genenoneNone3.3Actn3Actinin alpha 3NM 013456Actin-binding Multigene3.3family RepeatMm.202311Mus musculus, clone IMAGE: 1379624, mRNA,NAGTP-binding Lipoprotein3.3partial cdsMembrane Multigenefamily PalmitateTransducerGtpiinterferon-g induced GTPaseNM 019440None3.3Nat2N-acetyltransferase 2 (arylamine N-NM 010874Acyltransferase Multigene3.3acetyltransferase)family PolymorphismTransferaseEya2eyes absent 2 homolog (Drosophila)noneAlternative splicing3.3Developmental proteinMultigene family1110037N09RikRIKEN cDNA 1110037N09 genenoneNone3.25033414D02RikRIKEN cDNA 5033414D02 geneNM 026362None3.1Mm.26147ESTsNANone3.1Il4interleukin 4NM 021283B-cell activation Cytokine3.1Glycoprotein Growthfactor SignalUbap1ubiquitin-associated protein 1NM 023305None3.1Acox1acyl-Coenzyme A oxidase 1, palmitoylNM 015729FAD Fatty acid2.9metabolism FlavoproteinOxidoreductasePeroxisomeCcl5chemokine (C-C motif) ligand 5NM 013653Chemotaxis Cytokine2.9Inflammatory responseSignal T-cellAW457192expressed sequence AW457192NM 134084Cyclosporin Isomerase2.9Mitochondrion Multigenefamily Rotamase Transitpeptide2610016K11RikRIKEN cDNA 2610016K11 genenoneNone2.8Fzd4frizzled homolog 4 (Drosophila)NM 008055Developmental protein2.8G-protein coupled receptorGlycoprotein Multigenefamily SignalTransmembranePla2g4aphospholipase A2, group IVA (cytosolic,NM 008869Calcium Hydrolase Lipid2.8calcium-dependent)degradationPhosphorylationScinscinderinNM 009132None2.7NAAV239653 Mus musculus cDNA, 3AV239653None2.7end/clone = 4732435F04/clone_end = 3/gb = AV239653/gi = 6192160/ug = Mm.88313/len = 214/NOTE = replacement forprobe set(s) 96411_f_at on MG-U74A mRNATcf12transcription factor 12NM 011544Alternative splicing2.7Developmental proteinDNA-binding Nuclearprotein TranscriptionregulationMadh7MAD homolog 7 (Drosophila)NM 008543Alternative splicing2.7Multigene familyTranscription regulationGemGTP binding protein (geneNM 010276GTP-binding Membrane2.7overexpressed in skeletal muscle)PhosphorylationTpm1tropomyosin 1, alphaNM 0244273D-structure Acetylation2.7Alternative splicing Coiledcoil Multigene familyMuscle proteinPhosphorylation RepeatMap17membrane-associated protein 17NM 026018None2.7DcxdoublecortinNM 010025Neurogenesis Neurone2.7Phosphorylation RepeatIgk-V28immunoglobulin kappa chain variable 28 (V28)noneImmunoglobulin C region2.6Immunoglobulin domainRnf11ring finger protein 11NM 013876None2.6Nfixnuclear factor I/XNM 010906None2.6Lin7clin 7 homolog c (C. elegans)NM 011699None2.5Cln3ceroid lipofuscinosis, neuronal 3, juvenileNM 009907Glycoprotein Lysosome2.5(Batten, Spielmeyer-Vogt disease)TransmembraneHhexhematopoietically expressed homeoboxNM 008245Developmental protein2.5DNA-binding HomeoboxNuclear proteinGab1growth factor receptor bound proteinNM 021356None2.52-associated protein 1NonenonenoneNone2.5Kcnj3potassium inwardly-rectifying channel,NM 008426Ion transport Ionic channel2.5subfamily J, member 3Potassium transportTransmembrane Voltage-gated channelCraddCASP2 and RIPK1 domain containing adaptorNM 009950Apoptosis2.5with death domainMm.29914ESTsNANone2.4FosFBJ osteosarcoma oncogeneNM 010234DNA-binding Nuclear2.4protein PhosphorylationProto-oncogeneMm.24247ESTsNANone2.44930472G13RikRIKEN cDNA 4930472G13 geneNM 029447None2.4Ormdl3ORM1-like 3 (S. cerevisiae)NM 025661None2.4Umpkuridine monophosphate kinasenoneKinase Transferase2.4Cregcellular repressor of E1A-stimulated genesNM 011804None2.4UtrnutrophinnoneNone2.3Mm.27769ESTs, Weakly similar to RIKEN cDNA 0610011E17NANone2.3[Mus musculus] [M. musculus]Igtpinterferon gamma induced GTPaseNM 018738None2.3Arg2arginase type IINM 009705Arginine metabolism2.3Hydrolase ManganeseMitochondrion Transitpeptide Urea cyclePklrpyruvate kinase liver and red bloodNM 013631Alternative splicing2.2cellGlycolysis KinaseMagnesium Multigenefamily PhosphorylationTransferase1810010A06RikRIKEN cDNA 1810010A06 geneNM 026921None2.2Mm.532ESTs, Weakly similar to lysophospholipase 1;NANone2.2phospholipase 1a; lysophopholipase 1[Mus musculus] [M. musculus]Vamp5vesicle-associated membrane protein 5NM 016872Multigene family2.2Myogenesis Signal-anchorTransmembrane0710001O03RikRIKEN cDNA 0710001O03 geneNM 146094None2.22610003J05RikRIKEN cDNA 2610003J05 genenoneNone2.2Tde1ltumor differentially expressed 1, likeNM 019760None2.2Serpinf1serine (or cysteine) proteinase inhibitor,NM 011340Glycoprotein Serpin Signal2.1clade F), member 1Scotinscotin geneNM 025858None2.1G3bp2Ras-GTPase-activating protein (GAP<120>)NM 011816None2.1SH3-domain binding protein 21190002H23RikRIKEN cDNA 1190002H23 geneNM 025427None2.1Nsccn1non-selective cation channel 1NM 010940None2.1Tgoln2trans-golgi network protein 2NM 009444None2.1Ywhaetyrosine 3-monooxygenase/tryptophanNM 009536None2.15-monooxygenase activation protein,epsilon polypeptide4631408O11RikRIKEN cDNA 4631408O11 genenoneNone2.1Pou2af1POU domain, class 2, associating factor 1NM 011136Nuclear protein2.1Transcription regulationMm.220953Mus musculus, clone IMAGE: 4206769,NANone2.1mRNACasp6caspase 6NM 009811Apoptosis Hydrolase Thiol2.0protease ZymogenNonenonenoneGlycoprotein2.0Immunoglobulin C regionImmunoglobulin domainNr4a1nuclear receptor subfamily 4, group A,NM 010444DNA-binding Nuclear2.0member 1protein PhosphorylationReceptor Transcriptionregulation Zinc-finger1700023O11RikRIKEN cDNA 1700023O11 geneNM 029339None2.0Brca2breast cancer 2NM 009765Polymorphism Repeat2.0H2-T22histocompatibility 2, T region locus 22NM 010397None2.0

[0110]

5

TABLE 4

Genes With Upregulated Expression and Correlated Stem Cell Activity

Symbol or
Gene Description or similarity
Corrrelation

Acc. No.
to known proteins
to stem cell
Unigene No.

Rnps1
ribonucleic acid binding
1.000
Mm.1951

protein S1

Junb
Jun-B oncogene
1.000
Mm.1167

Hdac3
histone deacetylase 3
1.000
Mm.20521

Irf6
interferon regulatory factor 6
1.000
Mm.4179

Gata3
GATA binding protein 3
0.997
Mm.606

Xbp1
X-box binding protein 1
0.993
Mm.22718

Cited2
Cbp/p300-interacting
0.992
Mm.9524

transactivator, with Glu/Asp-

rich carboxy-terminal domain, 2

Nmyc1
neuroblastoma myc-related
0.986
Mm.16469

oncogene I

Zfp292
zinc finger protein 292
0.975
Mm.38193

Bdkrb1
bradykinin receptor, beta 1
1.000
Mm.57076

Map17
membrane-associated protein 17
0.995
Mm.30181

Ormdl3
ORM1-like 3 (S. cerevisiae)
0.990
Mm.180546

Fzd4
frizzled homolog 4
0.988
Mm.68712

(Drosophila)

Lgi4
leucine-rich repeat LGI
0.961
Mm.1662

family, member 4

Bdkrb1*
bradykinin receptor, beta 1
1.000
Mm.57076

Socs2
suppressor of cytokine
0.996
Mm.4132

signaling 2

Fzd4*
frizzled homolog 4
0.988
Mm.68712

(Drosophila)

Kit*
kit oncogene
0.961
Mm.4394

Inpp5d
inositol polyphosphate-5-
0.958
Mm.15105

phosphatase D

Fbxo9
f-box only protein 9
1.000
Mm.28584

Nedd4
neural precursor cell
0.993
Mm.16553

expressed, developmentally

down-regulted gene 4

Rnfl 1
ring finger protein 11
0.992
Mm.25228

Ian 1
immune associated nucleotide 1
0.999
Mm.28395

Iigp
interferon-inducible GTPase
0.997
Mm.29008

Ifi47
interferon gamma inducible
0.984
Mm.24769

protein

Tgtp
T-cell specific GTPase
0.994
Mm.15793

Igtp
interferon gamma induced GTPase
0.993
Mm.858

Gtpi
interferon-g induced GTPase
0.989
Mm.33902

Serpinb6a
serine (or cysteine) proteinase
0.996
Mm.2623

inhibitor, clade B, member 6a

Serpina3g
serine (or cysteine) proteinase
0.987
Mm.15085

inhibitor, clade A, member 3G

Camk2b
calcium/calmodulin-dependent
0.999
Mm.4857

protein kinase II, beta

Gab1
growth factor receptor bound
0.997
Mm.24573

protein 2-associated protein 1

Gabarapl1
gamma-aminobutyric acid
0.997
Mm.14638

(GABA(A)) receptor-

associated protein-like 1

Mtmr13
myotubularin related protein 13
0.996
Mm.200250

Mt2
metallothionein 2
0.999
Mm.147226

Car2
carbonic anhydrase 2
0.995
Mm.1186

Cdkn 1c
cyclin-dependent kinase
0.986
Mm.168789

inhibitor 1C (P57)

Lcn7
lipocalin 7
0.999
Mm.15801

A430017F18
No similar gene
1.000
Mm.44883

AU044919
No significant similar gene
1.000
Mm.14438

2310075M17Rik
Similar to S3543 GTP-binding
0.999
Mm.196592

protein (90%)

E112
Eleven-nineteen lysine-rich
0.998
Mm.21288

leukemia gene 2

LOC207685
Hypothetical protein
0.998
Mm.38214

2310061104Rik
No similar gene
0.998
Mm.5624

5830431A10Rik
Contain Corl/Xlr/Xmr
0.997
Mm.1148

conserved region

2700007P21Rik
Unknown protein
0.997
Mm.3587

B930086G17
No similar gene
0.992
Mm.24738

2410166105Rik
Hypothetical protein
0.989
Mm.30153

D10Ertd749e
Similar to ZW10 interacting
0.986
Mm.38994

protein-1

2210023F24Rik
Contain B-box Zn-finger and
0.983
Mm.5510

SPRY domain

Riken 4237666
No significant similar gene
0.978
Mm.276231

6230421P05Rik
No similar gene
0.978
Mm.26147

4631408O11Rik*
No significant similar gene
0.964
Mm.2935

1110054N06Rik*
Unknow protein with Ankyrin
0.960
Mm.15351

repeat

[0111]

6

TABLE 5

Genes down-regulated in CD38+CD34− Cells

Symbol or

Correlation
Unigene

Acc. No.
Description
to SC activity
No.

Satb1
Special AT-rich sequence binding protein 1
0.955
Mm.4381

Ptpro
Protein tyrosine phosphatase, receptor type, O
0.999
Mm.4715

Sell
Selectin, lymphocyte
0.988
Mm.1461

Ccl9
Chemokine (C-C motif) ligand 9
0.988
Mm.2271

Cnn3
Calponin 3, acidic
0.988
Mm.22171

Lgals3
Lectin, galactose binding, soluble 3
0.971
Mm.2970

Mki67
Antigen identified by monoclonal antibody Ki 67
0.998
Mm.4078

Bin1
Bridging integrator 1
0.977
Mm.4383

Sult4a1
Sulfotransferase family 4A, member 1
1.000
Mm.20451

Hdc
Histidine decarboxylase
0.996
Mm.18603

AI132321
Contain phospholipase D. active site motif
−1.000
Mm.203915

2610036L13Rik
No similar gene
−1.000
Mm.23526

BC018347
Similar to translation initiation factor 1F-2
−1.000
Mm.154309

X90778
Similar to Histone H2B
−1.000
Mm.21579

AW060549
Similar to Retrovirus-related POL polyprotein
−0.999
Mm.29177

X67863
Similar to Octapeptide-repeat protein T2
−0.995
Mm.35868

X15378
Similar to Myeloperoxidase and Eosinophil
−0.975
Mm.4668

peroxidase precursor

Plac8
Uncharacterized Cys-rich domain containing protein
−0.960
Mm.34609

D13Ertd275e
Hypothetical protein
−0.952
Mm.21231

[0112]

7

TABLE 6

Cassification and Characterization of Genes Upregulated in Mouse HSCs

Sequence
Unigene
Protein

Class
Name
Sequence Description
Code
Code
ID

Apoptosis
Birc5
baculoviral IAP repeat-containing 5
101521
Mm.8552
O70201

Cell cycle
Spin
spindlin
99563
Mm.42193

Chromosomal
Btg1

M. musculus
btg1 mRNA.
93104

P31607

Chromosomal
Calm2

Mus musculus
calmodulin synthesis (CaM)
93293

P02593

cDNA, complete cds.

Enzyme
Ctsl
cathepsin L
101963
Mm.930
P06797

Enzyme
Gdi1
guanosine diphosphate (GDP) dissociation
97313
Mm.205830
P50396

inhibitor 1

Enzyme
Hadh2
hydroxysteroid (17-beta) dehydrogenase 10
101045
Mm.6994
O08756

Enzyme
Mt2
Mouse metallothionein II (MT-II) gene.
101561

P02798

Enzyme
Pnp
purine-nucleoside phosphorylase
93290
Mm.17932
P23492

Enzyme
Vdu1
Vhlh-interacting deubiquitinating enzyme 1
160710
Mm.24383

Kinase
Csnk1e
casein kinase 1, epsilon
97925
Mm.30199
Q9QUI3

Kinase
Nme3
expressed in non-metastatic cells 3
94981_i
Mm.27278

Lectin
Lgals9
lectin, galactose binding, soluble 9
103335
Mm.18087
O08573

Metabolism
Aldh1a1
aldehyde dehydrogenase family 1,
100068
Mm.4514
P24549

subfamily A1

Metabolism
Aldh1a7
aldehyde dehydrogenase family 1,
94778
Mm.14609
O35945

subfamily A7

Metabolism
Cpo
coproporphyrinogen oxidase
98505_i
Mm.35820
P36552

Metabolism
Cpo
coproporphyrinogen oxidase
98506_r
Mm.35820
P36552

Metabolism
Ech1
enoyl coenzyme A hydratase 1, peroxisomal
93754
Mm.2112
O35459

Metabolism
Mtcp1

M. musculus
MTCP-1 gene.
103043

Q61908

Nuclear
Rbmx
RNA binding motif protein, X chromosome
97848
Mm.28275
Q9R0Y0

Nuclear
Snrpa
small nuclear ribonucleoprotein polypeptide
100101
Mm.4633
Q62189

A

Secreted
Iap
intracisternal A particles
97181_f
Mm.212712
P03975

Secreted
Tff2

Mus musculus
spasmolytic polypeptide
93302

Q03404

(mSP) gene, complete cds.

Signaling
Gnb4
guanine nucleotide binding protein, beta 4
93949
Mm.9336
P29387

Signaling
Tsc2
tuberous sclerosis 2
97953_g
Mm.30435
Q61037

Structural
Fscn1
fascin homolog 1, actin bundling protein
92838
Mm.13194
Q61553

(Strongylocentrotus) purpuratus)

Transcription
Irf1
Interferon regulatory factor 1
102401
Mm.1246
P15314

Transcription
Cited2
Cbp/p300-interacting transactivator, with
101973
Mm.9524
O35740

Glu/Asp-rich carboxy-terminal domain, 2

Transcription
Ncor1
nuclear receptor co-repressor 1
101536
Mm.88061
Q60974

Transcription
Sox6
SRY-box containing gene 6
92726
Mm.4656
P40645

Transcription
Hhex

Mus musculus
Hex(Prh) gene, exon4 and
98408
Mm.33896
Q9R1X2

complete cds.

Transcription
Trim30
tripartite motif protein 30
98030
Mm.3288
P15533

Transcription
Tieg
TGFB inducible early growth response
99602
Mm.4292
O89091

Transcription
Klf2
Kruppel-like factor 2 (lung)
96109
Mm.26938
Q60843

Transcription
Eif4a2
eukaryotic translation initiation factor 4A2
93089
Mm.16323
P10630

Transcription
H2a-615

Mus musculus
histone H2a.2-615 (H2a-
93068_r

P20670

615), and histone H3.2-615 (H3-615)

genes, complete cds.

Transcription
Nfe212

Mus musculus
p45 NF-E2 related factor 2
92562

Q60795

(NRF2) gene, exon 2 to exon 5 and complete cds.

Transcription
Fli1
Friend leukemia integration 1
94698
Mm.119781
P26323

Transcription
Mcmd5
mini chromosome maintenance deficient 5
100156
Mm.5048
P49718

(S. cerevisiae)

Transcription
H3f3b
H3 histone, family 3B
100708
Mm.18516
P06351

Transcription
Rev3l
REV3-like, catalytic subunit of DNA
103457
Mm.2167
Q61493

polymerase zeta RAD54 like (S. cerevisiae)

Transcription
Hoxb5
homeo box B5
103666
Mm.207
P09079

Transcription
Pbx1
pre B-cell leukemia transcription factor 1
94804
Mm.221246
P41778

Transcription
Zfp3611
zinc finger protein 36, C3H type-like 1
93324
Mm.18571
P23950

Transcription
Myb
myeloblastosis oncogene
92644_s
Mm.1202
P06876

Transcription
Sp4
trans-acting transcription factor 4
92992_i
Mm.5073

Transcription
Idb2

Mus musculus
helix-loop-helix protein Id2
93013

gene, 3′; region.

Transmembrane
Hiat1
hippocampus abundant gene transcript 1
160447
Mm.3792
P70187

Transmembrane
Igh-4
mouse gene for the constant part of gamma-
101870

P01869

1 immunogloblin.

Transmembrane
Ii
Ia-associated invariant chain
101054
Mm.7043
P04441

Transmembrane
H2-Aa
histocompatibility 2, class II antigen A,
92866
Mm.175310
P23150

alpha

Transmembrane
Epor
Mouse gene for erythropoietin receptor.
103997

P14753

Transmembrane
Irs2

Mus musculus
insulin receptor substrate-2
92205

O88970

(Irs2) gene, partial cds.

Transmembrane
H2-Eb1
histocompatibility 2, class 11 antigen E beta
94285
Mm.22564
Q61857

Transmembrane
Tnfrsf17
tumor necrosis factor receptor superfamily,
94190
Mm.12935
O88472

member 17

Transmembrane
Adcy9
adenylate cyclase 9
92527
Mm.4294
P51830

Transmembrane
Edg1
endothelial differentiation sphingolipid G-
161788_f
Mm.982

protein-coupled receptor 1

Transmembrane
Fzd4
frizzled homolog 4 (Drosophila)
93459_s
Mm.68712

Transport
Vps35
vacuolar protein sorting 35
92640
Mm.196201
Q9EQH3

Transport
Hbb-b2
Mouse gene for beta-1-globin.
103534

P02089

Transport
Kpnb1
karyopherin (importin) beta 1
93111
Mm.16710
P70168

Transport
Rab9
RAB9, member RAS oncogene family
95516
Mm.25306
Q9R0M6

Transport
Rac1
RAS-related C3 botulinum substrate 1
101555
Mm.889
P15154

Transport
Rab33b

Mus musculus
DNA for Rab33B, exon 2
103062

O35963

and complete cds.

Zinc Finger
Zfp216
zinc finger protein 216
160321
Mm.2904
O88878

Zinc Finger
Rnf11
ring finger protein 11
160205_f
Mm.25228
Q9QYK7

Zinc Finger
Nbr1
next to the Brca1
101484
Mm.784
P97432

Zinc Finger
pol

Mus musculus
clone MIA14 full-length
93907_f

P11365

intracisternal A-particle gag protein gene,

complete cds; and pol pseudogene,

complete sequence.

Zinc Finger
Gfi1b
growth factor independent 1B
102260
Mm.10804
O70237

Zinc Finger
Car1
carbonic anhydrase 1
98098
Mm.3471
P13634

Cul4a
cullin 4A
104288
Mm.22276

D7Wsu128e
DNA segment, Chr 7, Wayne State
103861_s
Mm.21103

University 128, expressed

Rhced
Rhesus blood group CE and D
103340
Mm.195461
Q9QX04

AU044919
expressed sequence AU044919
102823
Mm.14438

Igj
immunoglobulin joining chain
102372
Mm.1192

Lisch7
liver-specific bHLH-Zip transcription factor
162274_f
Mm.4067

Igh-VJ558
immunoglobulin heavy chain, (J558 family)
161486_f
Mm.157783

0910001L24
RIKEN cDNA 0910001L24 gene
161243_f
Mm.22637

Rik

Txnip
thioredoxin interacting protein
160547_s
Mm.77432

Dr1
down-regulator of transcription 1
160449
Mm.38184

4933429H19

Mus musculus
, Similar to translocation
160136_r
Mm.200980

Rik
protein 1, clone IMAGE: 5347105, mRNA,

partial cds

1500010B24
RIKEN cDNA 1500010B24 gene
160111
Mm.65264

Rik

IgM

Mus castaneus
IgK chain gene, C-region, 3′; end.
102156_f

AA409749
expressed sequence AA409749
100742
Mm.3628

D2Ertd63e
DNA segment, Chr 2, ERATO Doi 63,
95862
Mm.24965

expressed

Igk-V28

Mus musculus
anti-HIV-1 reverse
100322
Mm.220154

transcriptase single-chain variable fragment

mRNA, complete cds

5830431A10
RIKEN cDNA 5830431A10 gene
94136
Mm.1148

Rik

Igl-V1
Mouse Ig active lambda-1-chain C-region
93638_s

gene, 3′; end.

Imap38
immunity-associated protein, 38 kDa
92489
Mm.197478
P70224

92316_f
Mouse germline Ig lambda-2-chain C-
92316_f

region gene, 3′; end.

2700007P21
RIKEN cDNA 2700007P21 gene
92268
Mm.3587

Rik

104477
ESTs
104477
Mm.29940

0610012A05
RIKEN cDNA 0610012A05 gene
104206
Mm.27619

Rik

Atp6s1

Mus musculus
, clone MGC: 37615
103699_i
Mm.222723

IMAGE: 4989784, mRNA, complete cds

Gbp3
guanylate nucleotide binding protein 3
103202
Mm.1909

immunoglobulin
Mouse mRNA for immunoglobulin gamma-
102721

V region
3 V-D-J region and secreted constant

region, complete cds.

AI256744

Mus musculus
, clone IMAGE: 3500612,
102233
Mm.1043

mRNA, partial cds

Ptdss1
phosphatidylserine synthase 1
101931
Mm.9440
O55024

Gga1
golgi associated, gamma adaptin ear
98445
Mm.34525

containing, ARF binding protein 1

4121402D02
RIKEN cDNA 4121402D02 gene
97935
Mm.30252

Rik

Iigp
interferon-inducible GTPase
96764
Mm.29008
Q9Z1M3

2310022K15
RIKEN cDNA 2310022K15 gene
95622
Mm.28047

Rik

Vcl
vinculin
94963
Mm.12842

2610319K07
RIKEN cDNA 2610319K07 gene
104744
Mm.200479

Rik

Iga
Mouse Ig germline D-J-C region alpha gene
100583

and secreted tail; Mouse germ line gene for

immunoglobulin alpha H constant part

(coding for the last three exons)

Prpf8
pre-mRNA processing factor 8
98574
Mm.3757

Scotin
scotin gene
95102
Mm.196533

1110035L05
RIKEN cDNA 1110035L05 gene
95052
Mm.29140

Rik

3110001A13
RIKEN cDNA 3110001A13 gene
96640
Mm.200627

Rik

Vps26
vacuolar protein sorting 26 (yeast)
96665
Mm.27373

mu-
Mouse germ line gene fragment for mu-
93583_s

immunoglobulin
immunoglobulin C-terminus (secreted

form).

H19

M. musculus
H19 mRNA.
93028

Q61638

Car2
carbonic anhydrase 2
92642
Mm.1186

Rae1
RAE1 RNA export 1 homolog (S. pombe)
160466
Mm.4113

Map1lc3
microtubule-associated protein 1 light chain 3
160288
Mm.28357

1700008C22
RIKEN cDNA 1700008C22 gene
160123
Mm.177990

Rik

98254_f
un98f06.x1 NCI_CGAP_Mam6 Mus musculus
98254_f

cDNA clone IMAGE: 2581955 3′;

similar to gb: M10062 Mouse IgE-binding

factor mRNA, complete cds (MOUSE);

mRNA sequence.

Eef2
eukaryotic translation elongation factor 2
97559
Mm.27818
Q61509

Igk-V28
immunoglobulin kappa chain variable 28
99405
Mm.104747

(V28)

9030022E12
RIKEN cDNA 9030022E12 gene
104198
Mm.27519

Rik

D18362
expressed sequence D18362
103206
Mm.205433

Hey1

Mus musculus
6 days neonate head cDNA,
101913
Mm.222825

RIKEN full-length enriched library,

clone: 5430408K11: hairy/enhancer-of-split

related with YRPW motif 1, full insert

sequence

shrm
shroom
100024
Mm.46014

AW547365
expressed sequence AW547365
97425
Mm.30015

D8Ertd69e
DNA segment, Chr 8, ERATO Doi 69,
94922_i
Mm.26609

expressed

Frap1
FK506 binding protein 12-rapamycin
104708
Mm.21158

associated protein 1

4933434E20
RIKEN cDNA 4933434E20 gene
104038
Mm.21451

Rik

1810009A16
RIKEN cDNA 1810009A16 gene
104041
Mm.21458

Rik

Pex11a
peroxisomal biogenesis factor 11a
103660
Mm.20615
Q9Z211

AU044919
expressed sequence AU044919
102824_g
Mm.14438

MGC29044
hypothetical protein MGC29044
102375
Mm.1196

Mkm1
makorin, ring finger protein, 1
101070
Mm.7198

LOC207933
similar to Isopentenyl-diphosphate delta-
96269
Mm.29847

isomerase (IPP isomerase) (Isopentenyl

pyrophosphate isomerase)

Elp3
elongation protein 3 homolog (S. cerevisiae)
95717
Mm.29719

Add1
adducin 1 (alpha)
94535
Mm.29052

Pbef
pre-B-cell colony-enhancing factor
94461
Mm.28830

4930588A18

Mus musculus
, clone IMAGE: 4457493, mRNA
96717
Mm.233830

Rik

Dad1

Mus musculus
Defender against Apoptotic
96008

Death (Dad1) gene, exon 3.

2410015A15
RIKEN cDNA 2410015A15 gene
95433
Mm.24495

Rik

Xbp1
X-box binding protein 1
94821
Mm.22718

Net1
neuroepithelial cell transforming gene 1
94223
Mm.22261
Q9Z1L7

Igk-V28
immunoglobulin kappa chain variable 28
93086
Mm.104747

(V28)

LOC218490
similar to Transcription factor BTF3 (RNA
93057
Mm.1538

polymerase B transcription factor 3)

Lamc1
laminin, gamma 1
161706_f
Mm.1249

AI450287
expressed sequence AI450287
161596_f
Mm.222827

Sep15
15-kDa selenoprotein
160360
Mm.29812

LOC229906
similar to TRANSCRIPTION INITIATION
160225
Mm.27213

FACTOR IIB (TFIIB) (RNA

POLYMERASE II ALPHA INITIATION

FACTOR)

2810043O03
RIKEN cDNA 2810043O03 gene
98756
Mm.45532

Rik

96532
ESTs, Highly similar to nucleolar protein
96532
Mm.35019

GU2 [Mus musculus] [M. musculus]

Myt1l
myelin transcription factor 1-like
96495
Mm.2523
P97500

2010004A03
RIKEN cDNA 2010004A03 gene
94802
Mm.35302

Rik

C79248
expressed sequence C79248
94689
Mm.153895

Mylk
myosin, light polypeptide kinase
93482
Mm.27680

D1Ertd147e
DNA segment, Chr 1, ERATO Doi 147,
93191
Mm.5572

expressed

R75364
expressed sequence R75364
92397
Mm.89393

92245
ESTs, Highly similar to nucleolar protein
92245
Mm.35019

GU2 [Mus musculus] [M. musculus]

Ctse

Mus musculus
cathepsin E gene, exon 1,
104696

partial.

AA420392
expressed sequence AA420392
104670
Mm.32357

Acyp2
acylphosphatase 2, muscle type
104258
Mm.28407

Lrba
LPS-responsive beige-like anchor
104264
Mm.28458

Dock2
dedicator of cyto-kinesis 2
103462
Mm.2173

Gabpa
GA repeat binding protein, alpha
103440
Mm.18974

Nrip1
nuclear receptor interacting protein 1
103288
Mm.20895
Q9Z2K2

AI225904
expressed sequence AI225904
103200
Mm.1902

98438_f
Mouse Q4 class 1 MHC gene (exon 5).
98438_f

Q31220

2010012D11
RIKEN cDNA 2010012D11 gene
96231
Mm.140243

Rik

AU019574

Mus musculus
, Similar to hypothetical
96172
Mm.28395

protein FLJ11110, clone MGC: 11734

IMAGE: 3968418, mRNA, complete cds

9130415E20
RIKEN cDNA 9130415E20 gene
95020
Mm.40620

Rik

95021

Mus musculus
, clone IMAGE: 4502890,
95021
Mm.27476

mRNA

AW495846
expressed sequence AW495846
104549
Mm.23702

Gtpbp2
GTP binding protein 2
104144
Mm.22147

2310050N11
RIKEN cDNA 2310050N11 gene
104114
Mm.21954

Rik

Ormd13
ORM1-like 3 (S. cerevisiae)
98065
Mm.180546

2610003J05
RIKEN cDNA 2610003J05 gene
97491
Mm.31051

Rik

Map17
membrane-associated protein 17
96935
Mm.30181

Gabarapl2
GABA(A) receptor-associated protein like 2
96840
Mm.30017

2310050K10
RIKEN cDNA 2310050K10 gene
95743
Mm.29769

Rik

AI182287
expressed sequence AI182287
94469
Mm.28848

Nudel
nuclear distribution gene E-like
98884_r
Mm.31979

CpneI
copine I
97199
Mm.27660

Dnajb9
DnaJ (Hsp40) homolog, subfamily B,
96680
Mm.27432

member 9

95488

Mus musculus
, clone IMAGE: 3597827,
95488
Mm.25018

mRNA, partial cds

2700059C12
RIKEN cDNA 2700059C12 gene
93312
Mm.18485

Rik

Sdcbp
syndecan binding protein
93017
Mm.14744
O88601

[0113]

8

TABLE 7

Tansmembrane Proteins Enriched in Mouse HSCs

Classification
Description

surface
Histocompatibility 2, class II antigen

antigen
E beta

receptor
Gamma-aminobutyric acid (GABA) B

receptor, 1

oncogene
Myeloproliferative leukemia virus

oncogene (TPOR)

surface
Histocompatibility 2, class II antigen

antigen
A alpha

Cytotoxic T lymphocyte-associated

protein 2 beta

receptor
Erythropoietin receptor

oncogene
Kit oncogene

Coagulation factor II (thrombin)

receptor

Frizzled homolog 4 (Drosophila)

Membrane-associated protein 17

surface
ESTs similar to C211_Human putative

glycoprotein
surface glycoprotein

[0114]

9

TABLE 8

Transcription Factors Upregulated in Mouse HSCs

Symbol
Description
Fold change
Accession No.

Klf2
Kruppel-like factor 2 (lung)
44.9
NM 008452

Nmyc1
neuroblastoma myc-related
10.4
NM 008709

oncogene 1

Zfxlha
zinc finger homeobox 1 a
10.4
NM 011546

Gata3
GATA-binding protein 3
9.0
NM 008091

Tcfl5
transcription factor 15
8.6
NM 009328

Tall
T-cell acute lymphocytic
8.3
NM 011527

leukemia 1

Hoxb5
homeo box B5
7.2
NM 008268

Meis1
myeloid ecotropic viral
7.1
NM 010789

integration site 1

Pbx3b

Mus musculus
transcription
6.5
AF020200

factor PBX3b

Cited2
Cbp/p300-interacting
5.2
NM 010828

transactivator 2

Atf2
activating transcription factor 2
3.6
none

Pbx1
pre B-cell leukemia
4.7
NM 008783

transcription factor 1

None
chromatin remodeling factor
4.5
Mm.24637

None
EST similar to PRE-MRNA
3.4
Mm.29915

SPLICING FACTOR SRP20

Btf3
basic transcription factor 3
3.2
none

Tcfl2
transcription factor 12
2.7
NM 011544

Madh7
MAD homolog 7 (Drosophila)
2.7
NM 008543

Hhex
hematopoietically expressed
2.5
NM 008245

homeobox

Example 5

Hierarchical Clustering Analysis of Differential Expressed Genes

[0115] This Example describes study aimed at determining if genes differentially expressed with the HSC compartment are also expressed in other tissues. To perform this analysis we compared the gene expression levels of 210 differentially expressed HSC genes with a database composed of 45 normal tissue. Hierarchical clustering of these data was used to group both those tissues and genes with similar expression patterns. The three HSC cell subsets formed a distinct branch in this analysis, with LTR-enriched 38+34− cells forming a discrete branch compared to the STR cells (38+34+ and 38−34+). This clustering pattern is consistent with the stem cell activity pattern within the three subsets. Importantly, the HSC samples do not cluster near the bone or bone marrow samples suggesting that the differentially expressed HSC genes are not bone marrow related. This analysis also showed that the majority of these genes were not ubiquitously expressed although most were expressed at comparable levels in at least one other tissue.

[0116] Three of the genes were found to have their peak expression within the HSC compartment. These were the scaffolding protein Gab1 (GRB2-asssociated binding protein 1) and the uncharacterized gene A430017F18 which displayed the highest level expression in the LTR enriched CD38+CD34− cells, and the Pdgfrb gene (platelet derived growth factor receptor, beta polypeptide) which peaked within the 38+34+ STR HSC subset. Although the majority of these genes are also expressed at comparable levels in other tissues it is important to note that in many cases the level of expression in HSC subsets was at or near the peak expression determined for these genes across the entire 45 tissue panel. The high relative expression within HSCs of this subset of genes indicates that they likely to play an important role in the biology of HSCs.

[0117] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0118] All publications, GenBank sequences, patents and patent applications cited herein are hereby expressly incorporated by reference in their entirety and for all purposes as if each is individually so denoted.

Methods and compositions for modulating stem cells

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

US Classifications

International Classifications

Abstract

Description

Claims

CROSS-REFERENCE TO RELATED APPLICATIONS

Provisional Applications (1)