WO2011101550A1 - Method of detecting the differentiation status of a stem cell population - Google Patents

Method of detecting the differentiation status of a stem cell population Download PDF

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WO2011101550A1
WO2011101550A1 PCT/FI2011/050156 FI2011050156W WO2011101550A1 WO 2011101550 A1 WO2011101550 A1 WO 2011101550A1 FI 2011050156 W FI2011050156 W FI 2011050156W WO 2011101550 A1 WO2011101550 A1 WO 2011101550A1
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stem cells
cells
gene expression
expression
cell
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Suvi Natunen
Heidi Anderson
Jarno Tuimala
Jukka Partanen
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Suomen Punainen Risti, Veripalvelu
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5073—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, Konjac gum, Locust bean gum or Guar gum

Definitions

  • the present invention relates to methods utilizing novel target genes related to differentiation status of human stem cells.
  • the present invention provides a method for identification and/or quantification of a stem cell population comprising a step of preparing from a sample of the stem cell population a gene expression profile based on the expression level of at least one of the glycosyltransferase genes B3GNT5, GALNT1 and GALNT7.
  • the invention also relates to method for identifying compounds capable of modulating or hindering differentiation of human stem cells.
  • GALNT7 ( respectively) between CD34 positive cells and megakaryocytes.
  • WO2006/131599 discloses mRNA markers related to glycoproteins and/or glycosynthase proteins but no data on GALNT1 or 7 are given.
  • differentiated erythroid line (EC) but was not found in any other cell type, including undifferentiated HSC.
  • Figure 1 The expression of polypeptide-GalNAc-transferases GALNT1-3, 5-14 in different types of human stem cells as compared to differentiated cells, a) CD34+ hematopoietic stem cells compared to CD34- mononuclear cells, b) CD 133+ hematopoietic stem cells compared to CD 133- mononuclear cells, c) human embryonic stem cells (FES) compared to embryoid bodies (EB), d) cord blood mesenchymal stem cells (CB MSC) compared to cells
  • FES human embryonic stem cells
  • EB embryoid bodies
  • CB MSC cord blood mesenchymal stem cells
  • adipogenic adipogenic
  • osteogenic osteogenic
  • Figure 2. The top row shows the mean expression of GALNT1 and GALNT7 in the selected cell types. The bottom row shows the variation of the expression in the same cell types.
  • Figure 3. Stem cell populations can be identified using the combined knowledge of expression of GALNT1 and GALNT7. The data derived from Figure 2.
  • FIG. 1 Levels of B3GNT5 mRNA detected in various human tissues by using mRNA microarrays.
  • the data are derived from the public Genesapiens database.
  • the present invention provides a method of detecting the differentiation status of human stem cells.
  • the method can be applied to identify the presence of stem cells in a cell preparation. Also, the method can be applied to estimate the relative number of stem cells in a cell preparation or between cell preparations.
  • the cell preparation can be, for example, clinical grafts for stem cell transplantation.
  • the method comprises a step of preparing from a sample comprising human stem cells a gene expression profile based on the expression level of at least one of the genes selected from the group consisting of glycosyltransferase genes B3GNT5, GALNT1 and GALNT7. The level of gene expression in said sample can then be compared to the control samples not known to contain stem cells or containing known amounts of these cells. There are various methods described in the prior art for determination of the expression level, for example, the quantitative RT-PCR method.
  • the present invention further provides a method of identifying compounds that augment, modulate or hinder the differentiation of human stem cells, the method comprises the steps of:
  • step (b) incubating said cells for a time sufficient for the cells to change their differentiation status; (c) preparing a gene expression profile from the cells obtained from step (b), wherein said gene expression profile is based on the expression level of at least one of the genes selected from the group consisting of glycosyltransferase genes B3GNT5, GALNT1 and GALNT7; and
  • step (d) comparing gene expression profile obtained from step (c) to a gene expression profile made from corresponding stem cells not contacted with said compound, wherein the difference in the amount of expression of at least one of said genes in the presence of the compound indicates that the compound is a candidate compound for modulation or hindrance of the differentiation of human stem cells.
  • target nucleic acid refers to a nucleic acid (often derived from a biological sample), to which a polynucleotide probe is designed to specifically hybridize. It is either the presence or absence of the target nucleic acid that is to be detected, or the amount of the target nucleic acid that is to be quantified.
  • the target nucleic acid has a sequence that is
  • target nucleic acid can refer to the specific subsequence of a larger nucleic acid to which the probe is directed or to the overall sequence (e.g., gene or mRNA) whose expression level it is desired to detect.
  • the default parameters of the BLAST programs are suitable.
  • the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM 62 scoring matrix.
  • the TBLATN program (using protein sequence for nucleotide sequence) uses as defaults a word length (W) of 3, an expectation (E) of 10, and a BLOSUM 62 scoring matrix. (See, e.g., Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).
  • genes are differentially expressed in stem cells under
  • One or more of the target genes provided by the present invention can be used as part of an "an expression profile" that is representative of a particular state of a human stem cell. These new target genes can be applied in identification of stem cells or their quantification in more
  • heterogeneous cell preparations such as clinical grafts for stem cell transplantation.
  • the cellular status of stem cells e.g. how much they have 'sternness' or how much they have differentiated can be analyzed more reliably.
  • the present invention provides a novel tools for this purpose as well, since the number of cells remained as undifferentiated can be estimated by the gene expression profile.
  • Certain methods that are provided involve determining the expression level of one or more of the differentially expressed genes in a test cell population and comparing the result with the expression level of the same genes in a control cell population, or comparing the expression profile in one sample to that determined from another sample.
  • the level of expression of the differentially expressed nucleic acids can be determined at either the nucleic acid, that is, typically mRNA level or the protein level.
  • the phrases "determining the expression level,” "preparing a gene expression profile,” and other like phrases when used in reference to the differentially expressed nucleic acids mean that transcript levels and/or levels of protein encoded by the differentially encoded nucleic acids are detected.
  • the level can be determined qualitatively, but generally is determined quantitatively.
  • transcript levels of these genes can readily be determined based upon the sequence information that is disclosed herein and coupled with the nucleic acid and protein detection methods that are described herein and that are known in the art,. If transcript levels are determined, they can be determined using routine methods. For instance, the sequence information provided herein (e.g., GenBank sequence entries) can be used to construct nucleic acid probes using a number of conventional hybridization detection methods (e.g., Northern blots). Alternatively, the provided sequence information can be used to generate primers that in turn are used to amplify and detect differentially expressed nucleic acids that are present in a sample, e.g., using quantitative RT-PCR methods.
  • sequence information provided herein e.g., GenBank sequence entries
  • the provided sequence information can be used to generate primers that in turn are used to amplify and detect differentially expressed nucleic acids that are present in a sample, e.g., using quantitative RT-PCR methods.
  • encoded protein can be detected and optionally quantified using any of a number of established techniques.
  • One common approach is to use antibodies that specifically bind to the protein product in immunoassay methods. Additional details regarding methods of conducting differential gene expression are provided infra. Expression levels can be detected for one, some, or all of the differentially expressed nucleic acids that are listed herein.
  • Determination of expression levels is typically done with a test sample taken from a test cell population.
  • the term "population" when used in reference to a cell can mean a single cell, but typically refers to a plurality of cells (e.g., a tissue sample).
  • Certain screening methods are performed with test cells that are "capable of expressing” one or more of the differentially expressed nucleic acids.
  • the phrase "capable or expressing” means that the gene of interest is in intact form and can be expressed within the cell.
  • a number of the methods that are provided involve a comparison of expression levels for certain differentially expressed nucleic acids in a "test cell” with the expression levels for the same nucleic acids in a “control cell” (also sometimes referred to as a "control sample,” a “reference cell,” a “reference value,” or simply a “control”).
  • Other methods involve a comparison between one expression profile and a baseline expression profile. In either case, the expression level for the control cell or baseline expression profile essentially establishes a baseline against which an experimental value is compared.
  • the comparison of expression levels are meant to be interpreted broadly with respect to what is meant by: 1) the term “cell”, 2) the time at which the expression levels for test and control cells are determined, and 3) with respect to the measure of the expression levels.
  • test cell and “control cell” is used for convenience, the term “cell” is meant to be construed broadly.
  • a cell can also refer to a population of cells (e.g., a tissue sample), just as a population of cells can have a single member.
  • samples can be obtained from various sources, particularly sources of stem cells such as cord blood or bone marrow.
  • comparison of expression levels can be done contemporaneously (e.g., a test and control cell are each contacted with a test agent in parallel reactions).
  • the comparison alternatively can be conducted with expression levels that have been determined at temporally distinct times.
  • expression levels for the control cell can be collected prior to the expression levels for the test cell and stored for future use (e.g., expression levels stored on a computer compatible storage medium).
  • the expression level for a control cell or baseline expression profile can be a value for a single cell or it can be an average, mean or other statistical value determined for a plurality of cells.
  • the value for each expression level for the control cell is a range of values representative of the range observed for a particular population.
  • the particular control cell population can be e.g. human peripheral blood sample.
  • the test sample to be compared to this particular control could be e.g. stem cell graft prepared from human cord blood. The difference in the expression depicts the amount of stem cells in the said graft.
  • Expression level values can also be either qualitative or quantitative.
  • the values for expression levels can be normalized with respect to the expression level of a nucleic acid that is not one of the markers under analysis.
  • Comparison of the expression levels between test and control cells can involve comparing levels for a single marker or a plurality of markers (e.g., when expression profiles are compared).
  • the expression level for a single marker is determined, whether expression levels between the test and control cell are similar or different involves a comparison of the expression level of the single marker.
  • the comparison analysis can involve two analyses: 1) determination for each marker examined whether the expression level is similar between the test and control cells, and 2) determination of how many markers from the group of markers examined show similar or different expression levels.
  • Methods for Detecting Differential Gene Expression Assays to monitor the expression of a marker or markers as defined in the present invention may utilize any available means of monitoring changes in the expression level of the target genes.
  • the protein products encoded by the genes identified herein can also be assayed to determine the amount of expression. Any method for specifically and quantitatively measuring a specific protein or mRNA or DNA product can be used. However, methods and assays of the invention typically utilize PCR- or array hybridization- based methods when detecting the expression of genes.
  • the genes identified as being differentially expressed in stem cells may be used in a variety of nucleic acid detection assays to detect or quantify the expression level of a gene or multiple genes in a given sample. For example, traditional Northern blotting, dot blots, nuclease protection, RT-PCR, differential display methods, subtractive hybridization, and in situ hybridization may be used for detecting gene expression levels. Levels of mRNA expression may be monitored directly by hybridization of probes to the nucleic acids of the invention. Stem cell samples can be exposed to an agent to be tested under appropriate conditions and time and total RNA or mRNA is isolated by standard procedures such those disclosed in
  • the high density array will typically include a number of probes that specifically hybridize to the sequences of interest. See WO 99/32660 for methods of producing probes for a given gene or genes.
  • the array will include one or more control probes.
  • the present invention revealed a group of specific mRNA-level markers that are highly expressed in stem cells but not in the differentiated cell lineages and can be applied for the analysis of human stem cell populations.
  • the novel mRNA markers provide novel tools for identification and/or quantification of stem cells.
  • the invention can be applied to estimate the stem cell content in clinical grafts prepared for stem cell transplantation (e.g. Lanza et al (eds) Essentials of stem cell biology. 2 nd ed. Academic Press 2009). Transplantation of
  • hematopoietic stem cells can be used to cure various hematological malignancies, such as leukemias.
  • Transplantation of other stem cells, in particular mesenchymal stem cells can be applied as supportive therapy in various traumas and they also are immunomodulatory and can be applied in disease with distorted immune response, such as inflammatory bowel disease or autoimmune diseases.
  • the present invention can also be used for determining the status of stem cell-containing cell preparations for other purposes, such as use of embryonic stem cell or induced pluripotent stem cell (Takahashi et al Cell 2007; 131: 861 - 872) populations to produce differentiated tissues (e.g.
  • the invention can be used, e.g., to determine the amount of remaining undifferentiated cells, or to estimate the efficacy of differentiation process in different time points or between different inducing agents. It is also possible to perform further mRNA screening for example by array or RT-PCR methods to further verify the current results and/or screen additional mRNA markers. mRNA-analysis methods
  • the gene expression analysis is performed by at least one mRNA expression analysis method preferably selected from the group consisting of: a hybridization method, a gene array method, RT-PCR-method, qRT-PCR-method or SAGE-method (serial analysis of gene expression).
  • the detection of expression of one preferred mRNA species is performed.
  • mRNA expression analysis is performed for multiple mRNA species.
  • multiple mRNA species are analysed in a profiling method.
  • the profiling method is preferably directed to the analysis of the purity of a cell population or quantification of a cell population, i.e. estimation of the ratio of undifferentiated versus differentiated stem cells in a cell preparation.
  • the mRNA-profiling method is directed to the analysis of the differentiation status of a cell population.
  • the present invention is directed to the use of mRNA markers B3GNT5, GALNT1 and GALNT7 for identification or quantification of hematopoietic stem cells.
  • these cells are defined as CD34 or CD 133 surface marker positive cell population, but it is known that neither of the marker will identify solely hematopoietic stem cells.
  • the markers of the present invention can be used to identify more specific populations.
  • the hematopoietic stem cell population refers to cell populations included in the CD34 positive cells with a capacity to differentiate toward hematopoietic lineage, that is into the lymphoid, myeloid and erythroid types of cells.
  • the cell population is included in the CD133 positive cells.
  • the mRNA markers B3GNT5, GALNT1 and GALNT7 can be applied to estimate the amount of stem cells when
  • hematopoietic stem cells are expanded ex vivo to increase the cell number useful for therapy.
  • the present invention is directed to identification of human hematopoietic stem cell population that is not selectable by CD34 or CD 133 marker but has the capacity to differentiate into hematopoietic cells.
  • Mesenchymal stem cell related marker mRNAs are not selectable by CD34 or CD 133 marker but has the capacity to differentiate into hematopoietic cells.
  • the present invention is directed to the use of mRNA markers B3GNT5, GALNT1 and GALNT7, and in particular that of GALNT1 alone, for identification or quantification of mesenchymal stem cells.
  • MSCs Mesenchymal stem cells
  • MSCs Mesenchymal stem cells
  • MSCs are multipotent stem cells that can differentiate into a variety of cell types, typically to osteoblast, chondrocytes and adipocytes, both in vitro and in vivo.
  • MSCs are obtained, for example, from the cord blood and bone marrow.
  • the exact definitions for MSC or cell lineages differentiated thereof have not been established (Da Silva Meilleres et al. Stem Cells 2008; 26: 2287-99), some criteria for undifferentiated MSC are described in Dominici et al. (Cytotherapy 2006; 8: 315-317).
  • Dominici et al. MSCs express CD105, CD73 and CD90 and lack expression of CD45, CD34 and CD14. Therefore, the term "mesenchymal stem cells” relates in this invention to all human multipotent stromal cells.
  • MSCs have a capacity for self renewal while maintaining their multipotency.
  • a standard test to confirm multipotency is differentiation of the cells into osteoblasts, adipocytes, and chondrocytes.
  • the extent to which the cells will differentiate may vary depending on induction, e.g. chemical vs. mechanical.
  • the invention can be used to select the most effective method or agent to induce MSC toward the said lineages.
  • the capacity of cells to differentiate and proliferate decreases with the age of the donor, as well as with the time cultivated in vitro.
  • MSCs have the potential to differentiate into various cellular lineages and can be expanded in culture conditions without losing their multipotency, they present a valuable source for applications in cell therapy and tissue engineering.
  • MSC transplantation offers a promising approach for treating certain nonhematological malignant and nonmalignant diseases and for stem cell-mediated tissue regeneration. In particular, they can be applied to induce immunosuppression. This can be done as supportive therapy in hematological stem cell transplantations in which immunologically-mediated graft- versus-host disease is a major complication
  • the mRNA markers in any combination or any one alone, can be used in one embodiment as novel markers for identification of functionally relevant MSC populations.
  • embryonic stem cell also includes induced pluripotent progenitor (iPS) cells, which can be produced basically from any differentiated adult cell type to embryonic stem cell-like cells (Takahashi et al. Cell 131: 861-872, 2007). Their therapeutic potential has been predicted to be enormous because patient's own cells can be induced and hence, ethical and histocompatibility problems can be avoided. iPS cells can also be applied by pharma industry and research groups as patient- specific models and source for differentiated tissues.
  • iPS induced pluripotent progenitor
  • ESC cells refer to cells positive for cell surface markers SSEA3, SSEA4, Tra-1-60 and Tra-1-81, and positive for mRNA markers Oct-4 and Nanog (e.g. Lanza et al (eds) Essentials of stem cell biology. 2 nd ed. Academic
  • ESC or iPS cells have functional pluripotency. Their functional pluripotency can be demonstrated by the formation of teratomas and embryoid body using methods known in the art (Lanza et al (eds) Essentials of stem cell biology. 2 nd ed. Academic Press 2009).
  • ESC cells are obtained using methods (Klimanskaya et al Human embryonic stem cell lines derived from single blastomeres. Nature 2006; 444: 481-5), not destroying embyos or other ethically acceptable methods.
  • ESC and in particular iPS cells are currently focused to generation of particular tissues for drug screening and to generation of disease models for pharma industry and scientists, rather than their direct therapeutic use. Hence, good markers for distinct subpopulations are needed.
  • the present invention provides a method of detecting the differentiation status of human stem cells, i.e. a method of detecting markers related to differentiation status of human stem cells.
  • the method comprises a step of preparing from a sample comprising human stem cells a gene expression profile based on the expression level of at least one of the genes selected from the group consisting of glycosyltransferase genes B3GNT5, GALNT1 and GALNT7.
  • the method is used for identifying or quantifying human stem cells in said sample.
  • One aim of the invention is to confirm the undifferentiated state of a stem cell sample and thus confirm that the stem cell population under examination can be used, e.g., for a stem cell therapy.
  • Another aim of the invention is to test the purity of the human stem cell population.
  • the expression levels of both GALNT1 and GALNT7 are used together to distinguish between embryonic, mesenchymal and hematopoietic stem cells.
  • a high GALNT1 level similar to the level of about 500 - 2000 in the experiment described in Figure 3 of Example 2) with a low or intermediate level (below about 500 - 800) of GALNT7 indicates ESC
  • MSC have a high GALNT7 level (typically over 1000 in the experiment described in Figure 3 of Example 2) with a relatively low level of GALNT1.
  • stem cells in particular stem cell lines are examples of biological material with heterogeneous features, some overlap must be assumed.
  • some overlap must be assumed.
  • the mRNA markers of the present invention can be used in any combination for identification and/or quantification of human stem cell populations.
  • Another main embodiment of the invention is a method of identifying compounds that augment, modulate or hinder the differentiation of human stem cells. This method comprises the following steps:
  • step (c) preparing a gene expression profile from the cells obtained from step (b), wherein said gene expression profile is based on the expression level of at least one of the genes selected from the group consisting of glycosyltransferase genes B3GNT5, GALNT1 and GALNT7; and
  • step (d) comparing gene expression profile obtained from step (c) to a gene expression profile made from corresponding stem cells not contacted with said compound, wherein the difference in the amount of expression of at least one of said genes in the presence of the compound indicates that the compound is a candidate compound for modulation or hindrance of the differentiation of human stem cells.
  • the sample of step (a) comprises CD133 and/or CD34 positive cells.
  • suitable stem cells for the method comprise hematopoietic stem cells, mesenchymal stem cells, embryonic stem cells, and iPS cells. It is also advantageous for the method, if the sample of step (a) comprises undifferentiated stem cells.
  • the candidate compounds that are assayed in the above method can be randomly selected or rationally selected or designed.
  • An example of randomly selected compound is a chemical library or a peptide combinatorial library, or a growth broth of an organism.
  • the compounds used in the present method can be, as examples, peptides, small chemical molecules, vitamin derivatives, as well as carbohydrates, lipids, oligonucleotides and covalent and non-covalent combinations thereof.
  • the compounds may be contacted with cells by adding the compound to the culture media or buffer in which the stem cells are incubated.
  • the compound, such as a DNA vector may also be introduced directly into cells.
  • the present invention is further directed to indirect analysis of B3GNT5, GALNT1 and
  • the indirect analysis measures a factor correlating to the mRNA- expression.
  • the indirect analysis is directed to protein or glycan level markers. In a preferred embodiment the present invention is directed to
  • the protein level marker is that these markers can be observed by common analytic and diagnostic methods including use of diagnostic antibodies and antibody based technologies.
  • the direct gene products that is, proteins encoded by the genes B3GNT5, GALNT1 and GALNT7 are used as markers.
  • the activity of the enzymes encoded by genes B3GNT5, GALNTl and GALNT7 are known only partially (e.g., Varki et al. (eds) Essential of Glycobiology. 2 nd ed. Cold Spring Harbor Laboratory Press. 2009).
  • B3GNT5 encodes a UDP-GlcNAc
  • GALNTl and GALNT7 encode forms of transferases that add N-acetylgalactosamine from UDP-GalNAc onto polypeptides, hence starting O-glycosylation.
  • transferases that add N-acetylgalactosamine from UDP-GalNAc onto polypeptides, hence starting O-glycosylation.
  • sequences of the target or marker genes listed in the specification are available in the public databases such as in GenBank.
  • sequences of the genes in public databases, such as GenBank are herein expressly incorporated by reference in their entirety as of the filing date of this application (see www.ncbi.nim.nih.gov).
  • Fresh umbilical cord blood was obtained from informed and consented donors at the Helsinki University Central Hospital, Department of Obstetrics and Gynaecology, and Helsinki Maternity Hospital. The study protocol was approved by ethical review board of Helsinki University Central Hospital and the Finnish Red Cross Blood Service. Collection and processing of the fresh cord blood was performed as described earlier (Jaatinen et al. 2006). Mononuclear cells were isolated by Ficoll-Hypaque (Amersham Biosciences, Piscaway, NJ, USA) density gradient sentrifugation, and plated on fibronectin coated tissue culture plates at the density of 1 x 10 6 cells/cm 2 .
  • the proliferation medium consisted of minimum essential medium (ccMEM) with Glutamax (Gibco, Grand Island, NY, USA) and 10% fetal bovine serum (FCS) (Gibco) supplemented with 50 nM dexamethasone (Sigma, St Louis, MO, USA) 10 ng/mL epidermal growth factor (EGF; Sigma), 10 ng/mL recombinant human platelet- derived growth factor (rhPDGF-BB; R&D Systems, Minneapolis, MN, USA), 100 U penicillin and 1000 U streptomycin (Gibco). Cells were allowed to adhere overnight and nonadherent cells were washed out. Medium changes were performed twice weekly up to three weeks.
  • ccMEM minimum essential medium
  • FCS fetal bovine serum
  • MSCs Mesenchymal stem cells
  • cells from adipogenic and osteogenic differentiation were at 4 th or 5 th passage for microarray analysis.
  • MSCs from 4 th passage were analyzed for their cell surface molecule expression.
  • Cells were labeled with fluorochrome-conjugated monoclonal antibodies; allophycocyanin (APC)- conjugated CD13 (BD Pharmingen), phycoerythrin (PE)-conjugated CD14, CD19, CD34 and CD45 (BD Pharmingen), fluorescein isothiocyanate (FITC)-conjucated CD90 (clone 5E10, Stem Cell Technologies), FITC-CD105 (Abeam) and FITC-HLA-DR (BD Pharmingen).
  • APC allophycocyanin
  • PE phycoerythrin
  • FITC fluorescein isothiocyanate
  • CD90 clone 5E10, Stem Cell Technologies
  • FITC-CD105 Abeam
  • FITC-HLA-DR BD Pharmingen
  • FITC-, PE-and APC-conjugated isotypic controls were used. Labeling was carried out in ⁇ of 0,3% ultra pure bovine serum albumin (BSA) in phosphate buffered saline (PBS) on ice for 30 minutes. Flow cytometric analysis was performed on FACSAria (Becton Dickinson Biosciences) with a 488-nm blue laser for (PE and FITC) and a 633-nm red laser for (APC). Fluorescense was measured using 530/30-nm (FITC), 585/42-nm (PE) and 660/20-nm (APC) bandpass filters. Data were analysed using FACSDiva software (BD Biociences). All of the MSC lines were positive for CD105, CD90 and CD73, and negative for CD13, CD14, CD19, CD34, CD45 and HLA-DR.
  • BSA bovine serum albumin
  • PBS phosphate buffered saline
  • fourth or fifth-passage cells were treated with osteogenesis inducing medium up to 3 weeks with medium changes twice weekly.
  • Osteogenesis inducing medium consists of aMEM (Gibco), HEPES (Gibco) and FCS (Gibco) supplemented with 2 mM L-glutamine (Gibco), 100 nM dexamethasone, 10 mM glycerol-2- phosphate, 0.05 mM ascorbic acid-2-phosphate, 100 U penicillin and 1000 U streptomycin. Osteogenic differentiation was confirmed by the von Kossa stain demonstrating the calcium of mineralized tissue.
  • fourth or fifth-passage cells were treated with adipogenic induction medium for 2 days and then with terminal adipogenesis differentiation medium for up to 3 weeks with medium changes twice weekly. Both mediums consisted of a base of aMEM Glutamax (Gibco), 10% FCS (Gibco), 20 mM HEPES supplemented with 100 U penicillin and 1000 U streptomycin.
  • adipogenic induction medium was supplemented with 0.1 mM indomethasin (Sigma-Aldrich), 0.5 mM 3-isobutyl-l- methylxanthine, 0.4 g/ml dexamethasone DM-200 (PromoCell) and 0.5 g/ml insulin -0,25 (PromoCell), and terminal adipogenic differentiation medium was supplemented with 0.1 mM indomethasin (Sigma-Aldrich), 0.5 g/ml insulin -0.25 (PromoCell), and 3 ⁇ g/ml ciglitazone - 1,5 (Promocell). Adipogenic differentiation was confirmed by the Sudan III stain
  • Human embryonic stem cell lines FES21, FES22, FES29 and FES30 were derived from in vitro fertilized excess human embryos and cultured as previously described (Mikkola et al. 2006).
  • FES21 and FES22 cell lines were initially cultured on mouse embryonic fibroblast feeder cells, but were transferred on human foreskin fibroblast feeder cells after 10 and 28 passages, respectively.
  • FES29 and FES30 cells lines have been cultured on human foreskin fibroblast feeder cells only.
  • EB embryoid bodies
  • the hESC colonies were cultured for 10-14 days and disaggregated mechanically. The cells were then transferred to suspension cultures for 10 days to form EBs (Mikkola et al. 2006).
  • Cord blood was obtained from the Helsinki University Central Hospital, Department of Obstetrics and Gynaecology, and Helsinki Maternity Hospital. All donors gave informed consent and the study was approved by ethical review board of the Helsinki University Central Hospital and the Finnish Red Cross Blood Service. Collection and processing of the fresh cord blood was performed as described earlier (Jaatinen 2006). Ficoll-Hypaque density gradient (Amersham Biosciences, New Jersey, USA) was used to isolate mononuclear cells. Stem cell fraction was sorted from the mononuclear fraction with anti-CD 133 or anti-CD34 microbeads in magnetic affinity cell sorting (Miltenyi Biotec, Bergisch Gladbach, Germany) (Kekarainen 2006). Mature leukocytes (CD133- cells, CD34- cells) were collected for control purposes.
  • RNA from cord blood MSC, cells with osteogenic differentiation and adipogenic differentiation, human embryonic stem cells and embryoid bodies was purified with RNeasy Mini Kit (StemCell Technologies) directly from the cell culture plates or separated CD34+, CD34-, CD 133+ and CD 133- cells washed with PBS to remove the remaining medium according to the manufacturer's instructions.
  • RNA consentration was checked with Nanodrop ND-1000 (Thermo scientific, Wilmington, DE, US) and quality was controlled by Experion electrophoresis station (Bio-Rad Laboratories, Herkules, CA, US).
  • RNA from each sample was processed according to One-Cycle Target Labeling protocol from GeneChip Expression Analysis Technical manual (Specific Protocols for Using the GeneChip Hybridization, Wash and Stain Kit) and hybridized to GeneChip® Human Genome U133 Plus 2.0 Array. Concentration of cDNA was checked with Nanodrop ND-1000 (Thermo scientific) and quality was controlled by Experion electrophoresis station (Bio-Rad Laboratories). GeneChip Fluidics Station 450 was used to wash and stain the arrays and GeneChip Scanner 3000 with AutoLoader was used to scan the arrays.
  • GeneChip Fluidics Stations and Scanner were controlled with GeneChip Command Console (AGCC) software version 1.0.
  • AGCC GeneChip Command Console
  • the replicate results of hybridization data for MSCs, and cells with osteogenic differentiation and cells with adipogenetic differentiation were obtained from four different MSC line. Sample labelling and hybridization were carried out at the Finnish DNA Microarray Centre at Turku Centre for Biotechnology, Turku, Finland.
  • AffyReader a Microarray Pipeline component
  • the Affymetrix probeset expression data of transcripts were newly clustered to represent genes by recombining the probes that represented the same gene using GeneChip library files (Custom CDF. versio 10) with esembl gene ID (Dai et al. 2005, 2007). All samples were normalized using Robust- Multi-array Average background adjustment (RMA) for intensities, quantile normalization and median-polish summarization (Wu and Izarry, 2004).
  • RMA Robust- Multi-array Average background adjustment
  • the RMA normalization was implemented using Bioconductor R, package affy. Values were transformed into log2.
  • polypeptide-GalNAc-transferases 1-3 and 5-14 was analyzed in stem cells and differentiated cells ( Figure 1).
  • the expression in CD34+ and CD133+ hematopoietic stem cells was compared to the expression in CD34- and CD133- cells
  • the expression in cord blood mesenchymal stem cells was compared to the expression in adipogenically and osteogenically differentiated cells
  • the expression in embryonic stem cells was compared to the expression in embryoid bodies.
  • GALNT7 (GenelD: 51809) was overexpressed in stem cells as compared to differentiated cells in all of the cell types studied (fold change 2-8x).
  • GALNT1 (GenelD: 2589) was overexpressed in hematopoietic stem cells and GALNT3 in mesenchymal stem cells and embryonic stem cells.
  • Stem cells express a characteristic profile of polypeptide-GalNAc-transferases, which changes upon differentiation. Analysis of the expression levels of polypeptide-GalNAc-transferases, especially GALNT7, can be used to determine the undifferentiated state of cells.
  • Affymetrix dataset E-TABM-185 downloaded from the ArrayExpress database maintained by the European Bioinformatics Institute (EBI) was used for the analyses. This dataset has been normalized with the GCRMA algorithm using the probe annotations provided by Affymetrix. The probesets for GALNTl and GALNT7 were extracted from the dataset, and were used for further analyses. All probesets for these genes match exactly multiple transcripts, possibly originating from the known splice variants: Ensembl database, containing genomic data, release 56 reports two variants for both genes.
  • GALNTl and GALNT7 were analyzed in stem cells and differentiated cell types ( Figure 2).
  • the expression of GALNTl is significantly higher in mesenchymal stem cells than other stem cells or non-stem cells.
  • GALNTl is also slightly upregulated in embryonic stem cells when compared to non-stem cells.
  • GALNT7 is significantly over- expressed in embryonic stem cells.
  • the expression of GALNT7 is about at the steady same level in other stem cells or non-stem cells.
  • GALNTl and GALNT7 can be successfully employed together to differentiate the stem cell types from each other ( Figure 3).
  • Example 3 Expression of B3GNT5 Mesenchymal stem cells, RNA isolation and mRNA microarray analyses were done as described in Example 1. It was noted that the expression level of B3GNT5 (ENSG).
  • B3GNT5 The expression level of B3GNT5 (GenelD: 84002) as compared to other cell types was also analysed using the public 1ST database (www.genesapiens.org), a database based on data of nearly 10 000 Affymetrix microarrays described in more details by Kilpinen et al ⁇ Genome Biology 2008; 9: R139).
  • the mean of expression level of B3GNT5 (ENSG 00000176597) in hematopoietic stem cells was found to be at the level of about 3000 (arbitrary units), whereas the levels in most other cells were substantially lower (Figure 4).

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Abstract

The present invention relates to methods utilizing novel target genes related to differentiation status of human stem cells. In particular, the present invention provides a method of detecting the differentiation status of a human stem cell population comprising a step of preparing from a sample of the stem cells a gene expression profile based on the expression level of at least one of glycosyltransferase genes B3GNT5, GALNT1 and GALNT7. The invention also relates to method for identifying compounds capable of modulating or hindering differentiation of human stem cells.

Description

Method of detecting the differentiation status of a stem cell population
The present invention relates to methods utilizing novel target genes related to differentiation status of human stem cells. In particular, the present invention provides a method for identification and/or quantification of a stem cell population comprising a step of preparing from a sample of the stem cell population a gene expression profile based on the expression level of at least one of the glycosyltransferase genes B3GNT5, GALNT1 and GALNT7. The invention also relates to method for identifying compounds capable of modulating or hindering differentiation of human stem cells.
BACKGROUND OF THE INVENTION
Cheng et al., 2004, (Cell biology International 28:635-640) disclose expression levels of polypeptide GalNAc-transferases in hematopoietic stem cells and teach, e.g., on page 639 that "so Tl appears not to be correlated with the differentiation stage of cells" and in Table 2 that there are no clear differences in the expression levels in Tl or T7 (i.e. GALNT1 and
GALNT7, respectively) between CD34 positive cells and megakaryocytes.
WO2006/131599 discloses mRNA markers related to glycoproteins and/or glycosynthase proteins but no data on GALNT1 or 7 are given.
Liu et al., 2007, (J Leukocyte Biol 82: 1-17) disclose gene expression analysis in human hematopoietic stem cells, teaching in Table 2 that GALNT7 could be detected in
differentiated erythroid line (EC) but was not found in any other cell type, including undifferentiated HSC.
Kahai et al., 2009, (PloS ONE 4:e7535) disclose the role of GALNT7 in differentiation of osteoblasts in bone formation but teaches nothing about GALNT7 as a stem cell marker. In WO2007/028079, a method for culturing differentiated stem cells is disclosed with gene expression profiles comprising GALNT1. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. The expression of polypeptide-GalNAc-transferases GALNT1-3, 5-14 in different types of human stem cells as compared to differentiated cells, a) CD34+ hematopoietic stem cells compared to CD34- mononuclear cells, b) CD 133+ hematopoietic stem cells compared to CD 133- mononuclear cells, c) human embryonic stem cells (FES) compared to embryoid bodies (EB), d) cord blood mesenchymal stem cells (CB MSC) compared to cells
differentiated from them into adipogenic (adipo) and osteogenic (osteo) directions. The values are expressed as a median of 4 different cell lines for cord blood MSCs and hESCs, and as a median of CD34+/CD34- or CD133+/CD133- cells from 4 different cord blood units.
Figure 2. The top row shows the mean expression of GALNT1 and GALNT7 in the selected cell types. The bottom row shows the variation of the expression in the same cell types. Figure 3. Stem cell populations can be identified using the combined knowledge of expression of GALNT1 and GALNT7. The data derived from Figure 2.
Figure 4. Levels of B3GNT5 mRNA detected in various human tissues by using mRNA microarrays. The data are derived from the public Genesapiens database.
SUMMARY OF THE INVENTION
The present invention provides a method of detecting the differentiation status of human stem cells. The method can be applied to identify the presence of stem cells in a cell preparation. Also, the method can be applied to estimate the relative number of stem cells in a cell preparation or between cell preparations. The cell preparation can be, for example, clinical grafts for stem cell transplantation. The method comprises a step of preparing from a sample comprising human stem cells a gene expression profile based on the expression level of at least one of the genes selected from the group consisting of glycosyltransferase genes B3GNT5, GALNT1 and GALNT7. The level of gene expression in said sample can then be compared to the control samples not known to contain stem cells or containing known amounts of these cells. There are various methods described in the prior art for determination of the expression level, for example, the quantitative RT-PCR method. The present invention further provides a method of identifying compounds that augment, modulate or hinder the differentiation of human stem cells, the method comprises the steps of:
(a) contacting a compound with a sample of human stem cells;
(b) incubating said cells for a time sufficient for the cells to change their differentiation status; (c) preparing a gene expression profile from the cells obtained from step (b), wherein said gene expression profile is based on the expression level of at least one of the genes selected from the group consisting of glycosyltransferase genes B3GNT5, GALNT1 and GALNT7; and
(d) comparing gene expression profile obtained from step (c) to a gene expression profile made from corresponding stem cells not contacted with said compound, wherein the difference in the amount of expression of at least one of said genes in the presence of the compound indicates that the compound is a candidate compound for modulation or hindrance of the differentiation of human stem cells.
DESCRIPTION OF THE INVENTION Definitions The term "target nucleic acid" refers to a nucleic acid (often derived from a biological sample), to which a polynucleotide probe is designed to specifically hybridize. It is either the presence or absence of the target nucleic acid that is to be detected, or the amount of the target nucleic acid that is to be quantified. The target nucleic acid has a sequence that is
complementary to the nucleic acid sequence of the corresponding probe directed to the target. The term target nucleic acid can refer to the specific subsequence of a larger nucleic acid to which the probe is directed or to the overall sequence (e.g., gene or mRNA) whose expression level it is desired to detect.
For identifying whether a nucleic acid or polypeptide is within the scope of the invention, the default parameters of the BLAST programs are suitable. The BLASTN program (for nucleotide sequences) uses as defaults a word length (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 word length (W) of 3, an expectation (E) of 10, and the BLOSUM 62 scoring matrix. The TBLATN program (using protein sequence for nucleotide sequence) uses as defaults a word length (W) of 3, an expectation (E) of 10, and a BLOSUM 62 scoring matrix. (See, e.g., Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).
Overview
Many biological functions are controlled through changes in the expression of various genes by transcriptional (e.g., through control of initiation, RNA processing, etc.) and/or translational control. For example, fundamental biological processes such as cell cycle, cell differentiation and cell death, are often characterized by the variations in the expression levels of groups of genes (see e.g. WO02059271). Changes in gene expression are also associated with differentiation of stem cells. Thus, changes in the expression levels of particular genes can indicate the differentiation status of a cell. As there are no definitive, established markers for different stem cell populations, novel markers indicating the differentiation status, or "sternness" of a cell population are most useful and can be applied in various applications.
According to the invention, genes are differentially expressed in stem cells under
differentiation process and in stem cells which keep their undifferentiated status. One or more of the target genes provided by the present invention can be used as part of an "an expression profile" that is representative of a particular state of a human stem cell. These new target genes can be applied in identification of stem cells or their quantification in more
heterogeneous cell preparations such as clinical grafts for stem cell transplantation. Also, the cellular status of stem cells, e.g. how much they have 'sternness' or how much they have differentiated can be analyzed more reliably. There are various applications for the expression profiles. For clinical stem cell transplantation, a certain minimum number of stem cells need to given to the patient (typically about 2-4 x 10 nucleated cells/kg) for effective treatment
(e.g. Lungman et al., 2010, Bone Marrow Transplantation 45: 219-234). If the number of stem cells can be estimated more reliably than currently, one may assume to get better clinical responses. In certain cases, there may be need to differentiate the original stem cell population toward more differentiated cell lineages; the present invention provides a novel tools for this purpose as well, since the number of cells remained as undifferentiated can be estimated by the gene expression profile.
Certain methods that are provided involve determining the expression level of one or more of the differentially expressed genes in a test cell population and comparing the result with the expression level of the same genes in a control cell population, or comparing the expression profile in one sample to that determined from another sample. The level of expression of the differentially expressed nucleic acids can be determined at either the nucleic acid, that is, typically mRNA level or the protein level. Thus, the phrases "determining the expression level," "preparing a gene expression profile," and other like phrases when used in reference to the differentially expressed nucleic acids mean that transcript levels and/or levels of protein encoded by the differentially encoded nucleic acids are detected. When determining the level of expression, the level can be determined qualitatively, but generally is determined quantitatively.
Expression levels of these genes can readily be determined based upon the sequence information that is disclosed herein and coupled with the nucleic acid and protein detection methods that are described herein and that are known in the art,. If transcript levels are determined, they can be determined using routine methods. For instance, the sequence information provided herein (e.g., GenBank sequence entries) can be used to construct nucleic acid probes using a number of conventional hybridization detection methods (e.g., Northern blots). Alternatively, the provided sequence information can be used to generate primers that in turn are used to amplify and detect differentially expressed nucleic acids that are present in a sample, e.g., using quantitative RT-PCR methods. If instead expression is detected at the protein level, encoded protein can be detected and optionally quantified using any of a number of established techniques. One common approach is to use antibodies that specifically bind to the protein product in immunoassay methods. Additional details regarding methods of conducting differential gene expression are provided infra. Expression levels can be detected for one, some, or all of the differentially expressed nucleic acids that are listed herein.
Determination of expression levels is typically done with a test sample taken from a test cell population. As used herein, the term "population" when used in reference to a cell can mean a single cell, but typically refers to a plurality of cells (e.g., a tissue sample). Certain screening methods are performed with test cells that are "capable of expressing" one or more of the differentially expressed nucleic acids. As used in this context, the phrase "capable or expressing" means that the gene of interest is in intact form and can be expressed within the cell. A number of the methods that are provided involve a comparison of expression levels for certain differentially expressed nucleic acids in a "test cell" with the expression levels for the same nucleic acids in a "control cell" (also sometimes referred to as a "control sample," a "reference cell," a "reference value," or simply a "control"). Other methods involve a comparison between one expression profile and a baseline expression profile. In either case, the expression level for the control cell or baseline expression profile essentially establishes a baseline against which an experimental value is compared. The comparison of expression levels are meant to be interpreted broadly with respect to what is meant by: 1) the term "cell", 2) the time at which the expression levels for test and control cells are determined, and 3) with respect to the measure of the expression levels.
So, for example, although the term "test cell" and "control cell" is used for convenience, the term "cell" is meant to be construed broadly. A cell, for instance, can also refer to a population of cells (e.g., a tissue sample), just as a population of cells can have a single member. In general samples can be obtained from various sources, particularly sources of stem cells such as cord blood or bone marrow.
With respect to timing, comparison of expression levels can be done contemporaneously (e.g., a test and control cell are each contacted with a test agent in parallel reactions). The comparison alternatively can be conducted with expression levels that have been determined at temporally distinct times. As an example, expression levels for the control cell can be collected prior to the expression levels for the test cell and stored for future use (e.g., expression levels stored on a computer compatible storage medium).
The expression level for a control cell or baseline expression profile (e.g., baseline value) can be a value for a single cell or it can be an average, mean or other statistical value determined for a plurality of cells. As an example, the value for each expression level for the control cell is a range of values representative of the range observed for a particular population. The particular control cell population can be e.g. human peripheral blood sample. The test sample to be compared to this particular control could be e.g. stem cell graft prepared from human cord blood. The difference in the expression depicts the amount of stem cells in the said graft. Expression level values can also be either qualitative or quantitative. The values for expression levels can be normalized with respect to the expression level of a nucleic acid that is not one of the markers under analysis.
Comparison of the expression levels between test and control cells can involve comparing levels for a single marker or a plurality of markers (e.g., when expression profiles are compared). When the expression level for a single marker is determined, whether expression levels between the test and control cell are similar or different involves a comparison of the expression level of the single marker. When, however, expression levels for multiple markers are compared, the comparison analysis can involve two analyses: 1) determination for each marker examined whether the expression level is similar between the test and control cells, and 2) determination of how many markers from the group of markers examined show similar or different expression levels.
Methods for Detecting Differential Gene Expression Assays to monitor the expression of a marker or markers as defined in the present invention may utilize any available means of monitoring changes in the expression level of the target genes. The protein products encoded by the genes identified herein can also be assayed to determine the amount of expression. Any method for specifically and quantitatively measuring a specific protein or mRNA or DNA product can be used. However, methods and assays of the invention typically utilize PCR- or array hybridization- based methods when detecting the expression of genes.
The genes identified as being differentially expressed in stem cells may be used in a variety of nucleic acid detection assays to detect or quantify the expression level of a gene or multiple genes in a given sample. For example, traditional Northern blotting, dot blots, nuclease protection, RT-PCR, differential display methods, subtractive hybridization, and in situ hybridization may be used for detecting gene expression levels. Levels of mRNA expression may be monitored directly by hybridization of probes to the nucleic acids of the invention. Stem cell samples can be exposed to an agent to be tested under appropriate conditions and time and total RNA or mRNA is isolated by standard procedures such those disclosed in
Sambrook et al, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). One of skill in the art will appreciate that an enormous number of array designs are suitable for the practice of this invention. The high density array will typically include a number of probes that specifically hybridize to the sequences of interest. See WO 99/32660 for methods of producing probes for a given gene or genes. In addition, in a preferred embodiment, the array will include one or more control probes.
Novel gene markers for human stem cell populations and applications
The present invention revealed a group of specific mRNA-level markers that are highly expressed in stem cells but not in the differentiated cell lineages and can be applied for the analysis of human stem cell populations. As there is need for reliable and good markers for stem cell populations, the novel mRNA markers provide novel tools for identification and/or quantification of stem cells. In one embodiment, the invention can be applied to estimate the stem cell content in clinical grafts prepared for stem cell transplantation (e.g. Lanza et al (eds) Essentials of stem cell biology. 2nd ed. Academic Press 2009). Transplantation of
hematopoietic stem cells can be used to cure various hematological malignancies, such as leukemias. Transplantation of other stem cells, in particular mesenchymal stem cells can be applied as supportive therapy in various traumas and they also are immunomodulatory and can be applied in disease with distorted immune response, such as inflammatory bowel disease or autoimmune diseases. The present invention can also be used for determining the status of stem cell-containing cell preparations for other purposes, such as use of embryonic stem cell or induced pluripotent stem cell (Takahashi et al Cell 2007; 131: 861 - 872) populations to produce differentiated tissues (e.g. Zhao et al Biochem Biophys Res Commun 2002; 297; 177-184 for retina cells, or Brustle et al Science 1999; 285: 754 - 756 for glial cell tissues) for drug screening or studies of pathogenesis of diseases by pharma companies. In this embodiment, the invention can be used, e.g., to determine the amount of remaining undifferentiated cells, or to estimate the efficacy of differentiation process in different time points or between different inducing agents. It is also possible to perform further mRNA screening for example by array or RT-PCR methods to further verify the current results and/or screen additional mRNA markers. mRNA-analysis methods
In a preferred embodiment the gene expression analysis is performed by at least one mRNA expression analysis method preferably selected from the group consisting of: a hybridization method, a gene array method, RT-PCR-method, qRT-PCR-method or SAGE-method (serial analysis of gene expression). In an embodiment, the detection of expression of one preferred mRNA species is performed. In a preferred embodiment, mRNA expression analysis is performed for multiple mRNA species. Preferably, multiple mRNA species are analysed in a profiling method. The profiling method is preferably directed to the analysis of the purity of a cell population or quantification of a cell population, i.e. estimation of the ratio of undifferentiated versus differentiated stem cells in a cell preparation. In another embodiment, the mRNA-profiling method is directed to the analysis of the differentiation status of a cell population.
Hematopoietic stem cell related marker mRNAs
The present invention is directed to the use of mRNA markers B3GNT5, GALNT1 and GALNT7 for identification or quantification of hematopoietic stem cells. Typically, these cells are defined as CD34 or CD 133 surface marker positive cell population, but it is known that neither of the marker will identify solely hematopoietic stem cells. Hence, the markers of the present invention can be used to identify more specific populations. In the present invention, the hematopoietic stem cell population refers to cell populations included in the CD34 positive cells with a capacity to differentiate toward hematopoietic lineage, that is into the lymphoid, myeloid and erythroid types of cells. In one embodiment the cell population is included in the CD133 positive cells. In one embodiment, the mRNA markers B3GNT5, GALNT1 and GALNT7 can be applied to estimate the amount of stem cells when
hematopoietic stem cells are expanded ex vivo to increase the cell number useful for therapy.
In an embodiment the present invention is directed to identification of human hematopoietic stem cell population that is not selectable by CD34 or CD 133 marker but has the capacity to differentiate into hematopoietic cells. Mesenchymal stem cell related marker mRNAs
The present invention is directed to the use of mRNA markers B3GNT5, GALNT1 and GALNT7, and in particular that of GALNT1 alone, for identification or quantification of mesenchymal stem cells.
Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into a variety of cell types, typically to osteoblast, chondrocytes and adipocytes, both in vitro and in vivo. MSCs are obtained, for example, from the cord blood and bone marrow. The exact definitions for MSC or cell lineages differentiated thereof have not been established (Da Silva Meilleres et al. Stem Cells 2008; 26: 2287-99), some criteria for undifferentiated MSC are described in Dominici et al. (Cytotherapy 2006; 8: 315-317). According to Dominici et al. MSCs express CD105, CD73 and CD90 and lack expression of CD45, CD34 and CD14. Therefore, the term "mesenchymal stem cells" relates in this invention to all human multipotent stromal cells.
MSCs have a capacity for self renewal while maintaining their multipotency. A standard test to confirm multipotency is differentiation of the cells into osteoblasts, adipocytes, and chondrocytes. The extent to which the cells will differentiate may vary depending on induction, e.g. chemical vs. mechanical. In one embodiment, the invention can be used to select the most effective method or agent to induce MSC toward the said lineages. The capacity of cells to differentiate and proliferate decreases with the age of the donor, as well as with the time cultivated in vitro.
Since MSCs have the potential to differentiate into various cellular lineages and can be expanded in culture conditions without losing their multipotency, they present a valuable source for applications in cell therapy and tissue engineering.
MSC transplantation offers a promising approach for treating certain nonhematological malignant and nonmalignant diseases and for stem cell-mediated tissue regeneration. In particular, they can be applied to induce immunosuppression. This can be done as supportive therapy in hematological stem cell transplantations in which immunologically-mediated graft- versus-host disease is a major complication The mRNA markers, in any combination or any one alone, can be used in one embodiment as novel markers for identification of functionally relevant MSC populations.
Embryonic stem cells related mRNA markers
The present invention is directed to the use of mRNA markers B3GNT5, GALNT1 and GALNT7, and in particular that of GALNT7 alone, for identification or quantification of embryonic stem cells (ESC). Here, term "embryonic stem cell" also includes induced pluripotent progenitor (iPS) cells, which can be produced basically from any differentiated adult cell type to embryonic stem cell-like cells (Takahashi et al. Cell 131: 861-872, 2007). Their therapeutic potential has been predicted to be enormous because patient's own cells can be induced and hence, ethical and histocompatibility problems can be avoided. iPS cells can also be applied by pharma industry and research groups as patient- specific models and source for differentiated tissues. In the present invention, ESC cells refer to cells positive for cell surface markers SSEA3, SSEA4, Tra-1-60 and Tra-1-81, and positive for mRNA markers Oct-4 and Nanog (e.g. Lanza et al (eds) Essentials of stem cell biology. 2nd ed. Academic
Press 2009) . However, it is of note that there currently is no definite marker for ESC or iPS cells. Their functional pluripotency can be demonstrated by the formation of teratomas and embryoid body using methods known in the art (Lanza et al (eds) Essentials of stem cell biology. 2nd ed. Academic Press 2009). In a preferred embodiment, ESC cells are obtained using methods (Klimanskaya et al Human embryonic stem cell lines derived from single blastomeres. Nature 2006; 444: 481-5), not destroying embyos or other ethically acceptable methods.
The applications of ESC and in particular iPS cells are currently focused to generation of particular tissues for drug screening and to generation of disease models for pharma industry and scientists, rather than their direct therapeutic use. Hence, good markers for distinct subpopulations are needed.
Accordingly, the present invention provides a method of detecting the differentiation status of human stem cells, i.e. a method of detecting markers related to differentiation status of human stem cells. The method comprises a step of preparing from a sample comprising human stem cells a gene expression profile based on the expression level of at least one of the genes selected from the group consisting of glycosyltransferase genes B3GNT5, GALNT1 and GALNT7. Preferably, the method is used for identifying or quantifying human stem cells in said sample. One aim of the invention is to confirm the undifferentiated state of a stem cell sample and thus confirm that the stem cell population under examination can be used, e.g., for a stem cell therapy. Another aim of the invention is to test the purity of the human stem cell population. In a preferred embodiment, the expression levels of both GALNT1 and GALNT7 are used together to distinguish between embryonic, mesenchymal and hematopoietic stem cells. For example, a high GALNT1 level (similar to the level of about 500 - 2000 in the experiment described in Figure 3 of Example 2) with a low or intermediate level (below about 500 - 800) of GALNT7 indicates ESC, whereas MSC have a high GALNT7 level (typically over 1000 in the experiment described in Figure 3 of Example 2) with a relatively low level of GALNT1. Typically, it is assumed that barring single exceptions, no overlap between stem cell population are allowed. As stem cells, in particular stem cell lines are examples of biological material with heterogeneous features, some overlap must be assumed. In preferred
embodiments, the mRNA markers of the present invention can be used in any combination for identification and/or quantification of human stem cell populations.
Another main embodiment of the invention is a method of identifying compounds that augment, modulate or hinder the differentiation of human stem cells. This method comprises the following steps:
(a) contacting a compound with a sample of human stem cells;
(b) incubating said cells for a time sufficient for the cells to change their differentiation status, wherein the incubation time preferably varies from less than one day, to a few days or even to a few weeks, depending on the cell type and induction method used; as an example, cord blood derived MSC cells need 12-31 days to differentiate toward the osteogenic and adipocyte lineages when cultivated using a local standard protocol;
(c) preparing a gene expression profile from the cells obtained from step (b), wherein said gene expression profile is based on the expression level of at least one of the genes selected from the group consisting of glycosyltransferase genes B3GNT5, GALNT1 and GALNT7; and
(d) comparing gene expression profile obtained from step (c) to a gene expression profile made from corresponding stem cells not contacted with said compound, wherein the difference in the amount of expression of at least one of said genes in the presence of the compound indicates that the compound is a candidate compound for modulation or hindrance of the differentiation of human stem cells.
Preferably, the sample of step (a) comprises CD133 and/or CD34 positive cells. Examples of suitable stem cells for the method comprise hematopoietic stem cells, mesenchymal stem cells, embryonic stem cells, and iPS cells. It is also advantageous for the method, if the sample of step (a) comprises undifferentiated stem cells.
The candidate compounds that are assayed in the above method can be randomly selected or rationally selected or designed. An example of randomly selected compound is a chemical library or a peptide combinatorial library, or a growth broth of an organism. The compounds used in the present method can be, as examples, peptides, small chemical molecules, vitamin derivatives, as well as carbohydrates, lipids, oligonucleotides and covalent and non-covalent combinations thereof. The compounds may be contacted with cells by adding the compound to the culture media or buffer in which the stem cells are incubated. Alternatively, the compound, such as a DNA vector, may also be introduced directly into cells.
Indirect analysis of mRNA-level marker The present invention is further directed to indirect analysis of B3GNT5, GALNT1 and
GALNT7 mPvNA markers. The indirect analysis measures a factor correlating to the mRNA- expression. The indirect analysis is directed to protein or glycan level markers. In a preferred embodiment the present invention is directed to
1. defining the protein level expression corresponding to the mRNA markers B3GNT5, GALNT1 and GALNT7 and
2. correlating it with the respective mRNA-level expressions of B3GNT5, GALNT1 and GALNT7,
3. if correlation would exist, use of the correlating protein level marker instead of the mRNA marker.
The benefit of the use of the protein level marker is that these markers can be observed by common analytic and diagnostic methods including use of diagnostic antibodies and antibody based technologies. In one embodiment, the direct gene products, that is, proteins encoded by the genes B3GNT5, GALNT1 and GALNT7 are used as markers. The activity of the enzymes encoded by genes B3GNT5, GALNTl and GALNT7 are known only partially (e.g., Varki et al. (eds) Essential of Glycobiology. 2nd ed. Cold Spring Harbor Laboratory Press. 2009). B3GNT5 encodes a UDP-GlcNAc|3Gaip-l,3 - N- acetylglycosaminyltransferase, which adds GlcNAc sugar- moieties onto glycolipid molecules. GALNTl and GALNT7 encode forms of transferases that add N-acetylgalactosamine from UDP-GalNAc onto polypeptides, hence starting O-glycosylation. There are at least 21 similar genes identified in the human genome so far. Their expression levels, however, vary in different types of cells as do their fine- specificities. Consequently, there is no prior evidence for their function in relation to the stem cell biology.
It is noted that the sequences of the target or marker genes listed in the specification are available in the public databases such as in GenBank. The sequences of the genes in public databases, such as GenBank, are herein expressly incorporated by reference in their entirety as of the filing date of this application (see www.ncbi.nim.nih.gov).
EXPERIMENTAL SECTION
Example 1. Expression of polypeptide-GalNAc-transferases in stem cells
Materials and methods
Cells
Cord blood derived mesenchymal stem cell lines
Fresh umbilical cord blood was obtained from informed and consented donors at the Helsinki University Central Hospital, Department of Obstetrics and Gynaecology, and Helsinki Maternity Hospital. The study protocol was approved by ethical review board of Helsinki University Central Hospital and the Finnish Red Cross Blood Service. Collection and processing of the fresh cord blood was performed as described earlier (Jaatinen et al. 2006). Mononuclear cells were isolated by Ficoll-Hypaque (Amersham Biosciences, Piscaway, NJ, USA) density gradient sentrifugation, and plated on fibronectin coated tissue culture plates at the density of 1 x 106 cells/cm2. The proliferation medium consisted of minimum essential medium (ccMEM) with Glutamax (Gibco, Grand Island, NY, USA) and 10% fetal bovine serum (FCS) (Gibco) supplemented with 50 nM dexamethasone (Sigma, St Louis, MO, USA) 10 ng/mL epidermal growth factor (EGF; Sigma), 10 ng/mL recombinant human platelet- derived growth factor (rhPDGF-BB; R&D Systems, Minneapolis, MN, USA), 100 U penicillin and 1000 U streptomycin (Gibco). Cells were allowed to adhere overnight and nonadherent cells were washed out. Medium changes were performed twice weekly up to three weeks. Recovered colonies were replated and expanded in culture over several passages. The cells were subjected to adipogenic and osteogenic differentiation. Mesenchymal stem cells (MSCs) and cells from adipogenic and osteogenic differentiation were at 4th or 5th passage for microarray analysis.
Flow cytometry to characterize the MSC cell surface expression for critical MSC markers. MSCs from 4th passage were analyzed for their cell surface molecule expression. Cells were labeled with fluorochrome-conjugated monoclonal antibodies; allophycocyanin (APC)- conjugated CD13 (BD Pharmingen), phycoerythrin (PE)-conjugated CD14, CD19, CD34 and CD45 (BD Pharmingen), fluorescein isothiocyanate (FITC)-conjucated CD90 (clone 5E10, Stem Cell Technologies), FITC-CD105 (Abeam) and FITC-HLA-DR (BD Pharmingen). Appropriate FITC-, PE-and APC-conjugated isotypic controls (BD Biosciences) were used. Labeling was carried out in ΙΟΟμΙ of 0,3% ultra pure bovine serum albumin (BSA) in phosphate buffered saline (PBS) on ice for 30 minutes. Flow cytometric analysis was performed on FACSAria (Becton Dickinson Biosciences) with a 488-nm blue laser for (PE and FITC) and a 633-nm red laser for (APC). Fluorescense was measured using 530/30-nm (FITC), 585/42-nm (PE) and 660/20-nm (APC) bandpass filters. Data were analysed using FACSDiva software (BD Biociences). All of the MSC lines were positive for CD105, CD90 and CD73, and negative for CD13, CD14, CD19, CD34, CD45 and HLA-DR.
Multipotent differentiation
To induce osteogenic differentiation, fourth or fifth-passage cells were treated with osteogenesis inducing medium up to 3 weeks with medium changes twice weekly.
Osteogenesis inducing medium consists of aMEM (Gibco), HEPES (Gibco) and FCS (Gibco) supplemented with 2 mM L-glutamine (Gibco), 100 nM dexamethasone, 10 mM glycerol-2- phosphate, 0.05 mM ascorbic acid-2-phosphate, 100 U penicillin and 1000 U streptomycin. Osteogenic differentiation was confirmed by the von Kossa stain demonstrating the calcium of mineralized tissue.
To induce adipogenic differentiation, fourth or fifth-passage cells were treated with adipogenic induction medium for 2 days and then with terminal adipogenesis differentiation medium for up to 3 weeks with medium changes twice weekly. Both mediums consisted of a base of aMEM Glutamax (Gibco), 10% FCS (Gibco), 20 mM HEPES supplemented with 100 U penicillin and 1000 U streptomycin. In addition, adipogenic induction medium was supplemented with 0.1 mM indomethasin (Sigma-Aldrich), 0.5 mM 3-isobutyl-l- methylxanthine, 0.4 g/ml dexamethasone DM-200 (PromoCell) and 0.5 g/ml insulin -0,25 (PromoCell), and terminal adipogenic differentiation medium was supplemented with 0.1 mM indomethasin (Sigma-Aldrich), 0.5 g/ml insulin -0.25 (PromoCell), and 3 μg/ml ciglitazone - 1,5 (Promocell). Adipogenic differentiation was confirmed by the Sudan III stain
demonstrating triglycerides of adipogenic tissue. Human embryonic stem cell lines (hESC)
Human embryonic stem cell lines (hESC) FES21, FES22, FES29 and FES30 were derived from in vitro fertilized excess human embryos and cultured as previously described (Mikkola et al. 2006). FES21 and FES22 cell lines were initially cultured on mouse embryonic fibroblast feeder cells, but were transferred on human foreskin fibroblast feeder cells after 10 and 28 passages, respectively. FES29 and FES30 cells lines have been cultured on human foreskin fibroblast feeder cells only. To generate embryoid bodies (EB), the hESC colonies were cultured for 10-14 days and disaggregated mechanically. The cells were then transferred to suspension cultures for 10 days to form EBs (Mikkola et al. 2006).
Hematopoietic stem cells from cord blood
Cord blood was obtained from the Helsinki University Central Hospital, Department of Obstetrics and Gynaecology, and Helsinki Maternity Hospital. All donors gave informed consent and the study was approved by ethical review board of the Helsinki University Central Hospital and the Finnish Red Cross Blood Service. Collection and processing of the fresh cord blood was performed as described earlier (Jaatinen 2006). Ficoll-Hypaque density gradient (Amersham Biosciences, New Jersey, USA) was used to isolate mononuclear cells. Stem cell fraction was sorted from the mononuclear fraction with anti-CD 133 or anti-CD34 microbeads in magnetic affinity cell sorting (Miltenyi Biotec, Bergisch Gladbach, Germany) (Kekarainen 2006). Mature leukocytes (CD133- cells, CD34- cells) were collected for control purposes.
RNA Isolation
Total RNA from cord blood MSC, cells with osteogenic differentiation and adipogenic differentiation, human embryonic stem cells and embryoid bodies was purified with RNeasy Mini Kit (StemCell Technologies) directly from the cell culture plates or separated CD34+, CD34-, CD 133+ and CD 133- cells washed with PBS to remove the remaining medium according to the manufacturer's instructions. RNA consentration was checked with Nanodrop ND-1000 (Thermo scientific, Wilmington, DE, US) and quality was controlled by Experion electrophoresis station (Bio-Rad Laboratories, Herkules, CA, US).
Microarray
For the microarray, 2μ1 of total RNA from each sample was used. Samples were processed according to One-Cycle Target Labeling protocol from GeneChip Expression Analysis Technical manual (Specific Protocols for Using the GeneChip Hybridization, Wash and Stain Kit) and hybridized to GeneChip® Human Genome U133 Plus 2.0 Array. Concentration of cDNA was checked with Nanodrop ND-1000 (Thermo scientific) and quality was controlled by Experion electrophoresis station (Bio-Rad Laboratories). GeneChip Fluidics Station 450 was used to wash and stain the arrays and GeneChip Scanner 3000 with AutoLoader was used to scan the arrays. GeneChip Fluidics Stations and Scanner were controlled with GeneChip Command Console (AGCC) software version 1.0. The replicate results of hybridization data for MSCs, and cells with osteogenic differentiation and cells with adipogenetic differentiation were obtained from four different MSC line. Sample labelling and hybridization were carried out at the Finnish DNA Microarray Centre at Turku Centre for Biotechnology, Turku, Finland.
For the gene expression analysis, AffyReader, a Microarray Pipeline component, was used to extract gene expression measures from the Affymetrix CEL.files. The Affymetrix probeset expression data of transcripts were newly clustered to represent genes by recombining the probes that represented the same gene using GeneChip library files (Custom CDF. versio 10) with esembl gene ID (Dai et al. 2005, 2007). All samples were normalized using Robust- Multi-array Average background adjustment (RMA) for intensities, quantile normalization and median-polish summarization (Wu and Izarry, 2004). The RMA normalization was implemented using Bioconductor R, package affy. Values were transformed into log2.
Results and discussion
The expression of polypeptide-GalNAc-transferases 1-3 and 5-14 was analyzed in stem cells and differentiated cells (Figure 1). The expression in CD34+ and CD133+ hematopoietic stem cells was compared to the expression in CD34- and CD133- cells, the expression in cord blood mesenchymal stem cells was compared to the expression in adipogenically and osteogenically differentiated cells, and the expression in embryonic stem cells was compared to the expression in embryoid bodies. GALNT7 (GenelD: 51809) was overexpressed in stem cells as compared to differentiated cells in all of the cell types studied (fold change 2-8x). In addition, GALNT1 (GenelD: 2589) was overexpressed in hematopoietic stem cells and GALNT3 in mesenchymal stem cells and embryonic stem cells. Stem cells express a characteristic profile of polypeptide-GalNAc-transferases, which changes upon differentiation. Analysis of the expression levels of polypeptide-GalNAc-transferases, especially GALNT7, can be used to determine the undifferentiated state of cells.
Example 2. Analyses of public microarray data
Methods Preprocessed Affymetrix dataset E-TABM-185 downloaded from the ArrayExpress database maintained by the European Bioinformatics Institute (EBI) was used for the analyses. This dataset has been normalized with the GCRMA algorithm using the probe annotations provided by Affymetrix. The probesets for GALNTl and GALNT7 were extracted from the dataset, and were used for further analyses. All probesets for these genes match exactly multiple transcripts, possibly originating from the known splice variants: Ensembl database, containing genomic data, release 56 reports two variants for both genes.
R 2.9.0 was used for data analyses.
Results
The expression GALNTl and GALNT7 was analyzed in stem cells and differentiated cell types (Figure 2). The expression of GALNTl is significantly higher in mesenchymal stem cells than other stem cells or non-stem cells. GALNTl is also slightly upregulated in embryonic stem cells when compared to non-stem cells. GALNT7 is significantly over- expressed in embryonic stem cells. The expression of GALNT7 is about at the steady same level in other stem cells or non-stem cells. GALNTl and GALNT7 can be successfully employed together to differentiate the stem cell types from each other (Figure 3). GALNTl alone seems to able to differentiate mesenchymal stem cells from hematopoietic and embryonic stem cells, but not hematopoietic stem cells from embryonic stem cells. Adding information on GALNT7 expression enables one to tell the difference between hematopoietic and embryonic stem cells, also. Example 3. Expression of B3GNT5 Mesenchymal stem cells, RNA isolation and mRNA microarray analyses were done as described in Example 1. It was noted that the expression level of B3GNT5 (ENSG
00000176597) was highly expressed in undifferentiated mesenchymal stem cell population, but its expression decreased dramatically when the cells were differentiated. The expression level in undifferentiated mesenchymal stem cells was 3.4 times higher than that of cells differentiated toward osteogenic lineage and 2.3 times higher than that of cells differentiated toward adipocyte lineage.
The expression level of B3GNT5 (GenelD: 84002) as compared to other cell types was also analysed using the public 1ST database (www.genesapiens.org), a database based on data of nearly 10 000 Affymetrix microarrays described in more details by Kilpinen et al {Genome Biology 2008; 9: R139). The mean of expression level of B3GNT5 (ENSG 00000176597) in hematopoietic stem cells was found to be at the level of about 3000 (arbitrary units), whereas the levels in most other cells were substantially lower (Figure 4). The highest ones were for those annotated as "pancreas" (the mean about 1500), and two annotations "placenta" and "blood myeloid cells", that both most likely contain also hematopoietic stem cells. All other cell types were at the level of about 500 or below. It is of note that as the exact basis for each tissue annotation could not be traced, the exact content of each tissue annotation could not be known.
REFERENCES
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22:656-8

Claims

1. A method of detecting the differentiation status of human stem cells comprising a step of preparing from a sample comprising human stem cells a gene expression profile based on the expression level of at least one of the genes selected from the group consisting of
glycosyltransferase genes B3GNT5, GALNT1 and GALNT7.
2. The method according to claim 1, wherein said gene expression profile is based at least on the expression levels of GALNT1 and GALNT7.
3. The method according to claim 1, wherein said gene expression profile is based at least on the expression level of GALNT1.
4. The method according to claim 1, wherein said gene expression profile is based at least on the expression level of B3GNT5.
5. The method according to claim 1, wherein the method is to confirm the undifferentiated state of the stem cells.
6. The method according to claim 1, wherein the method is to estimate the amount of human stem cells in a cell preparation.
7. The method according to claim 1, wherein the method is for estimation of the relative proportions of undifferentiated and differentiated stem cells in said sample of human stem cells.
8. The method according to claim 1, wherein high expression level of glycosyltransferase genes B3GNT5, GALNT1 and/or GALNT7 indicates undifferentiated state of the stem cells.
9. The method according to claim 1, wherein the sample comprises hematopoietic stem cells, mesenchymal stem cells, and/or embryonic stem cells.
10. A method of identifying compounds that augment, modulate or hinder the differentiation of human stem cells, the method comprising the step of: (a) contacting a compound with a sample of human stem cells;
(b) incubating said cells for a time sufficient for the cells to change their differentiation status;
(c) preparing a gene expression profile from the cells obtained from step (b), wherein said gene expression profile is based on the expression level of at least one of the genes selected from the group consisting of glycosyltransferase genes B3GNT5, GALNT1 and GALNT7; and
(d) comparing gene expression profile obtained from step (c) to a gene expression profile made from corresponding stem cells not contacted with said compound, wherein the difference in the amount of expression of at least one of said genes in the presence of the compound indicates that the compound is a candidate compound for modulation or hindrance of the differentiation of human stem cells.
11. The method according to claim 10, wherein said gene expression profile is based at least on the expression levels of GALNT1 and GALNT7.
12. The method according to claim 10, wherein said gene expression profile is based at least on the expression level of GALNT1.
13. The method according to claim 10, wherein said gene expression profile is based at least on the expression level of B3GNT5.
14. The method according to claim 10, wherein the sample comprises hematopoietic stem cells, mesenchymal stem cells, and/or embryonic stem cells.
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