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WO2005075636A1 - Marqueurs moleculaires lies au developpement de metanephrine et aux progeniteurs renaux - Google Patents

Marqueurs moleculaires lies au developpement de metanephrine et aux progeniteurs renaux Download PDF

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WO2005075636A1
WO2005075636A1 PCT/AU2005/000162 AU2005000162W WO2005075636A1 WO 2005075636 A1 WO2005075636 A1 WO 2005075636A1 AU 2005000162 W AU2005000162 W AU 2005000162W WO 2005075636 A1 WO2005075636 A1 WO 2005075636A1
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protein
gene
receptor
solute carrier
carrier family
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PCT/AU2005/000162
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English (en)
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Melissa Little
Grant Challen
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The University Of Queensland
Monash University
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Priority claimed from AU2004900600A external-priority patent/AU2004900600A0/en
Application filed by The University Of Queensland, Monash University filed Critical The University Of Queensland
Publication of WO2005075636A1 publication Critical patent/WO2005075636A1/fr

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    • 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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • 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
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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

Definitions

  • TITLE MOLECULAR MARKERS ASSOCIATED WITH METANEPHRIC DEVELOPMENT AND RENAL PROGENITORS FIELD OF THE INVENTION relates to identifying, isolating and/or purifying metanephric mesenchyme cells and, in particular, renal progenitor cells, although without limitation thereto. More particularly, the invention relates to the use of specific molecular markers that correlate with particular stages of metanephric mesenchyme development to isolate and/or purify renal progenitor cells. This invention also relates to use of isolated and/or purified renal progenitor cells for kidney tissue repair and regeneration.
  • the central dogma of kidney development implies that the UB forms the ureter and collecting duct system of the mature kidney while the MM gives rise to the remaining portions of the nephrons, from Bowmans's capsule to distal tubule.
  • the undifferentiated MM can therefore be regarded as the renal progenitor population, a homogenous mass of cells with multipotent differentiation capacity, because it has the ability to differentiate into many more differentiated cell types than do the UB precursors (Herzlinger et al., 1992, Development 114 565-572). This suggests that of the two primordial tissues that interact to produce a mature kidney, the MM is more likely to be the source of a renal stem cell population rather than the UB.
  • the present invention broadly relates to defining a gene expression profile that facilitates identification, isolation and/or purification of committed but undifferentiated metanephric mesenchyme cells, including renal progenitor cells.
  • the invention provides a method of identifying a gene expression profile associated with metanephric mesenchyme development, said method including the step of identifying one or more genes that are differentially expressed by one or more metanephric mesenchyme cells at a particular stage of embryonic development compared to one or more intermediate mesoderm cells.
  • the invention provides a method of identifying a metanephric mesenchyme cell, said method including the step of determining a gene expression profile of said metanephric mesenchyme cell, wherein said gene expression profile comprises one or more genetic markers that are differentially expressed by one or more metanephric mesenchyme cells compared to one or more intermediate mesoderm cells.
  • the invention provides a method of isolating or purifying one or more metanephric mesenchyme cells including the step of identifying a gene expression profile that comprises one or more genetic markers that are differentially expressed by said one or more metanephric mesenchyme cells compared to one or more intermediate mesoderm cells.
  • gene expression is determined according to nucleic acid expression, such as mRNA expression.
  • gene expression is determined according to protein expression.
  • expressed genes that may be used in gene expression profiles associated with a particular stage of development of metanephric mesenchyme, which genes are differentially expressed with respect to DVI, are set forth in Tables 2 and 3.
  • the gene expression profile comprises one or more genetic markers set forth in Table 4.
  • advantageous cell surface markers include Neuropilin-1; CD 164 antigen; CD83 antigen; Stromal cell derived factor receptor 1; CD24a antigen; Serine protease inhibitor, Kunitz type 2; Tumor-associated calcium signal transducer 1; Receptor-like tyrosine kinase; Fibroblast growth factor receptor 2 ; Amyloid beta (A4) precursor protein; Bone morphogenetic protein receptor, type 1A; DNA segment, Chr 8, Wayne State University 49, expressed; Signal sequence receptor, alpha; Junction adhesion molecule 3; PTK7 protein tyrosine kinase 7; Cadherin 11; Syndecan binding protein; Integral membrane protein 2C; Tripartite motif-containing 59; NAD(P) dependent steroid dehydrogenase-like; DNA segment, Chr 3, University of California at Los Angeles 1; Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2;
  • Zinc finger Zinc finger, DHHC domain containing 6; Solute carrier family 37 (glycerol-3 -phosphate transporter), member 3; Sarcoma amplified sequence; RIKEN cDNA 1700022N24 gene; and Mid- 1 -related chloride channel
  • the invention provides a method of identifying a gene expression profile of a renal progenitor cell, said method including the step of identifying one or more genes that are differentially expressed by said renal progenitor cell compared to an intermediate mesenchyme cell.
  • the invention provides a method of identifying a renal progenitor cell, said method including the step of determining a gene expression profile of said renal progenitor cell, wherein the gene expression profile comprises one or more genetic markers differentially expressed compared to an intermediate mesenchyme cell.
  • the invention provides a method of isolating or purifying a renal progenitor cell, said method including the step of identifying a gene expression profile of said renal progenitor cell, wherein the gene expression profile comprises one or more genetic markers differentially expressed compared to an intermediate mesenchyme cell.
  • said renal progenitor cell is isolated from differentiating embryonic or adult stem cells in culture or from any adult tissue, including the kidney.
  • expressed genes that may be used in gene expression profiles associated with renal progenitor cells are set forth in Tables 2 and 3.
  • the gene expression profile comprises one or more genetic markers set forth in Table 4.
  • advantageous cell surface markers include Neuropilin-1; CD 164 antigen;
  • CD83 antigen Stromal cell derived factor receptor 1; CD24a antigen; Serine protease inhibitor, Kunitz type 2; Tumor-associated calcium signal transducer 1 ;
  • Receptor-like tyrosine kinase Fibroblast growth factor receptor 2 ; Amyloid beta (A4) precursor protein; Bone morpho genetic protein receptor, type 1A; DNA segment, Chr 8, Wayne State University 49, expressed; Signal sequence receptor, alpha; Junction adhesion molecule 3; PTK7 protein tyrosine kinase 7; Cadherin 11; Syndecan binding protein; Integral membrane protein 2C; Tripartite motif- containing 59; NAD(P) dependent steroid dehydrogenase-like; DNA segment, Chr 3, University of California at Los Angeles 1; Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2; RIKEN cDNA 1110018G07 gene; Purine rich element binding protein B; Solute carrier family 6
  • Neurotransmitter transporter taurine
  • member 6 Gap junction membrane channel protein alpha 1; Tumor differentially expressed 1; CD81 antigen; Solute carrier family 35, member 5; Solute carrier family 39 (zinc transporter), member 7; Gene rich cluster, C3f gene; Claudin 6; Solute carrier family 20, member 1; Solute carrier family 16 (monocarboxylic acid transporters), member 1; ADP- ribosylation factor-like 6 interacting protein 2; Autocrine motility factor receptor; Claudin 7; Calcitonin receptor-like; Tumor differentially expressed 2; Synaptophysin-like protein; Claudin 11 ; G protein-coupled receptor 89; ELOVL family member 6 (Elovl6), elongation of long chain fatty acids (yeast); Purinergic receptor (family A group 5); Non imprinted in Prader-Willi/Angelman syndrome
  • the cell surface markers profile may further comprise one or more cell surface markers together with one or more other stem cell markers as described in Table 6.
  • the one or more other stem cell markers are selected from the group consisting of CD34, c-kit and Sea 1, by virtue of their lack of, or low level of, expression.
  • a gene expression profile of a renal progenitor cell is defined as CD24a + cadherin 1 l + c-kit +/low Sca- j+ ⁇ ow CD34 -_
  • the invention provides use of metanephric mesenchyme cells, or more particularly renal progenitor cells, isolated or purified according to the invention, for in vitro and/or in vivo generation of renal tissue.
  • the invention provides a nucleic acid array comprising a plurality of isolated nucleic acid molecules described herein, for use according to a method of any preceding aspect.
  • the invention provides a protein array comprising a plurality of isolated protein molecules described herein, for use according to a method of any preceding aspect.
  • the gene expression profile comprises a plurality of genetic markers, each of the genetic markers displaying at least 1.8 fold higher levels of expression in metanephric mesenchyme cells relative to intermediate mesenchyme cells.
  • said metanephric mesenchyme cells, renal progenitor cells and/or renal stem cells are of mammalian origin.
  • said metanephric mesenchyme cells, renal progenitor cells and/or renal stem cells are of human origin.
  • said genetic markers are of mammalian origin.
  • said genetic markers are of human origin.
  • “comprise”, “comprises” and “comprising” are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers.
  • TABLE 1 Classes of predicted membrane organization.
  • TABLE 2 List of genes showing highest differential expression between intermediate mesoderm and metanephric mesenchyme at E10.5 genes from NIA
  • Microarrays according to a B-stat of >0 and a fold change >1.8 TABLE 3: List of genes showing highest differential expression between intermediate mesoderm and metanephric mesenchyme at El 0.5 genes from Compugen microarrays according to a B-stat of >0 and a fold change >1.8.
  • TABLE 4 Preferred markers of El 0.5 metanephric mesenchyme / renal progenitors based on data from Compugen and NIA chips taking into account fold change (>1.8), B-stat (>0) and verification by in situ hybridisation.
  • TABLE 5 Human gene equivalents of the mouse genes described in Table 4. Human genetic markers are listed in corresponding order to the mouse genetic markers in Table 4.
  • Human homologs of the mouse genetic markers were obtained using Homologene (available from the NCBI website: http://www.ncbi.nlm.nih.gov/) or BLAST to compare two potential homologous full length cDNA sequences.
  • FIG. 1 Analysis of microarray data. The three array comparison were normalised to each other to give a comparable range of log ratios.
  • a and B Boxplot representations of the individual hybridisations before (A) and after (B) print-tip lowess normalisation.
  • C to E Genespring scatterplots of each hybridisation. For replicate hybridisations (C and D), El 0.5 metanephric mesenchyme aRNA was labelled with Cy3 while El 0.5 intermediate mesoderm aRNA was labelled with Cy5. The sample labelling was reversed in a dye swap experiment to account for any dye bias (E). The outer lines represent 1.80-fold differences in expression between samples.
  • FIG. 2 RNA in situ hybridisation of genes differentially displayed between IM and MM at El 0.5 in the mouse.
  • Mt metanephros
  • a to D Isletl
  • E to H Gata3
  • I to L Ewing sarcoma homolog
  • M to P p53
  • Q to T 14-3-3-theta
  • U to X Retinoic acid receptor alpha
  • c to f HI 9
  • g to j Stearoyl-coenzyme A desaturase 2
  • k to n Enolase and (o to r) RIKEN cDNA 1600029D21 gene.
  • FIG. 3 RNA in situ hybridisations of cell surface proteins representing renal progenitor cell markers.
  • FIG. 4 Expression patterns of known stem cell markers during early kidney development.
  • FIG. 5 Wholemount RNA in situ hybridisation of genes enriched identified as enriched in El 0.5 MM from Compugen microarray analysis.
  • FIG. 6. In situ hybridisation analysis of CD24a and cadherin-11 throughout mouse kidney development.
  • FIG. 7 FACS analysis of embryonic and adult mouse kidneys for CD24a and cadherin-11 expressing cells demonstrating the presence of cell positive for both markers that decrease in population size over the course of kidney development.
  • the presence of adult CD24a + cadherinl 1 + cells suggests the possibility of an adult renal progenitor or stem cell.
  • FIG. 8. CD24a expression in embryonic and adult mouse kidney side population cells (filled histogram) compared to isotype control (open histogram).
  • FIG. 9. Nucleotide sequences for each of the human genetic markers set forth in Table 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is predicated, at least in part, by the present inventors' identification of differential gene expression by metanephric mesenchyme cells developing along a metanephric lineage or pathway.
  • MM metanephric mesenchyme
  • IM intermediate mesoderm
  • the present invention provides 554 non-redundant genes that are differentially expressed in committed but uninduced metanephric mesenchyme (MM) compared to the surrounding intermediate mesoderm tissue at El 0.5.
  • 51 non-redundant genes were found to encode transmembrane proteins expressed at the cell surface which could therefore be used both to identify and isolate renal progenitors.
  • Gene expression profiles may therefore be used to identify, isolate and/or purify renal progenitor cells during renal differentiation or upon induction or production of renal progenitors from stem cells.
  • Theiler Stages wherein El 0.5 corresponds to Theiler Stage 17. This corresponds to E32 in the developing human embryo. rhttp://www.ana.ed.ac.uk/anatomy/database/h ⁇ mat/MouseComp.html). It will be appreciated that the invention has initially been elucidated using a murine model of human kidney development. Accordingly, the invention described herein may advantageously be applied to human kidney development.
  • the murine gene expression profiles described herein and the developmental stages that they are associated with in mice will also apply to human renal development.
  • the murine genetic markers identified herein as associated with metanephric mesenchyme and, in particular, renal progenitor cells have human orthologs that may be readily utilized according to the invention in relation to human metanephric mesenchyme and, in particular, renal progenitor cells.
  • isolated is meant material that has been removed from its natural state or otherwise been subjected to human manipulation.
  • Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state.
  • purified and purification particularly in the context of cell purification from an initial cell population, is meant isolation of cells whereby the frequency or proportion of said cells in the isolated cell population is greater than in the initial cell population.
  • gene is used herein to describe a discrete, structural unit of a genome that may comprise one or more of introns, exons, open reading frames and regulatory sequences such as promoters and polyadenylation sequences.
  • a "gene expression profile” comprises one or more nucleic acid or protein products of gene expression (i.e genetic markers) that characterize a particular cell type and/or stage of development.
  • a “gene expression profile” comprises one or more nucleic acid or protein products of gene expression that are differentially expressed by one or more metanephric mesenchyme cells compared to one or more intermediate mesenchyme cells.
  • a “gene expression profile” comprises one or more nucleic acid or protein products of gene expression that are differentially expressed by one or more metanephric mesenchyme cells at a particular stage of development compared to metanephric mesenchyme cells at one or more other stages of development.
  • a gene expression profile of a renal progenitor cell or renal stem cell is a gene expression profile of a renal progenitor cell or renal stem cell.
  • gene expression profiles do not necessarily relate to the presence or absence of gene expression or to quantitatively measuring absolute levels of gene expression, but typically relate to relative or differential levels of gene expression.
  • a gene expression profile comprises a plurality of different nucleic acid or protein products of gene expression.
  • Tables 2 and 3 a plurality of genetic markers have been identified, at least some of which may be used to establish a gene expression profile of MM cells compared to IM cells.
  • the genetic markers set forth in Tables 2 and 3 display at least 1.8 fold higher levels of expression in MM cells relative to IM cells.
  • genetic markers set forth in Tables 2 and 3 may display at least 2, 3, 4, or 5-fold higher levels of expression in MM cells relative to IM cells.
  • statistical analyses and in situ hybridization studies have been performed to establish the degree of reliability and/or reproducibility of the genetic markers identified in Tables 2 and
  • Table 4 provides a list of murine genetic markers that have shown the highest reliability and/or reproducibility in terms of their association with MM cells compared to IM cells.
  • Table 5 provides a corresponding list of human genetic markers corresponding to the murine markers set forth in Table 4.
  • Human orthologs were identified using Homologene (available from the NCBI website: http://www.ncbi.nlm.nih.gov/) or BLAST to compare two potential homolog full length cDNA sequences.
  • the gene expression profile comprises one or more genetic markers selected from the group consisting of: Zinc finger protein 335; Ewing sarcoma homolog; t-complex protein 1; enolase 1, alpha non-neuron; tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide (CDK5 regulatory subunit associated protein 2); Cytoplasmic FMR1 interacting protein 1; Sine oculis-related homeobox 2 homolog (Drosophila); Minichromosome maintenance deficient 7 (S.
  • Neurotransmitter transporter taurine
  • Gap junction membrane channel protein alpha 1 Tumor differentially expressed 1; CD81 antigen; Solute carrier family 35, member F5; Solute carrier family 39 (zinc transporter), member 7; Gene rich cluster, C3f gene; Claudin 6; Solute carrier family 20, member 1; Solute carrier family 16 (monocarboxylic acid transporters), member 1; ADP- ribosylation factor-like 6 interacting protein 2; Autocrine motility factor receptor;
  • Claudin 7 Calcitonin receptor-like; Tumor differentially expressed 2; Synaptophysin-like protein; Claudin 11; G protein-coupled receptor 89; ELOVL family member 6, elongation of long chain fatty acids (yeast); Purinergic receptor (family A group 5); Non imprinted in Prader-Willi/Angelman syndrome 2 homolog (human); RIKEN cDNA 4930579A11 gene; RIKEN cDNA 2610311119 gene; Zinc finger, DHHC domain containing 6; Solute carrier family 37 (glycerol- 3-phosphate transporter), member 3; Sarcoma amplified sequence; RIKEN cDNA 1700022N24 gene; Mid- 1 -related chloride channel 1; Stearoyl-Coenzyme A desaturase 2; RIKEN cDNA 1110034A24 gene; Homeo box D13; retinol dehydrogenase 10 (all-trans) (RdhlO); Sal-like 4 (
  • examples of human nucleotide sequences corresponding to each of the genetic markers in Table 5 include examples of human nucleotide sequences corresponding to each of the genetic markers in Table 5.
  • the invention contemplates particular use of genetic markers in the form of one or more proteins expressed at the cell surface of a metanephric mesenchyme cell, inclusive or renal progenitor cells and renal stem cells.
  • Table 1 sets forth a classification of proteins that provides a code that is used in Tables 2-4.
  • preferred cell surface proteins are selected from the group consisting of:Neuropilin-l; CD 164 antigen; CD83 antigen; Stromal cell derived factor receptor 1; CD24a antigen; Serine protease inhibitor, Kunitz type 2; Tumor-associated calcium signal transducer 1; Receptor-like tyrosine kinase; Fibroblast growth factor receptor 2 ; Amyloid beta (A4) precursor protein; Bone morphogenetic protein receptor, type 1A; DNA segment, Chr 8, Wayne State
  • Solute carrier family 3 activators of dibasic and neutral amino acid transport
  • Purine rich element binding protein B Solute carrier family 6 (neurotransmitter transporter, taurine), member 6; Gap junction membrane channel protein alpha 1; Tumor differentially expressed 1; CD81 antigen; Solute carrier family 35, member 5; Solute carrier family 39 (zinc transporter), member 7; Gene rich cluster, C3f gene; Claudin 6; Solute carrier family 20, member 1; Solute carrier family 16 (monocarboxylic acid transporters), member 1; ADP-ribosylation factor
  • the present invention postulates that genetic markers differentially expressed by metanephric mesenchyme cells in earlier stages of development compared to later stages may be genetic markers associated with renal progenitors and thereby facilitate their identification and isolation with respect to other metanephric mesenchyme cells. While the invention as described herein makes reference to genetic markers set forth variously in Tables 2-5 and FIG.
  • the invention provides a principle capable of general application to the identification of other genetic markers that may be indicative of, or otherwise associated with metanephric mesenchyme development and, more particularly, renal progenitors inclusive of renal stem cells.
  • a particular feature of the invention is the identification and use of gene expression profiles for the identification, isolation and/or purification of renal progenitor cells and/or renal stem cells. It is postulated that renal progenitor cells may comprise renal stem cells.
  • a "renal progenitor cell” is a metanephric mesenchyme cell which is a developmental antecedent of one or more mature renal cell types.
  • a “stem cell” is a progenitor cell capable of self-renewal and differentiation into one or more mature cell types.
  • a “renal stem cell” is a progenitor cell capable of self- renewal and differentiation into one or more mature renal cell types. It will be appreciated that stem cells may be embryonic stem cells or adult stem cells.
  • the invention provides a gene expression profile of a renal progenitor cell in the form of a cell surface marker profile.
  • This profile may comprise cell surface markers as hereinbefore identified together with one or more other stem cell markers as described in Table 6.
  • the one or more other stem cell markers are selected from the group consisting of CD34, c-kit and Sea 1, by virtue of their lack of, or low level of, expression.
  • the cell surface marker profile of a renal progenitor cell is set forth as CD24a + cadherin l l + c-kit +/low Sca-l + low CD34 " .
  • Nucleic acid based determination of gene expression In particular embodiments of the present invention, gene expression profiles of metanephric mesenchyme, inclusive of renal progenitor cells and stem cells, may be determined by methods that employ nucleic acid detection.
  • such methods use gene-specific primers and/or probes for nucleic acid detection and include but are not limited to, nucleic acid arrays (in microarrays), nucleic acid sequence amplification and blotting techniques.
  • nucleic acid arrays in microarrays
  • nucleic acid sequence amplification for the purposes of determining one or more gene expression profiles of temporal stages of metanephric mesenchyme development, the invention contemplates particular embodiments of such methods which may be used alone or in combination. Generally, these methods of the invention measure nucleic acid expression levels of intermediate mesenchyme and metanephric mesenchyme cells or tissues.
  • nucleic acid ' ' designates single-or double-stranded mRNA, RNA, cRNA and DNA, said DNA inclusive of cDNA and genomic DNA.
  • a "probe” may be a single or double-stranded oligonucleotide or polynucleotide.
  • a “primer” is usually a single-stranded oligonucleotide, preferably having 15-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid "template” and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or SequenaseTM.
  • probes and/or primers may be labelled for the purpose of detecting amplification products and/or complementary sequences by hybridization and other uses as is well known in the art.
  • hybridize and hybridization are used herein to denote the pairing of at least partly complementary nucleotide sequences to produce a DNA- DNA, RNA-RNA or DNA-RNA hybrid.
  • Hybrid sequences comprising complementary nucleotide sequences occur through base-pairing between complementary purine and pyrimidine bases, or between modified purines (for example, inosine, methylinosine and methyladenosine) and modified pyrimidines
  • the invention contemplates use of one or more molecular makers set forth in any one of Tables 2, 3, 4 and/or 5, in nucleic acid form, for identification, isolation and/or purification of MM cells and/or renal progenitor cells inclusive of renal stem cells. More particularly, examples of human nucleic acid sequences are provided in Table 5 which may be utilized according to the invention. It will be appreciated that fragments of the aforementioned nucleic acid markers may be utilized, such as primers for nucleic acid sequence amplification and/or as probes for nucleic acid hybridization, although without limitation thereto.
  • Nucleic acid “fragments” may preferably comprise at least 20 contiguous nucleotides and up to 50, 100, 200, 300, 500 or more contiguous nucleotides, as required.
  • a nucleic acid amplification technique may include polymerase chain reaction (PCR) and ligase chain reaction (LCR) as for example described in Chapter 15 of Ausubel et al. supra; strand displacement amplification (SDA) as for example described in U.S. Patent No 5,422,252; rolling circle replication (RCR) as for example described in Liu et al, 1996, J.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • RCR rolling circle replication
  • NASBA nucleic acid sequence- based amplification
  • an "amplification product” refers to a nucleic acid product generated by any nucleic acid amplification technique.
  • quantitative PCR using primers corresponding to one or more genes expressed by MM or IM cells of particular stages of development may be used to quantify relative expression levels of one or many nucleic acids to thereby determine the relative gene expression for each MM or
  • expressed RNA is linearly amplified using a messageAMP to provide "aRNA" kit supplied by Ambion.
  • the aRNA is reverse transcribed using random hexamers (Promega) into cDNA incorporating either Cy5- or Cy3- labelled dUTPs (Amersham).
  • Nucleic acid arrays provide a particularly advantageous method of initially identifying or establishing a gene expression profile of a particular stage of metanephric mesenchyme development and also for subsequent detection of a gene expression profile when determining the stage of development of metanephric mesenchyme cells.
  • Nucleic acid arrays typically use libraries of genomic DNA or cDNA.
  • the invention provides a molecular library in the form of a nucleic acid array that comprises a substrate to which is immobilized, bound or otherwise coupled a plurality of nucleic acids that correspond to the expressed genes that are characteristic of a particular stage of metanephric mesenchyme development, or respective fragments thereof.
  • Each immobilized, bound or otherwise coupled nucleic acid has an "address" on the array that signifies the location and identity of said nucleic acid.
  • nucleic acid array technology has become well known in the art and examples of methods applicable to array technology are provided in Chapter 22 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds.
  • the array can have a density of at least 10, 50, 100, 200, 500, 1,000, 2,000, or 10,000 or more addresses/cm 2 , and ranges there between.
  • the substrate may be a two-dimensional substrate such as a glass slide, a wafer (e.g., silica or plastic), a mass spectroscopy plate, or a three-dimensional substrate such as a gel pad. Addresses in addition to the 15K mouse clone set of nucleic acids of the invention may also be disposed on the array.
  • At least one address of the plurality includes a nucleic acid capture probe that hybridizes specifically to a member of a nucleic acid library, e.g., the sense or anti-sense strand.
  • a subset of addresses of the plurality of addresses has a nucleic acid capture probe for a nucleic acid library member.
  • Each address of the subset can include a capture probe that hybridizes to a different region of a library member.
  • An array format may comprise glass slides having an immobilized, ordered grid of a plurality of cDNA fragments. In particular embodiments, said array has 5,000 to 19,000 or up to 40,000 cDNA fragments.
  • each said cDNA fragment corresponds to a particular gene or expressed sequence tag (EST) gene fragment.
  • An array can be generated by various methods, e.g., by photolithographic methods (see, e.g., U.S. Patent Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Patent No. 5,384,261), pin-based methods (e.g., as described in U.S. Pat. No. 5,288,514), and bead-based techniques (e.g., as described in PCT US/93/04145).
  • nucleic acid arrays were performed using chips arrayed with the National Institute of Health - National Institute of Aging 15K mouse cDNA clone set (http://lgsun.grc.nia.nih.gov/cDNA/15k.html) in addition to a selection of custom clones submitted from the Institute for Molecular Bioscience (University of Queensland, Brisbane) and contained 15,989 elements in total (SRC NIA v3.0 chips).
  • the National Institute of Ageing (NIA) array is a 15K mouse clone set containing 15,247 expressed sequence tags (ESTs) derived from pre-and periimplantation embryo E12.5 female gonad/mesonephros and newborn ovary cDNA libraries.
  • Compugen long oligonucleotide set used to create the additional Compugen lists are commercially available (http ://www.labonweb. com/chips/libraries .html) .
  • Other human sets may be obtained from Agilent (for long oligonucleotides), Affymetrix (for short oligonucleotides synthesized on a substrate) or cDNA microarrays as produced by the Ontario Cancer Institute (http://www.oci.utoronto.ca/services/microarray/).
  • gene expression is measured by isolating mRNA from samples MM and IM tissue and comparing expression with that of another sample (e.g. MM tissue of a different development stage or surrounding IM tissue).
  • complementary nucleotide sequences are identified by blotting techniques that include a step whereby nucleotides are immobilized on a matrix (preferably a synthetic membrane such as nitrocellulose), a hybridization step, and a detection step (eg. northern hybridization).
  • a matrix preferably a synthetic membrane such as nitrocellulose
  • Dot blotting and slot blotting can be used to identify complementary RNA/RNA, DNA/RNA or DNA/DNA polynucleotide sequences.
  • Such techniques are well known by those skilled in the art, and have been described in Ausubel et al, supra, at pages 2.9.1 through 2.9.20. Methods for detecting labelled nucleic acids hybridized to an immobilized nucleic acid are well known to practitioners in the art.
  • genes identified in Tables 2 and 3 may be used to determine whether a subpopulation of metanephric mesenchyme cells or stem cells from a variety of sources, including embryonic and adult stem cells from human tissues, is or has been, induced to become a renal progenitor based upon expression of one or more of these genes. This can be assessed in a variety of ways, including but not limited RT- PCR, Northern analysis and/or in situ hybridisation, such as hereinbefore described.
  • Protein based determination of gene expression and cell purification Gene expression profiles of metanephric mesenchyme, inclusive of renal progenitor cells and stem cells may be determined by methods that employ protein detection. Protein-based methods are also particularly useful for cell isolation and purification according to cell surface protein expression.
  • protein is meant an amino acid polymer. The amino acids may be natural or non-natural amino acids, D- or L- amino acids as are well understood in the art.
  • protein includes full-length proteins and fragments thereof including but not limited to, peptides, peptide-nucleic acid conjugates and epitopes capable of being recognized, bound or otherwise detected by an antibody.
  • identification of a gene expression profile may be performed using protein libraries or arrays.
  • a plurality of proteins may be used in a protein library displayed in any of a number of ways, e.g., in phage display or cell display systems, in protein arrays or by two-dimensional gel electrophoresis, or more specifically, differential two-dimensional gel electrophoresis (2D-DIGE).
  • 2D-DIGE differential two-dimensional gel electrophoresis
  • each of a plurality of expressed proteins of the invention is located at an identifiable address on the array.
  • the protein array comprises a substrate to which is immobilized, impregnated, bound or otherwise coupled a plurality of proteins described herein, or respective fragments thereof.
  • Each immobilized, impregnated bound or otherwise coupled protein is at an "address" on the array that signifies the location and identity of each said protein.
  • the substrate may be a chemically-derivatized aluminium chip, a synthetic membrane such as PVDF or nitrocellulose, a glass slide or microtiter plates.
  • Detection of substrate-bound proteins may be performed using known methods such as mass spectrometry, ELISA, immunohistochemistry, fluorescence microscopy or colorimetric detection.
  • Determination of protein expression may also conveniently be performed using antibodies or antibody fragments (such as Fab and Fab 2 fragments) directed to one or more proteins of a particular gene expression profile. These antibody-based methods may have particular efficacy in isolation and purification of renal progenitor cells.
  • the antibody or antibody fragment further comprises a label.
  • the label may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorophore, a chemiluminescent molecule, a lanthanide ion such as Europium (Eu 34 ), a radioisotope (e.g. 125 I) and a direct visual label.
  • a direct visual label use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.
  • a large number of enzymes useful as labels is disclosed in United States Patent Specifications U.S. 4,366,241, U.S.
  • Enzyme labels useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, ⁇ - galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like.
  • the enzyme label may be used alone or in combination with a second enzyme in solution.
  • the fluorophore may be fluorescein isothiocyanate (FITC), Oregon green, tetramethylrhodamine isothiocyanate (TRITL), allophycocyanin (APC), R-Phycoerythrin (RPE), Cy3 and/or Cy5 although without limitation thereto.
  • FITC fluorescein isothiocyanate
  • TRITL tetramethylrhodamine isothiocyanate
  • APC allophycocyanin
  • RPE R-Phycoerythrin
  • Cy3 and/or Cy5 although without limitation thereto.
  • one or more antibodies are used in conjunction with a cell isolation technique such as any technique that selects cells (i.e positive selection) or depletes cells (i.e negative selection) according to cell surface protein expression.
  • a non-exhaustive list includes panning, complement-mediated lysis, fluorescence-activated cell sorting (FACS) and magnetic activated cell sorting (MACS).
  • FACS fluorescence-activated cell sorting
  • MACS magnetic activated cell sorting
  • FACS enrichment fluorescently-labelled antibodies are bound to the cells of interest. These cells are then passed through the excitation laser in a single cell stream and measured for size, granularity and fluorescent activity. Specific parameters are set and cells that fall within those parameters (e.g. fluorescence, forward light scatter, side scatter) are collected by a cell sorter.
  • MACS enrichment monoclonal antibodies coupled to small magnetic particles are bound to the cells of interest. Using a magnet, the bound cells may be enriched from contaminating cells. Alternatively, contaminating cells may be removed with bound beads.
  • Protein based detection of gene expression profiles according to the invention may also utilize immunoassays, for example ELISA, immunohistochemistry or immunoblotting to detect relative expression levels of one or more proteins to determine the stage of metanephric mesenchyme development.
  • immunoassays for example ELISA, immunohistochemistry or immunoblotting to detect relative expression levels of one or more proteins to determine the stage of metanephric mesenchyme development.
  • Tables 2 and 3 identify genetic markers that are, or are likely to be, expressed at the cell surface.
  • a preferred group of cell surface markers include Neuropilin-1 (Nrpl); CD164 antigen; CD83 antigen (CD83); Stromal cell derived factor receptor 1 ; CD24a antigen; Serine protease inhibitor, Kunitz type 2 (Spint-2); Tumor-associated calcium signal transducer 1; Receptor-like tyrosine kinase; Fibroblast growth factor receptor 2; Amyloid beta (A4) precursor protein; Bone morphogenetic protein receptor, type 1A; DNA segment, Chr 8, Wayne
  • Solute carrier family 3 activators of dibasic and neutral amino acid transport
  • Purine rich element binding protein B purb
  • Solute carrier family 6 neurotransmitter transporter, taurine
  • Gap junction membrane channel protein alpha 1 Tumor differentially expressed 1; CD81 antigen; Solute carrier family 35, member F5; Solute carrier family 39 (zinc transporter), member
  • a gene expression profile particularly associated with renal progenitors and potentially renal stem cells is defined as CD24a + cadherin 1 l + c-kit +/low Sca-l +/low CD34 " .
  • CD34, Sca-1 and c-kit are additional cell surface markers that may be used with one or more of the genetic markers described herein that facilitate identification, isolation and/or purification of renal progenitors, inclusive of renal stem cells by virtue of their lack of, or low level of, expression.
  • Antibodies to the aforementioned cell surface markers are readily available commercially, either alone or conjugated to fluorochromes as hereinbefore described. It is also well within the scope of a person skilled in the art to produce antibodies by immunization of a production species (such as rabbits, mice, rats etc) to produce monoclonal or polyclonal antibodies according to standard methods in the art.
  • renal progenitor cells and renal stem cells purified according to the invention may find therapeutic use as adjunct therapy in the renal transplant procedures.
  • any source of embryonic or adult stem cell eg. human embryonic stem cell, neural stem cell, haematopoietic stem cell, mesodermal stem cell
  • embryonic or adult stem cell eg. human embryonic stem cell, neural stem cell, haematopoietic stem cell, mesodermal stem cell
  • mesodermal stem cell could be induced towards a mesodermal lineage using co-culture with growth factors or conditioned medias.
  • the expression of the cell markers may find utility in demonstrating which stem cells had adopted or were adopting a renal fate.
  • the skilled person may also appreciate the combination of cell surface markers which together define the renal progenitors allows antibodies to these cell surface markers to be used to enrich, purify and isolate specific subpopulations of renal progenitors.
  • This aspect of the invention provides utility in isolating renal progenitor cells from mixed populations in which some subpopulations adopt a renal fate and others do not. It will be appreciated that the above markers may also be used to identify and then isolate adult renal stem cell or progenitor populations from an adult kidney. It will be appreciated to the skilled person that the isolated progenitor cells may be used in adjunct therapy.
  • the progenitor cells may be introduced into the renal parenchyma of the kidney or the renal capsule to elicit repair. They may further be introduced via the generalised or renal vasculature, which may involve injection into the renal artery. Any introduction of renal progenitor cells may be accompanied by an adjunct insult or stress to the kidney, such as mild ischaemia or radiation or other stresses designed to stimulate the receptivity of the kidney. Introduction of renal progenitor cells may include growth factors, cytokine or other agents selected to stimulate the integration and onward differentiation of renal progenitors into the receiving kidney or reduce the rejection of the progenitor cells by the receiving kidney. They may further be used in combination with biomatrices and growth factors to generate a replacement kidney organ de novo.
  • MM and rostral IM tissue (nephrogenic cord including mesonephros and genital ridge) was dissected from El 0.5 embryos and snap- frozen on dry ice. Embryos were defined as El 0.5 by the presence of between 8 and 10 tail somites. Pooled tissue was stored at -80 °C. Dissections were performed in cold phosphate-buffered saline (PBS). Total RNA was prepared using Trizoll (GibcoBRL) extraction in combination with RNeasy mini kits
  • RNA was linearly amplified using the messageAMP aRNA kit (Ambion). Briefly, 1000 nanograms of total RNA was reverse transcribed into cDNA using a T7 promoter-dT primer and amplified through an in vitro transcription reaction (12 hours) using T7 RNA polymerase to produce antisense RNA (aRNA). The aRNA was reverse transcribed using random hexamers (Promega) into cDNA incorporating either Cy5- or Cy3-labelled dUTPs (Amersham). Labelled targets were hybridised to microarray chips for 16 hours at 45°C. Arrays were produced by the SRC Microarray Facility, University of Queensland (ARC Centre for Functional and Applied Genomics). Experiments were performed using chips arrayed with the National Institute of Health - National Institute of Aging (NIA) 15K mouse cDNA clone set
  • the NIA 15K mouse clone set contained 15,247 expressed sequence tags (ESTs) derived from pre- and peri-implantation embryo, E12.5 female gonad / mesonephros and newborn ovary cDNA libraries (Tanaka et al, 2000, Proc. Natl. Acad. Sci. USA 97 9127-9132) thus making it an ideal gene set for this experiment.
  • ESTs expressed sequence tags
  • RNA was linearly amplified using the Amino Allyl messageAMP aRNA kit (Ambion). Briefly, 1000 nanograms of total RNA was reverse transcribed into cDNA using a T7 promoter-dT primer and amplified through an in vitro transcription reaction (12 hours) using T7 RNA polymerase to produce antisense RNA (aRNA). 5-(3-aminoallyl)-UTP was incorporated into the aRNA during in vitro transcription.
  • a dye-coupling reaction was used to conjugate the amino allyl modified aRNA to mono-reactive NHS esters of either Cy3 or Cy5 moeities (Amersham). Labelled targets were hybridised to microarray chips for 16 hours at 42°C. Each array experiment was repeated in duplicate and included a dye reversal experiment to account for any dye bias. Hybridised slides were scanned with a GMS 418 array scanner (Genetic MicroSystems) and images were analysed with Imagene 5.5 (Biodiscovery). The microarray data was analysed with R statistical software using the LIMMA package (http://bioinf.wehi.edu.au/limma/) with scripts developed by Ola Spjuth of the Linnaeus Centre for Bioinformatics
  • Table 5 shows a subset of Table 3 whereby 35 non-redundant elements with a B-score >0 were shown to have an average increase in differential expression >1.80-fold greater in the uninduced MM compared to the surrounding IM.
  • Bioinformatics and membrane organization predictions Representative sequences for differentially expressed ESTs or oligonucleotides were extracted from the National Centre for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/). Using BLAST (Altshul et al, 1990, J. Mol. Biol., 215 403-410) each NIA EST sequence was mapped to an identical full-length RIKEN representative transcript / protein sequence (RIKEN RTPS 6.3 set).
  • the RIKEN RTPS 6.3 set was recently annotated using a number of bioinformatic approaches (Kanapin et al, 2003, Gen. Res. 13 1335-1344) including the prediction of the membrane organisation of individual full-length proteins into one of six categories based on the presence or absence of endoplasmic reticulum-retention signal peptides and helical transmembrane (Table 1).
  • RNA in situ hybridisation embryos were collected from outbred CD1 mice as above at days 10.5, 12.5 and 15.5 of gestation. E10.5 embryos were cut transversely below the forelimbs and longitudinally down the midline to expose the ND, UB and MM. At E12.5, complete urogenital (UG) tracts were collected.
  • metanephroi from El 5.5 embryos were dissected as above and fixed in 4% paraformaldehyde (PFA) in PBS for 2 hours at 4°C. Tissue was processed and embedded in paraffin. Sections were cut at 7 ⁇ m.
  • PFA paraformaldehyde
  • metanephroi were isolated from El 2.0 embryos and grown as explants for two days at 5% CO2, 37°C on 3.0 ⁇ m polycarbonate transwell filters
  • RNA in situ hybridisation Expression patterns were analysed by RNA in situ hybridisation using digoxigenin-labelled sense and antisense riboprobes. Probes were synthesized as described previously (Holmes et al, 1998, Mech. Dev. 79 57-72) using pSPORTl constructs containing the NIA EST of interest (SRC Microarray facility). Probes were not fragmented by hydrolysis and were purified using Sephadex columns (Roche) following digestion of the vector with DNasel (Promega) for 15 minutes at 37°C. Whole mount in situ hybridizations were performed as described by
  • RNA linear amplification to produce amplified RNA (aRNA).
  • aRNA amplified RNA
  • aRNA produced by in vitro transcription has been shown to have a correlation coefficient >0.95 to total RNA and aRNA produced using different amounts of template total RNA (Luo et al, 1999, Nat. Med. 5 117-122; Baugh et al, Nucl. Acid Res. 25 e29).
  • all samples were amplified under the exact same conditions at the same time.
  • E10.5 MM versus the adjacent IM determined based upon a variety of stringency criteria.
  • Thee criteria might include fold change or statistical score (B-stat) or a combination of the two.
  • Our preferred method of analysis is to take into account both B-stat and fold change.
  • Tables 2 and 3 represent a subset of the genes enriched in MM using either the NIA or Compugen gene sets. This includes all genes in which the fold change was greater than 1.8 fold and the B- stat was greater than 0. Both B-stat and fold change are indicated.
  • a total of 26 genes from NIA and 528 genes from Compugen are detailed in Tables 2 and 3. Hence, this set comprises 554 markers.
  • each differentially expressed EST or Compugen oligo was mapped to a full-length protein sequence from the RIKEN RTPS 6.3 set and the membrane organisation predictions were adopted for each gene of interest.
  • candidate genes from class C (type I membrane proteins), class D (type II membrane proteins) and class E (multi-span membrane proteins) for their potential utility in antibody-based fluorescence-activated cell sorting
  • CD24a was expressed in epithelial cells of both UB and MM lineages, although not in the lower limbs of the S-shaped bodies that give rise to the glomeruli.
  • Cadherin-11 showed expression in the mesoderm at El 0.5, but particularly the renal progenitor population. Expression of cadherin-11 became more widespread at E12.5 but was strongly expressed throughout the renal interstitium of the explant, particularly the cells surrounding the UB tips.
  • the full-length transcript representing the EST BG072301 encodes for a protein for which little information is currently known.
  • the transcription factors Oct-4 and nanog are markers of the pluripotential state. At El 0.5, expression of Oct-4 was restricted to primordial germ cells migrating through the urogenital ridge while nanog expression was observed in a non-specific fashion throughout the embryo, although seemingly higher in the El 0.5 MM. Neither of these genes showed any expression in metanephric explants. These stem cell markers would be unlikely to be of use according to the present invention The somatic stem cell markers CD34, podxl and nestin did not appear to mark the renal progenitor population. CD34 was expressed throughout the forming vasculature of the embryo and metanephric explant, but no expression was detected in El 0.5 MM.
  • haemangioblast marker podxl was expressed strongly in the aorta-gonad-mesonephros region at El 0.5, but not in the uninduced MM. In explants, expression of podxl was restricted to presumptive podocytes.
  • the neural progenitor marker nestin was strongly expressed in the ectoderm of the El 0.5 embryo but not in the MM, although expression of nestin was observed in the S-shaped bodies of metanephric explants. A lack of expression of CD34 would be of value to determine a lack of heamatopoietic origin.
  • the receptor tyrosine kinase c-kit CD 117
  • the surface glycoprotein stem cell antigen- 1 sca-1
  • both sca-1 and c-kit were expressed throughout the nephrogenic cord with sca-1 in particular showing an increase in expression in the El 0.5 MM.
  • sca-1 was expressed by the primitive nephron tubules of the while c-kit expression was observed throughout the primary renal interstitium.
  • renal progenitors would show a phenotype that was Oct-4 " , nanog lo/” , nestin “ , Sca + , c- kit Iow , CD34 " , EXAMPLE 2 Molecular Markers of Renal Cells Methods and Materials
  • Tissue was minced into a coarse slurry with scissors and digested in 10 mg/mL collagenase B (Roche), 1.2 U/mL dispase II (Roche), 0.01% DNase type I (Sigma) in HANKS for 20 minutes at 37°C with agitation. The concentration of collagenase was reduced to 1 mg/mL for dissociation of embryonic tissue.
  • Digested tissue was dissociated further using 23 gauge needles before being passed through a 40 ⁇ M cell strainer (BD Falcon). Red blood cells were lysed using Gey's solution and cells were resuspended in pre- warmed DMEM - phenol red (Gibco), 10 mM HEPES, 2% FCS at a concentration of 1 x 10 6 /mL.
  • DMEM - phenol red Gibco
  • 10 mM HEPES 10 mM HEPES
  • FCS 2% FCS at a concentration of 1 x 10 6 /mL.
  • For hoechst staining 5 ⁇ g/mL hoechst 33342 (Sigma) was added to each sample and incubated at 37°C for 90 minutes under protection from light. A control tube for each sample containing 50 ⁇ M verapamil (Sigma) was included in each preparation to set the side population gate.
  • Non-conjugated CD34 (Zymed), CD24a (Pharmingen) and cadherin-11 (Santa Cruz) primary antibodies were also used which were then subsequently stained with anti-mouse-FITC, anti-rat-FITC and anti-goat-PE secondary antibodies (Sigma) respectively. Finally, 2 ⁇ g/mL 7- aminoactinomycin D (7-AAD, Sigma) was added to each sample to identify viable cells. Cells were analysed and sorted on a FACS Vantage SE (Becton Dickinson) with both 488 nm argon (200 mW power) and 365 nm ultra-violet (50 mW power) lasers.
  • FACS Vantage SE Becton Dickinson
  • FITC and PE were excited with the 488 nm laser and emission signals were detected using 530/30 and 575/25 band pass filters respectively.
  • Hoechst and 7-AAD were excited with the 365 nm laser and emission detected using a 670/40 filter for 7-AAD and 424/44 (blue) and 660/20 (red) filters for hoechst. Compensation was adjusted using samples stained with one fluorochrome only and the side population gate was set using verapamil control samples. Data were acquired using CellQuest software (Becton Dickinson) and analysed with winMDI 2.8. Results One of the major goals of this screen was to identify cell surface markers that may be used to isolate potential renal stem cells based on their similarity to El 0.5 MM.
  • Section in situ analysis was conducted to resolve what cell types expressed these genes throughout subsequent stages of kidney development.
  • Cells co-expressing these molecules in the adult kidney may possess a phenotype more similar to that of the renal progenitor population and retain a degree of inherent differentiation capacity.
  • CD24a and cadherin-11 expression remains in the structures observed in metanephric explants, namely epithelial nephron segments and the interstitium respectively.
  • CD24a is expressed principally in distal convoluted tubules while cadherin-11 expression is also seen in distal tubules and loop of Henle segments. No expression of cadherin-11 is seen in any interstitial cell population in the adult.
  • Immunophenotyping was done by FACS analysis to determine the proportion of renal cells, if any, which retained expression of both CD24a and cadherin-11 during development (FIG. 7). Three timepoints were analysed, the El 0.5 MM, El 5.5 metanephroi and adult kidneys. As anticipated, the proportion of CD24a + cad-ll + cells in the kidney decreased throughout development, dropping from 16.22% of the total cell population at E10.5 to 8.13% at E15.5 to 4.39% in the adult. Specific markers of renal progenitor cells that continue to be expressed in a stem cell population should decrease in abundance as the kidney develops and differentiates and the progenitor pool becomes depleted.
  • kidney side population cells are a specific subpopulation of cells isolated by FACS on the basis of their ability to rapidly efflux the vital dye Hoechst 33342 that have been shown to be highly enriched for stem cells from a number of organs (Goodell MA.Multipotential stem cells and 'side population' cells. Cytotherapy. 2002;4(6):507-8).
  • the SP represent approximately 0.1-0.2% of the total cell population from El 5.5 and adult kidneys and approximately 91% and 67% of these embryonic and adult kidney SP cells respectively express CD24a when overlayed on isotype matched controls (FIG. 8).
  • CD24a has been identified as a potential renal stem cell marker by two independent experiments (microarray analysis at the renal progenitor timepoint, FACS analysis of kidney side population cells) enhances the likelihood that this molecule may mark a renal stem cell population. Discussion Of the genes identified as enriched in the uninduced MM from microarray analysis, CD24a antigen and cadherin-11 appear to be the best candidates for renal progenitor cell surface markers. CD24a was strongly and specifically expressed in all uninduced MM cells at E10.5 while cadherin-11 is also strongly expressed by this population. Although these molecules both appear to mark the renal progenitor population, their expression patterns diverged greatly as kidney development progressed.
  • CD24a expression was observed in all epithelial structures of the developing kidney except for the lower limbs of the S-shaped bodies while cadherin-11 was expressed by mesenchymal cells of the renal interstitium, most strongly by those surrounding the UB tips, but not in epithelial cells.
  • CD24a marks cell types of both MM and UB derivatives suggests that it identifies renal progenitors committed to differentiating into epithelial segments of the nephron while cadherin- 11 may identify MM cells destined to form the renal interstitium.
  • CD24a may mark a renal stem cell population.
  • the human ortholog, CD24 is strongly expressed in Wilms' tumours (Droz et al, 190, Hum. Pathol. 21 536-544) and renal cell carcinomas
  • CD24a / CD24 may represent a marker of renal progenitor cells conserved between murine and human systems but its expression is not restricted to the uninduced MM and it will be necessary to use other markers in combination with CD24a to specifically purify renal progenitors.
  • CD24a + was successfully used to isolate cells from murine embryonic and adult kidneys by FACS, demonstrating the utility of the cell surface markers in this invention.
  • CD83 is a marker of dendritic cells (Lechmann et al, 2002, Trends Immunol.
  • CD164 and CD81 are enriched in a population of bone marrow derived cells with multi-lineage potential (MIAMI cells) (DTppolito et al, 2004, J. Cell Sci. 1172971-81). While claudin-6 and spint-2 showed tremendous specificity of expression in the ND and UB, the UB has a much smaller differentiation spectrum than the MIAMI cells.
  • MM MM and is not likely to be the source of a stem cell population.
  • the existence of a single nephrogenic progenitor is not clear because it is uncertain whether all epithelial cell types in the adult kidney can be derived from a single precursor cell or whether each cell type has its own precursors (Al Awqati & Oliver, 2002, supra). Therefore, cellular therapy of kidney diseases may require isolation of two distinct progenitor populations, one from the MM and one from the UB, in which case these markers would prove useful.
  • the distinct expression of common stem cell markers in the uninduced MM at El 0.5 was not detected by microarray analysis or in situ hybridisation.
  • the pluripotency markers Oct-4 and nanog were not observed in the uninduced
  • MM or metanpehric explants which is as expected from a progenitor population restricted to mesodermal differentiation.
  • c-kit and sca-1 were expressed in the El 0.5 MM but also throughout the nephrogenic cord, the tissue that gives rise to all three mammalian excretory entities.
  • These cell surface proteins have traditionally been used to identify various lineages of bone-marrow derived stem cells (Ma et al, 2002, Br. J. Haematol. 116 401-408; Meirelles et al, 2003, Br. J. Haematol.
  • sca-1 was expressed by the primitive tubules of the nephrons.
  • this invention will facilitate purification of cells with this phenotype from mixed populations, such as kidneys at various stages of development or differentiating ES cell cultures, using antibody-based FACS.
  • renal progenitors would preferably show a phenotype that was CD24a + , cadherin l l + c-kit +/low Sca-l +/low CD34 " It will be appreciated by the skilled person that the present invention is not limited to the embodiments described in detail herein, and that a variety of other embodiments may be contemplated which are nevertheless consistent with the broad spirit and scope of the invention. Table 1

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Abstract

L'invention concerne des méthodes d'identification, d'isolation et/ou de purification de cellules mésenchymateuses métanéphriques et, plus spécifiquement, des cellules de progéniteurs rénaux. Ces méthodes utilisent un profil d'expression génique contenant des gènes qui sont exprimés différemment dans un mésenchyme déterminé mais non induit en comparaison avec le tissu mésodermique intermédiaire voisin. On a découvert huit gènes qui codent des protéines transmembranaires qui sont, notamment, bénéfiques dans l'isolation et la purification de cellules de progéniteurs rénaux. On a déterminé un phénotype de surface cellulaire des cellules de progéniteurs rénaux comme étant CD24a+cadhérine 11+c-kit +/basSca-1+/bas CD34-. Par ailleurs, on peut utiliser en vue d'une régénération in vivo et/ou in vitro du tissu rénal des cellules mésenchymateuses métanéphriques et, plus spécifiquement, des cellules de progéniteurs rénaux, isolées en fonction d'un profil d'expression génique.
PCT/AU2005/000162 2004-02-09 2005-02-09 Marqueurs moleculaires lies au developpement de metanephrine et aux progeniteurs renaux WO2005075636A1 (fr)

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