WO2008032905A1 - Genes involved in differentiation of human stem cell lines and the microarray kit containing these genes - Google Patents

Genes involved in differentiation of human stem cell lines and the microarray kit containing these genes Download PDF

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WO2008032905A1
WO2008032905A1 PCT/KR2006/005395 KR2006005395W WO2008032905A1 WO 2008032905 A1 WO2008032905 A1 WO 2008032905A1 KR 2006005395 W KR2006005395 W KR 2006005395W WO 2008032905 A1 WO2008032905 A1 WO 2008032905A1
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seq
genbank accession
human
genes
gene
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PCT/KR2006/005395
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French (fr)
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Chul Geun Kim
Byung-Rok Do
Jong Joo Lee
Jinseon Jeon
Hyen Seok Heo
Ho Chul Kang
Hyun-Soo Kim
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Hurim Biocell Co., Ltd.
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Priority claimed from KR1020060088351A external-priority patent/KR100800400B1/en
Priority claimed from KR1020060088354A external-priority patent/KR100807618B1/en
Application filed by Hurim Biocell Co., Ltd. filed Critical Hurim Biocell Co., Ltd.
Publication of WO2008032905A1 publication Critical patent/WO2008032905A1/en

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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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] 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
    • C12Q2600/158—Expression markers

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  • the present invention relates to maker genes and a microarray kit detecting differentiation of human stem cell lines.
  • This invention provides a method for monitoring stem cell differentiation.
  • Maker genes were selected by analyzing gene expression profiles of the differentiated and undifferentiated state of human embryonic stem cells (hESCs) and human adult stem cells (hASCs).
  • the genes involved in differentiation of human stem cell lines have been detected and confirmed by selecting the up-regulated genes and down-regulated genes in hESCs and hASCs.
  • the differentiation potential and stem cell characteristics of each stem cell lines were confirmed by examining the microscopic cell morphology, immunostaining with specific surface makers and RT-PCR with lineage specific marker genes to the undifferentiated and differentiating embryoid bodies in vitro.
  • the up-regulated genes and down-regulated genes in each stem cell line have been selected and confirmed by microarray and datamining procedures.
  • the genes up-regulated include a signaling protein (RHOG, a ras homolog gene family member G) 7 a transcription factor (HIFlA, hypoxia-inducible factor 1), and several proteins having the functions of protein transport (TLOCl) and the catabolic activity.
  • RHOG signaling protein
  • HIFlA hypoxia-inducible factor 1
  • TOCl protein transport
  • ESCs embryonic stem cells
  • ASCs adult stem cells
  • stem cells A wide variety of stem cells has been isolated, and embryonic, neural, and hematopoietic stem, cells are well characterized in vertebrates and their transcriptional profiles have been analyzed in mice (Ivanova et al., 2002; Ramalho-Santos et al., 2002). Theoretically, the self-renewal mechanism should be common to all of these stem cell lines, whereas commitment to specific lineages must be caused by differential gene expression.
  • ASCs have a potential advantage in cell therapies in that one can in principle overcome immunological hurdles by using the patients' own cells expanded in culture and then reintroduced into the tissue to be regenerated. Despite this theoretical advantage, it is technically challenging to use ASCs for this purpose due to their small numbers in mature tissues and the difficulty of expanding them in culture.
  • the PCT Publication WO 03/78589 A2 disclosed a method for identifying a genetic factor responsible for differentiation of a beginning cell to a target cell. Although this method had been disclosed, there is no specification of maker genes related to differentiation of the stem cells.
  • hESCs hematopoietic stem/ progenitor cells
  • hMSCs mesenchymal stem cells
  • the object of the present invention is to provide up-regulated genes during differentiation of hESCs and hASCs; a gene related with signaling pathway RHOG (GenBank Accession No. X61587); a gene related to the transcription factor HIFlA (GenBank Accession No. AA789181); genes related with metabolism PPIG (GenBank Accession No. AA954914), BCATl (GenBank Accession No. AI970531), PLOD2 (GenBank Accession No. U84573), PYGL (GenBank Accession No. AI091042), HUMAUANTIG (GenBank Accession No. AA902387), MANBA (GenBank Accession No. U60337), PTPN12 (GenBank Accession No.
  • Another object of this invention is to provide down-regulated genes during differentiation of hESCs and hASCs; a gene related with signal transduction FZD9 (GenBank Accession No. U82169); genes related with cell supporting protein COL4A3 (GenBank Accession No. M81379), MUCl (GenBank Accession No. AI922289), MGP (GenBank Accession No. AA484893); genes related with transportation TF (GenBank Accession No. S95936), AQPl (GenBank Accession No. S73482), FOLRl (GenBank Accession No. NM_016730); other genes of CLU (GenBank Accession No. X14723), OLFMl (GenBank Accession No. D82343), UMOD (GenBank Accession No. M15881), RNASEl (GenBank Accession No. AL046791).
  • the present invention also provides marker genes, comprising up-regulated and down-regulated expression genes, for detecting the profile and signature of human stem cell differentiation.
  • the third object of this invention is to provide a DNA microarray kit for analyzing the profile and signature of human stem cell differentiation, comprising: i ) A DNA chip plate wherein the marker genes of claim 3 are immobilized in the solid support; ii ) primer sets, polymerase and polymerization solution for amplifying the genes with the RT-PCR method from total RNA extracted from human stem cells; and iii) probes for detecting the hybridization between genes in the DNA chip and genes amplified from total RNA.
  • said primer sets comprise a primer set for the human PLEK gene (SEQ ID NO: 1 and SEQ ID NO: 2), a primer set for the human CD37 gene (SEQ ID NO: 3 and SEQ ID NO 4), a primer set for the human GATA2 gene (SEQ ID NO: 5 and SEQ ID NO: 6), a primer set for the human ELFl gene (SEQ ID NO: 7 and SEQ ID NO: 8), a primer set for the human PLOD2 (SEQ ID NO: 9 and SEQ ID NO: 10), a primer set for the human EVIl gene (SEQ ID NO: 11 and SEQ ID NO: 12), a primer set for the human HESl gene (SEQ ID NO: 13 and SEQ ID NO: 14), a primer set for the human USP9X gene (SEQ ID NO: 15 and SEQ ID NO: 16), a primer set for the human MAGEA4 gene (SEQ ID NO: 17 and SEQ ID NO: 18), a primer set for the human Oct4 gene (SEQ ID NO:
  • FIG. 1 shows schematic representation of the experimental procedure.
  • Total RNAs were extracted from each human stem cell population.
  • Universal human reference RNA (Clontech Lab. Inc., USA) was used as a baseline for total RNAs from each stem cell population.
  • fluorescent dye-labeled cDNA probes were prepared and hybridized to human cDNA array chips (TwinChipTM Human-8K).
  • FIG. 2 shows confirmation of human stem cells by morphology, immunohistochemistry, and semi-quantitative RT-PCR.
  • A Microscopic view of undifferentiated hESCs.
  • B Undifferentiated hESCs immunostained with anti-Oct4,
  • C anti-SSEA-1, and
  • D anti-SSEA-3 antibodies.
  • E Embryoid bodies of hESCs.
  • F Microscopic view of undifferentiated hMSCs.
  • G AP-stained undifferentiated hMSCs.
  • H hMSCs differentiating into osteogenic lineages.
  • I and
  • J MACS-sorted human CD133 + and CD34 + cells, respectively (200 x magnification for all figures).
  • K Semi-quantitative RT-PCR confirmation of each type of human stem cell by detecting expression of genes including Oct4, Nanog, ⁇ -FP, and ⁇ -globin.
  • FIG. 3 shows semi-quantitative RT-PCR analysis of several selected genes in undifferentiated and differentiating stem cells.
  • the semi-quantitative RT-PCR results (A) are in agreement with the microarray results (B).
  • the color scale for standardized signal intensities in the microarrays extends from brightest green (for down-regulation) to brightest red (up-regulation).
  • FIG. 4 shows overlapping gene expression in two hESC lines (MizhESl and SNU3). Up-regulated (A) and down-regulated (B) genes were selected by comparing microarray gene expression profiles in the hESC lines and the universal human reference RNA (Clontech Lab. Inc., USA).
  • FIG. 5 shows Expression of a set of 3,160 clones expressed in undifferentiated and differentiating (at days 5 and 9) human ESCs in vitro.
  • A Two-way hierarchical clustering using Pearson's correlation coefficient as the distance measure. Each row represents one of the three groups and each column represents one of the 3,160 cDNA probes (red, high expression; green, low expression).
  • FIG. 6 shows expression of MYCN, TFAP2C IMP-3, and MAGEA4 in hESCs on days 0, 5, and 10 of differentiation.
  • Real-Time RT-PCR was used to quantify MYCN, TFAP2C, IMP-3, and MAGEA4 expression.
  • Nanog and Oct3/4 mRNAs were used as controls. The average value for the expression of each gene was obtained from three independent experiments. The gene expression levels on days 5 and 10 were calibrated to the expression level on day 0 by the ⁇ Ct method.
  • FIG. 7 shows overlapping gene expression in CD34 + and CD133 + cells.
  • Up-regulated (A) and down-regulated (B) genes were selected by comparing the microarray profiles of the hHS/PCs and universal human reference RNA.
  • FIG. 8 shows two-way hierarchical clustering of a set of 4,666 clones expressed in hMSCs, hHS/PCs (CD34 + and CD133 + ), undifferentiated (SNU3 and Miz-hESl) and differentiating (d5 and d9 of Miz-hESl) hESCs.
  • Expression profiles were clustered by average linkage hierarchical clustering, using Pearson's correlation coefficient as the distance measure.
  • Each row represents one of the 7 samples; each column represents one of the 8,170 cDNA probes.
  • the color scale for the standardized signal intensities extends from brightest green (down-regulation) to brightest red (up-regulation).
  • Fig. 1 The entire experimental process is outlined in Fig. 1.
  • the stem cell lines were maintained in the appropriate media and the characteristic morphologies of the undifferentiated cells were monitored (Figs. 2 A, 2F, 21, and 2J).
  • the undifferentiated state of hESCs was regularly checked as Oct4 positive (Fig. 2B), SSEA-I negative (Fig. 2C), and SSEA-3 positive (Fig. 2D) by immunostaining.
  • the differentiation potential of hESCs was determined by inducing EBs generated by plating hESCs on non-tissue culture-treated dishes (Fig. 2E).
  • Total hESC RNAs were prepared for microarray analysis from undifferentiated hESC (Miz-hESl), EB (Miz-hESl) at day 5, and EB (Miz-hESl) at day 9.
  • the differentiation state of hESC EBs at each time point was monitored by semi-quantitative RT-PCR for hESC marker genes such as Oct4 and Nanog (Fig. 2K).
  • the transcript levels of ⁇ -fetoprotein and ⁇ -globin were also detected in each type of stem cell by semi-quantitative RT-PCR.
  • ⁇ -fetoprotein a plasma protein normally produced by the fetus, increased during hESC EB differentiation, while ⁇ -globin, one of major proteins in erythrocytes, was detected only in hHS/PCs (Fig. 2K).
  • hMSCs differentiated to the osteogenic lineage have different morphologies (Fig. 2H) than undifferentiated hMSCs (Fig. 2F), which are alkaline phophatase negative (Fig. 2G).
  • Total hMSC RNAs were extracted from cells passaged 3 to 8 times after isolation, and total hHS/PC RNA was obtained directly from isolated CD133 + (Fig. 21) and CD34 + (Fig. 2J) cells.
  • Stem cell cDNAs were labeled with Cy5 and hybridized along with Cy3-labeled cDNAs from human universal reference total RNA (Clontech Lab. Inc., USA) to cDNA microarrays (TwinChipTM Human-8K, Digital Genomics Co., Korea) containing 8,170 clones selected originally from a human cDNA library containing about 10,000 clones (Incyte Co., USA).
  • the TwinChipTM Human-8K microarray is widely used to study human gene expression profiles ranging from cancer cells (or stem cells) to adult tissues (Cho et al., 2004; Hong et al., 2004; Kim et al., 2005; Oh et al v 2004).
  • Human universal reference total RNA is a pool of total RNA extracts collected from different human tissues. It provides the broadest possible assay of gene expression, and is suitable for data normalization with any array or labeling method.
  • the gene-expression profiles obtained were analyzed to identify genes up- or down-regulated in each individual stem cell population and then compared with those sets of genes from other stem cell populations. Subsequently, several up- and down-regulated genes were selected for semi-quantitative RT-PCR to confirm the results from the cDNA microarrays.
  • the selected genes included PLEK, CD37, GATA2, ELFl, EVIl, USP9X, and MAGEA4 that showed similar expression patterns in the two hESC lines (Miz-hESl and SNU3) as detected from the microarray analysis.
  • the semiquantitative RT-PCR results were consistent with the expression profiles of selected genes (Fig. 3), confirming the reliability of our microarray data.
  • the highly up-regulated genes in the two lines that encode regulatory proteins such as transcription factors or signaling proteins as well as other functional proteins are shown in Table 1.
  • the 100 most highly up-regulated genes are classified by function in Table 1.
  • This list includes key hESC pluripotency genes such as EBAF (LeftyA), FGF2, and TDGFl (Smith, 2001), thus providing a positive control for the quality of the cells and the data obtained.
  • hESC pluripotency marker POU5F1 (Oct4), because of the absence of probes for this gene on the TwinChipTM Human-8K chip.
  • Table 1 Genes highly up-regulated in human stem cells.
  • Receptor EPHAl CD226, PTPRC, LILRB3, NRPl.
  • PDGFRA ACVR2B, CXADR TNFRSF IB, TLR2, TNFRSF12A, SLC7A11, GPR65, FPRI, LEPREL2 ITGA4
  • genes up-regulated in hESC lines are related to signal transduction pathways that regulate self-renewal in mouse and human ESCs (Rao, 2004). These include fibroblast growth factor 2 (FGF2) and its receptor FGFR4 involved in the FGF signaling pathway, a receptor transaldolase 1 (TDGFl) and a secreted inhibitor EBAF (LeftyA), in the TGF- ⁇ pathway, and FRAT2 (frequently rearranged in advanced T-cell lymphomas 2) in the Wnt signaling pathway.
  • FGF2 fibroblast growth factor 2
  • TDGFl receptor transaldolase 1
  • LeftyA secreted inhibitor EBAF
  • FRAT2 frequently rearranged in advanced T-cell lymphomas 2
  • hESC-specific genes (shown in Table 1) belonged to groups 5 (FRAT2, USP9X, USP9Y) or 6 (EBAF, MAGEA4, DNMT3B).
  • FRAT2, USP9X, USP9Y) or 6 EBAF, MAGEA4, DNMT3B.
  • EBAF EBAF, MAGEA4, DNMT3B
  • Table 2 genes involved in signaling pathways according to their expression during hESC differentiation.
  • FGF2 and FGFR4 were down-regulated during differentiation, verifying their potential role in the pluripotency of hESCs.
  • Components of the TGF signaling pathway were also observed to be down-regulated during differentiation of Miz-hESl cells; these included TDGFl, EBAF (LeftyA), MAP4K1, MAP4K7, and JUN.
  • LIF leukemia inhibitory factor
  • Positive or negative numbers indicate fold-increases or fold-decreases of gene expression, respectively, in comparison with reference RNA.
  • hESCs In addition to known genes specific to, or at least related to, hESCs, we identified several potential stem cell-specific genes that encode transcription factors (MYCN, SALL2, TFAP2C, and M96), components of signaling pathways (VRKl and TESl), proteins involved in other functions (IMP-3), and a functionally uncharacterized protein (MAGEA4) by comparing the expression profiles of undifferentiated hESCs and human universal reference total RNA (see Table 1). Most of these genes were down-regulated during hESC differentiation. They are members of Group 4 (TFAP2C), Group 5 (VRKl, M96) or Group 6 (MYCN 7 IMP-3, SALL2), based on k-means clustering (Fig. 5B).
  • TFAP2Q IMP3, MYCN, and MAGEA4 have been noted for their low expression in most human tissues (see GeneCards database: http://bioinfo.weizmann.ac.il/cards / index. shtml).
  • the expression pattern of these four genes in various tissues is closely comparable to that of SOX2, Nanog, and Oct4, the widely-recognized signature genes of hESCs.
  • TFAP2C, IMP3, MYCN, and MAGEA4 as putative hESC signature genes. The decreased expression of these four genes during hESC differentiation was confirmed by real-time RT-PCR (Fig. 6).
  • MYCN avian neuroblastoma derived V-myc myelocytomatosis virus related oncogene
  • MYCN avian neuroblastoma derived V-myc myelocytomatosis virus related oncogene
  • TFAP2C transcription factor activator protein-2 Y
  • AP-2 ⁇ transcription factor activator protein-2 Y
  • This transcription factor is a novel marker of testicular carcinoma in situ (CIS) and of CIS-derived tumors (Hoei-Hansen et al., 2004).
  • CIS testicular carcinoma in situ
  • TFAP2C did not decrease markedly during hESC differentiation unlike other candidates, suggesting an early- role in differentiation. Down-regulation of this gene occurred after the 9th day of differentiation.
  • IGF-II mRNA-binding protein 3 IMP-3 is known to be strongly expressed in both mouse and human embryos and to control IGF-II expression during late mammalian development (Nielsen et al v 1999).
  • MAGEA4 is a member of the melanoma antigen (MAGE) gene family, which is composed of more than 25 genes in humans (Chomez et al., 2001). MAGE genes are not expressed in most healthy adult tissues except for the testis, and various forms of cancer (Forslund and Nordqvist, 2001). Although some members of the MAGE family are known to play important roles in cell cycle control and apoptosis (Barker and Salehi, 2002), the their physiological functions remain mostly unknown. Further studies are needed to validate the identification of these four genes as hESC-specific markers.
  • MAGE melanoma antigen
  • CD34 + and CD133 + HS/ PCs were isolated from adult peripheral blood.
  • CD133 is solely expressed on CD34 bright stem/ progenitor cells.
  • CD133 + cells are much rarer in blood than CD34 + cells and are believed to be much more likely to be hHSCs than CD34 + cells (Kratz-Albers et al., 1998).
  • 653 and 601 genes were up-regulated more than 2-fold in the CD133 + and CD34 + cells, respectively (Fig. 7A).
  • the most highly up-regulated genes in both hHS/PCs are listed in Table 1. 842 and 1,007 genes were down-regulated more than 2-fold in the CD133 + and CD34 + cells, respectively (Fig. 7B). There were 500 overlaps in the two populations.
  • hHS/PCs contains several genes such as nuclear receptor interacting protein 1 (NRIPl), nuclear factor erythroid-derived 2 (NFE2), baculoviral IAP repeat-containing 3 (BIRC3), and CEBPB, that are also up-regulated in other hHS/PCs (Georgantas et al., 2004; Park et al., 2002).
  • NRIPl nuclear receptor interacting protein 1
  • NFE2 nuclear factor erythroid-derived 2
  • BIRC3 baculoviral IAP repeat-containing 3
  • CEBPB baculoviral IAP repeat-containing 3
  • Pleckstrin Pleckstrin (PLEK), the major PKC substrate in platelets (Tyers et al., 1988) with a potential role in blocking neoplastic transformation (Cmarik et al., 2000), was detected by RT-PCR and microarray analysis in both hHS/PCs, whereas CD37, GATA2 (a CD34 + marker), and ELFl (E74-like ets domain transcription factor) were detected only in CD34 + cells.
  • HSC markers were only up-regulated in one of the two hHS/PCs; for example, LIM domain only 2 (LMO2), fms-related tyrosine kinase 3 (FLT3), and CD34 in CD34 + cells, and POU domain, class 2 transcription factor (POU2F2) in CD133 + cells. Since CD133 + cells is the more primitive form with stem cell characteristics, the list of genes up-regulated only in CD133 + cells is informative (Table 3).
  • This list includes genes encoding transcription factors (POU2F2, MAFB), signal transduction proteins (SH2D2A, RCVl, GUCA2B, SOCS3, OPHNl, PNRCl, DLLl, NR5A1, HCK), a cell cycle related protein (STK17B), receptors/ surface markers/ membrane proteins (PILRA, CCRl, CCR5, C3AR1, CSFlR, AGCl, VNNl, ELA2, LCP2, NOTCH2, CEACAM8), and cytokines/ growth factors (EREG, XCLl).
  • Table 3 also summarizes genes up-regulated only in the CD34 + cells.
  • the hMSCs used in our invention were derived from human bone marrow cells. We identified 669 genes more than 2-fold increased in hMSCs compared to reference RNA. The most highly up-regulated genes are listed in Table 1. Among the 50 most up-regulated genes, transforming growth factor, betainduced (TGFBI), matrix metalloproteinase 2 (MMP2), fibronectin 1 (FNl), and collagen, type I, alpha 1 (COLlAl) were previously reported to be highly expressed in mouse bone marrow stromal cells (BMSCs) (Wieczorek et al, 2003) and in human BMSCs (Jia et al., 2002; Tremain et al., 2001), which are usually referred to as MSCs (Prockop, 1997).
  • TGFBI betainduced
  • MMP2 matrix metalloproteinase 2
  • FNl fibronectin 1
  • COLlAl collagen, type I, alpha 1
  • genes up-regulated in hMSCs but not in hESCs or hHSCs (Table 3) to list genes that may be involved in the hMSC-specific stem cell state. 819 genes were more than 2-fold down-regulated in hMSCs in comparison with reference RNA. These down-regulated genes may be molecular indicators involved in hMSCs differentiation.
  • up-regulated or down-regulated genes were identified, providing potential candidates for defining the stem cell state of human stem cells (Fig. 9A).
  • the genes up-regulated in all stem cells include those that encode a signaling protein (RHOG), a ras homolog gene family member G), a transcription factor (HIFlA, hypoxia-inducible factor 1), and several proteins with known functions such as protein transport (TLOCl) and catabolic activities, or with unknown functions (Table 1).
  • hESC lines Miz-hESl (registered in US NIH Stem Cell Bank in 2001) and SNU3 (Oh et al., 2005), were maintained on mitomycin G-inactivated primary mouse embryonic fibroblast (P-MEF) feeder layers, obtained from C57BL6 mice, in DMEM/F12 (1:1) (Invitrogen Co., USA) supplemented with 20% Knockout Serum Replacement (Invitrogen Co., USA), 0.1 mM non-essential amino acids (Sigma-Aldrich Co., USA), 0.1 mM ⁇ -mercaptoethanol (Invitrogen Co., USA), 4 ng/ml human basic fibroblast growth factor (Sigma-Aldrich Co., USA) and 100 U/ml penicillin/ streptomycin (Invitrogen Co., USA) at 37°C, in a 5% CO 2 humidified atmosphere.
  • P-MEF mitomycin G-inactivated primary mouse embryonic fibroblast
  • the culture medium was changed every day, and cells were passaged when they reached confluence by microdissection or using 200 U of collagenase IV (Sigma-Aldrich Co., USA).
  • In vitro differentiation was induced by culturing ESCs in suspension to form embryoid bodies (EBs) in differentiation medium; DMEM (Invitrogen Co., USA) supplemented with 10% fetal bovine serum (Hyclone, USA), 0.1 mM non-essential amino acids, 1 mM L-glutamine (Invitrogen Co., USA) and 0.1 mM ⁇ -mercaptoethanol.
  • the differentiation medium was changed every 2 to 3 days.
  • the EBs were harvested on day 5 and day 9 for future analysis.
  • Human bone marrow cells were aspirated from human iliac crest and separated by 70% Percoll-gradient centrifugation. The cells in the low density fraction were washed with control medium [DMEM-low glucose (Invitrogen Co., USA) supplemented with 10% FBS (Cambrex Co., USA) and 100 U/ ml penicillin/ streptomycin] and aliquots of 10 7 cells were seeded in 60 cm 2 culture dishes in control medium. After removing non-adherent cells, the adherent cells were replated into new control medium, and subcultured with 0.25% trypsin and 1 mM EDTA at near 100% confluence.
  • control medium DMEM-low glucose (Invitrogen Co., USA) supplemented with 10% FBS (Cambrex Co., USA) and 100 U/ ml penicillin/ streptomycin
  • Isolated hMSCs were grown in control medium from 5 ⁇ lO 3 cells per 100 mm culture dish at 37°C, in a 5% CO 2 humidified atmosphere (Song et al., 2005), and subcultured every 5.7 days after disaggregation with 0.05% typsin/0.53 mM EDTA.
  • mononuclear cells were isolated from adult peripheral blood by Ficoll-Hypaque (Sigma- Aldrich Co., USA) density gradient centrifugation. Isolated mononuclear cells were washed once with PBS buffer containing 5% BSA and filtered through 30 ⁇ m mesh. Collected mononuclear cells were labeled with CD34 or CD133 monoclonal antibodies using MicroBeads (Miltenyi Biotec Inc., USA). Finally, the labeled hematopoietic cells were isolated by High Gradient Immunomagnetic Separation using a MACS separator (Miltenyi Biotech Inc., USA).
  • Immunohistochemistry was performed as described previously (Ozono et al., 1997). hESCs grown on sterile cover slips were fixed with 1% paraformaldehyde in phosphate buffered saline (PBS) at room temperature. Fixed cells were incubated with appropriate primary antibodies against Oct4, SSEA-I, or SSEA-3 followed by biotinylated secondary antibody. Immunolabeled proteins were detected using a VECTASTAIN ABC-AP kit (Vector Laboratories, Inc., USA).
  • hMSCs were washed twice with PBS. After fixation with acetone/ methanol solution (1:1) at room temperature for 10 min, the cells were washed several times with PBS.
  • NBT/ BCIP staining solution was freshly made before use by mixing 3.3 mg of NBT (10 mg/ml in D.W.) and 1.5 mg of BCIP (25 mg/ml in dimethylformamide) in 10 ml of AP-substrate buffer (0.1 M Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl 2 ). The fixed cells were incubated with NBT/ BCIP staining solution in the dark at room temperature, for 10.15 min for hESCs and 40.45 min for hMSCs, washed twice with PBS and mounted for microscopic observation.
  • RNAs were extracted from each stem cell line with TRIZOL reagent (Invitrogen Co., USA) and purified with an RNeasy Mini Kit (Qiagen Inc., USA). Contaminating DNA was digested with RNase-free DNase I (Ambion Inc., USA) during RNA purification. Purified total RNA was quantified by spectrophotometer and its integrity assessed by running on 0.8% agarose gels.
  • First strand cDNAs were synthesized by incubating 3 ⁇ g of total RNA or 3 ⁇ g of Human Universal Reference RNA (Clontech Lab. Inc., USA) with 1 ⁇ i of 100 pmol/ ⁇ T7(dT)24 oligonucleotide (Bioneer Co., Korea) at 70 0 C for 10 min followed by incubation with 4 ⁇ l of 5 ⁇ first strand buffer, 2 ⁇ i of 0.1 M DTT, 1 ⁇ i of 10 mM dNTP, and 1 ⁇ i of 200 ⁇ J/ ⁇ i Superscript II RT (Invitrogen Co., USA) at 42 0 C for 2 h. The reaction was stopped by incubating for 15 min at 70 °C.
  • T7(dT)24 oligonucleotide was 5'-AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(24)-3 ⁇
  • Second strand cDNA synthesis began with the addition of 30 ⁇ l of 5 ⁇ second strand buffer (Invitrogen Co., USA), 3 ⁇ i of 10 mM dNTP, 4 ⁇ i of 10 U/ ⁇ i E. coli DNA polymerase I (Invitrogen Co., USA), 1 ⁇ i of 10 IJ / ⁇ i E.
  • RNAs were labeled as described elsewhere (Heo et al., 2005).
  • 5 ⁇ i of amplified RNA was incubated with 2 ⁇ i of 3 ⁇ g/ ⁇ i oligo(dT) primer at 70 °C for 10 min.
  • the reaction mixture was then incubated in the presence of 10 ⁇ i of 5 ⁇ first strand buffer, 5 ⁇ i of 0.1 M DTT, 1 ⁇ i of 5Ox amino-allyl-dNTP mix, and 2 ⁇ i of 200 U/ ⁇ i Superscript II RT. After 1 h incubation at 42 °C, 1 ⁇ l of Superscript II RT was added and incubation continued for an additional 1 h.
  • the reaction mixture was then concentrated using a Microcon YM-30 filtering unit (Millipore Co., USA). Coupling aminoallyl-cDNA to Cy dye ester was accomplished by the following steps: first, amino-allyl-cDNA was resuspended in 4.5 ⁇ i of 0.1 M sodium carbonate buffer, pH 9.0; then, 4.5 ⁇ i of appropriate NHS-ester Cy dyes (prepared in DMSO) was added; the coupling reaction mixture was incubated in the dark at room temperature for 1 h, and cleaned with a QIAquick PCR purification Kit (Qiagen Inc., USA). The labeling reaction was analyzed with a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, USA).
  • RNA was used for semi-quantitative RT-PCR and Realtime RT-PCR analyses.
  • the reverse transcription reaction was performed at 42 0 C for 1 h in a volume of 10 ⁇ i containing 300 ng of RNA, 100 U of M-MLV RT and 10 pmol of random hexamers.
  • the resulting cDNA served as template for 26 cycles of PCR with 0.2 U of AmpliTaq (Applied Biosy stems, USA).
  • the PCR products were separated on 5% polyacrylamide gels, and visualized by ethidium bromide staining.
  • Real-time RT-PCR was performed using iQ SYBR Green Supermix and an iCycler (Bio-Rad Laboratories Inc., USA) with typical amplification parameters (95 0 C for 5 min, followed by 40 cycles of 95 °C for 30 s, 62 °C for 30 s, and 72 °C for 30 s). Fold-differences were determined by comparing the ⁇ Ct of each gene on differentiation day 5 and day 10 to that on day 0 after normalization with hHPRT. All primers were designed with Perlprimer software, and checked by PCR to ensure that they generated single bands of the predicted size.
  • the synthetic oligonucleotide primer sets used in the PCR and Real-time RT-PCR are listed in Table 4.
  • CD37 X14046 F 5' -AGG AGA GCT GGG ACT ATG TGC-3'
  • GATA2 M68891 F 5' -ACG GAG AGC ATG AAG ATG GA-3' R: 5' -CAA TTT GCA CAA CAG GTG CC-3'
  • ELFl AA280194 F 5' -AAG TGA TGG AAA CAC AGC AG-3' R: 5' -TAG TAC CTG AGT GCT CTT CC-3'
  • HESl AI743113 F 5' -CGG AGC TGG TGC TGA TAA CA-3'
  • MAGEA4 NM_00236 F 5' -TGT GAG GAG TCA AGG TTC TG-3'
  • R 5' -CAA GTG AAG CTG ATG GTA GTG-3'
  • Oct4 BC069246 F 5'-CTG CAG TGT GGG TTT CGG GCA-3' R: 5'-CTT GCT GCA GAA GTG GGT GGA GGA-3 1
  • Nanog NM_024865 F 5'-GAG CTG GTT GCC TCA TGT TA-3' R: 5'-GAG GAA GGA TTC AGC CAG TG-3'
  • Sox2 NM_003106 F 5'-CAT CAC CCA CAG CAA ATG AC-3 1
  • R 5'-AAT TCA GCA AGA AGC CTC TC-3'
  • WDHDl BC063041 F 5'-ATG GTG TGG AAC TCT ATT GG-3' R: 5'-TCA AAT CCT GTA CCT CTG TG-3 1
  • JAK2 NM_004972 F 5'-AGA TAT GCA AGG GTA TGG AG-3 1
  • R 5'-AAA TGG AAC ACG ATC ATC TG-3' ⁇ -FP NM_001134
  • HPRT M31642 F 5' -GCT GGT GAA AAG GAC CCC A-3'

Abstract

The present invention relates to maker genes and a microarray kit for detecting genes involved in human stem cell differentiation. Further, we developed a DNA microarry kit containing the gene set that has been selected from up-regulated and down-regulated genes in analysis of stem cell expression profiles, to identify specific genes of hESCs and hASCs. This invention will provide a monitoring method for stem cell differentiation.

Description

Genes Involved in Differentiation of Human Stem Cell Lines and the Microarray Kit Containing These Genes
TECHNICAL FIELD
The present invention relates to maker genes and a microarray kit detecting differentiation of human stem cell lines. This invention provides a method for monitoring stem cell differentiation. Maker genes were selected by analyzing gene expression profiles of the differentiated and undifferentiated state of human embryonic stem cells (hESCs) and human adult stem cells (hASCs).
Particularly, the genes involved in differentiation of human stem cell lines have been detected and confirmed by selecting the up-regulated genes and down-regulated genes in hESCs and hASCs. Before performing microarray analyses, the differentiation potential and stem cell characteristics of each stem cell lines were confirmed by examining the microscopic cell morphology, immunostaining with specific surface makers and RT-PCR with lineage specific marker genes to the undifferentiated and differentiating embryoid bodies in vitro.
After detecting the common molecular regulatory pathways of hESCs and hASCs, the up-regulated genes and down-regulated genes in each stem cell line have been selected and confirmed by microarray and datamining procedures.
Total 22 up-regulated genes commonly in all stem cells have been selected and confirmed. The genes up-regulated include a signaling protein (RHOG, a ras homolog gene family member G)7 a transcription factor (HIFlA, hypoxia-inducible factor 1), and several proteins having the functions of protein transport (TLOCl) and the catabolic activity. Total 141 down-regulated genes commonly in all human stem cells have been also selected and confirmed.
BACKGROUND ART
Since Briggs and King demonstrated the totipotency of the adult cell nucleus by nuclear transplantation in the 1950's (Gurdon and Byrne, 2003), not much progress has been made in understanding the molecular nature of pluripotency. Recently, major interest has been stirred in stem cell research due to the great potential of pluripotent stem cells in regenerative medicine, as well as for understanding normal mammalian development. Stem cells are unspecialized cells that can generate cells with identical properties (self renewal) as well as give rise to differentiated cells (Smith, 2001).
There are basically two kinds of stem cells in animals including humans: embryonic stem cells (ESCs) and adult stem cells (ASCs). Each type has different functions and characteristics. ESCs derived from early stage embryos retain the ability to differentiate into all cell types. The ASCs found among the differentiated cells in a tissue or organ are similar to ESCs in that they maintain an undifferentiated state by self-renewal, but they differ in that their potencies are generally limited to the specific lineages found in that particular organ.
A wide variety of stem cells has been isolated, and embryonic, neural, and hematopoietic stem, cells are well characterized in vertebrates and their transcriptional profiles have been analyzed in mice (Ivanova et al., 2002; Ramalho-Santos et al., 2002). Theoretically, the self-renewal mechanism should be common to all of these stem cell lines, whereas commitment to specific lineages must be caused by differential gene expression.
ASCs have a potential advantage in cell therapies in that one can in principle overcome immunological hurdles by using the patients' own cells expanded in culture and then reintroduced into the tissue to be regenerated. Despite this theoretical advantage, it is technically challenging to use ASCs for this purpose due to their small numbers in mature tissues and the difficulty of expanding them in culture.
The use of hESCs (Reubinoff et al., 2000; Thomson et al., 1998) attracts tremendous public attention due to their pluripotency and the ease of generating large numbers in culture. Cell therapies using ESCs have been applied to immune-deficient Rag2 (Rideout IH et al., 2002) and cardiac defective Id^" (Fraidenraich et al., 2004) mouse models. However, there are still many limitations in using ESCs as therapeutic tools, notably the lack of reliable methods for generating specific cell lineages, and the difficulty of purifying specifically differentiated progeny and avoiding immunological rejection of the transplanted cells (Chien et al., 2004). Furthermore, once they are transplanted, it is almost impossible to control development of the transplanted ESCs into specific cell lineages. Despite these problems, the potential application of ESCs in regenerative medicine provides great impetus to studies of the molecular genetic nature of the stem cell state.
There is some evidence that ASCs can form differentiated cell types of another lineage (Forbes et al., 2002). There have also been efforts to understand the stem cell state of ESCs by analyzing transcriptional profiles using microarrays (Rao, 2004). Little is known, however, about the molecular programs and signaling genes involved in pluripotency and plasticity (Blau et al., 2001; Weissman, 2000). Although a few genes have been identified as potential stem cell molecular signatures from large scale transcriptional profiling of mouse stem cell populations, including embryonic, neural, and hematopoietic stem cells, the key molecular switches are still largely unknown (Fortunel et al., 2003; Ivanova et al., 2002; Ramalho-Santos et al., 2002).
Regarding the identification of genes for controlling differentiation, the PCT Publication WO 03/78589 A2 disclosed a method for identifying a genetic factor responsible for differentiation of a beginning cell to a target cell. Although this method had been disclosed, there is no specification of maker genes related to differentiation of the stem cells.
On the other hand, in the Korean Publication No. 2003-67871, a DNA microarray kit for detecting differentiation and specific marker genes for endoderm, mesoderm and ectoderm which were differentiated from embryos had been disclosed. However, there was no disclosure of common marker genes involved in differentiation of stem cells.
It is ceratin that understanding of the molecular mechanisms for self-renewal, pluripotency, plasticity, and differentiation of stem cells is a prerequisite not only for overcoming the limitations in using stem cells as therapeutic tools but also unlocking fundamental mysteries of mammalian development.
In the present invention, we identified genes that are up- or down-regulated in common in certain stem cell populations; i.e., hESCs, hematopoietic stem/ progenitor cells (hHS/PCs), and mesenchymal stem cells (hMSCs), and compared those sets of genes to one another to seek the molecular signatures of the stem cell state.
DISCLOSURE OF INVENTION
The object of the present invention is to provide up-regulated genes during differentiation of hESCs and hASCs; a gene related with signaling pathway RHOG (GenBank Accession No. X61587); a gene related to the transcription factor HIFlA (GenBank Accession No. AA789181); genes related with metabolism PPIG (GenBank Accession No. AA954914), BCATl (GenBank Accession No. AI970531), PLOD2 (GenBank Accession No. U84573), PYGL (GenBank Accession No. AI091042), HUMAUANTIG (GenBank Accession No. AA902387), MANBA (GenBank Accession No. U60337), PTPN12 (GenBank Accession No. D13380); other genes of SDCCAGl (GenBank Accession No. AA670455), KAB (GenBank Accession No. AB022657), KIAA0372 (GenBank Accession No. AB002370), PMAIPl (GenBank Accession No. D90070), ANKRD12 (GenBank Accession No. AI692537), PHC3 (GenBank Accession No. AA400519), MTHFD2 (GenBank Accession No. X16396), REGL (GenBank Accession No. D56495).
Another object of this invention is to provide down-regulated genes during differentiation of hESCs and hASCs; a gene related with signal transduction FZD9 (GenBank Accession No. U82169); genes related with cell supporting protein COL4A3 (GenBank Accession No. M81379), MUCl (GenBank Accession No. AI922289), MGP (GenBank Accession No. AA484893); genes related with transportation TF (GenBank Accession No. S95936), AQPl (GenBank Accession No. S73482), FOLRl (GenBank Accession No. NM_016730); other genes of CLU (GenBank Accession No. X14723), OLFMl (GenBank Accession No. D82343), UMOD (GenBank Accession No. M15881), RNASEl (GenBank Accession No. AL046791).
Further, the present invention also provides marker genes, comprising up-regulated and down-regulated expression genes, for detecting the profile and signature of human stem cell differentiation.
The third object of this invention is to provide a DNA microarray kit for analyzing the profile and signature of human stem cell differentiation, comprising: i ) A DNA chip plate wherein the marker genes of claim 3 are immobilized in the solid support; ii ) primer sets, polymerase and polymerization solution for amplifying the genes with the RT-PCR method from total RNA extracted from human stem cells; and iii) probes for detecting the hybridization between genes in the DNA chip and genes amplified from total RNA.
Finally, said primer sets comprise a primer set for the human PLEK gene (SEQ ID NO: 1 and SEQ ID NO: 2), a primer set for the human CD37 gene (SEQ ID NO: 3 and SEQ ID NO 4), a primer set for the human GATA2 gene (SEQ ID NO: 5 and SEQ ID NO: 6), a primer set for the human ELFl gene (SEQ ID NO: 7 and SEQ ID NO: 8), a primer set for the human PLOD2 (SEQ ID NO: 9 and SEQ ID NO: 10), a primer set for the human EVIl gene (SEQ ID NO: 11 and SEQ ID NO: 12), a primer set for the human HESl gene (SEQ ID NO: 13 and SEQ ID NO: 14), a primer set for the human USP9X gene (SEQ ID NO: 15 and SEQ ID NO: 16), a primer set for the human MAGEA4 gene (SEQ ID NO: 17 and SEQ ID NO: 18), a primer set for the human Oct4 gene (SEQ ID NO: 19 and SEQ ID NO: 20), a primer set for the human Nanog gene (SEQ ID NO: 21 and SEQ ID NO: 22), a primer set for the human Sox2 gene (SEQ ID NO: 23 and SEQ ID NO: 24), a primer set for the human WDHDl gene (SEQ ID NO: 25 and SEQ ID NO: 26), a primer set for the human JAK2 gene (SEQ ID NO: 27 and SEQ ID NO: 28), a primer set for the human α-FP gene (SEQ ID NO: 29 and SEQ ID NO: 30), a primer set for the human β-globin gene (SEQ ID NO: 31 and SEQ ID NO: 32) and a primer set for the human HPRT gene (SEQ ID NO: 33 and SEQ ID NO: 34).
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows schematic representation of the experimental procedure. Total RNAs were extracted from each human stem cell population. Universal human reference RNA (Clontech Lab. Inc., USA) was used as a baseline for total RNAs from each stem cell population. After amplifying RNA samples, fluorescent dye-labeled cDNA probes were prepared and hybridized to human cDNA array chips (TwinChip™ Human-8K).
FIG. 2 shows confirmation of human stem cells by morphology, immunohistochemistry, and semi-quantitative RT-PCR. (A) Microscopic view of undifferentiated hESCs. (B) Undifferentiated hESCs immunostained with anti-Oct4, (C) anti-SSEA-1, and (D) anti-SSEA-3 antibodies. (E) Embryoid bodies of hESCs. (F) Microscopic view of undifferentiated hMSCs. (G) AP-stained undifferentiated hMSCs. (H) hMSCs differentiating into osteogenic lineages. (I) and (J) MACS-sorted human CD133+ and CD34+ cells, respectively (200 x magnification for all figures). (K) Semi-quantitative RT-PCR confirmation of each type of human stem cell by detecting expression of genes including Oct4, Nanog, α-FP, and β-globin.
FIG. 3 shows semi-quantitative RT-PCR analysis of several selected genes in undifferentiated and differentiating stem cells. The semi-quantitative RT-PCR results (A) are in agreement with the microarray results (B). The color scale for standardized signal intensities in the microarrays extends from brightest green (for down-regulation) to brightest red (up-regulation).
FIG. 4 shows overlapping gene expression in two hESC lines (MizhESl and SNU3). Up-regulated (A) and down-regulated (B) genes were selected by comparing microarray gene expression profiles in the hESC lines and the universal human reference RNA (Clontech Lab. Inc., USA).
FIG. 5 shows Expression of a set of 3,160 clones expressed in undifferentiated and differentiating (at days 5 and 9) human ESCs in vitro. (A) Two-way hierarchical clustering using Pearson's correlation coefficient as the distance measure. Each row represents one of the three groups and each column represents one of the 3,160 cDNA probes (red, high expression; green, low expression). (B) K-means clustering analysis (k = 7) of the 3,160 genes showing altered expression during hESC differentiation.
FIG. 6 shows expression of MYCN, TFAP2C IMP-3, and MAGEA4 in hESCs on days 0, 5, and 10 of differentiation. Real-Time RT-PCR was used to quantify MYCN, TFAP2C, IMP-3, and MAGEA4 expression. To monitor the state of hESC differentiation, Nanog and Oct3/4 mRNAs were used as controls. The average value for the expression of each gene was obtained from three independent experiments. The gene expression levels on days 5 and 10 were calibrated to the expression level on day 0 by the ΔΔCt method. Significant differences (Student's t-test, p < 0.05) in relative gene expression between day 0 and day 5, or day 0 and day 10, are marked by * (open bar, day 0; hatched bar, day 5; closed bar, day 10).
FIG. 7 shows overlapping gene expression in CD34+ and CD133+ cells. Up-regulated (A) and down-regulated (B) genes were selected by comparing the microarray profiles of the hHS/PCs and universal human reference RNA.
FIG. 8 shows two-way hierarchical clustering of a set of 4,666 clones expressed in hMSCs, hHS/PCs (CD34+ and CD133+), undifferentiated (SNU3 and Miz-hESl) and differentiating (d5 and d9 of Miz-hESl) hESCs. Expression profiles were clustered by average linkage hierarchical clustering, using Pearson's correlation coefficient as the distance measure. Each row represents one of the 7 samples; each column represents one of the 8,170 cDNA probes. The color scale for the standardized signal intensities extends from brightest green (down-regulation) to brightest red (up-regulation).
BEST MODE FOR CARRYING OUT THE INVENTION
Microarray analysis of human stem cells To identify the molecular signatures specific to various stem cell lines, we established transcriptional gene expression profiles for hESCs, hHS/PCs, and hMSCs using cDNA microarrays. The entire experimental process is outlined in Fig. 1. The stem cell lines were maintained in the appropriate media and the characteristic morphologies of the undifferentiated cells were monitored (Figs. 2 A, 2F, 21, and 2J). The undifferentiated state of hESCs was regularly checked as Oct4 positive (Fig. 2B), SSEA-I negative (Fig. 2C), and SSEA-3 positive (Fig. 2D) by immunostaining. The differentiation potential of hESCs was determined by inducing EBs generated by plating hESCs on non-tissue culture-treated dishes (Fig. 2E). Total hESC RNAs were prepared for microarray analysis from undifferentiated hESC (Miz-hESl), EB (Miz-hESl) at day 5, and EB (Miz-hESl) at day 9. The differentiation state of hESC EBs at each time point was monitored by semi-quantitative RT-PCR for hESC marker genes such as Oct4 and Nanog (Fig. 2K). The transcript levels of α-fetoprotein and β-globin were also detected in each type of stem cell by semi-quantitative RT-PCR. The expression of α-fetoprotein, a plasma protein normally produced by the fetus, increased during hESC EB differentiation, while β-globin, one of major proteins in erythrocytes, was detected only in hHS/PCs (Fig. 2K). hMSCs differentiated to the osteogenic lineage have different morphologies (Fig. 2H) than undifferentiated hMSCs (Fig. 2F), which are alkaline phophatase negative (Fig. 2G). Total hMSC RNAs were extracted from cells passaged 3 to 8 times after isolation, and total hHS/PC RNA was obtained directly from isolated CD133+ (Fig. 21) and CD34+ (Fig. 2J) cells. Stem cell cDNAs were labeled with Cy5 and hybridized along with Cy3-labeled cDNAs from human universal reference total RNA (Clontech Lab. Inc., USA) to cDNA microarrays (TwinChip™ Human-8K, Digital Genomics Co., Korea) containing 8,170 clones selected originally from a human cDNA library containing about 10,000 clones (Incyte Co., USA). The TwinChip™ Human-8K microarray is widely used to study human gene expression profiles ranging from cancer cells (or stem cells) to adult tissues (Cho et al., 2004; Hong et al., 2004; Kim et al., 2005; Oh et alv 2004). Human universal reference total RNA is a pool of total RNA extracts collected from different human tissues. It provides the broadest possible assay of gene expression, and is suitable for data normalization with any array or labeling method. The gene-expression profiles obtained were analyzed to identify genes up- or down-regulated in each individual stem cell population and then compared with those sets of genes from other stem cell populations. Subsequently, several up- and down-regulated genes were selected for semi-quantitative RT-PCR to confirm the results from the cDNA microarrays. The selected genes included PLEK, CD37, GATA2, ELFl, EVIl, USP9X, and MAGEA4 that showed similar expression patterns in the two hESC lines (Miz-hESl and SNU3) as detected from the microarray analysis. The semiquantitative RT-PCR results were consistent with the expression profiles of selected genes (Fig. 3), confirming the reliability of our microarray data.
Gene expression profiling of undifferentiated hESC lines
In this invention, we used two hESC lines, SNU3 and Miz-hESl, for the cDNA microarray analyses. A total of 637 and 1,261 genes were up-regulated more than 2-fold in SNU3 and Miz-hESl, respectively (Fig. 4A). The number of overlapping genes in the two hESC lines was 518, comprising a potential pool for defining the stem cell state in hESCs. These results were obtained from four different microarrays to avoid experimental errors due to differences in microarray sensitivity. Thus, the differences/ between the profiles of the two hESC lines should be due to real differences between the lines (Abeyta et al., 2004). The highly up-regulated genes in the two lines that encode regulatory proteins such as transcription factors or signaling proteins as well as other functional proteins are shown in Table 1. The 100 most highly up-regulated genes are classified by function in Table 1. This list includes key hESC pluripotency genes such as EBAF (LeftyA), FGF2, and TDGFl (Smith, 2001), thus providing a positive control for the quality of the cells and the data obtained. However, we were unable to detect another well-known hESC pluripotency marker, POU5F1 (Oct4), because of the absence of probes for this gene on the TwinChip™ Human-8K chip.
Table 1. Genes highly up-regulated in human stem cells.
Common in three human
Functional category hESC hHS/PC hMSC stem cells
Signaling pathway USP9X, USP9Y, TNFAIP3, MAIL, DKKl, CSPG2, RHOG TDGFl, FRAT2, BCL2A1, WAS, FZD2 YESl, VRKl HCLSl
Transcription factor MYCN, SALL2, HlFlA, STAT5A, HlFlA, STATl HlFlA TFAP2C, M96 ZNF292
Transcription regulation DNMT3B, SMAKCA5, NRIPl SMC4L1
DNA binding TERFl, ZNF195, CHDl, H2AFY, HRB2 DEK, TlFl, SMARCAS BCLI lA, HMGB2, TCEAl , WDHDl, TOP2A, TOP2B
Cell cycle CENPE, DLG7, CDK6 COL8A1 BUBlB, GPC4, CDC2, ECT2, CHEKl
Receptor EPHAl, CD226, PTPRC, LILRB3, NRPl. PDGFRA, ACVR2B, CXADR TNFRSF IB, TLR2, TNFRSF12A, SLC7A11, GPR65, FPRI, LEPREL2 ITGA4
Metabolism UGP2, INDO, ARHGDIB, PPIG PLOD2, ARHE, PPIG. BCAT1, AASS, ENTPD3, LOXLl1 ASPH, PLOD2, PYGL, ALPl, PPAT, NT5E, EXTL2, HUMAUANTIG, TK1, EPRS, HDC, HAS2, MANBA, PTPN 12 P1P5K2A, MTHFD2, GBEl GYG2, TKT, ALDH7A1
Unknown MAGEA4 FLJ23231, SRPUL, LOC90355, SDCCAGl. KAB, PMAIPl, KIAAOlOl, PMAIPl, KIAA0650, IFI35, ESTs KIAA0372, PMAIPl, GJAl, LRPPRC, ANKRD12. ABL1, ANKRD 12, PHC3, KIAA1915, MGC16491, TCTElL, ESTs MTHFD2, REGL KIAA0056, LOC83690, ESTs
The listed genes were selected from the 50-100 most highly up-regulated genes in each type of human stem cell. Several genes up-regulated in hESC lines are related to signal transduction pathways that regulate self-renewal in mouse and human ESCs (Rao, 2004). These include fibroblast growth factor 2 (FGF2) and its receptor FGFR4 involved in the FGF signaling pathway, a receptor transaldolase 1 (TDGFl) and a secreted inhibitor EBAF (LeftyA), in the TGF-β pathway, and FRAT2 (frequently rearranged in advanced T-cell lymphomas 2) in the Wnt signaling pathway. These results are identical to those observed in early studies (Amit et al., 2000; Ramalho-Santos et al., 2002; Rao et al., 2004; Sato et al., 2003; Sperger et al., 2003; Thomson et al., 1998). Recently, a study using chromatin immunoprecipitation coupled with DNA microarrays demonstrated that the hESC-specific transcription factors Oct4, SOX2, and Nanog co-occupied the promoters of a large number of target genes (Boyer et al., 2005). TDGF-I, EBAF, and FRAT2 were identified as the active genes whose promoters were co-occupied. These findings point to an important contribution of these ESC-specific regulators, Oct4, SOX2, and Nanog, as well as signaling pathways such as TGF-β and Wnt to the pluripotency and self-renewal of hESCs. Our microarray data also revealed that a gene encoding a DNA methyl transferase, DNMT3B, was the most highly up-regulated in both hESC populations, as shown in previous reports (Rao et al., 2004; Sperger et al., 2003).
We also identified 581 and 1,231 genes more than 2-fold down-regulated in SNU3 and Miz-hESl, respectively (Fig. 4B). The total of 445 overlaps between the two cell lines represents another potential set of molecular signatures for the stem cell state in hESCs. Interestingly, the highly down-regulated genes are mostly lineage specific genes that are not related to signaling pathways, DNA/ RNA binding, or cell cycle/ differentiation. The simplest ex-planation of this is that many of these genes act during tissue specific differentiation, and need to be down-regulated in hESCs until the differentiation program is mobilized. To clarify the proposed roles of the various groups of genes, further functional studies are called for.
Expression profiling of genes in signaling pathways in differentiating hESCs
Next, we looked into differences in gene expression patterns during differentiation of hESCs (Miz-hESl) in vitro. Although the two hESC lines, Miz-hESl and SNU3, share statistically significant affects on gene expression (Fig. 4A), we employed Miz-hESl in our invention since this line is registered in the US NIH Stem Cell Bank and validated as a universal standard. To induce differentiation EBs were generated by maintaining Miz-hESl cells in differentiation medium in the absence of feeder cells and basic fibroblast growth factor (bFGF). Cells were collected on day 5 and day 9 after EB formation, and RNAs were extracted for subsequent microarray analyses (see Fig. 1). Using the microarray results, we performed 2-way hierarchical clustering of a set of 3,160 clones expressed in undifferentiated and differentiated (day 5 and day 9) Miz-hESl cells (Fig. 5A). Seven clusters of genes were observed, and k-means clustering was carried out to group the 3,160 clones into 7 distinct clusters based on similarities of expression pattern (Fig. 5B). From each clustergram we were able to compare the relative component gene expression levels at three different stages of differentiation (days 0, 5, and 9). Genes in clusters 5 and 6 were highly expressed in undifferentiated hESCs. Indeed most hESC-specific genes (shown in Table 1) belonged to groups 5 (FRAT2, USP9X, USP9Y) or 6 (EBAF, MAGEA4, DNMT3B). We also analyzed genes involved in signaling pathways according to their expression during hESC differentiation (Table 2). In the FGF pathway, both FGF2 and FGFR4 were down-regulated during differentiation, verifying their potential role in the pluripotency of hESCs. Components of the TGF signaling pathway were also observed to be down-regulated during differentiation of Miz-hESl cells; these included TDGFl, EBAF (LeftyA), MAP4K1, MAP4K7, and JUN. Several genes of the Wnt pathway including FRAT2, HDACl, HDAC2, TCF3, USP9X, USP9Y, and MYC were highly expressed in undifferentiated Miz-hESl cells, and their expression decreased during differentiation. It is noteworthy that leukemia inhibitory factor (LIF), an external signal of the JAK/STAT pathway, was more highly expressed in undifferentiated Miz-hESl cells, but the downstream components of this pathway were mostly unchanged during differentiation (Table 2). This finding is consistent with a previous report that STAT3 activation is not sufficient to block hESC differentiation (Humphrey et al, 2004), indicating that JAK/STAT independent pathways operate in parallel to maintain the stem cell state in hESCs, although the JAK/STAT pathway is known to be crucial for stem cell maintenance in mice.
Table 2. Expression patterns of genes involved in major signaling pathways during the differentiation of hESCs (Miz-hESl) in vitro.
Clone ID Gene symbol dO d5 d9 Clone ID Gene symbol dO d5 d9
Wnt pathway Shh pathway
1690530 FZDl -1.54 -1.29 -1.39 2881837 SMO 2.28 1.38 1.46
1843837 FZD6 1.33 1.16 1.76 1627426 PRKARIA -2.02 -1.18 -1.50
2214002 FZD2 2.05 2.49 3.13 1516301 FOXMl 5.23 2.40 1.96
2736837 FZD7 1.46 πd -1.22 2056290 DYRKlA 1.38 1.37 1.55
4422958 FZD9 -99.32 -26.21 -21.56
4290851 LRP6 2.51 1.14 -1.05
2017133 WNTIl -1.45 -1.13 1.19 JAK/STAT
2622566 WNT7A 2.23 1.84 1.97 pathway
1356168 CSNK2A1 2.58 1.48 1.22
1621349 CSNK2A2 2.19 1.28 1.29 2987878 LIF 4.32 1.78 1.56
1649713 CSNKlE 2.44 1.92 1.60 1461950 LlFR 1.33 1.33 2.03
1861707 DVL2 1.96 1.24 1.31 2172334 IL6ST -1.66 -1.40 -1.21
1405911 DVL3 -1.50 1.08 1.37 1926442 GAB2 -2.42 -1.50 1.32
3871545 FRAT2 4.14 3.18 1.55 1220385 SOCS3 -3.21 1.07 -1.06
1648251 HDAC1 3.24 1.46 1.48 2554841 AKT2 -1.39 -4.12 -3.13
2360075 HDAC2 4.72 2.01 2.09 487291 STAT3 -1.38 -1.41 -1.60
640174 TCF3 3.99 1.98 1.72 2275583 CEBPB -4.14 -1.74 -1.73
1001245 USP9X 11.24 6.29 2.20 444605 NFKBl -2.00 -1.21 -1.18
2844339 USP9Y 11.63 6.09 2.70 1969563 JUN 2.15 1.26 1.35
1673876 MYC 4.17 1.17 1.03 591358 TIMP1 -2.72 -1.37 -1.41
2057653 CCNDl 2.18 1.83 2.49 2679117 PIMl 2.47 1.46 1.61
1668572 PPARG 2.06 1.24 1.46 1673876 MYC 4.17 1.17 1.03
FGF pathway
1711206 FGF2 4.47 1.65 nd
1211126 FGFR4 2.23 1.16 -1.19
TGF/BMP pathway
1275388 TDGFl 8.77 2.56 1.08
1865634 EBAF 2.42 -2.94 -1.49
1746640 MAP4K1 2.70 1.29 1.34
2921723 MAP3K7 2.03 1.38 1.30
1969563 JUN 2.15 1.26 1.35
Positive or negative numbers indicate fold-increases or fold-decreases of gene expression, respectively, in comparison with reference RNA.
Identification of potential hESC signature genes
In addition to known genes specific to, or at least related to, hESCs, we identified several potential stem cell-specific genes that encode transcription factors (MYCN, SALL2, TFAP2C, and M96), components of signaling pathways (VRKl and TESl), proteins involved in other functions (IMP-3), and a functionally uncharacterized protein (MAGEA4) by comparing the expression profiles of undifferentiated hESCs and human universal reference total RNA (see Table 1). Most of these genes were down-regulated during hESC differentiation. They are members of Group 4 (TFAP2C), Group 5 (VRKl, M96) or Group 6 (MYCN7 IMP-3, SALL2), based on k-means clustering (Fig. 5B).
Among the potential hESC-specific genes highly up-regulated in both hESC lines (SNU3 and Miz-hESl), TFAP2Q IMP3, MYCN, and MAGEA4 have been noted for their low expression in most human tissues (see GeneCards database: http://bioinfo.weizmann.ac.il/cards / index. shtml). The expression pattern of these four genes in various tissues is closely comparable to that of SOX2, Nanog, and Oct4, the widely-recognized signature genes of hESCs. Thus, according to their distinctive hESC-specific expression, we propose TFAP2C, IMP3, MYCN, and MAGEA4 as putative hESC signature genes. The decreased expression of these four genes during hESC differentiation was confirmed by real-time RT-PCR (Fig. 6).
MYCN (avian neuroblastoma derived V-myc myelocytomatosis virus related oncogene) is a component of a transcription regulatory network related to cell growth and proliferation. Rapid down-regulation of MYCN has been observed as tissues become terminally differentiated and growth arrested (Thomas et al., 2004). The mechanisms causing persistence of embryonic cells that later give rise to tumors are unknown (Hansford et al., 2004). TFAP2C (transcription factor activator protein-2 Y), also known as AP-2γ, has been reported to be developmentally regulated and associated with the undifferentiated state in germ cells. It also has been proposed that TFAP2C is involved in self-renewal and survival of immature germ cells and tissue-specific stem cells. This transcription factor is a novel marker of testicular carcinoma in situ (CIS) and of CIS-derived tumors (Hoei-Hansen et al., 2004). Interestingly, expression of the transcription factor TFAP2C did not decrease markedly during hESC differentiation unlike other candidates, suggesting an early- role in differentiation. Down-regulation of this gene occurred after the 9th day of differentiation. IGF-II mRNA-binding protein 3 (IMP-3) is known to be strongly expressed in both mouse and human embryos and to control IGF-II expression during late mammalian development (Nielsen et alv 1999). MAGEA4 is a member of the melanoma antigen (MAGE) gene family, which is composed of more than 25 genes in humans (Chomez et al., 2001). MAGE genes are not expressed in most healthy adult tissues except for the testis, and various forms of cancer (Forslund and Nordqvist, 2001). Although some members of the MAGE family are known to play important roles in cell cycle control and apoptosis (Barker and Salehi, 2002), the their physiological functions remain mostly unknown. Further studies are needed to validate the identification of these four genes as hESC-specific markers.
Expression profiling of human adult stem cells by cDNA microarrays
The use of adult stem cells isolated from patients can provide an excellent solution to immunological problems raised in cell therapies. The main problem with this approach is that adult stem cells are rare and hard to expand in culture. A promising solution to this problem comes from guiding development of hESCs to adult stem cells, which requires extensive studies of hESC development both in vivo and in culture. Comparative analyses of gene expression profiles of various adult stem cells might generate insights into genetic pathways involved in self-renewal as well as multilineage specific differentiation. To this end, we analyzed the transcription profiles of two hHS/PCs (CD133+ and CD34+) and the hMSCs using cDNA microarrays. Expression profiling of hHS/PCs
For human hematopoietic stem cell (hHSC) microarray analyses, human CD34+ and CD133+ HS/ PCs were isolated from adult peripheral blood. CD133 is solely expressed on CD34 bright stem/ progenitor cells. Thus CD133+ cells are much rarer in blood than CD34+ cells and are believed to be much more likely to be hHSCs than CD34+ cells (Kratz-Albers et al., 1998). 653 and 601 genes were up-regulated more than 2-fold in the CD133+ and CD34+ cells, respectively (Fig. 7A). The most highly up-regulated genes in both hHS/PCs are listed in Table 1. 842 and 1,007 genes were down-regulated more than 2-fold in the CD133+ and CD34+ cells, respectively (Fig. 7B). There were 500 overlaps in the two populations.
Our up-regulated gene list for hHS/PCs contains several genes such as nuclear receptor interacting protein 1 (NRIPl), nuclear factor erythroid-derived 2 (NFE2), baculoviral IAP repeat-containing 3 (BIRC3), and CEBPB, that are also up-regulated in other hHS/PCs (Georgantas et al., 2004; Park et al., 2002). We also carried out semiquantitative RT-PCR for several genes up-regulated in the hematopoietic cells to further confirm our microarray results for the hHS/PCs (see Fig. 3). Pleckstrin (PLEK), the major PKC substrate in platelets (Tyers et al., 1988) with a potential role in blocking neoplastic transformation (Cmarik et al., 2000), was detected by RT-PCR and microarray analysis in both hHS/PCs, whereas CD37, GATA2 (a CD34+ marker), and ELFl (E74-like ets domain transcription factor) were detected only in CD34+ cells.
Several known HSC markers were only up-regulated in one of the two hHS/PCs; for example, LIM domain only 2 (LMO2), fms-related tyrosine kinase 3 (FLT3), and CD34 in CD34+ cells, and POU domain, class 2 transcription factor (POU2F2) in CD133+ cells. Since CD133+ cells is the more primitive form with stem cell characteristics, the list of genes up-regulated only in CD133+ cells is informative (Table 3). This list includes genes encoding transcription factors (POU2F2, MAFB), signal transduction proteins (SH2D2A, RCVl, GUCA2B, SOCS3, OPHNl, PNRCl, DLLl, NR5A1, HCK), a cell cycle related protein (STK17B), receptors/ surface markers/ membrane proteins (PILRA, CCRl, CCR5, C3AR1, CSFlR, AGCl, VNNl, ELA2, LCP2, NOTCH2, CEACAM8), and cytokines/ growth factors (EREG, XCLl). Table 3 also summarizes genes up-regulated only in the CD34+ cells.
Table 3. Genes up-regulated in only one type of adult stem cell.
Functional category CD34+ CD133* hMSC
Transcription facuor ELFl, MAFQ BCL3, MEISl, POU2F2, MAFB PMXl1 MDFI, TAZ
NHLHl1 FLIl
Signal transduction SOCS2 SH2D2A, RCVl, GUCA2B, WNT5A, AXL1 DOKl, BDNF,
SOCS3, OPHNl, PNRCl, MADH3, MADH7, JAG2,
DLL1, NR5A1, HCK SELlL
Cell cycle LMO2 STK 17B C0L8A1 , CALDl, UBE2C
Receptor/surface PLEK, ITGA2B, CD37, COROlA, PILRA, CCRl, CCR5, C3AR1, NRPl, ANXA7, TNC1 ESDN, marker/membrane protein LGALS9, EVI2B, FLT3, CD34 CSFlR, AGCl, VNNl, ELA2, EFEMPl1 EDG2, COL16A1,
LCP2, N0TCH2, CEACAM8 SRPX1 COL6A2, COL2A1,
IL6ST, CDH2, DCBLDl, LUM
Cytokines/growth factor EREG, XCLl CTGF, FAM3C, CYR61
D isease/cancer/unknown TULPl SLC22A1L, CDlB1 BAGE PKD2, BCAR3, SSAl
Expression profiling of hMSCs
The hMSCs used in our invention were derived from human bone marrow cells. We identified 669 genes more than 2-fold increased in hMSCs compared to reference RNA. The most highly up-regulated genes are listed in Table 1. Among the 50 most up-regulated genes, transforming growth factor, betainduced (TGFBI), matrix metalloproteinase 2 (MMP2), fibronectin 1 (FNl), and collagen, type I, alpha 1 (COLlAl) were previously reported to be highly expressed in mouse bone marrow stromal cells (BMSCs) (Wieczorek et al, 2003) and in human BMSCs (Jia et al., 2002; Tremain et al., 2001), which are usually referred to as MSCs (Prockop, 1997). We also summarize genes up-regulated in hMSCs but not in hESCs or hHSCs (Table 3) to list genes that may be involved in the hMSC-specific stem cell state. 819 genes were more than 2-fold down-regulated in hMSCs in comparison with reference RNA. These down-regulated genes may be molecular indicators involved in hMSCs differentiation.
Identification of potential signature genes in human adult stem cells
Several genes for signaling molecules and transcription factors whose functions are not clearly defined were highly expressed in human adult stem cells (Table 1). From our microarray analyses we identified signaling molecules such as TNFAIP3, MAIL, BCL2A1, CSPG2, WAS, and HCLSl, and transcription factors such as ZNF292 and STAT5A, which were highly expressed in hHS/PCs, while the signaling molecules DKKl, CSPG2 and FZD2 and a transcription factor STATl were highly expressed in hMSCs.
Signature genes of human stem cells which are overlapped among the three types of human stem cells
To identify similarities between human stem cell types, a set of 4,666 clones expressed in hMSCs, hHS/PCs (CD34+ and CD133+), undifferentiated hESCs (SNU3 and Miz-hESl), and differentiated hESCs (day 5 and day 9 of Miz-hESl) were analyzed for 2-way hierarchical clustering. This demonstrated that each type of stem cell is distinguished from others by its pattern of gene expression. Interestingly, the overall gene expression pattern of hESCs is more similar to hMSCs than hHS/PCs (Fig. 8). Indeed, 124 up-regulated genes overlapped in hESCs and hMSCs, compared with 72 in hESCs and hHS/PCs (Fig. 9A). Similar clustering results were observed in mouse stem cell types, in which mESCs were more similar to mouse neural stem cells than to mHS/PCs (Ramalho-Santos et al., 2002). Our clustering results imply that the generation of hMSCs may be easier than hHS/PCs from hESCs, since hESCs already contain many genes related to the regulation of hMSC function.
To gain insight into molecular regulatory pathways shared by all the human stem cells investigated in this invention, we analyzed the overlaps of up-regulated or down-regulated genes in the various stem cell types. 22 up-regulated genes were identified, providing potential candidates for defining the stem cell state of human stem cells (Fig. 9A). The genes up-regulated in all stem cells include those that encode a signaling protein (RHOG), a ras homolog gene family member G), a transcription factor (HIFlA, hypoxia-inducible factor 1), and several proteins with known functions such as protein transport (TLOCl) and catabolic activities, or with unknown functions (Table 1).
We also identified 141 genes that were down-regulated in common in all human stem cells (Fig. 9B).
Conclusion
Despite the limitations of microarrays for identifying stem cell state genes (Fortunel et al., 2003), our findings may provide molecular clues to the fundamental biological properties of human stem cells: self-renewal and pluripotency. In this invention, we examined the expression of over 8,000 genes in hESCs and adult stem cells, and identified candidate genes involved in the common self-renewing phenotype of stem cells, and differentially expressed genes correlated with specific differentiation lineages. Further functional studies of these candidate genes may aid in understanding stem cell development in vivo, and provide useful tools for controlling hESC development into specific adult stem cells.
EXAMPLES
Materials
i ) hESC culture and differentiation
hESC lines, Miz-hESl (registered in US NIH Stem Cell Bank in 2001) and SNU3 (Oh et al., 2005), were maintained on mitomycin G-inactivated primary mouse embryonic fibroblast (P-MEF) feeder layers, obtained from C57BL6 mice, in DMEM/F12 (1:1) (Invitrogen Co., USA) supplemented with 20% Knockout Serum Replacement (Invitrogen Co., USA), 0.1 mM non-essential amino acids (Sigma-Aldrich Co., USA), 0.1 mM β-mercaptoethanol (Invitrogen Co., USA), 4 ng/ml human basic fibroblast growth factor (Sigma-Aldrich Co., USA) and 100 U/ml penicillin/ streptomycin (Invitrogen Co., USA) at 37°C, in a 5% CO2 humidified atmosphere. The culture medium was changed every day, and cells were passaged when they reached confluence by microdissection or using 200 U of collagenase IV (Sigma-Aldrich Co., USA). In vitro differentiation was induced by culturing ESCs in suspension to form embryoid bodies (EBs) in differentiation medium; DMEM (Invitrogen Co., USA) supplemented with 10% fetal bovine serum (Hyclone, USA), 0.1 mM non-essential amino acids, 1 mM L-glutamine (Invitrogen Co., USA) and 0.1 mM β-mercaptoethanol. The differentiation medium was changed every 2 to 3 days. The EBs were harvested on day 5 and day 9 for future analysis.
ii ) Isolation and maintenance of hMSCs
Human bone marrow cells were aspirated from human iliac crest and separated by 70% Percoll-gradient centrifugation. The cells in the low density fraction were washed with control medium [DMEM-low glucose (Invitrogen Co., USA) supplemented with 10% FBS (Cambrex Co., USA) and 100 U/ ml penicillin/ streptomycin] and aliquots of 107 cells were seeded in 60 cm2 culture dishes in control medium. After removing non-adherent cells, the adherent cells were replated into new control medium, and subcultured with 0.25% trypsin and 1 mM EDTA at near 100% confluence. Isolated hMSCs were grown in control medium from 5χlO3 cells per 100 mm culture dish at 37°C, in a 5% CO2 humidified atmosphere (Song et al., 2005), and subcultured every 5.7 days after disaggregation with 0.05% typsin/0.53 mM EDTA.
iii) Isolation of hHS/PCs
To isolate human CD34+ and CD133+ HS/ PCs, mononuclear cells were isolated from adult peripheral blood by Ficoll-Hypaque (Sigma- Aldrich Co., USA) density gradient centrifugation. Isolated mononuclear cells were washed once with PBS buffer containing 5% BSA and filtered through 30 μm mesh. Collected mononuclear cells were labeled with CD34 or CD133 monoclonal antibodies using MicroBeads (Miltenyi Biotec Inc., USA). Finally, the labeled hematopoietic cells were isolated by High Gradient Immunomagnetic Separation using a MACS separator (Miltenyi Biotech Inc., USA).
(Example 1) Immunohistochemistry and alkaline phosphatase staining
Immunohistochemistry was performed as described previously (Ozono et al., 1997). hESCs grown on sterile cover slips were fixed with 1% paraformaldehyde in phosphate buffered saline (PBS) at room temperature. Fixed cells were incubated with appropriate primary antibodies against Oct4, SSEA-I, or SSEA-3 followed by biotinylated secondary antibody. Immunolabeled proteins were detected using a VECTASTAIN ABC-AP kit (Vector Laboratories, Inc., USA).
For alkaline phosphatase staining, hMSCs were washed twice with PBS. After fixation with acetone/ methanol solution (1:1) at room temperature for 10 min, the cells were washed several times with PBS. NBT/ BCIP staining solution was freshly made before use by mixing 3.3 mg of NBT (10 mg/ml in D.W.) and 1.5 mg of BCIP (25 mg/ml in dimethylformamide) in 10 ml of AP-substrate buffer (0.1 M Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2). The fixed cells were incubated with NBT/ BCIP staining solution in the dark at room temperature, for 10.15 min for hESCs and 40.45 min for hMSCs, washed twice with PBS and mounted for microscopic observation.
(Example 2) Total RNA extraction
Total RNAs were extracted from each stem cell line with TRIZOL reagent (Invitrogen Co., USA) and purified with an RNeasy Mini Kit (Qiagen Inc., USA). Contaminating DNA was digested with RNase-free DNase I (Ambion Inc., USA) during RNA purification. Purified total RNA was quantified by spectrophotometer and its integrity assessed by running on 0.8% agarose gels.
(Example 3) RNA amplification
First strand cDNAs were synthesized by incubating 3 μg of total RNA or 3 μg of Human Universal Reference RNA (Clontech Lab. Inc., USA) with 1 μi of 100 pmol/μβ T7(dT)24 oligonucleotide (Bioneer Co., Korea) at 700C for 10 min followed by incubation with 4 μl of 5χ first strand buffer, 2 μi of 0.1 M DTT, 1 μi of 10 mM dNTP, and 1 μi of 200 \J/μi Superscript II RT (Invitrogen Co., USA) at 420C for 2 h. The reaction was stopped by incubating for 15 min at 70 °C. The sequence of the T7(dT)24 oligonucleotide was 5'-AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(24)-3\ Second strand cDNA synthesis began with the addition of 30 μl of 5χ second strand buffer (Invitrogen Co., USA), 3 μi of 10 mM dNTP, 4 μi of 10 U/μi E. coli DNA polymerase I (Invitrogen Co., USA), 1 μi of 10 IJ /μi E. coli DNA ligase (Invitrogen Co., USA), 1 μi of 2 \J/μi of RNase H (Invitrogen Co., USA), and 91 μi of nuclease-free water followed by incubation at 160C for 2 h. After incubation, 2 μi of T4 DNA polymerase (5 U/μi) (Invitrogen Co., USA) was added followed by 15 min incubation at 16 °C. Samples were purified using a MinElute PCR Purification Kit (Qiagen Inc., USA). After cDNA synthesis, 5 μi of cDNA was incubated with 15 μi of transcription mixture (MEGAscript T7 Kit, Ambion Inc., USA), consisting of 2 μi of 10x reaction buffer, 2 μi of each 4 mM ATP, CTP, GTP, and UTP solution, and 2 μi of enzyme mix at 37 °C for 4 h. Immediately following incubation, 2 μi of RNase-free DNase I was added and the reaction mixture was incubated at 370C for 15 min. After in vitro transcription, the amplified RNA was purified with an RNeasy Mini Kit.
(Example 4) Microarray analysis and data mining
Total cellular RNAs were labeled as described elsewhere (Heo et al., 2005). For fluorescent target labeling, 5 μi of amplified RNA was incubated with 2 μi of 3 μg/ μi oligo(dT) primer at 70 °C for 10 min. The reaction mixture was then incubated in the presence of 10 μi of 5χ first strand buffer, 5 μi of 0.1 M DTT, 1 μi of 5Ox amino-allyl-dNTP mix, and 2 μi of 200 U/μi Superscript II RT. After 1 h incubation at 42 °C, 1 μl of Superscript II RT was added and incubation continued for an additional 1 h. The reaction mixture was then concentrated using a Microcon YM-30 filtering unit (Millipore Co., USA). Coupling aminoallyl-cDNA to Cy dye ester was accomplished by the following steps: first, amino-allyl-cDNA was resuspended in 4.5 μi of 0.1 M sodium carbonate buffer, pH 9.0; then, 4.5 μi of appropriate NHS-ester Cy dyes (prepared in DMSO) was added; the coupling reaction mixture was incubated in the dark at room temperature for 1 h, and cleaned with a QIAquick PCR purification Kit (Qiagen Inc., USA). The labeling reaction was analyzed with a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, USA). Labeled samples from each stem cell line were co-hybridized to a TwinChip human 8K cDNA microarray (Digital Genomics Co., Korea) which includes 8,170 independent human cDNA sequences. Hybridization was repeated four times with dye-swapping and data were normalized in an intensitydependent manner using the scatter plot smoother 'LOWESS' (Yang et al., 2002). Genes with significant differences in expression were selected using significance analysis of microarray software (SAM) (Tusher et al., 2001) in one-class response format. The statistical significance of differential expression was assessed by computing a q-value for each gene. Genes were considered up-regulated or down-regulated when the logarithm of the gene expression ratio in four independent hybridizations was more than 1 or less than -1, that is, a 2 fold difference in expression level, where the q-value was less than 0.5.
(Example 5) Semi-quantitative RT-PCR and Real-time RT-PCR assays
Total RNA was used for semi-quantitative RT-PCR and Realtime RT-PCR analyses. The reverse transcription reaction was performed at 420C for 1 h in a volume of 10 μi containing 300 ng of RNA, 100 U of M-MLV RT and 10 pmol of random hexamers. The resulting cDNA served as template for 26 cycles of PCR with 0.2 U of AmpliTaq (Applied Biosy stems, USA). The PCR products were separated on 5% polyacrylamide gels, and visualized by ethidium bromide staining.
Real-time RT-PCR was performed using iQ SYBR Green Supermix and an iCycler (Bio-Rad Laboratories Inc., USA) with typical amplification parameters (950C for 5 min, followed by 40 cycles of 95 °C for 30 s, 62 °C for 30 s, and 72 °C for 30 s). Fold-differences were determined by comparing the ΔCt of each gene on differentiation day 5 and day 10 to that on day 0 after normalization with hHPRT. All primers were designed with Perlprimer software, and checked by PCR to ensure that they generated single bands of the predicted size. The synthetic oligonucleotide primer sets used in the PCR and Real-time RT-PCR are listed in Table 4.
Table 4. Origins, NCBI gene accession numbers and sequences of synthetic oligonucleotide PRC primer. Gene Accession number Primer sequence
PLEK X07743 F: 5' -CTA CTA CTT TCC AGA CAG TGG G-3'
R: 5' -AAA TAG TGC ACT TCA TCT GCT G-3'
CD37 X14046 F: 5' -AGG AGA GCT GGG ACT ATG TGC-3'
R: 5' -AGG AGA GCT GGG ACT ATG TGC-3'
GATA2 M68891 F: 5' -ACG GAG AGC ATG AAG ATG GA-3' R: 5' -CAA TTT GCA CAA CAG GTG CC-3'
ELFl AA280194 F: 5' -AAG TGA TGG AAA CAC AGC AG-3' R: 5' -TAG TAC CTG AGT GCT CTT CC-3'
EVIl AK025934 F: 5' -TAT CCA CGA AGA ACG GCA AT-3'
R: 5' -AAA GGG CTT CAC ACT GCT GT-3'
HESl AI743113 F: 5' -CGG AGC TGG TGC TGA TAA CA-3'
R: 5' -TCG TTC ATG CAC TCG CTG AA-3'
USP9X X98296 F: 5' -TTC TTA CAG ATG AAG CAG TGA G-3'
R: 5' -AGA AAC TCA TAG CAT TGC CC-3'
MAGEA4 NM_00236 F: 5' -TGT GAG GAG TCA AGG TTC TG-3' R: 5' -CAA GTG AAG CTG ATG GTA GTG-3'
Oct4 BC069246 F: 5'-CTG CAG TGT GGG TTT CGG GCA-3' R: 5'-CTT GCT GCA GAA GTG GGT GGA GGA-31
Nanog NM_024865 F: 5'-GAG CTG GTT GCC TCA TGT TA-3' R: 5'-GAG GAA GGA TTC AGC CAG TG-3'
Sox2 NM_003106 F: 5'-CAT CAC CCA CAG CAA ATG AC-31 R: 5'-AAT TCA GCA AGA AGC CTC TC-3'
WDHDl BC063041 F: 5'-ATG GTG TGG AAC TCT ATT GG-3' R: 5'-TCA AAT CCT GTA CCT CTG TG-31
JAK2 NM_004972 F: 5'-AGA TAT GCA AGG GTA TGG AG-31 R: 5'-AAA TGG AAC ACG ATC ATC TG-3' α-FP NM_001134 F: 5'-TGA AAA CCC TCT TGA ATG CC-3' R: 5'-TCT TGC TTC ATC GTT TGC AG-3' β-globin BC007075 F: 5'-CAT GGT GCA TCT GAC TCC TG-31
R: 5'-GCC ACC ACT TTC TGA TAG GC-31
HPRT M31642 F: 5' -GCT GGT GAA AAG GAC CCC A-3'
R: 5' -AGC TCT ACT AAG CAG ATG GC~3'

Claims

WHAT IS CLAIMED IS :
1. Up-regulated genes during differentiation of hESCs and hASCs comprising: a gene related with signaling pathway RHOG (GenBank Accession No. X61587); a gene related with transcription factor HIFlA (GenBank Accession No. AA789181); genes related with metabolism PPIG (GenBank Accession No. AA954914), BCATl (GenBank Accession No. AI970531), PLOD2 (GenBank Accession No. U84573), PYGL (GenBank Accession No. AI091042), HUMAUANTIG (GenBank Accession No. AA902387), MANBA (GenBank Accession No. U60337), PTPN12 (GenBank Accession No. D13380); other genes of SDCCAGl (GenBank Accession No. AA670455), KAB (GenBank Accession No. AB022657), KIAA0372 (GenBank Accession No. AB002370), PMAIPl (GenBank Accession No. D90070), ANKRD12 (GenBank Accession No. AI692537), PHC3 (GenBank Accession No. AA400519), MTHFD2 (GenBank Accession No. X16396), REGL (GenBank Accession No. D56495).
2. Down-regulated genes during differentiation of hESCs and hASCs comprising: a gene related with signal transduction FZD9 (GenBank Accession No. U82169); genes related with cell supporting protein COL4A3 (GenBank Accession No. M81379), MUCl (GenBank Accession No. AI922289), MGP (GenBank Accession No. AA484893); genes related with transportation TF (GenBank Accession No. S95936), AQPl (GenBank Accession No. S73482), FOLRl (GenBank Accession No. NM_016730); other genes of CLU (GenBank Accession No. X14723), OLFMl (GenBank Accession No. D82343), UMOD (GenBank Accession No. M15S81), RNASEl (GenBank Accession No. AL046791).
3. The marker genes for detecting the profile and signature of human stem cell differentiation comprising: i ) up-regulated genes of claim 1; and ii ) down-regulated genes of claim 2.
4. A DNA microarray kit for detecting the profile and signature of human stem cell differentiation comprising: i ) A DNA chip plate wherein the marker genes of claim 3 are immobilized in solid support; ii ) primer sets, polymerase and polymerization solution for amplifying the genes with the RT-PCR method from total RNA extracted from human stem celsl; and iii) probes for detecting the hybridization between genes in the DNA chip and genes amplified from total RNA.
5. The DNA microarray kit for detecting the profile and signature of differentiation of human stem cell according to claim 4, comprising a primer set for the human PLEK gene (SEQ ID NO: 1 and SEQ ID NO: 2), a primer set for the human CD37 gene (SEQ ID NO: 3 and SEQ ID NO 4), a primer set for the human GATA2 gene (SEQ ID NO: 5 and SEQ ID NO: 6), a primer set for the human ELFl gene (SEQ ID NO: 7 and SEQ ID NO: 8), a primer set for the human PLOD2 (SEQ ID NO: 9 and SEQ ID NO: 10), a primer set for the human EVIl gene (SEQ ID NO: 11 and SEQ ID NO: 12), a primer set for the human HESl gene (SEQ ID NO: 13 and SEQ ID NO: 14), a primer set for the human USP9X gene (SEQ ID NO: 15 and SEQ ID NO: 16), a primer set for the human MAGEA4 gene (SEQ ID NO: 17 and SEQ ID NO: 18), a primer set for the human Oct4 gene (SEQ ID NO: 19 and SEQ ID NO: 20), a primer set for the human Nanog gene (SEQ ID NO: 21 and SEQ ID NO: 22), a primer set for the human Sox2 gene (SEQ ID NO: 23 and SEQ ID NO: 24), a primer set for the human WDHDl gene (SEQ ID NO: 25 and SEQ ID NO: 26), a primer set for the human JAK2 gene (SEQ ID NO: 27 and SEQ ID NO: 28), a primer set for the human α-FP gene (SEQ ID NO: 29 and SEQ ID NO: 30), a primer set for the human β-globin gene (SEQ ID NO: 31 and SEQ ID NO: 32) and a primer set for the human HPRT gene (SEQ ID NO: 33 and SEQ ID NO: 34).
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