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Reproductionresearch REPRODUCTIONRESEARCH Expression profiles of protein tyrosine kinase genes in human embryonic stem cells Mi-Young Son, Janghwan Kim, Hyo-Won Han, Sun-Mi Woo, Yee Sook Cho, Yong-Kook Kang and Yong-Mahn Han1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), Center for Regenerative Medicine, 52 Eoeun- Dong, Yuseong-Gu, Daejeon 305-806, South Korea and 1Department of Biological Sciences and Center for Stem Cell Differentiation, KAIST, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, South Korea Correspondence should be addressed to Y-K Kang; Email: [email protected] Y-M Han; Email: [email protected] Abstract Complex signaling pathways operate in human embryonic stem cells (hESCs) and are coordinated to maintain self-renewal and stem cell characteristics in them. Protein tyrosine kinases (PTKs) participate in diverse signaling pathways in various types of cells. Because of their functions as key molecules in various cellular processes, PTKs are anticipated to have important roles also in hESCs. In this study, we investigated the roles of PTKs in undifferentiated and differentiated hESCs. To establish comprehensive PTK expression profiles in hESCs, we performed reverse transcriptase PCR using degenerate primers according to the conserved catalytic PTK motifs in both undifferentiated and differentiated hESCs. Here, we identified 42 different kinases in two hESC lines, including 5 non-receptor tyrosine kinases (RTKs), 24 RTKs, and 13 dual and other kinases, and compared the protein kinase expression profiles of hESCs and retinoic acid- treated hESCs. Significantly, up- and downregulated kinases in undifferentiated hESCs were confirmed by real-time PCR and western blotting. MAP3K3, ERBB2, FGFR4, and EPHB2 were predominantly upregulated, while CSF1R, TYRO3, SRC, and GSK3A were consistently downregulated in two hESC lines. Western blot analysis showed that the transcriptional levels of these kinases were consistent with the translational levels. The obstruction of upregulated kinases’ activities using specific inhibitors disturbed the undifferentiated status and induced the differentiation of hESCs. Our results support the dynamic expression of PTKs during hESC maintenance and suggest that specific PTKs that are consistently up- and downregulated play important roles in the maintenance of stemness and the direction of differentiation of hESCs. Reproduction (2008) 136 423–432 Introduction differentiation, and development (Hunter 1987). These proteins are among the key molecules that regulate Human embryonic stem cells (hESCs) have the ability signaling pathways, but only account for less than 10% of to self-renew and differentiate into a variety of cell the total cellular kinases (Hunter 1994). PTKs are types within the three embryonic germ layers classified into receptor tyrosine kinases (RTK) and (Thomson et al. 1998). To identify the key molecules cytoplasmic non-RTKs (NRTK). In humans, 90 PTKs involved in maintaining stemness of hESCs, transcrip- have been identified to date, comprising 58 receptor and tome characterization procedures, such as microarray 32 NRTKs (Robinson et al. 2000). Expression of most (Ramalho-Santos et al. 2002, Sato et al. 2003, Sperger PTKs may be tightly regulated to retain unique features of et al. 2003, Dvash et al. 2004), serial analysis of gene a specific cell type. However, limited information is expression (Richards et al.2004), and expressed available on the molecular behavior of PTKs in hESCs. sequence tags (ESTs; Brandenberger et al. 2004), have Here, we present the PTK-focused expression profiles been employed. Molecules participating in several of hESCs. The transcriptional profiles of protein kinases signaling pathways, including fibroblast growth factor (PKs) from hESCs are compared with those of retinoic (FGF), WNT, NODAL, PI3K/AKT, MAP kinase (MAPK)/ acid (RA)-treated hESCs by an RT-PCR-based cloning ERK, and NF-kB, may be involved in maintaining the technique using degenerate primers corresponding to the stemness of hESCs (Sato et al. 2003, Brandenberger highly conserved catalytic domains of PTKs. We et al. 2004, Armstrong et al. 2006, Rho et al. 2006). analyzed 1298 clones and identified 42 kinase genes in Generally, protein tyrosine kinases (PTKs) play pivotal two genetically independent hESC lines. Our PTK roles in cell proliferation, apoptosis, oncogenesis, profiling data should be valuable in understanding the q 2008 Society for Reproduction and Fertility DOI: 10.1530/REP-08-0080 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 10/02/2021 02:29:41AM via free access 424 M-Y Son and others molecular mechanisms underlying the stemness, devel- oping the novel biomarkers for the pluripotency, and identifying the potential regulators for differentiation in hESCs. Results Identification of the tyrosine kinase family in hESCs To elucidate the molecular mechanisms of PTKs regulat- ing the stemness of hESCs, differentiated cells were derived from the hESCs used as controls. To induce differentiation, hESCs were treated with RA for 5 days. As shown in Fig. 1A, alkaline phosphatase (AP) activity was considerably weak in RA-treated hESCs, compared with control hESCs. Moreover, transcriptional expression of stem cell marker genes, such as POU5F1 (OCT4) and NANOG, was remarkably reduced as assessed by real- time PCR (Fig. 1B). To assess the degree of diversity in differentiation capacity, we performed the real-time PCR analyses of the lineage markers for endoderm (amylase, albumin), mesoderm (GATA2), and ectoderm (NCAM). The RA-treated hESCs used in this study expressed diverse lineage markers (Supplementary Fig. 1, which can be viewed online at www.reproduction-online.org/supple- mental). Thus, the RA treatment appeared to result in the loss of stemness with no preference for differentiation towards a specific lineage, consistent with a previous report (Draper et al. 2002). To determine the transcrip- tional patterns of PTKs, degenerate RT-PCR was per- formed in both control and RA-treated hESCs. Degenerate primers were designed on the basis of conserved motifs within the catalytic domains of tyrosine kinase, DFG and DVW. The DFG motif is present in all PK types; while DVW is primarily encoded by tyrosine kinases, but a small subset of serine/threonine kinases also contain the motif (Robinson et al. 1996, Lin et al. 1998). In this study, the forward (F1, F2, and F3) and reverse primer (R) are based on DFG and DVW motifs respectively (Supple- mentary Table 1, which can be viewed online at www.reproduction-online.org/supplemental). DNA fragments of 150–170 bp were successfully amplified by degenerate PCR (Fig. 1C). The F1-R combination yielded the least PCR products in two hESC lines. Similar results have been observed with other tissues and cell lines (Lin et al. 1998, Wu et al. 2000). We performed two independent degenerate PCRs for each hESC lines and pooled the amplification products together respectively, Figure 1 Induction of hESC differentiation by retinoic acid (RA). (A) Alkaline phosphatase staining of hESC colonies. (B) Expression of hESC markers, POU5F1 and NANOG, by real-time PCR analysis. Statistical significance of data was assessed using Student’s t-test (*P!0.01). (C) Representative gel electrophoresis of amplified protein tyrosine kinase RT-PCR products (150–170 bp). RT-PCR using a combination of degenerate primers, F1, F2, F3, and R, was performed, as described in the Materials and Methods section. Reproduction (2008) 136 423–432 www.reproduction-online.org Downloaded from Bioscientifica.com at 10/02/2021 02:29:41AM via free access Expression profiles of tyrosine kinases in hESCs 425 for subsequent analysis. The pooled amplified products EPHA2, EPHB3, and MERTK) were commonly up- and were eluted, cloned, and sequenced. BLASTN analyses downregulated in both hESC lines respectively. The revealed that 90.0% (743/826 clones) and 94.3% (445/ cellular functions of the kinase genes identified in this 472 clones) genes from CHA-hES3 and SNUhES3 study are presented in Table 3. These PK profiles encoded human PKs respectively (Table 1). Thus, we obtained using gene frequency analyses provide presented a high fidelity procedure for identifying PKs in valuable information on the dynamic behavior of hESCs. Among the identified genes, there were 36 and 31 these proteins between undifferentiated and differen- non-redundant human kinase genes in CHA-hES3 and tiated hESCs. SNUhES3 respectively. Non-PKs were additionally detected, possibly as a result of PCR-related mispriming. Validation of selected tyrosine kinases by real-time PCR Murine PKs were observed at a low frequency in both and western blotting hESC types, which appear to be derived from feeder cells. Twenty-three human PTK genes and two serine/threonine To validate the differential expression of PK genes kinases were commonly expressed in the two hESCs between normal and differentiated hESCs, further experi- (Table 2). ments were performed on several selected genes. Prior to choosing the genes for ensuing analyses, we needed to set up selection criteria because the two different hESC lines Differential expression of PK genes in hESCs of different genetic backgrounds that we analyzed To identify the candidate genes associated with the showed some variations in gene expression patterns and stemness of hESCs, expression profiles of PK genes in possibly in preferential differentiation potential, as hESCs were compared with those of RA-treated hESCs by reported previously (Abeyta et al. 2004, Skottman et al. analyzing gene frequency (Table 2). In total, 386 clones 2005). We selected the genes using the following criteria: from CHA-hES3 cells and 357 clones from RA-treated (i) The expression of genes should be commonly up- CHA-hES3 cells were assessed. Out of the 36 PKs and downregulated in both hESC lines respectively. (ii) Among commonly up- and downregulated genes, identified in CHA-hES3 cells, 15 were transcriptionally the significant P values of the genes from the gene upregulated and 19 were downregulated, compared frequency analysis on PK expression profile should be with RA-treated CHA-hES3 cells. Two PK genes were lower than 0.01 in at least one of the two hESC lines.
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