FLT3 signaling augments macrophage production from human pluripotent stem cells.

Recent advances in cell engineering technologies enable immune cells to be utilized for adoptive cell transfer (ACT) immunotherapy against cancers. Macrophages have the potential to directly and indirectly exterminate cancers and are therefore an attractive option for therapies. To develop new ACT therapies using macrophages, a great number of macrophages are required. Human induced pluripotent stem cells (iPSCs) are expected to be a source of macrophages; therefore, a system to efficiently produce macrophages from human iPSCs is needed. Here, we demonstrated that human iPSCs were robustly differentiated into macrophages by enforced FMS-like tyrosine kinase-3 (FLT3) signaling via the introduction of exogenous FLT3 into iPSCs and the addition of its ligand FLT3L to the macrophage induction culture. These iPSC-derived macrophages were identical to those obtained by standard differentiation induction methods. Thus, our novel system enables the preparation of scalable macrophages from human iPSCs. We believe that this system will be useful to develop a novel ACT therapy using macrophages.

An Interferon-γ/FLT3 Axis Positively Regulates Hematopoietic Progenitor Cell Expansion from Human Pluripotent Stem Cells.

Since it became possible to differentiate human pluripotent stem cells (hPSCs) into hematopoietic cells in vitro, great efforts have been made to obtain highly potent hematopoietic stem/progenitor cells (HSPCs) from hPSCs. Immunophenotypical HSPCs can be obtained from hPSCs, but their repopulating potential in vivo is low. Here, we developed a novel hematopoietic differentiation method for human-induced pluripotent stem cells (hiPSCs) to determine why the existing hPSC differentiation systems are inadequate. hiPSC-derived CD45+CD34+ cells in our system were mostly CD38- immunophenotypical HSPCs. The vast majority of human CD45+CD34+ cells in umbilical cord blood, fetal liver, and bone marrow are CD38+ hematopoietic progenitor cells (HPCs); therefore, the poor production of CD38+ HPCs was indicative of a systematic problem. hiPSC-derived CD45+CD34+ cells did not express FLT3, a receptor tyrosine kinase. Exogenous FLT3 activity significantly enhanced the production of CD38+ HPCs from hiPSCs. Thus, poor production of CD38+ HPCs was due to a lack of FLT3 expression. Interferon-γ upregulated expression of FLT3 and increased the number of CD38+ HPCs among hiPSC-derived CD45+CD34+ cells. These results suggest that the poor production of CD38+ HPCs with hPSC differentiation systems is due to a lack of FLT3 expression, and that the addition of interferon-γ can solve this problem.

Nitric Oxide and a Conditioned Medium Affect the Hematopoietic Development in a Microfluidic Mouse Embryonic Stem Cell/OP9 Co-Cultivation System.

A microfluidic co-culture system, consisting of mouse embryonic stem cells (mESCs)/OP9 cells, was evaluated as a platform for studying hematopoietic differentiation mechanisms in vitro. mESC differentiation into blood cells was achieved in a microchannel that had the minimum size necessary to culture cells. The number of generated blood cells increased or decreased based on the nitric oxide (NO) donor or inhibitor used. Conditioned medium from OP9 cell cultures also promoted an increase in the number of blood cells. The number of generated blood cells under normal medium flow conditions was lower than that observed under the static condition. However, when using a conditioned medium, the number of generated blood cells under flow conditions was the same as that observed under the static condition. We conclude that secreted molecules from OP9 cells have a large influence on the differentiation of mESCs into blood cells. This is the first report of a microfluidic mESC/OP9 co-culture system that can contribute to highly detailed hematopoietic research studies by mimicking the cellular environment.

In vitro differentiation of mouse T cell-derived hybrid cells obtained through cell fusion with embryonic stem cells.

Nuclear reprogramming is an innovative advance in cell biology. An important research initiative in this field is cell fusion-mediated nuclear reprogramming, wherein the nuclei of somatic cells, such as thymocytes, are initialized through cell fusion with embryonic stem cells (ESCs). However, hybrid cells obtained through cell fusion between ESCs and thymocytes failed to contribute to the embryo proper when injected into blastocysts, which suggested that there are fundamental defects in such hybrid cells. Here, we performed side-by-side comparative analyses of the in vitro growth and differentiation capacities of ESCs and ESC-T hybrid cells. We found that the hybrid cells were larger and proliferated more slowly than the ESCs in 2i/LIF medium. Upon in vitro induction of differentiation, hybrid cells gave rise to cells of the three germ layers. Under culture conditions for hematopoietic differentiation, hybrid cells successively differentiated into lateral mesodermal cells, hemogenic endothelial cells, and various types of hematopoietic cells, including erythroid, myeloid, and lymphoid cells, although T cell maturation in the CD4/CD8 double-negative fraction was delayed. These results verified the multi-lineage differentiation capacity of ESC-T hybrid cells. The minimal contribution of hybrid cells to chimeric embryos may be due to their slow growth.

Inhibitory action of an ERK1/2 inhibitor on primitive endoderm cell differentiation from mouse embryonic stem cells.

A combination of extracellular signal-regulated kinase 1/2 (ERK1/2) and glycogen synthase kinase 3ß (GSK3ß) inhibitors, called 2i, is widely used for maintaining the pluripotency of mouse embryonic stem cells (ESCs) in vitro. Without 2i, a few mouse ESCs spontaneously gives rise to primitive endoderm (PrE) cells, whereas 2i completely blocks this PrE cell differentiation. However, the molecular mechanisms underlying the inhibitory action of 2i on PrE cell differentiation remain unclear. Robust PrE cell induction is achieved by enforced expression of the transcription factor Gata4. Here, we analyzed how 2i inhibits the PrE cell differentiation using mouse ESCs carrying an inducible Gata4 expression cassette. We found that 2i effectively inhibited the Gata4-induced PrE cell differentiation and the ERK1/2 inhibitor was responsible for this effect. We further revealed that the transcriptional activation ability of Gata4 was necessary for PrE cell induction and its disruption by the ERK1/2 inhibitor. The phosphorylation of Ser105, Ser266, and Ser411 of the Gata4 protein was not involved in the PrE cell induction. Overexpression of Klf4, an ERK1/2 substrate, inhibited the Gata4-mediated transcriptional activation. Our data indicated that ERK1/2 supported the PrE cell induction via the indirect transcriptional activation of Gata4.

Domain-specific biological functions of the transcription factor Gata2 on hematopoietic differentiation of mouse embryonic stem cells.

The generation of mouse hematopoietic stem cells from hemogenic endothelial cells (HECs) in the aorta/gonad/mesonephros region of developing embryos requires a zinc finger transcription factor Gata2. In the previous study, an enforced expression of Gata2 in vitro promoted the production of HECs from mesodermal cells differentiated from mouse embryonic stem cells (ESCs). Our research group has previously demonstrated that the enforced expression of Gata2 in ESC-derived HECs enhances erythroid and megakaryocyte differentiation and inhibits macrophage differentiation. However, the manner in which the multiple functions of Gata2 are regulated remains unclear. Mouse ESCs differentiate into various types of hematopoietic cells when cocultured with OP9 stromal cells (OP9 system). Using this system and the inducible gene cassette exchange system, which facilitates the establishment of ESCs carrying inducible transgenes under an identical gene expression regulatory unit, the domain-specific functions of Gata2 were systematically dissected in this study. We determined that the N-terminal (amino acid 1-110) region of Gata2 was an erythroid-inducing region, both the middle (amino acid 111-200) and C-terminal (amino acid 413-480) regions were megakaryocyte-inducing regions. Furthermore, the present data strongly suggest that intramolecular antagonistic interactions between each of these regions fine-tune the biological functions of Gata2.

Overexpression of Lhx2 suppresses proliferation of human T cell acute lymphoblastic leukemia-derived cells, partly by reducing LMO2 protein levels.

T cell acute lymphoblastic leukemia (T-ALL) is a malignant cancer with poor prognosis. The transcriptional co-factor LIM domain only 2 (LMO2) and its target gene HHEX are essential for self-renewal of T cell precursors and T-ALL etiology. LMO2 directly associates with LDB1 in a large DNA-containing nuclear complex and controls the transcription of T-ALL-related genes. Recently, we reported that overexpression of the LIM-homeodomain transcription factor, Lhx2, results in liberation of the Lmo2 protein from the Lmo2-Ldb1 complex, followed by ubiquitin proteasome mediated degradation. Here, we found that proliferation of five human T-ALL-derived cell lines, including CCRF-CEM, was significantly suppressed by retroviral overexpression of Lhx2. The majority of Lhx2-transduced CCRF-CEM cells arrested in G0 phase and subsequently underwent apoptosis. Expression of LMO2 protein as well as HHEX, ERG, HES1 and MYC genes was repressed in CCRF-CEM cells by transduction with Lhx2. Lhx2-mediated growth inhibition was partially rescued by simultaneous overexpression of Lmo2; however, both the C-terminal LIM domain and the homeodomain of Lhx2 were required for its growth-suppressive activity. These data indicate that Lhx2 is capable of blocking proliferation of T-ALL-derived cells by both LMO2-dependent and -independent means. We propose Lhx2 as a new molecular tool for anti-T-ALL drug development.

Efficient production of platelets from mouse embryonic stem cells by enforced expression of Gata2 in late hemogenic endothelial cells.

Platelets are essential for blood circulation and coagulation. Previous study indicated that overexpression of Gata2 in differentiated mouse embryonic stem cells (ESCs) resulted in robust induction of megakaryocytes (Mks). To evaluate platelet production capacity of the Gata2-induced ESC-derived Mks, we generated iGata2-ESC line carrying the doxycycline-inducible Gata2 expression cassette. When doxycycline was added to day 5 hemogenic endothelial cells in the in vitro differentiation culture of iGata2-ESCs, c-Kit(-)Tie2(-)CD41(+) Mks were predominantly generated. These iGata2-ESC-derived Mks efficiently produced CD41(+)CD42b(+)CD61(+) platelets and adhered to fibrinogen-coated glass coverslips in response to thrombin stimulation. Transmission electron microscopy analysis demonstrated that the iGata2-ESC-derived platelets were discoid-shaped with α-granules and an open canalicular system, but were larger than peripheral blood platelets in size. These results demonstrated that an enforced expression of Gata2 in late HECs of differentiated ESCs efficiently promotes megakaryopoiesis followed by platelet production. This study provides valuable information for ex vivo platelet production from human pluripotent stem cells in future.

GSK3ß inhibition activates the CDX/HOX pathway and promotes hemogenic endothelial progenitor differentiation from human pluripotent stem cells.

WNT/ß-CATENIN signaling promotes the hematopoietic/endothelial differentiation of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs). The transient addition of a GSK3ß inhibitor (GSKi) has been found to facilitate in vitro endothelial cell differentiation from hESCs/hiPSCs. Because hematopoietic and endothelial cells are derived from common progenitors (hemogenic endothelial progenitors [HEPs]), we examined the effect of transient GSKi treatment on hematopoietic cell differentiation from hiPSCs. We found that transient GSKi treatment at the start of hiPSC differentiation induction altered the gene expression profile of the cells. Multiple CDX/HOX genes, which are expressed in the posterior mesoderm of developing embryos, were significantly upregulated by GSKi treatment. Further, inclusion of the GSKi in a serum- and stroma-free culture with chemically defined medium efficiently induced HEPs, and the HEPs gave rise to various lineages of hematopoietic and endothelial cells. Therefore, transient WNT/ß-CATENIN signaling triggers activation of the CDX/HOX pathway, which in turn confers hemogenic posterior mesoderm identity to differentiating hiPSCs. These data enhance our understanding of human embryonic hematopoietic/endothelial cell development and provide a novel in vitro system for inducing the differentiation of hematopoietic cells from hiPSCs.

Efficient production of T cells from mouse pluripotent stem cells by controlled expression of Lhx2.

LIM-homeobox transcription factor Lhx2 induces ex vivo amplification of adult hematopoietic stem cells (HSCs) in mice. We previously showed that engraftable HSC-like cells are generated from mouse embryonic stem cells (ESCs) and induced pluripotent stem cells by enforced expression of Lhx2. However, when these HSC-like cells were transplanted into irradiated congenic mice, donor-derived T cells were barely detectable, whereas other lineages of hematopoietic cells were continuously produced. Here we investigated T-cell differentiation potential of the Lhx2-induced HSC-like cells using ESCs carrying doxycycline (dox)-inducible Lhx2 expression cassette. Dox-mediated over-expression of Lhx2 conferred a self-renewing activity to ESC-derived c-Kit(+) CD41(+) embryonic hematopoietic progenitor cells (HPCs), thereby converting them to HSC-like cells. When these HSC-like cells were transplanted into irradiated immunodeficient mice and they were supplied with a dox-containing water, CD4/8 double negative T cells were detected in their thymi. Once the Lhx2 expression was terminated, differentiation of CD4/8 double positive and single positive T cells was initiated in the thymi of transplanted mice and mature T cells were released in the peripheral blood. These results showed that engraftable HSC-like cells with full hematopoietic potential can be obtained from ESCs by the conditional expression of Lhx2.

LIM homeobox transcription factor Lhx2 inhibits skeletal muscle differentiation in part via transcriptional activation of Msx1 and Msx2.

LIM homeobox transcription factor Lhx2 is known to be an important regulator of neuronal development, homeostasis of hair follicle stem cells, and self-renewal of hematopoietic stem cells; however, its function in skeletal muscle development is poorly understood. In this study, we found that overexpression of Lhx2 completely inhibits the myotube-forming capacity of C2C12 cells and primary myoblasts. The muscle dedifferentiation factors Msx1 and Msx2 were strongly induced by the Lhx2 overexpression. Short interfering RNA-mediated knockdown of Lhx2 in the developing limb buds of mouse embryos resulted in a reduction in Msx1 and Msx2 mRNA levels, suggesting that they are downstream target genes of Lhx2. We found two Lhx2 consensus-binding sites in the -2097 to -1189 genomic region of Msx1 and two additional sites in the -536 to +73 genomic region of Msx2. These sequences were shown by luciferase reporter assay to be essential for Lhx2-mediated transcriptional activation. Moreover, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that Lhx2 is present in chromatin DNA complexes bound to the enhancer regions of the Msx1 and Msx2 genes. These data demonstrate that Msx1 and Msx2 are direct transcriptional targets of Lhx2. In addition, overexpression of Lhx2 significantly enhanced the mRNA levels of bone morphogenetic protein 4 and transforming growth factor beta family genes. We propose that Lhx2 is involved in the early stage of skeletal muscle development by inducing multiple differentiation inhibitory factors.

Endothelial cell-specific expression of roundabout 4 is regulated by differential DNA methylation of the proximal promoter.

OBJECTIVE: The molecular basis of endothelial cell (EC)-specific gene expression is poorly understood. Roundabout 4 (Robo4) is expressed exclusively in ECs. We previously reported that the 3-kb 5'-flanking region of the human Robo4 gene contains information for lineage-specific expression in the ECs. Our studies implicated a critical role for GA-binding protein and specificity protein 1 (SP1) in mediating overall expression levels. However, these transcription factors are also expressed in non-ECs. In this study, we tested the hypothesis that epigenetic mechanisms contribute to EC-specific Robo4 gene expression. METHODS AND RESULTS: Bisulfite sequencing analysis indicated that the proximal promoter of Robo4 is methylated in non-ECs but not in ECs. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine increased Robo4 gene expression in non-ECs but not in ECs. Proximal promoter methylation significantly decreased the promoter activity in ECs. Electrophoretic mobility shift assays showed that DNA methylation of the proximal promoter inhibited SP1 binding to the -42 SP1 site. In DNase hypersensitivity assays, chromatin condensation of the Robo4 promoter was observed in some but not all nonexpressing cell types. In Hprt (hypoxanthine phosphoribosyltransferase)-targeted mice, a 0.3-kb proximal promoter directed cell-type-specific expression in the endothelium. Bisulfite sequencing analysis using embryonic stem cell-derived mesodermal cells and ECs indicated that the EC-specific methylation pattern of the promoter is determined by demethylation during differentiation and that binding of GA-binding protein and SP1 to the proximal promoter is not essential for demethylation. CONCLUSIONS: The EC-specific DNA methylation pattern of the Robo4 proximal promoter is determined during cell differentiation and contributes to regulation of EC-specific Robo4 gene expression.

Molecular functions of the LIM-homeobox transcription factor Lhx2 in hematopoietic progenitor cells derived from mouse embryonic stem cells.

We previously demonstrated that hematopoietic stem cell (HSC)-like cells are robustly expanded from mouse embryonic stem cells (ESCs) by enforced expression of Lhx2, a LIM-homeobox domain (LIM-HD) transcription factor. In this study, we analyzed the functions of Lhx2 in that process using an ESC line harboring an inducible Lhx2 gene cassette. When ESCs are cultured on OP9 stromal cells, hematopoietic progenitor cells (HPCs) are differentiated and these HPCs are prone to undergo rapid differentiation into mature hematopoietic cells. Lhx2 inhibited differentiation of HPCs into mature hematopoietic cells and this effect would lead to accumulation of HSC-like cells. LIM-HD factors interact with LIM domain binding (Ldb) protein and this interaction abrogates binding of LIM-only (Lmo) protein to Ldb. We found that one of Lmo protein, Lmo2, was unstable due to dissociation of Lmo2 from Ldb1 in the presence of Lhx2. This effect of Lhx2 on the amount of Lmo2 contributed into accumulation of HSC-like cells, since enforced expression of Lmo2 into HSC-like cells inhibited their self-renewal. Expression of Gata3 and Tal1/Scl was increased in HSC-like cells and enforced expression of Lmo2 reduced expression of Gata3 but not Tal1/Scl. Enforced expression of Gata3 into HPCs inhibited mature hematopoietic cell differentiation, whereas Gata3-knockdown abrogated the Lhx2-mediated expansion of HPCs. We propose that multiple transcription factors/cofactors are involved in the Lhx2-mediated expansion of HSC-like cells from ESCs. Lhx2 appears to fine-tune the balance between self-renewal and differentiation of HSC-like cells.

Tumor-suppressive function of protein-tyrosine phosphatase non-receptor type 23 in testicular germ cell tumors is lost upon overexpression of miR142-3p microRNA.

Protein-tyrosine phosphatase non-receptor type 23 (PTPN23) is a candidate tumor suppressor involved in the tumorigenesis of various organs. However, its physiological role(s) and detailed expression profile(s) have not yet been elucidated. We investigated the function and regulation of PTPN23 in the formation of testicular germ cell tumors (TGCTs). Expression of PTPN23 in human TGCT cell lines was significantly lower than that in spermatogonial stem cells in mice. Overexpression of PTPN23 in NEC8, a human TGCT cell line, suppressed soft agar colony formation in vitro and tumor formation in nude mice in vivo. These data indicate that PTPN23 functions as a tumor suppressor in TGCTs. Multiple computational algorithms predicted that the 3' UTR of human PTPN23 is a target for miR-142-3p. A luciferase reporter assay confirmed that miR-142-3p bound directly to the 3' UTR of PTPN23. Introduction of pre-miR-142 in the PTPN23 transfectant of NEC8 led to suppressed expression of PTPN23 and increased soft agar colony formation. Quantitative RT-PCR data revealed a significantly higher expression of miR-142-3p in human seminomas compared with normal testes. No difference in mRNA expression between seminoma and non-seminoma samples was detected by in situ hybridization. Both quantitative RT-PCR and immunohistochemical analyses revealed that PTPN23 expression was significantly lower in TGCTs than in normal testicular tissues. Finally, a lack of PTPN23 protein expression in human TGCTs correlated with a relatively higher miR-142-3p expression. These data suggest that PTPN23 is a tumor suppressor and that repression of PTPN23 expression by miR-142-3p plays an important role in the pathogenesis of TGCTs.

Embryonic stem cell differentiation system for evaluating gene functions involved in physiological megakaryocytic differentiation.

Megakaryocytic differentiation is accompanied by marked morphological changes induced by endomitosis and proplatelet formation. Molecular mechanisms underlying this unique cell differentiation process have been investigated by gain/loss-of-function studies using leukemic cell lines. However, these cell lines cannot completely mimic physiological megakaryocytic differentiation, including the morphological changes, and sometimes lead to contradictory results between cell lines. The goal of this study was to establish a novel cell differentiation system that completely mimics physiological megakaryocytic differentiation for analyzing gene function. To that end, we used homologous recombination to prepare an embryonic stem (ES) cell line containing a GFP-transgene driven by the PF4 promoter at the Hprt locus. Differentiation of these cells resulted in megakaryocytes and proplatelets, suggesting physiological megakaryocytic differentiation. However, the number of GFP-expressing cells was low (1.7% GFP(+) cells among CD41(+) cells). Insertion of full-length or small core ß-globin insulators on either side of the transgene significantly increased the number of GFP-expressing cells (∼60% GFP(+) cells among CD41(+) cells), and GFP-expression was specifically observed in megakaryocytic cells. Similar results were obtained with other ES cells containing a GPIIb-GFP transgene. Altogether, we have succeeded in efficiently expressing exogenous genes specifically in differentiating megakaryocytes and in establishing a novel ES cell differentiation system for analyzing gene function involved in physiological megakaryocytic differentiation.

Multiple ETS family proteins regulate PF4 gene expression by binding to the same ETS binding site.

In previous studies on the mechanism underlying megakaryocyte-specific gene expression, several ETS motifs were found in each megakaryocyte-specific gene promoter. Although these studies suggested that several ETS family proteins regulate megakaryocyte-specific gene expression, only a few ETS family proteins have been identified. Platelet factor 4 (PF4) is a megakaryocyte-specific gene and its promoter includes multiple ETS motifs. We had previously shown that ETS-1 binds to an ETS motif in the PF4 promoter. However, the functions of the other ETS motifs are still unclear. The goal of this study was to investigate a novel functional ETS motif in the PF4 promoter and identify proteins binding to the motif. In electrophoretic mobility shift assays and a chromatin immunoprecipitation assay, FLI-1, ELF-1, and GABP bound to the -51 ETS site. Expression of FLI-1, ELF-1, and GABP activated the PF4 promoter in HepG2 cells. Mutation of a -51 ETS site attenuated FLI-1-, ELF-1-, and GABP-mediated transactivation of the promoter. siRNA analysis demonstrated that FLI-1, ELF-1, and GABP regulate PF4 gene expression in HEL cells. Among these three proteins, only FLI-1 synergistically activated the promoter with GATA-1. In addition, only FLI-1 expression was increased during megakaryocytic differentiation. Finally, the importance of the -51 ETS site for the activation of the PF4 promoter during physiological megakaryocytic differentiation was confirmed by a novel reporter gene assay using in vitro ES cell differentiation system. Together, these data suggest that FLI-1, ELF-1, and GABP regulate PF4 gene expression through the -51 ETS site in megakaryocytes and implicate the differentiation stage-specific regulation of PF4 gene expression by multiple ETS factors.

In vitro generation of HSC-like cells from murine ESCs/iPSCs by enforced expression of LIM-homeobox transcription factor Lhx2.

Identification of genes involved in in vitro differentiation induction of embryonic stem cells (ESCs) into hematopoietic stem cells (HSCs) has been challenged during last decade. To date, a homeobox transcription factor Hoxb4 has been only demonstrated to possess such an effect in mice. Here, we show that HSC-like cells were efficiently induced from mouse ESCs by enforced expression of Lhx2, a LIM-homeobox transcription factor. Transduction of Lhx2 into ESC-derived mesodermal cells resulted in robust differentiation of c-Kit(+)/Sca-1(+)/Lineage(-) (KSL) cells in vitro. The KSL cell induction frequency was superior to the case of Hoxb4. Furthermore, transplantation of Lhx2-transduced hematopoietic cells into lethally irradiated mice resulted in multilineage repopulation of hematopoietic cells over 4 months. Transduction of Lhx2 into induced pluripotent stem cells (iPSCs) was also effective in generating KSL cells in vitro, as well as HSC-like activities in vivo. These results demonstrate that ectopic expression of Lhx2 confers an in vivo engrafting capacity to ESC/iPSC-derived hematopoietic cells and in vivo behavior of iPSC-derived hematopoietic cells is almost identical to that of ESC-derived cells.

Disabled-2 is required for mesoderm differentiation of murine embryonic stem cells.

A variety of signaling networks are implicated in the control of mesoderm differentiation. Previous studies demonstrated that Disabled-2 (DAB2) is a multifunctional protein involved in growth factor signaling and embryonic development. In this study, we investigated DAB2 expression and function during in vitro mesoderm differentiation of murine embryonic stem cells (ESCs). We found that DAB2 was up-regulated when ESCs were co-cultured with OP9 stromal cells for mesoderm differentiation. DAB2 was also up-regulated when ESCs were induced for embryoid body formation. Expression of DAB2 short hairpin small interfering RNA (shDAB2) did not alter the puripotency of ESCs. However, shDAB2 disrupted ESCs cell-cell adhesion and affected embryoid body and colony formation that subsequently impeded mesoderm differentiation of ESCs. Immunofluorescent staining revealed that disorganization of beta-catenin and plakoglobin cellular distribution may account for the aberrant cell-cell adhesion in DAB2-deficient cells. Accordingly, DAB2 was identified as a plakoglobin-binding partner with the interaction mediated by the phosphotyrosine binding domain of DAB2 and the Asn-Pro-Asp-Tyr (NPDY) motif of plakoglobin. Molecular analysis and transcriptome profiling also revealed that DAB2 was involved in the regulation of insulin-like growth factor 2-mediated signaling and in the expression of p53, asparagine synthetase and glutathione peroxidase 2. Expression screening of 52 ESCs-related miRNAs further unveiled the interplay between DAB2 and the signaling networks associated with cell death, differentiation and development. This study thereby defines a role of DAB2 in fate determination of ESCs and suggests the presence of a DAB2-associated regulatory circuit in the control of mesoderm differentiation.

Novel role for the intraflagellar transport protein CMG-1 in regulating the transcription of cyclin-D2, E-cadherin and integrin-alpha family genes in mouse spermatocyte-derived cells.

Capillary morphogenesis gene (CMG)-1 is a mammalian homologue of the intraflagellar transport protein IFT-74/72 of Chlamydomonas. CMG-1 is abundantly expressed in immature stages of male germ-line cells of the adult mouse testis and is required for the expression of cyclin-D2 in GC-2, a mouse premeiotic spermatocyte-derived cell line. In this study, we show that the knockdown of CMG-1 in GC-2 cells leads to down-regulation of E-cadherin, integrin-alpha1, alpha2, alpha10, and alpha11 expression. The ability of the CMG-1-knockdown GC-2 cells to adhere to type-I collagen-coated plates was consequently impaired. Inducible expression of an siRNA-resistant CMG-1 cDNA in these cells rescued the expression of E-cadherin and the integrin-alpha family genes and partially restored adherence to type-I collagen. CMG-1 participates in the transcriptional regulation of cyclin-D2 via a genomic DNA region between -250 and -216 of the mouse cyclin-D2 gene. Closely related sequences were found in the enhancer/promotor regions of E-cadherin and the four integrin-alpha family genes. Based on these data, we propose that CMG-1 serves as a transcriptional regulator of proliferation and adhesion-associated genes in early stage male germ-line cells in the testis.

Increase of hematopoietic progenitor and suppression of endothelial gene expression by Runx1 expression during in vitro ES differentiation.

OBJECTIVE: Runx1 is essential for both the establishment of hematopoiesis during development and maintenance of adult hematopoiesis. To reveal the roles of Runx1, we examined how and when Runx1 functions during development of hematopoiesis, and revealed the genes controlled by Runx1. MATERIALS AND METHODS: A combined in vitro approach involving in vitro hematopoietic differentiation of embryonic stem cells and conditional gene expression of Runx1 was utilized for this study. Then we analyzed the effects of Runx1 on the differentiation and proliferation of hematopoietic cells and carried out DNA microarray analysis. RESULTS: Pulse expression of Runx1 prior to the emergence of hematopoietic cells caused immature hematopoietic cell increase but did not have any effects on the induction of hemogenic cells. During this process, the mRNA level of several endothelial cell-specific genes was downregulated. CONCLUSION: Runx1 expression play important roles on the proliferation of emerging immature hematopoietic progenitors or the transition process from endothelial to hematopoietic cells presumably by suppressing the genes related to endothelial phenotype.