Kenji Kitajima

GATA-2 and GATA-2/ER display opposing activities in the development and differentiation of blood progenitors

GATA‐2 is a zinc finger transcription factor essential for the development of hematopoiesis. While GATA‐2 is generally considered to play an important role in the biology of hematopoietic stem and progenitor cells, its function within these compartments is not well understood. Here we have employed both conditional expression of GATA‐2 and conditional activation of a GATA‐2/estrogen receptor (ER) chimera to examine the effect of enforced GATA‐2 expression in the development and differentiation of hematopoietic progenitors from murine embryonic stem cells. Consistent with the phenotype of GATA‐2 null animals, conditional expression of GATA‐2 from a tetracycline‐inducible promoter enhanced the production of hematopoietic progenitors. Conditional activation of a GATA‐2/ER chimera produced essentially opposite effects to those observed with conditional GATA‐2 expression. GATA‐2 and GATA‐2/ER differ in their binding activities and transcriptional interactions from other hematopoietic‐associated transcription factors such as c‐Myb and PU.1. While we have exploited these differences in activity to explore the transcriptional networks underlying hematopoietic cell fate determination, our results suggest that care should be taken in interpreting results obtained using only chimeric proteins.

In Vitro Differentiation of Mouse Embryonic Stem Cells to Hematopoietic Cells on an OP9 Stromal Cell Monolayer

Publisher Summary Although the in vitro differentiation capacity of the embryonic stem cells is rather limited, as compared to their in vivo totipotency, they can be readily induced to differentiate into hematopoietic lineage cells and constitute a powerful tool for investigating hematopoietic development and differentiation. Embryoid body (EB) formation is the conventional method most frequently used for in vitro differentiation from ES cells. The induction of differentiation into cells of the lympho-hematopoietic lineage and dopamine neurons can be induced efficiently by coculture with stromal cell lines. This chapter describes the method established for induction of differentiation, the OP9 system. The OP9 system is a unique method that uses the macrophage colony stimulating factor (M-CSF)-deficient stromal cell line OP9. The OP9 system can be used for hematopoietic development, hematopoietic differentiation, B cell formation, and megakaryocyte formation, and it can also be used for analysis of gene function using the tetracycline (Tet)-regulatory gene expression system. In addition, the merits and demerits of the OP9 system in comparison with the conventional EB formation method are described here.

Efficient Derivation of Embryonic Stem Cells by Inhibition of Glycogen Synthase Kinase‐3

Embryonic stem (ES) cells are derived from the inner cell mass (ICM) of blastocysts. The use of ES cells as a source of differentiated cells holds great promise for cell transplantation therapy. The efficiency of ES cell derivation is affected by genetic variation in mice; that is, some mouse strains, such as C57BL/6, are amenable to ES cell derivation, whereas others, such as BALB/c, are refractory. Developing an efficient method to establish ES cells from strains of various genetic backgrounds should be valuable for derivation of ES cells in various mammalian species, including human. Although it is well‐established that various signaling pathways, including phosphoinositide 3‐kinase (PI3K)/Akt and Wnt/β‐catenin, regulate the maintenance of ES cell pluripotency, little is known about the signaling pathways involved in the derivation of ES cells from ICMs. In this study, we demonstrated that inhibition of glycogen synthase kinase‐3 (GSK‐3), one of the crucial molecules in the regulation of the Wnt/β‐catenin, Hedgehog, and Notch signaling pathways, dramatically augmented ES cell derivation from both C57BL/6 and BALB/c mouse strains. In contrast, Akt signaling activation enhanced the growth of ICM but did not increase the efficiency of ES cell derivation. Our study establishes an efficient means for ES cell derivation by pharmacological inhibition of GSK‐3.

Cross talk between retinoic acid signaling and transcription factor GATA-2.

ABSTRACT All-trans-retinoic acid (RA) stimulates differentiation of normal hematopoietic progenitors and acute myeloid leukemia cells. GATA-2 is a transcription factor expressed in early progenitor cells and implicated in the control of the fate of hematopoietic stem cells and progenitor cells. We have investigated the possibility that the GATA and nuclear hormone receptor pathways are functionally linked through direct protein-protein interaction. Here we demonstrate that in human myeloid KG1 cells, RA receptor alpha (RARα), the major RAR expressed in hematopoietic cells, associates with GATA-2. This association is mediated by the zinc fingers of GATA-2 and the DNA-binding domain of RARα. As a consequence of this interaction, RARα is tethered to the DNA sites that are recognized and bound by GATA-2, and the transcriptional activity of GATA-2 becomes RA responsive. The RA responsiveness of GATA-dependent transcription is eliminated by expression of either a dominant negative form of RARα or a GATA-2 mutant that fails to interact with RARα. Overexpression of RXRα inhibits RARα binding to the GATA-2-DNA complex, thus resulting in attenuation of the effects of RARα on GATA-2 activity. In addition, inhibition by RA of GATA-2-dependent hematopoietic colony formation in an embryonic stem cell model of hematopoietic differentiation provided biological evidence for functional cross talk between RA and GATA-2-dependent pathways.

Alteration of cell adhesion and cell cycle properties of ES cells by an inducible dominant interfering Myb mutant

The Myb transcription factors, c-Myb, A-Myb, and B-Myb, regulate cell differentiation and/or proliferation. To investigate the role of B-Myb in embryogenesis, we introduced an inducible dominant interfering Myb protein (MERT) into embryonic stem (ES) cells, which express B-Myb as an exclusive member of Myb family. Disruption of normal B-Myb function by the conditional activation of MERT caused a drastic morphological alteration of ES cells and G1-S cell cycle arrest. The inhibition of B-Myb function by MERT dissociated tightly packed ES cell colonies into dispersed single cells that subsequently detached from the culture dish. Cell adhesion analyses revealed that suppression of B-Myb function reduced the adhesion with extracellular matrix proteins, such as laminin, collagen, and fibronectin. This reduction was presumably due to decreased cell surface expression of β1 integrin. Embryoid body formation was also severely retarded by the activation of MERT. This impairment was attributed to reduced expression of E-cadherin, which functions as a homophilic intercellular adhesion molecule. Simultaneously, blocking B-Myb function did not alter the expression of differentiation markers. Our data indicate that B-Myb plays important roles in regulating cell adhesion and cell cycle progression. These results are well consistent with the recent report on the phenotype of B-Myb null mice and show that the regulation of cell adhesion is an important B-Myb function that has not yet been assumed.

Tumor-suppressive Function of Protein-tyrosine Phosphatase Non-receptor Type 23 in Testicular Germ Cell Tumors Is Lost upon Overexpression of miR142–3p microRNA

Background: The PTPN23 gene is a candidate tumor suppressor involved in the tumorigenesis of various organs. Results: Expression of PTPN23 in testicular germ cell tumor cells is negatively regulated by miR-142-3p. Conclusion: A lack of PTPN23 protein expression in human TGCTs is inversely correlated with miR-142-3p expression. Significance: Loss of PTPN23 expression mediated by miR-142--3p may be a key event in the pathogenesis of TGCTs. 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.

Differentiation status dependent function of FOG‐1

The molecular interactions between transcription factors and cofactors play crucial roles in various biological processes, including haematopoiesis. FOG‐1 is a cognate cofactor of GATA‐1, and the FOG‐1/GATA‐1 complex is essential for the haematopoietic differentiation of erythroid cells and megakaryocytes. In order to elucidate the biological functions of FOG‐1 in the different contexts of cell differentiation, we analysed the effects of FOG‐1 expression on haematopoietic cell differentiation, using a combination of in vitro differentiation of mouse embryonic stem (ES) cells and conditional gene expression. FOG‐1 suppressed the proliferation of primitive and definitive erythroid cells in all stages of differentiation. However, FOG‐1 inhibited and enhanced megakaryopoiesis in the early and late differentiation stages, respectively, through different molecular mechanisms. In addition, FOG‐1 inhibited the proliferation of ES cells, the molecular mechanism of which differs from those of erythroid and megakaryocytic cells. These results suggest that FOG‐1 functions in a cell differentiation context‐dependent manner.

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. Stem Cells 2013;31:2680–2689

GSK3β inhibition activates the CDX/HOX pathway and promotes hemogenic endothelial progenitor differentiation from human pluripotent stem cells

WNT/β-CATENIN signaling promotes the hematopoietic/endothelial differentiation of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs). The transient addition of a GSK3β 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/β-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.

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.