Search

PDF file - 415K, In vivo toxicological assays including histological and serum biochemical analysis of mice administered with CVB3.

PDF file - 62K, In vitro large-scale screening of 28 enteroviruses for promising oncolytic virus candidates (MOI = 0.01, 0.1).

PDF file - 120K, In vitro and in vivo oncolytic effects of CVB3 against TC-1 mouse lung cancer cells.

PDF file - 43K, Inhibition of CAR expression of A549 cells by siRNA transduction.

PDF file - 96K, Remarkable CVB3 induced suppression of H1299 human tumor growth in mice.

PDF file - 107K

Highlights • Self-renewal of common marmoset embryonic stem cells (CM ESCs) is promoted by bFGF. • bFGF activates the PI3K-AKT pathway in CM ESCs on feeder cells. • bFGF and TGFβ in combination support culture of CM ESCs without feeder cells. • CM ESCs show phenotypes similar to those of human ESCs and mouse epiblast SCs., Common marmoset (CM) is widely recognized as a useful non-human primate for disease modeling and preclinical studies. Thus, embryonic stem cells (ESCs) derived from CM have potential as an appropriate cell source to test human regenerative medicine using human ESCs. CM ESCs have been established by us and other groups, and can be cultured in vitro. However, the growth factors and downstream pathways for self-renewal of CM ESCs are largely unknown. In this study, we found that basic fibroblast growth factor (bFGF) rather than leukemia inhibitory factor (LIF) promoted CM ESC self-renewal via the activation of phosphatidylinositol-3-kinase (PI3K)-protein kinase B (AKT) pathway on mouse embryonic fibroblast (MEF) feeders. Moreover, bFGF and transforming growth factor β (TGFβ) signaling pathways cooperatively maintained the undifferentiated state of CM ESCs under feeder-free condition. Our findings may improve the culture techniques of CM ESCs and facilitate their use as a preclinical experimental resource for human regenerative medicine.

We designed a phase I trial to investigate the safety, immune responses and clinical benefits of a five-peptide cancer vaccine in combination with chemotherapy. Study subjects were patients positive for HLA-A2402 with locally advanced, metastatic, and/or recurrent gastrointestinal, lung or cervical cancer. Eighteen patients including nine cases of colorectal cancer were treated with escalating doses of cyclophosphamide 4days before vaccination. Five HLA-A2402-restricted, tumor-associated antigen (TAA) epitope peptides from KOC1, TTK, URLC10, DEPDC1 and MPHOSPH1 were injected weekly for 4weeks. Treatment was well tolerated without any adverse events above grade 3. Analysis of peripheral blood lymphocytes showed that the number of regulatory T cells dropped from baseline after administration of cyclophosphamide and confirmed that TAA-specific T cell responses were associated significantly with longer overall survival. This phase I clinical trial demonstrated safety and promising immune responses that correlated with vaccine-induced T-cell responses. Therefore, this approach warrants further clinical studies.

Although oncolytic virotherapy is a promising anticancer therapy, antitumor efficacy is hampered by low tumor selectivity. To identify a potent and selective oncolytic virotherapy, we carried out large-scale two-step screening of 28 enteroviral strains and found that coxsackievirus B3 (CVB3) possessed specific oncolytic activity against nine human non–small cell lung cancer (NSCLC) cell lines. CVB3-mediated cytotoxicity was positively correlated with the expression of the viral receptors, coxsackievirus and adenovirus receptor, and decay-accelerating factor, on NSCLC cells. In vitro assays revealed that the CVB3 induced apoptosis and phosphoinositide 3-kinase/Akt and mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) survival signaling pathways, leading to cytotoxicity and regulation of CVB3 replication. Intratumoral injections of CVB3 elicited remarkable regression of preestablished NSCLC tumors in vivo. Furthermore, administrations of CVB3 into xenografts on the right flank resulted in significantly durable regression of uninjected xenografts on the left flank, where replication-competent CVB3 was detected. All treatments with CVB3 were well tolerated without treatment-related deaths. In addition, after CVB3 infection, NSCLC cells expressed abundant cell surface calreticulin and secreted ATP as well as translocated extranuclear high-mobility group box 1, which are required for immunogenic cell death. Moreover, intratumoral CVB3 administration markedly recruited natural killer cells and granulocytes, both of which contributed to the antitumor effects as shown by depletion assays, macrophages, and mature dendritic cells into tumor tissues. Together, our findings suggest that CVB3 is a potent and well-tolerated oncolytic agent with immunostimulatory properties active against both localized and metastatic NSCLC. Cancer Res; 72(10); 2609–21. ©2012 AACR.

We report the development of a new method to induce glioblastoma multiforme in adult immunocompetent mice by injecting Cre-loxP-controlled lentiviral vectors expressing oncogenes. Cell type- or region-specific expression of activated forms of the oncoproteins Harvey-Ras and AKT in fewer than 60 glial fibrillary acidic protein-positive cells in the hippocampus, subventricular zone or cortex of mice heterozygous for the gene encoding the tumor suppressor Tp53 were tested. Mice developed glioblastoma multiforme when transduced either in the subventricular zone or the hippocampus. However, tumors were rarely detected when the mice were transduced in the cortex. Transplantation of brain tumor cells into naive recipient mouse brain resulted in the formation of glioblastoma multiforme-like tumors, which contained CD133(+) cells, formed tumorspheres and could differentiate into neurons and astrocytes. We suggest that the use of Cre-loxP-controlled lentiviral vectors is a novel way to generate a mouse glioblastoma multiforme model in a region- and cell type-specific manner in adult mice.

Drosophila tumor suppressor WARTS (Wts) is an evolutionally conserved serine / threonine kinase and participates in a signaling complex that regulates both proliferation and apoptosis to ensure the proper size and shape of the fly. Human counterparts of this complex have been found to be frequently downregulated or mutated in cancers. WARTS, a human homolog of Wts, is also known as tumor suppressor and mitotic regulator, but its molecular implications in tumorigenesis are still obscure. Here, we show that WARTS binds via its C-terminus to the PDZ domain of a proapoptotic serine protease Omi / HtrA2. Depletion of WARTS inhibited Omi / HtrA2-mediated cell death, whereas overexpression of WARTS promoted this process. Furthermore, WARTS can enhance the protease activity of Omi / HtrA2 both in vivo and in vitro. Activation of Omi / HtrA2-mediated cell death is thus a potential mechanism for the tumor suppressive activity of WARTS.

Aurora-A is a centrosomal serine-threonine kinase that regulates mitosis. Over-expression of Aurora-A has been found in a wide range of tumors and has been implicated in oncogenic transformation. However, how Aurora-A over-expression contributes to promotion of carcinogenesis remains elusive. Immunohistochemical analysis of breast tumors revealed that over-expressed Aurora-A is not restricted to the centrosomes but is also found in the cytoplasm. This over-expressed Aurora-A appeared to be phosphorylated on Thr288, which is known to be required for its enzymatic activation. In analogy to Aurora-A's role in oocyte maturation and the early embryonic cell cycle, here we investigated whether ectopically over-expressed Aurora-A can similarly stimulate polyadenylation of mRNA in human somatic cultured cells by interacting with a human ortholog of cytoplasmic polyadenylation element binding protein, h-CPEB. In vitro experiments revealed that Aurora-A binds directly to, and phosphorylates, h-CPEB. We found that polyadenylation of mRNA tails of cyclin B1 and Cdk1 was synergistically stimulated when Aurora-A and h-CPEB were over-expressed, and they were further promoted in the presence of an Aurora-A activator Ajuba. Our results suggest a function of ectopically over-expressed Aurora-A that might be relevant for carcinogenesis.

Mitotic catastrophe is an important mechanism for the induction of cell death in cancer cells by antineoplastic agents that damage DNA. This process is facilitated by defects in the G1 and G2 checkpoints of the cell cycle that are apparent in most cancer cells and which allow the cells to enter mitosis with DNA damage. We have now characterized the dynamics of mitotic catastrophe induced by DNA-damaging agents in p53-deficient cancer cells. Cells that entered mitosis with DNA damage transiently arrested at metaphase for more than 10 h without segregation of chromosomes and subsequently died directly from metaphase. In those metaphase arrested precatastrophic cells, anaphase-promoting complex appeared to be inactivated and BubR1 was persistently localized at kinetochores, suggesting that spindle checkpoint is activated after the DNA damage. Furthermore, suppression of spindle checkpoint function by BubR1 or Mad2 RNA interference in the DNA damaged cells led to escape from catastrophic death and to subsequent abnormal mitosis. Dysfunction of the spindle checkpoint in p53-deficient cancer cells is thus likely a critical factor in resistance to DNA-damaging therapeutic agents.

Defects in chromosomes or mitotic spindles activate the spindle checkpoint, resulting in cell cycle arrest at prometaphase. The prolonged activation of spindle checkpoint generally leads to mitotic exit without segregation after a transient mitotic arrest and the consequent formation of tetraploid G(1) cells. These tetraploid cells are usually blocked to enter the subsequent S phase by the activation of p53/pRb pathway, which is referred to as the G(1) tetraploidy checkpoint. A human homologue of the Drosophila warts tumor suppressor, WARTS, is an evolutionarily conserved serine-threonine kinase and implicated in development of human tumors. We previously showed that WARTS plays a crucial role in controlling mitotic progression by forming a regulatory complex with zyxin, a regulator of actin filament assembly, on mitotic apparatus. However, when WARTS is activated during cell cycle and how the loss of WARTS function leads to tumorigenesis have not been elucidated. Here we show that WARTS is activated during mitosis in mammalian cells, and that overexpression of a kinase-inactive WARTS in Rat1 fibroblasts significantly induced mitotic delay. This delay resulted from prolonged activation of the spindle assembly checkpoint and was frequently followed by mitotic slippage and the development of tetraploidy. The resulting tetraploid cells then abrogated the G(1) tetraploidy checkpoint and entered S phase to achieve a DNA content of 8N. This impairment of G(1) tetraploidy checkpoint was caused as a consequence of failure to induce p53 expression by expressing a kinase-inactive WARTS. WARTS thus plays a critical role in maintenance of ploidy through its actions in both mitotic progression and the G(1) tetraploidy checkpoint.

Although pluripotent stem cells are well recognized as a potential source of cell therapy, it is still needed to improve efficiency to differentiate into target cell lineages. Tumor suppressor P53 regulates multiple signaling pathways triggered by diverse cellular stresses including DNA damages, oncogenic stimulations, and hypoxic stress, resulting in cell-cycle arrest, apoptosis, and senescence. P53 signaling is also important for double-stranded DNA breaks (DSBs) induced during physiologic events, i.e., rearrangement of antigen-specific receptors. It has been reported that P53-mediated DSB checkpoint contribute to normal murine T lymphopoiesis, especially at the double-negative (DN) stage which is defined as CD4-CD8- fraction in thymus and requires rearrangements of the T cell receptor (TCR) β locus and successful pre-TCR signaling. Here we defined the role of P53 on lymphopoiesis from human embryonic stem cells (ESCs).Firstly we modified P53 gene of human ESC H1 by utilizing of zinc finger nuclease targeting the P53 gene, kindly provided by Sangamo BioSciences. Sequencing analysis of the P53 knockout (KO) ES cells showed the successful deletion which induced the frame shift of the downstream sequence in both of its alleles. Western blot analysis of P53 phosphorylation status in P53 KO ESCs showed undetectable levels of phosphorylated or non-phosphorylated P53 proteins when cultured in the presence or absence of apoptotic signal triggered by mitomycin C (MMC). In consistent with this, P53 KO ESCs showed significant resistance to MMC-induced cell death. In addition, P53 KO ESCs lacked apoptotic stimulation-induced upregulation of P53 downstream target genes including P53 up-regulated modulator of apoptosis (PUMA).We then induced hematopoietic differentiation of P53 KO ESCs through embryoid body formation. Erythroid lineage cells developed from human ESCs were significantly suppressed in the absence of P53 signaling during embryoid body maturation. Pharmacological inhibition of P53 had the same effect as genetic disruption of P53 gene. CD34+ hematopoietic precursors were isolated from embryoid bodies originated from H1 and P53 KO ECSs, plated on OP9-DL1 stromal cells, and cultured in the presence of stem cell factor (SCF), FLT3 ligand, and interleukin (IL)-7. After 3-4 weeks of culture, CD45+CD3+ T lineage cells were induced from both H1 and P53 KO ECSs-derived CD34+ cells. Among these cells, most of the cells were in CD4+CD8+ double-positive (DP) stage, with increase in the yield of DP cells in the absence of P53 signaling (H1: 343 cells/1 × 10^6 input CD34+ cells; P53 KO: 2476 cells /1 × 10^6 input CD34+ cells; FigureFigure). Whether pharmacological inhibition of P53 had the similar effect on T lymphopoiesis as genetic disruption of P53 gene needs to be investigated furthermore.Our data indicate that P53 mediated signaling regulate in vitro early T lymphopoiesis from human pluripotent stem cells, especially at the transition from double negative into DP stage. These observations promoted us to perform high throughput transcriptome analysis including cDNA microarray analysis between early T lineage cells derived from H1 and P53 KO ESCs. Genes associated with the early T lymphopoiesis from human ESCs were identified and currently under further characterization.View Large Image | Download PowerPoint Slide

Background: Various mitotic events are controlled by Cdc2-cyclin B and other mitotic kinases. Aurora/Ipl1-related mitotic kinases were proved to play key roles in mitotic progression in diverse lower organisms. Aurora-A is a mammalian counterpart of aurora/Ipl1-related kinases and is thought to be a potential oncogene. However, the regulation of aurora-A activation and the commitment of aurora-A in the progression of G2-M phase are largely unknown in mammalian cells. Results: We demonstrated that aurora-A is activated depending on the activation of Cdc2-cyclin B in mammalian cells. Since Cdc2-cyclin B does not directly phosphorylate aurora-A, indirect pathways such as the inhibition of PP1 by Cdc2-cyclin B may act for the activation of aurora-A kinase. Microinjection of anti-aurora-A antibodies into HeLa cells at late G2 phase caused a significant delay in mitotic entry. Furthermore, aurora-A activation at G2-M transition was inhibited by DNA damage, and the over-expression of aurora-A induced the abrogation of the DNA damage-induced G2 checkpoint. Conclusions: Aurora-A is activated downstream of Cdc2-cyclin B and plays crucial roles in proper mitotic entry and G2 checkpoint control. Dysregulation of aurora-A induces abnormal G2-M transition in mammalian cells and may lead to chromosome instability, which results in the development and progression of malignant tumours.

Identification of physiological substrates for Cdc2/cyclin B is crucial for understanding the functional link between mitotic events and Cdc2/cyclin B activation. A human homologue of the Drosophila warts tumor suppressor, termed WARTS, is a serine/threonine kinase and a dynamic component of the mitotic apparatus. We have found that Cdc2/cyclin B forms a complex with a fraction of WARTS in the centrosome and phosphorylates the Ser613 site of WARTS during mitosis. Immunocytochemical analysis has shown that the S613-phosphorylated WARTS appears in the spindle poles at prometaphase and disappears at telophase. Our findings suggest that Cdc/cyclin B regulates functions of WARTS on the mitotic apparatus.

We identified a human homolog of Drosophila warts tumor suppressor gene, termed h-warts, which was mapped at chromosome 6q24-25.1. The h-warts protein has a serine/threonine kinase domain and is localized to centrosomes in interphase cells. However, it becomes localized to the mitotic apparatus, including spindle pole bodies, mitotic spindle, and midbody, in a highly dynamic manner during mitosis. Furthermore, h-warts is specifically phosphorylated in cells at mitotic phase, most likely by Cdc2 kinase. These findings suggest that h-warts functions as a component of the mitotic apparatus and is involved in proper progression of mitosis.

Recent generation of induced pluripotent stem (iPSCs) has made a significant impact on the field of human regenerative medicine. Prior to the clinical application of iPSCs, testing of their safety and usefulness must be carried out using reliable animal models of various diseases. In order to generate iPSCs from common marmoset (CM; Callithrix jacchus), one of the most useful experimental animals, we have lentivirally transduced reprogramming factors, including POU5F1 (also known as OCT3/4), SOX2, KLF4, and c-MYC into CM fibroblasts. The cells formed round colonies expressing embryonic stem cell markers, however, they showed an abnormal karyotype denoted as 46, X, del(4q), +mar, and formed human dysgerminoma-like tumors in SCID mice, indicating that the transduction of reprogramming factors caused unexpected tumorigenesis of CM cells. Moreover, CM dysgerminoma-like tumors were highly sensitive to DNA-damaging agents, irradiation, and fibroblast growth factor receptor inhibitor, and their growth was dependent on c-MYC expression. These results indicate that DNA-damaging agents, irradiation, fibroblast growth factor receptor inhibitor, and c-MYC-targeted therapies might represent effective treatment strategies for unexpected tumors in patients receiving iPSC-based therapy.

Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumor in humans. Here we show that gliomas can originate from differentiated cells in the central nervous system (CNS), including cortical neurons. Transduction by oncogenic lentiviral vectors of neural stem cells (NSCs), astrocytes, or even mature neurons in the brains of mice can give rise to malignant gliomas. All the tumors, irrespective of the site of lentiviral vector injection (the initiating population), shared common features of high expression of stem or progenitor markers and low expression of differentiation markers. Microarray analysis revealed that tumors of astrocytic and neuronal origin match the mesenchymal GBM subtype. We propose that most differentiated cells in the CNS upon defined genetic alterations undergo dedifferentiation to generate a NSC or progenitor state to initiate and maintain the tumor progression, as well as to give rise to the heterogeneous populations observed in malignant gliomas.

Glioblastoma (GBM) is the most aggressive form of glioma. Despite ceaseless efforts by researchers and physicians to find new therapeutic strategies, there have been no significant advances in the treatment of GBMs for several decades and most patients with GBM die within one and half years of diagnosis. Undoubtedly, one reason for this is the insufficient understanding of the initiation and progression of GBMs at the molecular level. However, recent information regarding the genetic and epigenetic alterations and the microRNAs that are aberrantly activated or inactivated in GBMs has helped elucidate the formation of GBM in more detail. Here, we describe recent advances in the understanding of the biology of GBMs.

Glioblastoma (GBM) is the most aggressive form of glioma. Despite ceaseless efforts by researchers and physicians to find new therapeutic strategies, there have been no significant advances in the treatment of GBMs for several decades and most patients with GBM die within one and half years of diagnosis. Undoubtedly, one reason for this is the insufficient understanding of the initiation and progression of GBMs at the molecular level. However, recent information regarding the genetic and epigenetic alterations and the microRNAs that are aberrantly activated or inactivated in GBMs has helped elucidate the formation of GBM in more detail. Here, we describe recent advances in the understanding of the biology of GBMs.

Hematopoietic stem cells (HSCs) have the capacity to self-renew as well as to differentiate into all blood cell types, and they can reconstitute hematopoiesis in recipients with bone marrow ablation. In addition, transplantation therapy using HSCs is widely performed for the treatment of various incurable diseases such as hematopoietic malignancies and congenital immunodeficiency disorders. For the safe and successful transplantation of HSCs, their genetic and epigenetic integrities need to be maintained properly. Therefore, understanding the molecular mechanisms that respond to various cellular stresses in HSCs is important. The tumor suppressor protein, p53, has been shown to play critical roles in maintenance of “cell integrity” under stress conditions by controlling its target genes that regulate cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. In this paper, we summarize recent reports that describe various biological functions of HSCs and discuss the roles of p53 associated with them.

Tumor suppressor P53 regulates multiple signaling pathways triggered by diverse cellular stresses including DNA damages, oncogenic stimulations, and hypoxic stress, resulting in cell-cycle arrest, apoptosis, and senescence. P53 signaling is also important for double-stranded DNA breaks (DSBs) induced during physiologic events, i.e., rearrangement of antigen-specific receptors. It has been reported that P53-mediated DSB checkpoint contribute to normal murine T lymphopoiesis, especially at the double-negative (DN) stage which is defined as CD4-CD8- fraction in thymus and requires rearrangements of the T cell receptor (TCR) b locus and successful pre-TCR signaling (Guidos CJ et al., Genes Dev, 1996; Jiang D et al., J Exp Med 2006; HaksMC et al., Immunity, 1999). Here we defined the role of P53 on hematopoietic development, especially lymphopoiesis, from human embryonic stem cells (ESCs). Firstly we modified P53 gene of human ESC H1 by utilizing genome editing tool of zinc finger nuclease (ZFN) targeting the 5th exon of the P53 gene, kindly provided by Sangamo BioSciences. Sequencing analysis of the P53 knockout (KO) ES cells showed the successful deletion at the 5th exon which induced the frame shift of the downstream sequence in both of its alleles. qRT-PCR showed no stable expression of full length P53 mRNA and western blot analysis of P53 phosphorylation status in P53 KO ESCs showed undetectable levels of phosphorylated or non-phosphorylated P53 proteins when cultured in the presence or absence of apoptotic signal triggered by mitomycin C (MMC). In consistent with this, P53 KO ESCs showed significant resistance to MMC-induced cell death. In addition, P53 KO ESCs lacked apoptotic stimulation-induced upregulation of P53 downstream target genes including P53 up-regulated modulator of apoptosis (PUMA). On the other hand induction of P53 target gene P21 was not observed both in H1 and P53 KO ESCs, as reported previously by other groups (Ginis I et al., Dev Biol, 2004; Barta T et al., Stem Cells, 2010; Garc'a CP et al., Stem Cell Res, 2014; World J et al., Stem Cells, 2014). We then induced hematopoietic differentiation of P53 KO ESCs through embryoid body formation. Erythroid lineage cells developed from human ESCs were significantly suppressed in the absence of P53 signaling during embryoid body maturation. Pharmacological inhibition of P53 had the same effect as genetic disruption of P53 gene. CD34+ hematopoietic precursors were isolated from embryoid bodies originated from H1 and P53 KO ECSs, plated on OP9-DL1 stromal cells, and cultured in the presence of stem cell factor (SCF), FLT3 ligand, and interleukin (IL)-7. After 3-4 weeks of culture, CD45+CD3+ T lineage cells were induced from both H1 and P53 KO ECSs-derived CD34+ cells. Among these cells, most of the cells were in CD4+CD8+ double-positive (DP) stage, with increase in the yield of DP cells in the absence of P53 signaling (H1: 343 cells/1 x 106 input CD34+ cells; P53 KO: 2476 cells/1 x 106 input CD34+ cells; Figure). Whether pharmacological inhibition of P53 had the similar effect on T lymphopoiesis as genetic disruption of P53 gene needs to be investigated furthermore. Our data indicate that P53 mediated signaling regulate in vitro early T lymphopoiesis from human pluripotent stem cells, especially at the transition from double negative into DP stage. These observations promoted us to perform high throughput transcriptome analysis including cDNA microarray analysis between early T lineage cells derived from H1 and P53 KO ESCs. Genes associated with the early T lymphopoiesis from human ESCs were identified and currently under further characterization. Disclosures No relevant conflicts of interest to declare.

Glioblastoma (GBM) is the most malignant brain tumor and is highly resistant to intensive combination therapies and anti-VEGF therapies. To assess the resistance mechanism to anti-VEGF therapy, we examined the vessels of GBMs in tumors that were induced by the transduction of p53 +/− heterozygous mice with lentiviral vectors containing oncogenes and the marker GFP in the hippocampus of GFAP-Cre recombinase (Cre) mice. We were surprised to observe GFP + vascular endothelial cells (ECs). Transplantation of mouse GBM cells revealed that the tumor-derived endothelial cells (TDECs) originated from tumor-initiating cells and did not result from cell fusion of ECs and tumor cells. An in vitro differentiation assay suggested that hypoxia is an important factor in the differentiation of tumor cells to ECs and is independent of VEGF. TDEC formation was not only resistant to an anti-VEGF receptor inhibitor in mouse GBMs but it led to an increase in their frequency. A xenograft model of human GBM spheres from clinical specimens and direct clinical samples from patients with GBM also showed the presence of TDECs. We suggest that the TDEC is an important player in the resistance to anti-VEGF therapy, and hence a potential target for GBM therapy.

The mechanism that regulates the terminal maturation of hematopoietic stem cells into erythroid cells is poorly understood. Therefore, identifying genes and surface markers that are restricted to specific stages of erythroid maturation will further our understanding of erythropoiesis. To identify genes expressed at discrete stages of erythroid development, we screened for genes that contributed to the proliferation and maturation of erythropoietin (EPO)-dependent UT-7/EPO cells. After transducing erythroid cells with a human fetal liver (FL)-derived lentiviral cDNA library and culturing the cells in the absence of EPO, we identified 17 candidate genes that supported erythroid colony formation. In addition, the mouse homologues of these candidate genes were identified and their expression was examined in E12.5 erythroid populations by qRT-PCR. The expression of candidate erythroid marker was also assessed at the protein level by immunohistochemistry and ELISA. Our study demonstrated that expression of the Apoa-1 gene, an apolipoprotein family member, significantly increased as hematopoietic stem cells differentiated into mature erythroid cells in the mouse FL. The Apoa-1 protein was more abundant in mature erythroid cells than hematopoietic stem and progenitor cells in the mouse FL by ELISA. Moreover, APOA-1 gene expression was detected in mature erythroid cells from human peripheral blood. We conclude that APOA-1 is a novel marker of the terminal erythroid maturation of hematopoietic stem cells in both mice and humans.

Hematopoietic stem/progenitor cells (HSPCs) derived from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have potential therapeutic applications in humans. To assess the safety and efficacy of ESC/iPSC-based therapies, reliable animal models are required prior to their clinical application. The common marmoset (CM) was recently found to be a useful nonhuman primate animal model for drug development and safety assessment. However, a method for the efficient hematopoietic differentiation of CM ESCs has not been established. In this study, we developed a novel and efficient method for differentiating CM ESCs into hematopoietic cells by transiently inhibiting the phosphoinositide 3-kinase (PI3K)-Protein kinase B (AKT) pathway, a critical pathway that maintains the undifferentiated state of CM ESCs during embryoid body (EB) formation. Compared with controls, transient inhibition of the P13K-AKT pathway resulted in a threefold increase in the proportion of enriched CD34⁺ cells (p0.001) and an increase in the number of hematopoietic colonies on day 8 of CM EB cultures. Moreover, number of blast colonies, number of hematopoietic progenitor cell populations of CD34⁺CD117⁺, CD34⁺CD45⁺, and CD43⁺CD45⁺ cells, and expression of hematopoietic genes were increased by transient inhibition of the PI3K-AKT pathway. We also demonstrated that the hematopoietic progenitor cell population was increased by inhibition of PI3K in a human system. Our novel and efficient ESC differentiation method might be useful for preclinical research on human hematopoietic disorders and may be efficiently translated to human ESC/iPSC-based regenerative medicine.

Various kinds of functional cells differentiated from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have recently been developed and expected for use in human regenerative medicine. However, the safety and efficacy of ESC/iPSC-based therapies must be carefully evaluated prior to clinical application, by using reliable animal models. The common marmoset (CM, Callithrix jacchus) is known to be a suitable preclinical model for clinical translation studies, and CM ESCs have already been established by us. Hematopoietic stem/progenitor cells (HSCs/HPCs) are one of very useful cells for transplantation therapy to treat various diseases including leukemia. However the shortage of their donors becomes a huge social problem and the expansion of HSCs/HPCs in vitro is known to be very difficult. We have previously demonstrated that CM ESCs showing indefinite self-renewal can be differentiated into hematopoietic lineages by the forced expression of hematopoietic transcription factor (TAL1/SCL). However the efficiency of their hematopoietic differentiation was quite low (less than 5%). Therefore the development of new method to promote hematopoietic differentiation of CM ESCs more efficiently is needed. To promote hematopoietic differentiation of CM ESCs, we focused on self-renewal pathway of CM ESCs and oxygen levels during EB formation. We have reported that self-renewal of CM ESCs is regulated by phosphoinositide 3-kinases (PI3Ks)-protein kinase B (AKT) pathway that is known to regulate cell cycle and cell proliferation as well as cell survival (Nii et al., 2014). On the other hand, the differentiation of mouse ESCs to hematopoietic precursors such as hemangioblasts, bipotential progenitors of endothelial and hematopoietic cells, can be enhanced by hypoxic condition (Ramírez-Bergeron et al., 2004). In addition, expansion of HSCs/HPCs can be increased by hypoxic condition in vitro (Danet et al., 2003). Thus, we hypothesized that the suppression of ESC self-renewal by the inhibition of PI3K-AKT pathway under hypoxic condition would improve hematopoietic differentiation of CM ESCs. To test our hypothesis that the inhibition of self-renewal pathway of CM ESCs could promote their hematopoietic differentiation, we treated CM ESCs with PI3K inhibitor (LY: LY294002) for the first 4 days of EB formation and examined the proportion of CD34+ cells by flow cytometric analysis, and found that the populations of CD34+ cells were significantly increased in the presence of LY. Moreover, the day8-EBs treated with LY gave rise to significantly more hematopoietic colonies than controls in colony forming unit (CFU) assay. These results indicated that hematopoietic differentiation was significantly enhanced by the inhibition of PI3K-AKT pathway in the process of EB formation. To further promote hematopoietic differentiation of CM ESCs, we conducted EB formation assay of CM ESCs and induced their differentiation into HPCs under hypoxic condition. We found that the hypoxic condition (5% O2) significantly increased the proportion of both CD34+ and CD34+/CD117+ cells in day8-EBs especially when PI3K-AKT pathway was inhibited by the LY treatment. These results were also obtained from human ESCs. In the present study, we demonstrated that transient treatment of PI3K inhibitor during EB formation under hypoxia condition promoted hematopoietic differentiation of human and CM ESCs, which might contribute to the development of the valuable experimental system using CM ESCs in order to test new strategies of human regenerative medicine. Disclosures No relevant conflicts of interest to declare.

Aurora-A is a centrosomal serine-threonine kinase that regulates mitosis. Over-expression of Aurora-A has been found in a wide range of tumors and has been implicated in oncogenic transformation. However, how Aurora-A over-expression contributes to promotion of carcinogenesis remains elusive. Immunohistochemical analysis of breast tumors revealed that over-expressed Aurora-A is not restricted to the centrosomes but is also found in the cytoplasm. This over-expressed Aurora-A appeared to be phosphorylated on Thr288, which is known to be required for its enzymatic activation. In analogy to Aurora-A's role in oocyte maturation and the early embryonic cell cycle, here we investigated whether ectopically over-expressed Aurora-A can similarly stimulate polyadenylation of mRNA in human somatic cultured cells by interacting with a human ortholog of cytoplasmic polyadenylation element binding protein, h-CPEB. In vitro experiments revealed that Aurora-A binds directly to, and phosphorylates, h-CPEB. We found that polyadenylation of mRNA tails of cyclin B1 and Cdk1 was synergistically stimulated when Aurora-A and h-CPEB were over-expressed, and they were further promoted in the presence of an Aurora-A activator Ajuba. Our results suggest a function of ectopically over-expressed Aurora-A that might be relevant for carcinogenesis.

The three human homologues of Aurora kinases (A, B and C) are essential for proper execution of various mitotic events and are important for maintaining genomic integrity. Aurora-A is mainly localized at spindle poles and the mitotic spindle during mitosis, where it regulates the functions of centrosomes, spindles and kinetochores required for proper mitotic progression. Recent studies have revealed that Aurora-A is frequently overexpressed in various cancer cells, indicating its involvement in tumorigenesis. What are the normal physiological roles of Aurora-A, how are these regulated and how might the enzyme function during tumorigenesis?

Aurora-A, a serine/threonine mitotic kinase, was reported to be overexpressed in various human cancers, and its overexpression induces aneuploidy, centrosome amplification and tumorigenic transformation in cultured human and rodent cells. However, the underlying mechanisms and pathological settings by which Aurora-A promotes tumorigenesis are largely unknown. Here, we created a transgenic mouse model to investigate the involvement of Aurora-A overexpression in the development of mammary glands and tumorigenesis using a Cre-loxP system. The conditional expression of Aurora-A resulted in significantly increased binucleated cell formation and apoptosis in the mammary epithelium. The surviving mammary epithelial cells composed hyperplastic areas after a short latency. Induction of Aurora-A overexpression in mouse embryonic fibroblasts prepared from the transgenic mice also led to aberrant mitosis and binucleated cell formation followed by apoptosis. The levels of p53 protein were remarkably increased in these Aurora-A-overexpressing cells, and the apoptosis was significantly suppressed by deletion of p53. Given that no malignant tumor formation was found in the Aurora-A-overexpressing mouse model after a long latency, additional factors, such as p53 inactivation, are required for the tumorigenesis of Aurora-A-overexpressing mammary epithelium. Our findings indicated that this mouse model is a useful system to study the physiological roles of Aurora-A and the genetic pathways of Aurora-A-induced carcinogenesis.

Calpains form a superfamily of Ca(2+)-dependent intracellular cysteine proteases with various isoforms. Two isoforms, micro- and m-calpains, are ubiquitously expressed and known as conventional calpains. It has been previously shown that the mammalian calpains are activated during mitosis by transient increases in cytosolic Ca(2+) concentration. However, it is still unknown whether the activation of calpains contributes to particular events in mitosis. With the use of RNA interference (RNAi), we investigated the roles of calpains in mitosis. Cells reduced the levels of m-calpain, but not mu-calpain, arrested at prometaphase and failed to align their chromosomes at the spindle equator. Specific peptidyl calpain inhibitors also induced aberrant mitosis with chromosome misalignment. Although both m-calpain RNAi and calpain inhibitors affected neither the separation of centrosomes nor the assembly of bipolar spindles, Mad2 was detected on the kinetochores of the misaligned chromosomes, indicating that the prometaphase arrest induced by calpain inhibition is due to activation of the spindle assembly checkpoint. Furthermore, when calpain activity was inhibited in cells having monopolar spindles, chromosomes were clustered adjacent to the centrosome, suggesting that calpain activity is involved in a polar ejection force for metaphase alignment of chromosomes. Based on these findings, we propose that activation of m-calpain during mitosis is required for cells to establish the chromosome alignment by regulating some molecules that generate polar ejection force.

The Aurora (Ipl1)-related kinases are universal regulators of mitosis. We now show that Aurora-A, in addition to Aurora-B, regulates kinetochore function in human cells. A two-hybrid screen identified the kinetochore component CENP-A as a protein that interacts with Aurora-A. Aurora-A phosphorylated CENP-A in vitro on Ser-7, a residue also known to be targeted by Aurora-B. Depletion of Aurora-A or Aurora-B by RNA interference revealed that CENP-A is initially phosphorylated in prophase in a manner dependent on Aurora-A, and that this reaction appears to be required for the subsequent Aurora-B-dependent phosphorylation of CENP-A as well as for the restriction of Aurora-B to the inner centromere in prometaphase. Prevention of CENP-A phosphorylation also led to chromosome misalignment during mitosis as a result of a defect in kinetochore attachment to microtubules. Our observations suggest that phosphorylation of CENP-A on Ser-7 by Aurora-A in prophase is essential for kinetochore function.

Aurora-A, a member of the Aurora/Ipl1-related kinase family, is overexpressed in various types of cancer and considered to play critical roles in tumorigenesis. To better understand the pathological effect of Aurora-A activation, it is first necessary to elucidate the physiological functions of Aurora-A. Here, we have investigated the roles of Aurora-A in mitotic progression with the small interfering RNA, antibody microinjection, and time lapse microscopy using human cells. We demonstrated that suppression of Aurora-A by small interfering RNA caused multiple events to fail in mitosis, such as incorrect separation of centriole pairs, misalignment of chromosomes on the metaphase plate, and incomplete cytokinesis. Antibody microinjection of Aurora-A into late G2 cells induced dose-dependent failure in separation of centriole pairs at prophase, indicating that Aurora-A is essential for proper separation of centriole pairs. When we injected anti-Aurora-A antibodies into prometaphase cells that had separated their centriole pairs, chromosomes were severely misaligned on the metaphase plate, indicating that Aurora-A is required for proper movement of chromosomes on the metaphase plate. Furthermore, inhibition of Aurora-A at metaphase by microinjected antibodies prevented cells from completing cytokinesis, suggesting that Aurora-A also has important functions in late mitosis. These results strongly suggest that Aurora-A is essential for many crucial events during mitosis and that the phosphorylation of a series of substrates by Aurora-A at different stages of mitosis may promote diverse critical events in mitosis to maintain chromosome integrity in human cells.

Various mitotic events are controlled by Cdc2-cyclin B and other mitotic kinases. Aurora/Ipl1-related mitotic kinases were proved to play key roles in mitotic progression in diverse lower organisms. Aurora-A is a mammalian counterpart of aurora/Ipl1-related kinases and is thought to be a potential oncogene. However, the regulation of aurora-A activation and the commitment of aurora-A in the progression of G2-M phase are largely unknown in mammalian cells.We demonstrated that aurora-A is activated depending on the activation of Cdc2-cyclin B in mammalian cells. Since Cdc2-cyclin B does not directly phosphorylate aurora-A, indirect pathways such as the inhibition of PP1 by Cdc2-cyclin B may act for the activation of aurora-A kinase. Microinjection of anti-aurora-A antibodies into HeLa cells at late G2 phase caused a significant delay in mitotic entry. Furthermore, aurora-A activation at G2-M transition was inhibited by DNA damage, and the over-expression of aurora-A induced the abrogation of the DNA damage-induced G2 checkpoint.Aurora-A is activated downstream of Cdc2-cyclin B and plays crucial roles in proper mitotic entry and G2 checkpoint control. Dysregulation of aurora-A induces abnormal G2-M transition in mammalian cells and may lead to chromosome instability, which results in the development and progression of malignant tumours.

The mitotic apparatus plays a pivotal role in dividing cells to ensure each daughter cell receives a full set of chromosomes and complement of cytoplasm during mitosis. A human homologue of the Drosophila warts tumor suppressor, h-warts/LATS1, is an evolutionarily conserved serine/threonine kinase and a dynamic component of the mitotic apparatus. We have identified an interaction of h-warts/LATS1 with zyxin, a regulator of actin filament assembly. Zyxin is a component of focal adhesion, however, during mitosis a fraction of cytoplasmic-dispersed zyxin becomes associated with h-warts/LATS1 on the mitotic apparatus. We found that zyxin is phosphorylated specifically during mitosis, most likely by Cdc2 kinase, and that the phosphorylation regulates association with h-warts/LATS1. Furthermore, microinjection of truncated h-warts/LATS1 protein, including the zyxin-binding portion, interfered with localization of zyxin to mitotic apparatus, and the duration of mitosis of these injected cells was significantly longer than that of control cells. These findings suggest that h-warts/LATS1 and zyxin play a crucial role in controlling mitosis progression by forming a regulatory complex on mitotic apparatus.

Hematopoietic stem cell (HSC) transplantation is the most successful cellular therapy for the malignant hematopoietic diseases such as leukemia, and early recovery of host’s hematopoiesis after HSC transplantation has eagerly been expected to reduce the regimen related toxicity for many years. For the establishment of the safer and more efficient cell source for allogeneic or autologous HSC transplantation, HSCs differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that show indefinite proliferation in an undifferentiated state and pluripotency, are considered to be one of the best candidates. Unfortunately, despite many recent efforts, the HSC-specific differentiation from ESCs and iPSCs remains poor [Kaufman, DS et al., 2001][Ledran MH et al., 2008]. In this study, we developed the new method to differentiate HSC from non-human primate ESC/iPSC. It has been reported that common marmoset (CM), a non-human primate, is a suitable experimental animal for the preclinical studies of HSC therapy [Hibino H et al., 1999]. We have been investigated the hematopoietic differentiation of CM ESCs into HSCs, and previously reported that the induction of CD34+ cells having a blood colony forming capacity from CM ESCs were promoted by lentiviral transduction of TAL1 cDNA [Kurita R et al., 2006]. However, those CD34+ cells did not have a bone marrow reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγnull) mice, suggesting that transduction of TAL1 gene was not sufficient to induce functional HSCs which have self-renewal capability and multipotency. Thus, we tried to find other hematopoietic genes being able to promote hematopoietic differetiation more efficiently than TAL1. We selected 6 genes (LYL1, HOXB4, BMI1, GATA2, c-MYB and LMO2) as candidates for factors that induce the differentiation of ESCs into HSCs, based on the previous study of hematopoietic differentiation from human and mouse ESCs. And CM ESCs (Cj11) lentivirally transduced with the respective candidate gene were processed for embryoid body (EB) formation to induce their differentiation into HSCs for 9 days. We found that lentiviral transduction of LYL1 (lymphoblastic leukemia 1), a basic helix-loop-helix transcription factor, in EBs markedly increased the proportion of cells positive for CD34 (approximately 20% of LYL1-transduced cells). RT-PCR showed that LYL1-transduced EBs expressed various hematopoietic genes, such as TAL1, RUNX1 and c-KIT. To examine whether these CD34+ cells have the ability to differentiate into hematopoietic cells in vitro, we performed colony-forming unit (CFU) assay, and found that CD34+ cells in LYL1-transduced EBs could form multi-lineage blood colonies. Furthermore the number of blood colonies originated from CD34+CD45+ cells in LYL1-transduced EBs was almost the same as that from CD34+CD45+ cells derived from CM bone marrow. These results suggested that enforced expression of LYL1 in CM ESCs promoted the emergence of HSCs by EB formation in vitro. The LYL1 was originally identified as the factor of a chromosomal translocation, resulting in T cell acute lymphoblastic leukemia [Mellentin JD et al., 1989]. The Lyl1-deficient mice display the reduction of B cells and impaired long-term hematopoietic reconstitution capacity [Capron C et al., 2006]. And, transduction of Lyl1 in mouse bone marrow cells induced the increase of HSCs and lymphocytes in vitro and in vivo [Lukov GL et al., 2011]. Therefore we hypothesized that LYL1 may play essential roles in bone marrow reconstitution by HSCs differentiated from CM ESCs. To examine this, we transplanted CD34+ cells derived from LYL1-transduced CM ESCs into bone marrow of sublethally irradiated NOG mice, and found that about 7% of CD45+ cells derived from CM ESCs were detected in peripheral blood (PB) of recipient mice at 8 weeks after transplant (n=4). Although CM CD45+ cells disappeared at 12 weeks after transplant, CD34+ cells (about 3%) were still found in bone marrow at the same time point. Given that TAL1-transduced EBs derived from CM ESCs could not reconstitute bone marrow of irradiated mice at all, LYL1 rather than TAL1 might be a more appropriate transcription factor that can give rise to CD34+ HSCs having the enhanced capability of bone marrow reconstitution from CM ESCs. We are planning to do in vivo study to prove this hypothesis in CM. Disclosures: No relevant conflicts of interest to declare.

Abstract 2311 The human regenerative medicine by the transplantation of the functional cells differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) have great potential of contributing to the treatments for various diseases, and thus have attracted huge public attention. However, the risk of unwelcome tumor formation originated from transplanted cells in recipients remains to be solved. Therefore the safety and efficacy of ESC/iPSC-based therapies should be carefully evaluated using reliable animal disease models before their clinical application. Among experimental animal models, common marmoset (CM, Callithrix jacchus), one of NEW WORLD monkeys, has recently been recognized as a useful non-human primate because they are small, easy to handle, highly reproductive and genetically very similar to humans. We have continuously investigated the characteristics of ESCs and iPSCs derived from CM. Understanding the self-renewal pathways in ESCs/iPSCs is crucial for the development of improved technology to culture and differentiate them into functional cells of potential therapeutic use. It has been reported that the maintenance of self-renewal in human or mouse ESCs/iPSCs require basic fibroblast growth factor (bFGF) or leukemia Inhibitory factor (LIF) respectively, however the growth factors required for the culture of CM ESCs/iPSCs have not been clearly determined. To clarify whether LIF or bFGF is more appropriate to maintain self-renewal of CM ESCs in culture, we examined the proliferation rate of CM40, a CM ESC line, maintained in the presence or absence of LIF or bFGF. CM ESCs were passaged at a ratio of 1:3 every 3 to 4 days. We found that the number of OCT3/4+cells was significantly increased by the addition of bFGF but not of LIF compared to control (w/o cytokines). Similar results were obtained when Cj11, another CM ESC line, was used. These results indicate that bFGF is essential for culturing CM ESCs, but LIF is dispensable. It has been reported that bFGF and its downstream PI3K-AKT and MEK-ERK pathways are important for maintenance of ESCs in human. Thus we examined whether PI3K-AKT and MEK-ERK pathway play crucial roles in the maintenance of self-renewal in CM ESCs. CM40 was cultured in the medium containing bFGF in the presence of PI3K inhibitor (LY294002) or MEK inhibitor (PD0325901). We found that the percentage and number of OCT3/4+ cells were gradually decreased in the presence of LY294002 (10 μM or 20 μM), suggesting that PI3K-AKT pathway is essential for the self-renewal of CM ESCs. Furthermore, the percentage and number of OCT3/4+cells were gradually decreased by addition of PD0325901 (1 μM or 5 μM) in the course of 4 passages, indicating that MEK-ERK pathway also plays a role in the self-renewal of CM ESCs. Next we examined if inhibition of self-renewal pathway such as PI3K-AKT or MEK-ERK promote hematopoietic differentiation in CM ESCs. One of methods for inducing hematopoietic cells from ESCs is embryoid body (EB) formation which is a conventional technique frequently used for in vitro differentiation of ESCs. Thus to induce hematopoietic differentiation, we performed EB formation assay by plating single-cell suspension of CM ESCs (3 × 105 cells) in StemLine II supplemented with 50 ng/ml BMP4 and 50 ng/ml VEGF with or without 10 μM LY294002 or 5 μM PD0325901 for 2 days. Then we removed half the medium and added fresh medium with the same final concentrations of BMP4, VEGF, LY294002 and PD0325901, plus 25 ng/ml SCF, 25 ng/ml TPO and 25 ng/ml FLT3L to expand the hematopoietic progenitors. We found that addition of LY294002 or PD0325901 increased the population of cells positive for CD34, a marker for hematopoietic stem/progenitor and endothelial cells, in day4-EBs. These CD34+cells showed hematopoietic differentiation potential proved by colony forming unit (CFU) assay Taken together, inhibition of self-renewal pathway such as PI3K-AKT or MEK-ERK in CM ESCs is thought to promote their hematopoietic differentiation by EB formation. Our findings might be useful to develop a better technology of the culture and hematopoietic differentiation of CM ESCs as well as to test efficacy and safety of ESC-derived hematopoietic cells using CM disease models for the future ESC/iPSC-based human regenerative medicine. Disclosures: No relevant conflicts of interest to declare.

Background Antitumor immunotherapy to target tumor antigens and evade immune tolerance is considered reasonable and promising. However, no combined immunotherapy has been established. We designed a phase I trial of cancer vaccine combined with chemotherapy to investigate safety as the primary endpoint, and immune responses and clinical benefits as secondary endpoints. Methods Patients positive for HLA-A2402 who showed locally advanced, metastatic, and/or recurrent gastrointestinal, lung or cervical cancer were evaluated in a phase I clinical trial of cancer vaccine and chemotherapy. Cyclophosphamide (CPM) was administered 4 days before vaccination to eliminate regulatory T cells (Treg). 18 patients were treated in cohorts of six patients, each with escalating CPM dose (150, 300 and 600 mg/m2). Five HLA-A2402-restricted tumor-associated antigen (TAA) epitope peptides from KOC1, TTK, URLC10, DEPDC1, and MPHOSPH1 were injected once a week for 4 weeks. Patients without gastrointestinal bleeding, pleural effusion or ascites also received low dose IL-2 every after vaccination. Results The treatment was tolerated well without any associated adverse events above grade 3. The study included primary disease with 9 cases of colorectal cancer, 3 cholangiocellular carcinoma, 3 lung cancer, and one each of esophageal, gastric and cervical cancer. Nine of 13 patients (69%) assessed by Elispot assay showed cytotoxic T lymphocytes (CTL) specific for TAA to more than one of five antigens after vaccination. Log-rank test among these demonstrated that CTL responses induced by vaccine contributed significantly to overall survival (MST: 9.2 vs 3.9 months, P = 0.003). In addition, a significantly broader antigenic repertoire was found in longer survivors (p = 0.033). Treg number dropped from baseline with dose just after CPM administration. This seems to reflect direct inhibition of CPM on Treg. Interestingly, reduced Treg correlated significantly with longer overall survival (P = 0.024), suggesting CPM augmented, vaccine-induced CTL responses through Treg reduction. Conclusion This phase I clinical study demonstrated promising survival associated with Treg inhibition, as well as CTL responses that warrants further clinical studies. Disclosure K. Yoshida: I am a research staff of the company, OncoTherapy Science Inc. providing the peptide vaccines used in this study. T. Tsunoda: I am the President and CEO of the company, OncoTherapy Science, Inc. providing the peptide vaccines used in this study. Y. Nakamura: I am one of the major stock holders of the company, OncoTherapy Science, Inc. K. Tani: Our department obtained the partial research funding in 2007 from the comapany, OncoTherapy Science, Inc. All other authors have declared no conflicts of interest.

Although oncolytic virotherapy is a promising anticancer therapy, antitumor efficacies are hampered by low tumor selectivity. To identify a potent and selective oncolytic virus, we performed large-scale two-step screening of 28 enteroviral strains and found that coxsackievirus B3 (CVB3) possessed specific oncolytic activity against nine human non-small cell lung cancer (NSCLC) cell lines. CVB3-mediated cytotoxicity was positively correlated with the expression of viral receptors, CAR and DAF, on NSCLC cells, and attenuated by loss of CAR expression by siRNA transfection on NSCLC cells. In vitro assays revealed that CVB3 induced apoptosis and PI3K/Akt and MEK/ERK survival signaling pathways, leading to cytotoxicity and regulation of CVB3 replication. Intratumoral injections of CVB3 elicited remarkable regression of pre-established tumors in vivo. Furthermore, administration of CVB3 into xenografts on the right flank resulted in significantly durable regression of uninjected xenografts on the left flank, where replication-competent CVB3 was detected. All treatments with CVB3 were well-tolerated without treatment-related deaths. In addition, intratumoral CVB3 administration markedly recruited NK cells and granulocytes, both of which expressed cytolytic degranulation marker CD107a and contributed to the antitumor effects as demonstrated by depletion assays, macrophages, and mature dendritic cells (DCs) into tumor tissues. Furthermore, we found that CVB3 infection induced abundant calreticulin (CRT) exposure, known as a major checkpoint of immunogenic cell death, on the cell surface of NSCLC cells, as well as extranuclear high mobility group box 1 (HMGB1) translocation from the nuclei to the cytosol in NSCLC cells. Altogether, our findings suggest that CVB3 is a potent and well-tolerated oncolytic agent with immunostimulatory properties active against both localized and metastatic NSCLC and may yield a unique and more effective antitumor activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-369. doi:1538-7445.AM2012-LB-369

Malignant gliomas remain one of the most aggressive tumors of the central nervous system. Different interpretations have been proposed about the nature of the neural cell type that is targeted by transformation and results in tumorigenesis. The identification of the cellular origin of gliomas presents an opportunity for improving our understanding of this type of cancer. We recently developed a mouse glioma model using Cre-inducible lentiviral vectors that faithfully recapitulate the pathophysiology of human glioblastoma multiforme (GBM). Injection of a single lentiviral vector expressing H-RasV12-shp53 in the cortex of Synapsin I-Cre (SynI-Cre) mice led to tumor formation after 6-8 weeks of injection. SynI-Cre mice primarily and specifically express the Cre recombinase transgene in differentiated neurons. Tumors were also obtained when CamK2a-Cre mice, also expressing Cre specifically in neurons, were injected with the same virus. We also aim to target astrocytes by injecting the virus either in the cortex or the stratium of GFAP-Cre mice, and tumors presenting the classical characteristics of GBM developed, suggesting that astrocytes can also serve as the glioma cell of origin. We made sections of these brains at various time points following injection of the lentiviral vector and, using high resolution large-scale mosaic imaging, we examined the expression of different markers. Notably, tumors start out to be GFAP+, but by eight weeks are largely Nestin+ and Sox2+. We believe that either astrocytes or neurons can be reprogrammed by the introduction of oncogenes/tumor suppressors to form cancer iPS-like stem cells that can give rise to all the cell lineages and heterogeneity observed in GBM. To further explore this hypothesis, we transduced primary cortical astrocytes and neurons obtained from GFAP-Cre and SynI-Cre mice, respectively. The transduced cells when switched to neural stem cell (NSC) media displayed: i) neurosphere-like structures, ii) robust NSC marker expression (Nestin and Sox2), iii) self-renewal capacity, iv) strong tumor initiating capacity, v) expression of reprogramming factors, and vi) capacity to differentiate into different lineages. Finally, we assessed the human relevance of our findings by comparing the transcriptome profile of tumors in our model with the molecular signatures of human glioma samples. The data from the molecular signatures and histopathology of tumors originating in the cortex where the primary target is astrocytes in the GFAP cre mice and Neurons in the Synapsin Cre mice show both are mesenchymal GBM subtype. We obtained Neural subtype mostly when the virus was injected in the hippocampus of Nestin-Cre mice (aim to target NSC/progenitor cells). Together, our results suggest that any cell in the brain, whether terminally differentiated or neural stem cell, can be the glioma cell of origin and the biological behavior of these tumors depends on the dysregulation of specific genetic elements. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-191. doi:1538-7445.AM2012-LB-191

Recent generation of induced pluripotent stem cells (iPSCs) has made a great impact on the field of regenerative medicine. Before the clinical application of iPSCs, preclinical testing of the safety and usefulness of them must be performed using reliable animal models of various diseases. The Common Marmoset (CM, callithrix jacchus), has recently been considered one of the most useful experimental animals for medical research because of close phylogenetic similarity to humans. To generate iPSCs from CM cells, we transduced reprogramming factors including Oct3/4, Klf4, Sox2 and c-Myc into CM fibroblasts. We found that several round-shaped colonies similar to embryonic stem cells (ESCs) were formed. These cells expressed ES markers such as SSEA4 and TRA-1-60, and showed alkaline phosphatase activity. Moreover RT-PCR revealed that the cells were also positive for other ES markers such as LIN28, SALL1 and DPPA4, suggesting that these cells have been reprogrammed. Next we performed karyotype analysis, and found that these cells contained 46, X, del(4q),+mar. Although parental CM fibroblasts had abnormal chromosome called mar, majority of chromosomes were intact, indicating that chromosome 4q was lost in the process of reprogramming. We called these reprogrammed cells abnormally reprogrammed cells (ARCs). We next injected one million cells of ARCs into the testis of SCID mice. Tumors were formed approximately 6 weeks after the injection, and HE staining showed that the tumor was composed of nests and sheets of uniform round or polygonal cells with abundant, clear to faintly eosinophilic cytoplasm with well-demarcated cytoplasmic borders. In addition, immunohistochemical analysis demonstrated that the tumor cells were focally and weakly immunopositive for vimentin, and immunonegative for cytokeratin, S100, Desmin, α-smooth muscle actin and Neuron-specific enolase, and the tumor tissue appeared to be c-kit+/CD30-/CD45- by quantitative RT-PCR, all of which were characteristics of human dysgerminoma. Thus we called the tumor common marmoset dysgerminoma (CM DGs). Next we tried to culture CM DGs in vitro, and found that they could proliferate in a semifloating condition. Western blotting revealed that these tumor cells showed continuous expression of exogenous reprogramming factors. We also examined the sensitivity of CM DGs to irradiation and DNA damage agents such as mitomicin C and cisplatin, and found that they were highly sensitive to all the DNA damage treatments tested compared to the parental cells. We also found that knockdown of Sox2 or c-Myc inhibited the proliferation of CM DGs due to the increase of cell death. These results indicate that irradiation, chemotherapy using DNA damaging agents or targeting reprogramming factors such as Sox2 and c-Myc might be effective for unexpected tumors accidentally found in patients treated with the functional cells derived from iPSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2564. doi:1538-7445.AM2012-2564

Abstract 2206 Recently various kinds of functional cells differentiated from embryonic stem cells and induced pluripotent stem cells (ESCs/iPSCs) are expected to be utilized for cell therapy in clinical medicine. Among the transplantable functional cells differentiated from ESCs/iPSCs, endothelial progenitor cells (EPCs) and hematopoietic stem cells (HSCs) are considered to be strong candidate cells for regenerative medicine to cure various diseases such as ischemic disease and hematopoietic malignancy. Although the transplantation of EPCs and HSCs derived from human bone marrow, mobilized peripheral blood, and umbilical cord blood is commonly conducted in clinical settings, their availability for clinical use has often been hampered by both the lack of HLA compatible donor and the insufficient number of the cells. As the in vitro expansion of EPCs and HSCs derived from above sources is very difficult using current technology, it may be easier to expand EPCs and HSCs derived from ESCs/iPSCs in vitro. Hemangioblasts have the ability to differentiate into both EPCs and HSCs. Thus the technology to differentiate hemangioblast from ESCs/iPSCs that possess indefinite proliferative capacity is strongly expected. Differentiation of ESCs/iPSCs to hemangioblasts is best exemplified in recent studies that have used two step procedures to enhance hemangioblast differentiation with embryoid body (EB) formation and blast colony forming cell (BL-CFC) assay (Lu SJ et al., Nat Methods 4: 501–509, 2007). However the efficiency of hemangioblast differentiation by this method was quite low (approximately 0.35 ± 0.01%). PI3K-AKT pathway is well known to regulate various cell functions. In ESCs, PI3K-AKT pathway plays an important role in maintaining the undifferentiated state (Armstrong L et al., Hum Mol Genet 15: 1894–1913, 2006), suggesting that inhibition of PI3K may promote the differentiation of ESCs/iPSCs. Previously, we demonstrated that common marmosets (CM) are suitable laboratory animal models for preclinical studies of hematopoietic stem cell therapies (Hibino H et al., blood 1: 2839–2848, 1999). To develop the method for the more efficient generation of hemangioblasts from ESCs/iPSCs, we promoted the hemangioblast differentiation by the inhibition of PI3K-AKT pathway with the inhibitor, LY294002. CM-ESCs (Cj11 and CM40) were differentiated by EB formation in the presence of LY294002 for 4 days, and the EBs were trypsinized, and the dissociated individual cells were processed for BL-CFC assay in the methylcellulose medium containing various cytokines without LY294002 for 7 days. The number of blast colonies found in the BL-CFC assay significantly increased (approximately 10-fold; 3.5 ± 0.3%, p < 0.001) with the treatment of LY294002 during EB formation compared with control. The colonies formed in the BL-CFC assay were homogeneous and looked like a tuft of grapes which is one of hemangioblast characters, and expressed hemagioblast markers (FLK1+, VE-cadherin+, CD31+ and CD45−), suggesting that the inhibition of PI3K during EB formation promoted the generation of hemangioblast-like cells from CM-ESCs. To determine endothelial potential of these hemangioblast-like cells derived from CM-ESCs, we grew them as adherent layers on gelatin-coated plates in EGM-2 medium. The adherent cells derived from hemangioblast-like cells expressed endothelial cell markers (CD31 and vWF). Next, we also examined hematopoietic potential of hemangioblast-like cells by colony forming unit (CFU) assay. Unexpectedly no colonies were formed regardless of whether LY294002 was added or not during EB formation, indicating that hemangioblast-like cells derived from CM-ESC might be endothelial progenitors rather than hemangioblasts. Our novel technology is 10-fold more efficient in inducing endothelial differentiation from ESCs than previously reported methods. It should be emphasized that these endothelial progenitors are morphologically homogenous and expressed endothelial cell markers in a defined adherent cell culture condition, suggesting that our novel technology will be useful for an efficient generation of homogeneous EPCs for future regenerative medicine against ischemic diseases. Disclosures: No relevant conflicts of interest to declare.

Abstract 3122 Pioneering work by Yamanaka's group has demonstrated that mammalian somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by the ectopic expression of Oct3/4 (also called Pou5f1), Klf4, Sox2 with or without c-Myc (OKSM) [Takahashi, K., and Yamanaka, S. (2006). Cell 126, 663–676, Takahashi et al. (2007). Cell 131, 861–872, Nakagawa et al., (2008) Nat Biotechnol 26, 739–740.]. The iPSCs are similar to embryonic stem cells (ESCs) in their morphology, and their abilities of differentiation to three germ layers in vitro and in vivo. Comparing with ESCs, iPSCs have less concerns regarding the ethically controversial limitation or immune rejection. And iPSCs have great potential of clinical application for personalized stem cell-based therapies. However, the efficiency of iPSC generation is quite low (0.01∼1%), which is considered to be an impediment of the clinical utility of iPSCs. Although the molecular mechanisms of reprogramming process during the generation of iPSCs have not been fully understood, senescence and apoptosis induced by the ectopic expression of OKSM have been considered to be the major road block in the process of iPSC generation. Therefore the new strategy of iPSC generation which can alleviate such hurdles during the reprogramming process should be developed. The signaling pathway mediated by the activation of phosphoinositide 3-kinase (PI3K) has been shown to play crucial roles for various aspects of cell biology including cell survival, proliferation, migration, metabolism and vesicular trafficking in variety of cell types. In addition previous studies have shown that PI3K pathway is an important regulator of ES cell cycle by promoting G0 to G1 transition and ES cell proliferation. In this study we hypothesized that activation of PI3K pathway might facilitate the reprogramming process for iPSC generation. To test this hypothesis we first induced the expression of OKSM in mouse embryonic fibroblasts (MEFs) lacking Pten which is a negative regulator of PI3K pathway. About 7 days after the retroviral transduction of OKSM into Pten−/− MEFs, ES like round-shaped colonies were found (Figure 1). Twenty-two days after the retroviral transduction, the efficiency of iPSC generation was examined by the number of colonies having alkaline phosphatase (AP) activity. We found that significantly higher number of AP+ colonies were formed from Pten−/− MEFs (74 ± 15) compared to Pten+/− or wild-type MEFs (17 ± 5 and 16 ± 2, respectively). Moreover the number of SSEA1+ colonies induced by the expression of OKSM significantly increased from Pten−/− MEFs (103 ± 2) in comparison to Pten+/− or wild-type MEFs (40 ± 9 and 21 ± 9, respectively). Similar results were obtained when OKS were transduced without c-Myc. Next we tested the hematopoietic differentiation in vitro with the reprogrammed MEFs by using embryoid bodies (EBs) formation assay. After the following colony forming unit (CFU) assay, we observed the higher percentage of hematopoietic cells from reprogrammed Pten−/− MEF group comparing with controls, indicating that reprogrammed Pten−/− MEFs had the higher potential to differentiate into hematopoietic cells. Continuous activation of PI3K pathway may cause the transformation of the cells; therefore to efficiently and safely generate iPSCs transient activation of the pathway combined with the transduction of OKSM would be desirable. To establish transient activation of the PI3K pathway we used Pten inhibitor, Dipotassium Bisperoxo(5-hydroxypyridine-2-carboxyl) oxovanadate (V) (bpV), during the generation of iPSCs. The efficiency of AP+ cell-generation from MEFs by transducing Yamanaka factors with bpV was approximately 9%. This was 2 times higher than that by transducing Yamanaka factors without bpV in our hand. These AP+ cells showed a normal karyotype and an ability of differentiation into three germ layers in immunodeficient mice. Therefore ectopic expression of OKSM in the presence of Pten inhibitor can establish the efficient generation of iPSCs. Overall, transient activation of PI3K pathway by Pten inhibition offers a highly efficient method generating iPSCs, and might contribute to the establishment of human regenerative medicine with iPSCs. Disclosures: No relevant conflicts of interest to declare.

Abstract 2348 Since the successful establishment of human embryonic stem cells (ESCs) in 1998, transplantation of functional cells differentiated from ESCs to the specific impaired organ has been expected to cure its defective function [Thomson JA et al., Science 282:1145–47, 1998]. For the establishment of the regenerative medicine using ESCs, the preclinical studies utilizing animal model systems including non-human primates are essential. We have demonstrated that non-human primate of common marmoset (CM) is a suitable experimental animal for the preclinical studies of hematopoietic stem cells (HSCs) therapy [Hibino H et al., Blood 93:2839–48, 1999]. Since then we have continuously investigated the in vitro and in vivo differentiation of CM ESCs to hematopoietic cells by the exogenous hematopoietic gene transfer. In earlier study, we showed that the induction of CD34+ cells having a blood colony forming capacity from CM ESCs is promoted by lentiviral transduction of TAL1 cDNA [Kurita R et al., Stem Cells 24:2014-22,2006]. However those CD34+ cells did not have a bone marrow reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγnull) mice, suggesting that transduction of TAL1 gene is not enough to induce functional HSCs which have self-renewal capability and multipotency. Thus we tried to find other hematopoietic genes being able to promote hematopoietic differetiation more efficiently than TAL1. We selected 6 genes (LYL1, HOXB4, BMI1, GATA2, c-MYB and LMO2) as candidates for factors that induce the differentiation from ESCs to HSCs, based on the comparison of gene expression level between human ESCs and HSCs by Digital Differential Display from the Uni-Gene database at the NCBI web site (http://www.ncbi.nlm.nih.gov/UniGene/). Then, we transduced the respective candidate gene in CM ESCs (Cj11), and performed embryoid body (EB) formation assay to induce their differentiation to HSCs for 9 days. We found that lentiviral transduction of LYL1, a basic helix-loop-helix transcription factor, in EBs derived from Cj11, one of CM ESC lines, markedly increased the number of cells positive for CD34, a marker for hematopoietic stem/progenitors. The lymphoblastic leukemia 1 (LYL1) was originally identified as the factor of a chromosomal translocation, resulting in T cell acute lymphoblastic leukemia [Mellentin JD et al., Cell 58:77-83.1989]. These class II bHLH transcription factors regulate gene expression by binding to target gene sequences as heterodimers with E-proteins, in association with Gata1 and Gata2 [Goldfarb AN et al., Blood 85:465-71.1995][Hofmann T et al., Oncogene 13:617-24.1996][Hsu HL et al., Proc Natl Acad Sci USA 91:5947-51.1994]. The Lyl1-deficient mice display the reduction of B cells and impaired long-term hematopoietic reconstitution capacity [Capron C et al., Blood 107:4678-4686. 2006]. And, overexpression of Lyl1 in mouse bone marrow cells induced the increase of HSCs, HPCs and lymphocytes in vitro and in vivo [Lukov GL et al., Leuk Res 35:405-12. 2011]. These information indicate that LYL1 plays important roles in hematopoietic differentiation in primate animals including human and common marmoset. To examine whether overexpression of LYL1 in EBs can promote hematopoietic differentiation in vitro we performed colony-forming unit (CFU) assay, and found that LYL1-overexpressing EBs showed the formation of multi-lineage blood cells consisting of erythroid cells, granulocytes and macrophages. Next, we analyzed gene expression level by RT-PCR, and found that the transduction of LYL1 induced the expression of various hematopoietic genes. These results suggested that the overexpression of LYL1 can promote the differentiation of CM ESCs to HSCs in vitro. Furthermore we found that the combined overexpression of TAL1 and LYL1 could enhance the differentiation of CD34+ cells from CM ESCs than the respective overexrpession of TAL1 or LYL1. Collectively, our novel technology to differentiate hematopoietic cells from ESCs by the transduction of specific transcription factors is novel, and might be applicable to expand human hematopoietic stem/progenitor cells in vitro for future regenerative medicine to cure human hematopoietic cell dyscrasias. Disclosures: No relevant conflicts of interest to declare.

Glioblastomas are the most common and lethal form of intracranial tumors. In the last century we have accumulated tremendous amounts of data on this type of cancer, but we have achieved very little improvement in its treatment. This inadequate progress led us to reexamine the gliomagenesis theory and reconsider the cell of origin of this deadly disease. We recently developed a mouse glioma model using Cre-inducible lentiviral vectors that faithfully recapitulate the pathophysiology of human glioblastoma multiforme (GBM) (1). Injection of a single lentiviral vector expressing H-RasV12 and si_p53 into the brain of GFAP-Cre or Nestin-Cre mice led to tumor formation after 6-8 weeks of injection. Tumors were also obtained when the virus was injected either in the cortex or the stratium in the GFAP-Cre mice, suggesting that astrocytes can also serve as the glioma cell of origin. We made sections of these brains at various time points following injection of the lentiviral vector and, using high resolution large-scale mosaic imaging (2), we examined the expression of different markers. Notably, tumors start out to be GFAP+, but by eight weeks are largely Nestin+ and Sox2+. We believe that astrocytes can be reprogrammed by the introduction of oncogenes/tumor suppressors to form cancer iPS-like stem cells that can give rise to all the cell lineages and heterogeneity observed in GBM. To further explore this hypothesis, we transduced primary cortical astrocytes obtained from GFAP-Cre P2 mice. Only when both H-Ras and si_p53 were in the viral vector were the cells able to reprogram and give rise to neurospheres, a property ascribed to neural stem cells. We were also able to obtain tumors when we aimed to transduce neurons by injecting our lentiviral vector in Syn-Cre mice, suggesting that the reprogramming process is not restricted to glial cells. So far we have used H-Ras as surrogate for EGFR amplification and loss of NF1 observed in patients, but recently we have generated a lenti vector that combines both tumor suppressor genes si_NF1 and si_p53, and the preliminary results confirmed our findings using H-Ras-si_p53 lentivector. Together, our results suggest that any cell in the brain, whether terminally differentiated or neural stem cell, can be the glioma cell of origin and the biological behavior of these tumors depends on the dysregulation of specific genetic elements. (1) Marumoto T, Tashiro A, Friedmann-Morvinski D, Scadeng M, Soda Y, Gage FH, Verma IM. Development of a novel mouse glioma model using lentiviral vectors. Nat Med 15 (1), 110-116 (2009). (2) Price DL, Chow SK, Maclean NA, Hakozaki H, Peltier S, Martone ME, Ellisman MH. High-resolution large-scale mosaic imaging using multiphoton microscopy to characterize transgenic mouse models of human neurological disorders. Neuroinformatics. 2006 Winter;4(1):65-80. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 975. doi:10.1158/1538-7445.AM2011-975

Abstract 1477 Poster Board I-500 Human embryonic stem (ES) cells differentiate into three lineages in vitro and in vivo as mouse ES cells. They are therefore highly promising source of various cells/tissues in the regenerative medicine. The current protocols, however, remain to be optimized for the induction of the cells/tissues required. We have recently reported that the lentiviral transduction of TAL1/SCL gene to ES cells derived from the common marmoset, a small nonhuman primate, enables efficient differentiation into hematopoietic progenitor cells even in the absence of stromal cells (Kurita et al. Stem Cells.24:2014-22, 2006). Such culture condition without any stromal cells is considered to facilitate clinical application of ES cell-derived cell/tissues therapy in the regenerative medicine. The present study addressed whether the strategy is also effective in human ES cells. First, we determined optimal culture conditions to induce multilineage hematopoietic differentiation in a human ES cell line, khES-1, kindly provided by Dr. Nakatsuji, Kyoto University, Japan, as assessed by the expression of Brachyury, Flk1 and CD34. We found that the addition of BMP4 and VEGF augmented hematopoietic differentiation of embryoid bodies, and determined optimal concentrations of the cytokines. We established four human ES cell lines stably expressing TAL1/SCL gene by lentiviral transduction. The TAL1/SCL transduction further increased the hematopoietic differentiation under the optimal culture condition as assessed by the expression of CD34, CD235a and CD133. We also observed increased number of hematopoietic progenitor cells derived from two of the TAL1/SCL expressing human ES cell lines by colony-forming assays. Hematopoietic differentiation of the TAL1/SCL expressing ES cells in vivo is also being investigated by transplantation into irradiated immune deficient mice. These results suggest that the combination of optimal culture conditions and lentiviral TAL1/SCL gene transduction is a highly effective strategy to obtain hematopoietic stem cells from human ES cells in the absence of any stromal cells. Disclosures: No relevant conflicts of interest to declare.