Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency.

Efficient gene transfer into human pluripotent stem cells (hPSCs) holds great promise for regenerative medicine and pharmaceutical development. In the past decade, various methods were developed for gene transfer into hPSCs; however, hPSCs form tightly packed colonies, making gene transfer difficult. In this study, we established a stable culture method of hPSCs at a single-cell state to reduce cell density and investigated gene transfection efficiency followed by gene editing efficiency. hPSCs cultured in a single-cell state were transfected using nonliposomal transfection reagents with plasmid DNA or mRNA encoding enhanced green fluorescent protein. We found that most cells (DNA > 90%; mRNA > 99%) were transfected without the loss of undifferentiated PSC marker expression or pluripotency. Moreover, we demonstrated an efficient gene editing method using transcription activator-like effector nucleases (TALENs) targeting the adenomatous polyposis coli (APC) gene. Our new method may improve hPSC gene transfer techniques, thus facilitating their use for human regenerative medicine.

Phase I clinical trial of a five-peptide cancer vaccine combined with cyclophosphamide in advanced solid tumors.

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.

Improved hematopoietic differentiation of primate embryonic stem cells by inhibition of the PI3K-AKT pathway under defined conditions.

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 (p < 0.001) 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.

Analysis of essential pathways for self-renewal in common marmoset embryonic stem cells.

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 ß (TGFß) 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.

Characterization of common marmoset dysgerminoma-like tumor induced by the lentiviral expression of reprogramming factors.

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.

Inhibition of PTEN tumor suppressor promotes the generation of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) can be generated from patients with specific diseases by the transduction of reprogramming factors and can be useful as a cell source for cell transplantation therapy for various diseases with impaired organs. However, the low efficiency of iPSC derived from somatic cells (0.01-0.1%) is one of the major problems in the field. The phosphoinositide 3-kinase (PI3K) pathway is thought to be important for self-renewal, proliferation, and maintenance of embryonic stem cells (ESCs), but the contribution of this pathway or its well-known negative regulator, phosphatase, and tensin homolog deleted on chromosome ten (Pten), to somatic cell reprogramming remains largely unknown. Here, we show that activation of the PI3K pathway by the Pten inhibitor, dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate, improves the efficiency of germline-competent iPSC derivation from mouse somatic cells. This simple method provides a new approach for efficient generation of iPSCs.

Dedifferentiation of neurons and astrocytes by oncogenes can induce gliomas in mice.

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.

Roles of p53 in various biological aspects of hematopoietic stem cells.

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.

Molecular biology of glioma.

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.

Coxsackievirus B3 is an oncolytic virus with immunostimulatory properties that is active against lung adenocarcinoma.

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.

Transdifferentiation of glioblastoma cells into vascular endothelial cells.

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.

APOA-1 is a novel marker of erythroid cell maturation from hematopoietic stem cells in mice and humans.

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.

Development of a novel mouse glioma model using lentiviral vectors.

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.

The tumor suppressor WARTS activates the Omi / HtrA2-dependent pathway of cell death.

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.

Over-expression of Aurora-A targets cytoplasmic polyadenylation element binding protein and promotes mRNA polyadenylation of Cdk1 and cyclin B1.

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.

RNA interference targeting aurora kinase a suppresses tumor growth and enhances the taxane chemosensitivity in human pancreatic cancer cells.

AURKA/STK15/BTAK, the gene encoding Aurora A kinase that is involved in the regulation of centrosomes and segregation of chromosomes, is frequently amplified and overexpressed in various kinds of human cancers, including pancreatic cancer. To address its possibility as a therapeutic target for pancreatic cancer, we employed the RNA interference technique to knockdown AURKA expression and analyzed its phenotypes. We found that the specific knockdown of AURKA in cultured pancreatic cancer cells strongly suppressed in vitro cell growth and in vivo tumorigenicity. The knockdown induced the accumulation of cells in the G(2)-M phase and eventual apoptosis. Furthermore, we observed a synergistic enhancement of the cytotoxicity of taxanes, a group of chemotherapeutic agents impairing G(2)-M transition, by the RNA interference-mediated knockdown of AURKA. These results indicate that inhibition of AURKA expression can result in potent antitumor activity and chemosensitizing activity to taxanes in human pancreatic cancer.

Aurora-A - a guardian of poles.

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?

Cre-loxP-controlled periodic Aurora-A overexpression induces mitotic abnormalities and hyperplasia in mammary glands of mouse models.

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.