Michiyo Okada

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.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.

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.

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 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.

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.

631. Novel Polymer-Coated Stealth Oncolytic Measles Virus Overcame Immune Suppression and Induced Stronger Antitumor Activity

Background: Although there have recently been much progress in cancer treatment including surgery, chemotherapy and radiotherapy, cancers have long been the leading cause of death in Japan in the last 30 years and the development of new therapeutic modalities is imminently required. As a new modality, there has recently been great interest in oncolytic virotherapy. Particularly, measles virus is one of the candidate viruses expected for strong antitumor effects. The efficacy of virotherapy, however, was strongly limited by the immune response of the host in previous clinical trials.Methods: To enhance and prolong the antitumor activity of the virotherapy in vivo, we combined newly developed tools of the genetically engineered measles virus (MV-NPL) and multilayer virus coating method of layer-by-layer deposition of ionic polymers. We compared the oncolytic effects of this polymer-coated MV-NPL with naked MV-NPL in vitro and in vivo.Results: In the presence of anti-MV neutralizing antibodies, the polymer-coated virus showed more enhanced oncolytic activity than naked MV-NPL in vitro. We also examined the antitumor immunity in virus treated mice. Complement-dependent cytotoxicity activities in mice treated with polymer-coated MV-NPL were higher than those with naked virus and stronger antitumor activity was observed in these mice.Conclusion: This novel polymer-coated MV-NPL may be promising for future clinical cancer therapy.

Abstract 2564: Characterization of dysgerminoma-like cells emerged in the process of iPSC generation from common Marmoset fibroblasts

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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