Jiyuan Liao

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.

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.

236. Role of P53 on T Lymphopoiesis from Human Embryonic Stem Cells

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

Angiotensin II type I receptor blocker, Losartan, inhibits fibrosis in liver by suppressing TGF-beta1 production

Renin-angiotensin system is involved in liver fibrogenesis through activating hepatic stellate cells (HSCs). Losartan, which is an angiotensin II type 1 receptor antagonist, could function as a selective peroxisome proliferator-activated receptor c activator. Here we studied the effect of losartan on liver fibrosis in vivo. In vivo study, we used the Concanavalin A (Con A)-induced mouse liver fibrosis model through T cell activation and upregulation of TGF-β1. The mice were administrated ConA for 4 weeks to induce liver fibrosis, and then co-administrated with losartan. Losartan prevented liver fibrogenesis and downregulated TGF-β1 expression. Also, Losartan inhibited HSCs activation and proliferation. These results suggested that Losartan prevent liver fibrosis through suppressing TGF-β1 expression. Abbreviations: AT1-R, angiotensin II type 1 receptor; ARB: angiotensin II receptor blocker; RAS: renin-angiotensin system; TGF-β1: transforming growth factor β1; NASH: non-alcoholic steatohepatitis Introduction Liver fibrosis is the common histological feature of most chronic liver diseases, and leads to cirrhosis and hepatocellular carcinoma (HCC) [1]. Liver cirrhosis is the end stage of chronic liver diseases. Liver fibrosis is the common pathological basis of numerous chronic liver diseases including viral liver disease, alcoholic, non-alcoholic steatohepatitis (NASH), autoimmune hepatitis, primary biliary cirrhosis, and metabolic disease. Liver cirrhosis is characterized by the increase and excessive deposition of the liver extracellular matrix (ECM) [2-4]. Excessive activation of transforming growth factor-β (TGF-β) increases the synthesis and decreases the degradation of ECM proteins with a gradual destruction of organ tissue and structure [5]. The major source of ECM deposition in the liver is hepatic stellate cells (HSCs). After liver injury, TGF-β1 promotes the activation and proliferation of hepatic HSCs [6]. High levels of TGF-β1 are often found in liver fibrosis and there may be a positive correlation between the elevation of TGF-β1 mRNA level and fibrogenic activity [7]. Thus, synthesis of ECM proteins increases in the liver due to excessive activation of the TGF-β1 signal transduction pathway. Therefore, this pathway has become a potential target for the treatment of the liver fibrosis. The renin-angiotensin system (RAS) plays an important role in controlling liver fibrosis [8]. It is well known that the RAS influences cell differentiation, nutrition, and fibrosis, and it has been reported to play a role in heart and kidney disease [9]. Moreover, recent studies have revealed the RAS plays an important role in the progression of many chronic liver diseases [10]. Furthermore, angiotensin II receptor blocker (ARB) modulation of the RAS to treat liver fibrosis has been reported in animal study [11]. ARBs, such as Losartan are largely regarded as a means to block vasoconstriction and inhibit the cell proliferation and fibrosis that are mediated by the angiotensin II type 1 receptor (AT1-R) [10]. Yang et al. [12] suggested that AT1-R play an important role in the development of fibrosis. An increase in hepatic transforming growth factor β1 (TGF-β1) and pro-inflammatory cytokine levels was attenuated in AT1-R knockout mice compared to WT mice [13]. Furthermore, Bataller et al.[14] demonstrated that increased systemic Angiotensin II augments hepatic fibrosis and promotes inflammation, oxidative stress, and thrombogenic events. Losartan was reported to inhibit CCl4-induced Correspondence to: Hisanobu Ogata, Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan, Tel: +81-92-642-5256; Fax: +81-92-642-5271; E-mail: h-ogata@sentan.med.kyushu-u.ac.jp

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