A nonhuman primate model of liver fibrosis towards cell therapy for liver cirrhosis.

Orthotopic liver transplantation (OLT) is the only curative treatment for refractory chronic liver failure in liver cirrhosis. However, the supply of donated livers does not meet the demand for OLT due to donor organ shortage. Cell therapy using hepatocyte-like cells derived from human induced pluripotent stem cells (hiPSC-HLCs) is expected to mitigate the severity of liver failure, postpone OLT and ameliorate the insufficient liver supply. For the successful clinical translation of hiPSC-based cell therapy against liver cirrhosis, realistic animal models are required. In this study, we created a nonhuman primate (NHP) liver fibrosis model by repeated administrations of thioacetamide (TAA) and evaluated the short-term engraftment of hiPSC-HLCs in the fibrotic liver. The NHP liver fibrosis model reproduced well the pathophysiology of human liver cirrhosis including portal hypertension. Under immunosuppressive treatment, we transplanted ALBUMIN-GFP reporter hiPSC-HLC aggregates into the fibrotic livers of the NHP model via the portal vein. Fourteen days after the transplantation, GFP-expressing hiPSC-HLC clusters were detected in the portal areas of the fibrotic livers. These results will facilitate preclinical studies using the NHP liver fibrosis model and help establish iPSC-based cell therapies against liver cirrhosis.

Modelling urea-cycle disorder citrullinemia type 1 with disease-specific iPSCs.

Citrullinemia type 1 (CTLN1) is a urea cycle disorder (UCD) caused by mutations of the ASS1 gene, which is responsible for production of the enzyme argininosuccinate synthetase (ASS), and classically presented as life-threatening hyperammonemia in newborns. Therapeutic options are limited, and neurological sequelae may persist. To understand the pathophysiology and find novel treatments, induced pluripotent stem cells (iPSCs) were generated from a CTLN1 patient and differentiated into hepatocyte-like cells (HLCs). CTLN1-HLCs have lower ureagenesis, recapitulating part of the patient's phenotype. l-arginine, an amino acid clinically used for UCD treatment, improved this phenotype in vitro. Metabolome analysis revealed an increase in tricarboxylic acid (TCA) cycle metabolites in CTLN1, suggesting a connection between CTLN1 and the TCA cycle. This CTLN1-iPSC model improves the understanding of CTLN1 pathophysiology and can be used to pursue new therapeutic approaches.

Retinoic acid regulates erythropoietin production cooperatively with hypoxia-inducible factors in human iPSC-derived erythropoietin-producing cells.

Erythropoietin (EPO) is a crucial hormone for erythropoiesis and produced by adult kidneys. Insufficient EPO production in chronic kidney disease (CKD) can cause renal anemia. Although hypoxia-inducible factors (HIFs) are known as a main regulator, the mechanisms of EPO production have not been fully elucidated. In this study, we aimed to examine the roles of retinoic acid (RA) in EPO production using EPO-producing cells derived from human induced pluripotent stem cells (hiPSC-EPO cells) that we previously established. RA augmented EPO production by hiPSC-EPO cells under hypoxia or by treatment with prolyl hydroxylase domain-containing protein (PHD) inhibitors that upregulate HIF signals. Combination treatment with RA and a PHD inhibitor improved renal anemia in vitamin A-depleted CKD model mice. Our findings using hiPSC-EPO cells and CKD model mice may contribute to clarifying the EPO production mechanism and developing efficient therapies for renal anemia.

Differentiation and isolation of iPSC-derived remodeling ductal plate-like cells by use of an AQP1-GFP reporter human iPSC line.

Cholangiocytes are the epithelial cells that line bile ducts, and ductal plate malformation is a developmental anomaly of bile ducts that causes severe congenital biliary disorders. However, because of a lack of specific marker genes, methods for the stepwise differentiation and isolation of human induced pluripotent stem cell (hiPSC)-derived cholangiocyte progenitors at ductal plate stages have not been established. We herein generated an AQP1-GFP reporter hiPSC line and developed a combination treatment with transforming growth factor (TGF) ß2 and epidermal growth factor (EGF) to induce hiPSC-derived hepatoblasts into AQP1+ cells in vitro. By confirming that the isolated AQP1+ cells showed similar gene expression patterns to cholangiocyte progenitors at the remodeling ductal plate stage around gestational week (GW) 20, we established a differentiation protocol from hiPSCs through SOX9+CK19+AQP1- ductal plate-like cells into SOX9+CK19+AQP1+ remodeling ductal plate-like cells. We further generated 3D bile duct-like structures from the induced ductal plate-like cells. These results suggest that AQP1 is a useful marker for the generation of remodeling ductal plate cells from hiPSCs. Our methods may contribute to elucidating the differentiation mechanisms of ductal plate cells and the pathogenesis of ductal plate malformation.

Novel hybrid three-dimensional artificial liver using human induced pluripotent stem cells and a rat decellularized liver scaffold.

INTRODUCTION: Liver transplantation is currently the only curative therapy for end-stage liver failure; however, establishment of alternative treatments is required owing to the serious donor organ shortage. Here, we propose a novel model of hybrid three-dimensional artificial livers using both human induced pluripotent stem cells (hiPSCs) and a rat decellularized liver serving as a scaffold. METHODS: Rat liver harvesting and decellularization were performed as reported in our previous studies. The decellularized liver scaffold was recellularized with hiPSC-derived hepatocyte-like cells (hiPSC-HLCs) through the biliary duct. The recellularized liver graft was continuously perfused with the culture medium using a pump at a flow rate of 0.5 mL/min in a standard CO2 (5%) cell incubator at 37 °C. RESULTS: After 48 h of continuous perfusion culture, the hiPSC-HLCs of the recellularized liver distributed into the parenchymal space. Furthermore, the recellularized liver expressed the albumin (ALB) and CYP3A4 genes, and secreted human ALB into the culture medium. CONCLUSION: Novel hybrid artificial livers using hiPSCs and rat decellularized liver scaffolds were successfully generated, which possessed human hepatic functions.

Identification of negative transcriptional factor E4BP4-binding site in the mouse circadian-regulated gene Mdr2.

The hepatic transporter Mdr2 is an ATP-binding cassette transporter which excretes phosphatidylcholine into the bile. We showed that the level of Mdr2 mRNA oscillated in circadian fashion in mouse liver whereas such oscillation was dampened in the liver of Clock mutants. To examine transcriptional regulation of the Mdr2 gene we performed luciferase reporter assays using plasmid constructs containing the 5'-flanking region of the Mdr2 gene. Reporter assays using deletion constructs demonstrated that E4BP4 represses the transcriptional activity of the promoter including the D1 and D2 sites within four putative E4BP4-binding sites. Chromatin immunoprecipitation and gel shift assays showed that E4BP4 binds to the D2 site, but not to the D1 site. These data suggested that E4BP4 is a negative transcription factor for circadian Mdr2 mRNA expression.

Adrenergic receptor agonists induce the differentiation of pluripotent stem cell-derived hepatoblasts into hepatocyte-like cells.

Current induction methods of hepatocytes from human induced pluripotent stem cells (hiPSCs) are neither low cost nor stable. By screening a chemical library of 1,120 bioactive compounds and known drugs, we identified the α1-adrenergic receptor agonist methoxamine hydrochloride as a small molecule that promotes the differentiation of hiPSC-derived hepatoblasts into ALBUMIN+ hepatocyte-like cells. Other α1-adrenergic receptor agonists also induced the differentiation of hepatocyte-like cells, and an α1-receptor antagonist blocked the hepatic-inducing activity of methoxamine hydrochloride and that of the combination of hepatocyte growth factor (HGF) and Oncostatin M (OsM), two growth factors often used for the induction of hepatoblasts into hepatocyte-like cells. We also confirmed that treatment with methoxamine hydrochloride activates the signal transducer and activator of transcription 3 (STAT3) pathway downstream of IL-6 family cytokines including OsM. These findings allowed us to establish hepatic differentiation protocols for both mouse embryonic stem cells (mESCs) and hiPSCs using small molecules at the step from hepatoblasts into hepatocyte-like cells. The results of the present study suggest that α1-adrenergic agonists induce hepatocyte-like cells by working downstream of HGF and OsM to activate STAT3.

Human pluripotent stem cell-derived erythropoietin-producing cells ameliorate renal anemia in mice.

The production of erythropoietin (EPO) by the kidneys, a principal hormone for the hematopoietic system, is reduced in patients with chronic kidney disease (CKD), eventually resulting in severe anemia. Although recombinant human EPO treatment improves anemia in patients with CKD, returning to full red blood cell production without fluctuations does not always occur. We established a method to generate EPO-producing cells from human induced pluripotent stem cells (hiPSCs) by modifying previously reported hepatic differentiation protocols. These cells showed increased EPO expression and secretion in response to low oxygen conditions, prolyl hydroxylase domain-containing enzyme inhibitors, and insulin-like growth factor 1. The EPO protein secreted from hiPSC-derived EPO-producing (hiPSC-EPO) cells induced the erythropoietic differentiation of human umbilical cord blood progenitor cells in vitro. Furthermore, transplantation of hiPSC-EPO cells into mice with CKD induced by adenine treatment improved renal anemia. Thus, hiPSC-EPO cells may be a useful tool for clarifying the mechanisms of EPO production and may be useful as a therapeutic strategy for treating renal anemia.

Efficient Detection and Purification of Cell Populations Using Synthetic MicroRNA Switches.

Isolation of specific cell types, including pluripotent stem cell (PSC)-derived populations, is frequently accomplished using cell surface antigens expressed by the cells of interest. However, specific antigens for many cell types have not been identified, making their isolation difficult. Here, we describe an efficient method for purifying cells based on endogenous miRNA activity. We designed synthetic mRNAs encoding a fluorescent protein tagged with sequences targeted by miRNAs expressed by the cells of interest. These miRNA switches control their translation levels by sensing miRNA activities. Several miRNA switches (miR-1-, miR-208a-, and miR-499a-5p-switches) efficiently purified cardiomyocytes differentiated from human PSCs, and switches encoding the apoptosis inducer Bim enriched for cardiomyocytes without cell sorting. This approach is generally applicable, as miR-126-, miR-122-5p-, and miR-375-switches purified endothelial cells, hepatocytes, and insulin-producing cells differentiated from hPSCs, respectively. Thus, miRNA switches can purify cell populations for which other isolation strategies are unavailable.

[Discussions on home care of solely living people receiving nursing services at the station].

The number of solely living people receiving nursing services at the station has continuously increased to 23 people in the period of four years and six months from September 1998 to February 2003. We plan to survey the condition of home care of solely living people, which is considered to give hints for future nursing.

[Discussions on home care of solely living people receiving nursing services at the station].

The number of solely living people receiving nursing services at the station has continuously increased to 23 people in the period of four years and six months from September 1998 to February 2003. We plan to survey the condition of home care of solely living people, which is considered to give hints for future nursing.