Human liver organoids generated with single donor-derived multiple cells rescue mice from acute liver failure.

BACKGROUND: Acute liver failure (ALF) is a life-threatening disease with a high mortality rate. However, there are limited treatments or devices available for ALF therapy. Here, we aimed to develop a new strategy for ALF treatment by transplanting functional liver organoids (LOs) generated from single donor-derived human induced pluripotent stem cell (hiPSC) endoderm, endothelial cells (ECs), and mesenchymal cells (MCs). METHODS: First, we isolated ECs and MCs from a single donor umbilical cord (UC) through enzyme digestion and characterized the UC-ECs and UC-MCs by flow cytometry. Second, using a nonviral reprogramming method, we generated same donor-derived hiPSCs from the UC-ECs and investigated their hepatic differentiation abilities. Finally, we simultaneously plated EC-hiPSC endoderm, UC-ECs, and UC-MCs in a three-dimensional (3D) microwell culture system, and generated single donor cell-derived differentiated LOs for ALF mouse treatment. RESULTS: We obtained ECs and MCs from a single donor UC with high purity, and these cells provided a multicellular microenvironment that promoted LO differentiation. hiPSCs from the same donor were generated from UC-ECs, and the resultant EC-hiPSCs could be differentiated efficiently into pure definitive endoderm and further into hepatic lineages. Simultaneous plating of EC-hiPSC endoderm, UC-ECs, and UC-MCs in the 3D microwell system generated single donor cell-derived LOs (SDC-LOs) that could be differentiated into functional LOs with enhanced hepatic capacity as compared to that of EC-hiPSC-derived hepatic-like cells. When these functional SDC-LOs were transplanted into the renal subcapsules of ALF mice, they rapidly assumed hepatic functions and improved the survival rate of ALF mice. CONCLUSION: These results demonstrate that functional LOs generated from single donor cells can improve the condition of ALF mice. Functional SDC-LO transplantation provides a promising novel approach for ALF therapy.

[Heparin-induced thrombocytopenia with pseudo-pulmonary embolism in a patient who was newly introduced to hemodialysis treatment].

Pseudo-pulmonary embolism (PPE) superimposed on heparin-induced thrombocytopenia (HIT) is an important complication in patients undergoing hemodialysis (HD) treatment. We report the clinical profile of an HD patient with acute respiratory distress induced by PPE and HIT. A 67-year-old man with diabetic nephropathy and end-stage renal failure developed congestive heart failure. He was admitted to Kitasato University Hospital. He was introduced to HD treatment using low-molecular-weight heparin as an anticoagulant for an HD session on day 1 of admission. On day 11 after admission, he suddenly developed respiratory distress and hypoxia at 30 min after the start of the fifth HD session. The HD session was immediately discontinued, and oxygen inhalation improved his complaints and hypoxia. The platelet count decreased from 220 x 10(9)/L at the start of the HD session to 80 x 10(9)/L at the end of the HD session. We suspected HIT when blood clotting occurred in his hemodialyzer and blood circuit for HD during the HD session on day 12. Chest X-ray, electrocardiogram, echocardiography, and pulmonary microcirculation scintigraphy were normal. Serum analysis was positive for heparin-platelet factor 4 (PF4) antibody. We then diagnosed him with PPE superimposed on HIT. After the anticoagulant agent for HD was changed from low-molecular-weight heparin to nafamostat mesilate, his clinical symptoms and thrombocytopenia disappeared. PPE superimposed on HIT appeared approximately 7-10 days after the initial use of heparin for the HD session. PPE also led to acute respiratory distress, blood coagulation in the hemodialyzer and blood circuit for HD, as well as thrombocytopenia with less than a 50% decrease in platelet counts. The prognosis of PEE and HIT is good after discontinuing the use of heparin.

Prediction of perinatal outcomes based on primary symptoms in women with placental abruption.

AIMS: Placental abruption is an important cause of perinatal mortality and morbidity. Although there are many reports on the risk factors for placental abruption, there are few on its classification. Our aim is to evaluate the associations between primary symptoms and the outcomes of placental abruption. MATERIAL AND METHODS: We carried out a retrospective cohort study of 12,474 births at the Perinatal Center for Maternity and Neonates of the Yokohama City University Medical Center between January 2000 and December 2012. There were 151 women with placental abruption, 136 of whom were included in this study. The subjects were classified into two groups according to their primary symptoms: those with bleeding (external bleeding group) and those with abdominal pain (abdominal pain group). Maternal and neonatal outcomes were compared between the two groups. RESULTS: Both fetal and maternal outcomes were significantly poorer in the abdominal pain group than in the external bleeding group in terms of intrauterine fetal death (6.5% vs 33.3%, P < 0.001), perinatal mortality (8.1% vs 33.3%, P = 0.001), umbilical arterial pH < 7.1 (15.7% vs 57.1%, P < 0.001), bleeding volume, rate of blood transfusion, and disseminated intravascular coagulation incidence. CONCLUSIONS: This classification based on primary symptoms was found to be useful for predicting both maternal and neonatal outcomes of placental abruption.

Heme oxygenase-1 mediates the anti-allergic actions of quercetin in rodent mast cells.

OBJECTIVE AND DESIGN: We investigated the involvement of heme oxygenase (HO)-1 in the anti-allergic action of quercetin against degranulation of rat basophilic leukemia (RBL-2H3) cells, rat peritoneal mast cells, and mouse bone marrow-derived mast cells. METHODS: The strength of allergic reaction was evaluated by the extent of degranulation in mast cells sensitized with various stimulants. The levels of HO-1, HO-2, and nuclear factor erythroid 2-related factor 2 (Nrf2) expressions were determined by quantitative RT-PCR, western blotting, or immunocytochemistry. RESULTS: Heme oxygenase activity was upregulated after short exposure to quercetin, followed by the induction of HO-1 expression after long exposure to quercetin. The inhibition of degranulation by quercetin was reversed using tin protoporphyrin IX (SnPP), an HO-1 inhibitor. HO-1 metabolites, bilirubin and CO, led to inhibit degranulation, and quercetin translocated Nrf2 from cytoplasm into nucleus in RBL-2H3 cells. CONCLUSION: These results strongly suggest that quercetin exerted anti-allergic actions via activation of Nrf2-HO-1 pathway.

Rines/RNF180, a novel RING finger gene-encoded product, is a membrane-bound ubiquitin ligase.

We identified and characterized a novel RING finger gene, Rines/RNF180, which is well conserved among vertebrates. Putative Rines gene product (Rines) contains a RING finger domain, a basic coiled-coil domain, a novel conserved domain (DSPRC) and a C-terminal hydrophobic region that is predicted to be a transmembrane domain. N-terminally epitope tagged-Rines (Nt-Rines) was detected in the endoplasmic reticulum membrane/nuclear envelope in cultured mammalian cells. Nt-Rines was not extracted by high salt or alkaline buffers and was degraded in intact endoplasmic reticulum treated with proteinase K, indicating that Nt-Rines is an integral membrane protein with most of its N-terminal regions in the cytoplasm. Rines was expressed in brain, kidney, testis and uterus of adult mice, and in developing lens and brain, particularly in the ventricular layer of the cerebral cortex at embryonic stages. In cultured cells, Nt-Rines can bind another protein and promoted its degradation. The degradation was inhibited by proteasomal inhibitors. In addition, Nt-Rines itself was heavily ubiquitinated and degraded by proteasome. The involvement of Rines in the ubiquitin-proteasome pathway was further supported by its binding to the UbcH6 ubiquitin-conjugating enzyme and by its trans-ubiquitination enhancing activities. These results suggest that Rines is a membrane-bound E3 ubiquitin ligase.

Zic1 and Zic3 regulate medial forebrain development through expansion of neuronal progenitors.

The medial telencephalon is a source of neurons that follow distinct tangential trajectories of migration to various structures such as the cerebral cortex, striatum, and olfactory bulb. In the present study, we characterized the forebrain anomalies in Zic1/Zic3 compound mutant mice. Zic1 and Zic3 were strongly expressed in the medial structures, including the septum, medial cerebral cortex, and choroid plexus. Mice homozygous for the Zic1 mutant allele together with the null Zic3 allele showed medial forebrain defects, which were not obvious in either Zic1 or Zic3 single mutants. Absence of both Zic1 and Zic3 caused hypoplasia of the hippocampus, septum, and olfactory bulb. Analysis of the cell cycle revealed that the cell cycle exit rate was increased in the septa of double mutants. Misexpression of Zic3 in the ventricular layer of the cerebral cortex inhibited neuronal differentiation. These results indicated that both Zic1 and Zic3 function in maintaining neural precursor cells in an undifferentiated state. The functions of these genes may be essential to increasing neural cell numbers regionally in the medial telencephalon and to proper mediolateral patterning of the telencephalon.

Elucidation of penetrance variability of a ZIC3 mutation in a family with complex heart defects and functional analysis of ZIC3 mutations in the first zinc finger domain.

We studied a series of 42 cases of transposition of the great arteries (TGA), a complex heart defect (CHD) that is two times more prevalent in males than in females. A mutation in the X chromosome at the ZIC3 gene was found in two affected siblings (one male, one female) and their unaffected mother. A second factor, skewed X-inactivation pattern explained the discrepancy between the daughter/mother phenotype. In this family, the missense mutation (p.W255G) was found in the first zinc finger of ZIC3, a domain that is relatively specific to each of the five human ZIC genes. It was tested further along with two other mutations of this domain (p.C253S and p.H286R). In transfected 3T3 cells, mutants p.W255G and p.H286R expressed lower protein levels, and an increased protein degradation (p.W255G only). Moreover, mutants p.C253S and p.W255G had a decreased transcription activation of the TK-luciferase reporter gene. Nuclear translocation of the three ZIC3 mutants varied considerably depending on the experimental models. Finally, p.W255G and p.H286R showed diminished activities for both left-right axis disturbance and neural crest induction in Xenopus embryos. These results suggest that mutations in the first zinc finger of ZIC3 mildly affect several functions of the protein.

Locomotor and oculomotor impairment associated with cerebellar dysgenesis in Zic3-deficient (Bent tail) mutant mice.

We examined the adult neural phenotypes of the Bent tail mutant mouse. The Bent tail mutant mouse was recently shown to lack a submicroscopic part of the X chromosome containing the Zic3 gene, which encodes a zinc-finger protein controlling vertebrate neural development. While nearly one-fourth of hemizygous Bent tail (Bn/Y, Zic3-deficient) mice developed neural tube defects in their midbrain and hindbrain region, the other Bn/Y mice showed apparently normal behaviour in a C57BL/6 genetic background. A battery of behavioural and eye movement tests revealed impaired spontaneous locomotor activity, reduction of muscle tone and impairments of vestibuloocular and optokinetic eye movements in these mice. Morphological examination of the mutant brain showed a significant reduction in the cell numbers in the cerebellar anterior lobe and paraflocculus-flocculus complex. Our results indicate that the cerebellar dysgenesis characterized by subregional hypoplasia affects the locomotor activity, muscle tone and eye movement control of the mice. These findings may have some clinical implications in relation to disorders characterized by cerebellar dysgenesis, such as Joubert syndrome.

Myogenic repressor I-mfa interferes with the function of Zic family proteins.

Zinc finger proteins belonging to the Zic family control several developmental processes such as patterning of the axial skeleton. Here we mapped the transcriptional regulatory domains in Zic2 protein and identified a protein which specifically binds to one of them. In the mapping experiments, an amino-terminal region was identified as transcriptional regulatory domains. A search for proteins binding to the amino terminal domain of Zic2 revealed that inhibitor of MyoD family (I-mfa) protein, which has been identified as a repressor of myogenic helix-loop-helix class transcription factors, can physically interact with the amino terminal domain. When Zic1-3 and I-mfa proteins were co-expressed in cultured cells, nuclear import of the Zic proteins was inhibited. Consequently, I-mfa inhibited transcriptional activation by the Zic proteins in cultured cells. These results suggest that the physical and functional interaction between Zic and I-mfa proteins can play a role in the vertebrate development.

Novel method for quantification of brain cell swelling in rat hippocampal slices.

We have developed a novel device for the quantification of edematous morphology changes in acute brain slices. We can also carry out real-time monitoring of detailed hippocampal cells. The device we developed is based on infrared differential interference contrast microscopy (IR-DIC) and a custom-made real-time computerized image-analysis system for quantification of the morphological dynamics of cells in slice preparations. We applied the coefficient of variation (CV) of light intensity in IR-DIC images to evaluate the change in morphological dynamics. We examined three kinds of edema in the CA1 region of rat hippocampal acute slices under conditions of hypotonic, strong excitation, and experimental ischemia, together with field excitatory postsynaptic potential (fEPSP) recording from radiatum in CA1 the region. There were notable close relationships among the edema formations, the light transmittance, the extent of changes in CV, and features of fEPSP during the three different insults. The present results indicate that CV is a reliable quantification index for edema formation in brain tissue and confirm that applying CV for the analysis in addition to the light transmittance analysis presents additional important information on brain tissue swelling.

Postnatal development of the mouse volatile papilla taste bud cells.

In the present study, we examined specific markers for taste bud cells in the mouse and the postnatal development of volatile papilla taste bud cells in ddY mice. We examined the immunoreactivity of 4 types of carbonic anhydrase isoenzymes, CA I, CA II, CA III and CA VI, as specific markers for taste bud cells, and K8.13 cytokeratin antibody as a specific marker for the lingual epithelial cells. Of the carbonic anhydrase isoenzymes, only CA III immunoreactivity was clearly detected in the spindle shaped gustatory cells. CA VI immunoreactivity was detectable in suspentacular cells. CA I and CA II antibodies did not recognize any taste bud cell specifically. K8.13 cytokeratin immunoreactivity was detected in the lingual epithelial cells, but not in taste bud cells. At 7 days after birth, the suckling phase, very small taste buds developed from the anaplastic gustatory cells. At 14 days after birth, the taste buds showed larger size than those at 7 days after birth. At 21 days birth, after the weaning phase, taste bud structure approximated the mature structure. These results demonstrate the specificity of anti-CA III and anti-CA VI for gustatory cells and suspentacular cells, respectively. These markers should be useful for an analysis of taste bud development in mice.

Distributional changes of BrdU, PCNA, E2F1 and PAL31 molecules in developing murine palatal rugae.

The distribution of cells incorporating bromodeoxyuridine (BrdU) and the expression of molecules involved in the control of cell proliferation (proliferating cell nuclear antigen [PCNA], a cellular factor in F9 teratocarcinoma cells that recognizes an adenovirus E1A inducible promoter 1 [E2F1] and proliferation-related acidic nuclear protein 31 [PAL31]) during morphogenesis of the murine palatine rugae (PR) was examined histochemically. Pattern formation of the PR rudiment was initiated with cell cycle related molecules in the epithelium of the primary palate. Cells which had incorporated BrdU were detected at the outer areas of the presumptive epithelial placode (EP) and the EP at 11.5-13.5 days post coitum (dpc) and the outer areas of the PR protrusion after 14.5 dpc. The number of PCNA-positive cells at the central area of the PR protrusion decreased after 16.5 dpc. E2F-positive cells were detected at the outer areas of the PR protrusion at 15.5 and 16.5 dpc. The number of PAL31-positive cells at the presumptive EP area and the already-formed EP area was decreased at 11.5-13.5 dpc. In two dimensional histological reconstructions, PAL31 expression approximately corresponded to the distribution of BrdU-positive cells at 11.5 and 13.5 dpc. EP placode formation might be regulated by spatiotemporal cell proliferation control involving the expression of the PAL31 molecule. Following EP formation, PR development and growth control involved the expression of E2F1 and PCNA molecules.

Chain-growth polycondensation for well-defined aramide. Synthesis of unprecedented block copolymer containing aramide with low polydispersity.

Poly(p-benzamide) with a defined molecular weight and a low polydispersity and a block copolymer containing this well-defined aramide was synthesized. Phenyl 4-aminobenzoate, which would yield poly(p-benzamide), did not polymerize under the conditions of chain-growth polycondensation. However, phenyl 4-(4-octyloxybenzylamino)benzoate (1b) polymerized at room temperature in the presence of base and phenyl 4-nitrobenzoate (2) as an initiator in a chain-growth polycondensation manner to give well-defined aromatic polyamides having the 4-octyloxybenzyl groups as a protecting group on nitrogen in an amide. It was confirmed by a model reaction that deprotection of this protecting group proceeded completely with trifluoroacetic acid (TFA) without breaking the amide linkage. The utility of this approach to poly(p-benzamide) with a low polydispersity was demonstrated by the synthesis of block copolymers. Thus, phenyl 4-(octylamino)benzoate (1a) polymerized in the presence of 2 and base, followed by addition of 1b and base to the reaction mixture of the prepolymer to yield the block copolymer of 1a and 1b with a controlled molecular weight and a low polydispersity. The block copolymer was treated with TFA, resulting in a soluble block copolymer of poly(N-octyl-p-benzamide) and poly(p-benzamide). The SEM images of the supramolecular assemblies of the block copolymer showed mum-sized bundles and aggregates of flake structures.

A mouse with a point mutation in plasma membrane Ca2+-ATPase isoform 2 gene showed the reduced Ca2+ influx in cerebellar neurons.

We analyzed mutant mice showing behavioral defects such as severe tremor, up-and-down and side-to-side wriggling of neck without coordination, and found that the gene causing the defects was located between 46 and 60.55 centimorgans (cM) on the mouse chromosome 6. In this region, nucleotide transition of the plasma membrane Ca2+-ATPase isoform 2 (PMCA2) gene was found, which caused a glutamic acid to change into lysine. Since PMCA2 is expressed in the cerebellum and plays an important role to maintain the homeostasis of the intracellular Ca2+ as a Ca2+ pump, the behavioral defect can be ascribed to the impairment of Ca2+ regulation in neurons of the cerebellum. To confirm the defect of Ca2+ homeostasis in the mutant mice, we measured high K+-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in the cerebellar neurons. Contrary to our expectation, the extent of the [Ca2+]i increase in all the regions tested in the cerebellar slice was far smaller than that of the wild type mice, while the resting [Ca2+]i remained almost unaltered. The rate of rise in [Ca2+]i during high K+-induced depolarization was significantly reduced, and the extrusion rate of increased [Ca2+]i was also reduced. These results suggested that voltage-gated Ca2+ channels were down-regulated in the mutant mice in order to regulate [Ca2+]i toward the normal homeostasis. The behavioral defects may be ascribed to the down-regulated Ca2+ homeostasis since dynamic changes in [Ca2+]i are important for various neuronal functions.

Distribution of apoptotic cells and apoptosis-related molecules in the developing murine palatine rugae.

Distribution of apoptotic cells and expression of the apoptosis-related factors p53, bcl-2 and bad during morphogenesis of the murine palatine rugae (PR) were examined histochemically using the terminal deoxynucleotidyl transferase-mediated UTP nick end-labeling (TUNEL) technique and specific antibodies against apoptosis and cell cycle-related molecules. Formation of the PR rudiment was controlled by cell proliferation and apoptosis in the palatal epithelium. TUNEL-positive cells were detected only at the epithelial placode area at 12.5-13.5 days post coitus (dpc), but only a few cells were positive at the protruding PR area at 14.5-16.5 dpc. Bcl-2 protein was expressed mainly in the areas outside of those containing TUNEL-positive cells at 15.5 -6.5 dpc. P53 protein was not detected throughout gestation. Bad was detected in the epithelial layer at 13.5 and 15.5 dpc and overlapping the apoptotic area at 13.5-15.5 dpc. Apoptosis of palatal epithelial cells might therefore involve spatiotemporally regulated expression of bad during murine PR development.