Generation of chimpanzee induced pluripotent stem cell lines for cross-species comparisons.

As humans' closest living relatives, chimpanzees offer valuable insights into human evolution. However, technical and ethical limitations hinder investigations into the molecular and cellular foundations that distinguish chimpanzee and human traits. Recently, induced pluripotent stem cells (iPSCs) have emerged as a novel model for functional comparative studies and provided a non-invasive alternative for studying embryonic phenomena. In this study, we generated five new chimpanzee iPSC lines from peripheral blood cells and skin fibroblasts with SeV vectors carrying four reprogramming factors (human OCT3/4, SOX2, KLF4, and L-MYC) and characterized their pluripotency and differentiation potential. We also examined the expression of a human-specific non-coding RNA, HSTR1, which is predicted to be involved in human brain development. Our results show that the chimpanzee iPSCs possess pluripotent characteristics and can differentiate into various cell lineages. Moreover, we found that HSTR1 is expressed in human iPSCs and their neural derivatives but not in chimpanzee counterparts, supporting its possible role in human-specific brain development. As iPSCs are inherently variable due to genetic and epigenetic differences in donor cells or reprogramming procedures, it is essential to expand the number of chimpanzee iPSC lines to comprehensively capture the molecular and cellular properties representative of chimpanzees. Hence, our cells provide a valuable resource for investigating the function and regulation of human-specific transcripts such as HSTR1 and for understanding human evolution more generally.

A Case of Mixed Connective Tissue Disease That Transformed Into Systemic Lupus Erythematosus After a Long Clinical Course.

Mixed connective tissue disease (MCTD), a multisystem autoimmune disease that was first proposed in 1972, has overlapping features with other autoimmune diseases. In recent studies, mixed connective tissue disease has been reported to change into other connective tissue diseases (CTD; such as systemic lupus erythematosus [SLE], polymyositis, and systemic sclerosis [SSc]) in the long term. We report the case of a 58-year-old Japanese man diagnosed with mixed connective tissue disease 15 years ago. During his clinical course, he developed discoid lupus erythematosus, pancytopenia, a low complement titer, proteinuria, and hematuria. He also turned positive for the anti-double-stranded deoxyribonucleic acid (dsDNA) antibody. A kidney biopsy revealed lupus nephritis (LN) class IV. Therefore, we considered this to be a shift from mixed connective tissue disease to systemic lupus erythematosus. We changed his treatment to lupus nephritis, after which he remained in remission. Our case suggests that mixed connective tissue disease may shift to other connective tissue diseases over a long period; therefore, it is necessary to identify whether patients with mixed connective tissue disease fulfill the diagnostic criteria for other connective tissue diseases when new manifestations appear.

The dual origin of the peripheral olfactory system: placode and neural crest.

BACKGROUND: The olfactory epithelium (OE) has a unique capacity for continuous neurogenesis, extending axons to the olfactory bulb with the assistance of olfactory ensheathing cells (OECs). The OE and OECs have been believed to develop solely from the olfactory placode, while the neural crest (NC) cells have been believed to contribute only the underlying structural elements of the olfactory system. In order to further elucidate the role of NC cells in olfactory development, we examined the olfactory system in the transgenic mice Wnt1-Cre/Floxed-EGFP and P0-Cre/Floxed-EGFP, in which migrating NC cells and its descendents permanently express GFP, and conducted transposon-mediated cell lineage tracing studies in chick embryos. RESULTS: Examination of these transgenic mice revealed GFP-positive cells in the OE, demonstrating that NC-derived cells give rise to OE cells with morphologic and antigenic properties identical to placode-derived cells. OECs were also positive for GFP, confirming their NC origin. Cell lineage tracing studies performed in chick embryos confirmed the migration of NC cells into the OE. Furthermore, spheres cultured from the dissociated cells of the olfactory mucosa demonstrated self-renewal and trilineage differentiation capacities (neurons, glial cells, and myofibroblasts), demonstrating the presence of NC progenitors in the olfactory mucosa. CONCLUSION: Our data demonstrates that the NC plays a larger role in the development of the olfactory system than previously believed, and suggests that NC-derived cells may in part be responsible for the remarkable capacity of the OE for neurogenesis and regeneration.

Schwann-spheres derived from injured peripheral nerves in adult mice--their in vitro characterization and therapeutic potential.

Multipotent somatic stem cells have been identified in various adult tissues. However, the stem/progenitor cells of the peripheral nerves have been isolated only from fetal tissues. Here, we isolated Schwann-cell precursors/immature Schwann cells from the injured peripheral nerves of adult mice using a floating culture technique that we call "Schwann-spheres." The Schwann-spheres were derived from de-differentiated mature Schwann cells harvested 24 hours to 6 weeks after peripheral nerve injury. They had extensive self-renewal and differentiation capabilities. They strongly expressed the immature-Schwann-cell marker p75, and differentiated only into the Schwann-cell lineage. The spheres showed enhanced myelin formation and neurite growth compared to mature Schwann cells in vitro. Mature Schwann cells have been considered a promising candidate for cell-transplantation therapies to repair the damaged nervous system, whereas these "Schwann-spheres" would provide a more potential autologous cell source for such transplantation.

Sox10-Venus mice: a new tool for real-time labeling of neural crest lineage cells and oligodendrocytes.

BACKGROUND: While several mouse strains have recently been developed for tracing neural crest or oligodendrocyte lineages, each strain has inherent limitations. The connection between human SOX10 mutations and neural crest cell pathogenesis led us to focus on the Sox10 gene, which is critical for neural crest development. We generated Sox10-Venus BAC transgenic mice to monitor Sox10 expression in both normal development and in pathological processes. RESULTS: Tissue fluorescence distinguished neural crest progeny cells and oligodendrocytes in the Sox10-Venus mouse embryo. Immunohistochemical analysis confirmed that Venus expression was restricted to cells expressing endogenous Sox10. Time-lapse imaging of various tissues in Sox10-Venus mice demonstrated that Venus expression could be visualized at the single-cell level in vivo due to the intense, focused Venus fluorescence. In the adult Sox10-Venus mouse, several types of mature and immature oligodendrocytes along with Schwann cells were clearly labeled with Venus, both before and after spinal cord injury. CONCLUSIONS: In the newly-developed Sox10-Venus transgenic mouse, Venus fluorescence faithfully mirrors endogenous Sox10 expression and allows for in vivo imaging of live cells at the single-cell level. This Sox10-Venus mouse will thus be a useful tool for studying neural crest cells or oligodendrocytes, both in development and in pathological processes.

Norepinephrine does not alter NPY and POMC mRNA expression in neonatal chicks.

Norepinephrine (NE), synthesized in both the central and peripheral nervous system, is involved in food intake regulation of both mammals and chickens. Neuropeptide Y (NPY), a potent orexigenic peptide, is colocalized with NE neurons in the central and peripheral nervous system, suggesting an interaction. Proopiomelanocortin (POMC) is the precursor of alpha-melanocyte stimulating hormone, a potent anorexigenic peptide synthesized in the hypothalamus. In this study, two experiments were conducted to examine the effect of intracerebroventricular (ICV) injection of NE on appetite mediators in neonatal chicks (Gallus gallus). Experiment 1 was done to confirm the effect of centrally administered NE (0, 25, 50, and 100 microg) on food intake following a 3h fast, and to determine the change in NPY mRNA expression in the central nervous system (CNS). In Experiment 2, chicks fed ad libitum were treated ICV with NE (50 microg) to determine if changes occurred in brain NPY and POMC mRNA levels. In Experiment 1, the ICV injection of NE dose-dependently reduced food intake, but there was no change in NPY mRNA expression in the CNS. In Experiment 2, there was no significant change in NPY and POMC mRNA expression between the control and NE-treated group, indicating that ICV injection of NE may not be associated with changes in NPY or POMC gene expression.

Oral administration of Excitin-1 (beta-alanyl-L-leucine) alters behavior and brain monoamine and amino acid concentrations in rats.

We previously demonstrated that beta-alanyl-branched chain amino acids have excitatory effects. Therefore, we named beta-alanyl-L-leucine, beta-alanyl-L-isoleucine and beta-alanyl-L-valine as Excitin-1, -2, and -3 , respectively. Since there is little known about the effects of Excitins, we clarified whether oral administration of Excitin-1 affects behavior in rats, alters the monoamine and amino acid levels in the central nervous system, whether Excitin-1 is incorporated into the brain, and how long it remains in the blood. Excitin-1 increased motor behavior, increasing the distance of path and number of rearings in the open field. Excitin-1 influenced some monoamine and amino acid levels in the cerebral cortex and hypothalamus. Following oral administration, Excitin-1 was detected in the cerebral cortex, hypothalamus, hippocampus and olfactory bulb. In the plasma, Excitin-1 and its metabolites beta-alanine and L-leucine were recorded. The present study demonstrated that Excitin-1 was incorporated in the brain and promoted behavioral changes in rats.

Change in hepatic and plasma bile acid contents and its regulatory gene expression in the chicken embryo.

To investigate changes in bile acid biosynthesis in chicken (Gallus gallus) during embryonic stages, we studied the contribution of hepatic and plasma total bile acid levels, mRNA expression of cholesterol 7 alpha-hydroxylase (CYP7A1), and the expression of its regulatory genes in two embryo models (i.e., broilers and layers) differing in lipid metabolism. Total bile acid levels in plasma and liver were low during embryonic stages, as well as expression of CYP7A1. At hatch (P0), hepatic and plasma total bile acid levels and CYP7A1 mRNA expression in liver were markedly increased in both models. The hepatic mRNA expression of liver X receptor (LXR)alpha, a regulator of CYP7A1 gene expression gradually decreased with developmental stages of both broilers and layers. The hepatic mRNA expression of farnesoid X receptor (FXR), a repressor of CYP7A1 gene expression, also decreased with embryonic development. The present results showed that the mRNA expression of CYP7A1 and synthesis of bile acids was low in embryonic stages, suggesting that FXR might be a key regulator of CYP7A1 gene expression in the chicken embryo.

Intracerebroventricular injection of neuropeptide Y modifies carbohydrate and lipid metabolism in chicks.

The purpose of the present study was to investigate whether intracerebroventricular (ICV) injection of neuropeptide Y (NPY) affects heat production (HP), body temperature, and plasma concentrations of metabolic fuels in chicks. ICV injection of NPY (0, 188 or 375 pmol) did not affect HP, but significantly lowered respiratory quotient as well as the rectal temperature. These data suggest that the energy sources for HP were modified by NPY in the body. This idea was confirmed by subsequent experiments in which ICV injection of NPY significantly reduced plasma glucose and triacylglycerol concentrations but increased non-esterified fatty acid concentrations. The effect of NPY on the utilization of metabolic fuels was not associated changes in plasma catecholamine and corticosterone concentrations. In summary, the present study demonstrated that central NPY modifies peripheral carbohydrate and lipid metabolism in chicks.

Heat production and lipid metabolism in broiler and layer chickens during embryonic development.

We compared heat production (HP) and lipid metabolism in broiler and layer chickens (Gallus gallus) during embryonic development. To investigate HP and respiratory quotient (RQ), oxygen (O2) consumption and carbon dioxide (CO2) production were measured using an open-circuit calorimeter system. HP consistently had a tendency (P = 0.06) to be lower in broilers than in layers during embryonic development, and HP gradually decreased with developmental stage in both strains. RQ values of both strains were approximately 0.7 at every embryonic stage investigated. These results suggest that chicken embryos mainly use lipid for energy, and the RQ was significantly lower in broilers than in layers during embryonic development. Consumption of the yolk sac as a lipid source was faster in broilers than in layers. Plasma D-3-hydroxybutyrate (D3HB) and glycerol concentrations, associated with fatty acid oxidation, were lower in broiler than layer embryos. These results demonstrate that HP and lipid metabolism are different between the strains during embryonic development, and may be one factor for the growth difference between broiler and layer embryos.

Involvement of CRF on the anorexic effect of GLP-1 in layer chicks.

Glucagon-like peptide-1 (GLP-1) is recognized as an anorexic peptide in the brain of chicks. However, the mechanism underlying the inhibition of feeding has not been well studied. It is reported that GLP-1 activates neurons containing corticotrophin-releasing factor (CRF) in the brain of mammals. Since CRF is also an anorexic peptide, it is possible that the anorexic effect of GLP-1 is mediated by CRF in chicks. The present study was carried out to test this. First, we determined plasma corticosterone (CORT) concentrations after intracerebroventricular (ICV) injection of GLP-1 and found that this treatment increased CORT release in layer chicks. The CORT-releasing effect was partly attenuated by co-injection of astressin, a CRF receptor antagonist, demonstrating that GLP-1 stimulated CORT secretion by activation of CRF neurons. CRF neurons also appear to be involved in mediating the inhibition of food intake by GLP-1 because this effect was also partly attenuated by astressin. Furthermore, we demonstrated that the anorexic effect of GLP-1 was weaker in broiler than layer chicks. The present results suggest that the anorexic effect of GLP-1 might be mediated by CRF neurons in the chick brain and that the sensitivity of the inhibitory response to GLP-1 differs between chick strains.

Nitric oxide involves in carnosine-induced hyperactivity in chicks.

Carnosine has been characterized as a putative neurotransmitter and implicated as having a possible role in neuron-glia cell interactions. We previously confirmed that central administration of carnosine induced hyperactivity in chicks. In the present study, we investigated the effects of nitric oxide (NO) synthase (NOS) inhibitors on carnosine-induced hyperactivity in chicks. Carnosine-induced (3.2 micromol) hyperactivity was attenuated by intracerebroventricular (i.c.v.) co-administration with a non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester HCl (200 and 400 nmol) in a dose-dependent manner, while the hyperactivity was not attenuated by the inactive isomer of the NOS inhibitor N(G)-nitro-D-arginine methyl ester HCl (400 nmol). The i.c.v. injection of a selective inhibitor of inducible NOS (iNOS) L-N(6)-(1-iminoethyl) lysine HCl (400 nmol) did not affect carnosine-induced hyperactivity. These results suggest that carnosine-induced hyperactivity may be linked to the constitutive NOS (cNOS), rather than iNOS, in the brain. Central carnosine may regulate brain function and/or behaviors by NO generation via cNOS in chicks.

Gonadotropin-inhibiting hormone stimulates feeding behavior in chicks.

Neuropeptides containing a C-terminal Arg-Phe-NH2 motif (RFamide peptides) are suggested to be involved in the control of feeding behavior in both invertebrates and vertebrates. Gonadotropin-inhibitory hormone (GnIH) is the first identified avian RFamide peptide that inhibits gonadotropin release from the pituitary. The GnIH precursor encodes one GnIH and its related peptides (GnIH-RP-1 and -RP-2) that shared the same C-terminal motif, Leu-Pro-Xaa-Arg-Phe-NH2 (Xaa = Leu or Gln) (LPXRFamide). GnIH neurons are localized in the paraventricular nucleus, with their fibers visible in multiple brain locations including the median eminence and brainstem. In this study, we therefore investigated the action of GnIH and its related peptides on feeding behavior. Intracerebroventricular (ICV) injection of GnIH, GnIH-RP-1 and GnIH-RP-2 significantly stimulated food intake in chicks. The chicken pentapeptide LPLRFamide, a degraded C-terminus of GnIH and GnIH-RP-1, did not stimulate feeding thereby demonstrating the importance of the N-terminus of GnIH and its related peptides for the orexigenic effect. Anti-GnIH antiserum suppressed appetite induced by fasting, but did not modify feeding under ad libitum conditions. The present study suggests that GnIH and its related peptides act as endogenous orexigenic factors in the brain of chicks.