Tumor suppressor let-7 acts as a key regulator for pluripotency gene expression in Muse cells.

In embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), the expression of an RNA-binding pluripotency-relevant protein, LIN28, and the absence of its antagonist, the tumor-suppressor microRNA (miRNA) let-7, play a key role in maintaining pluripotency. Muse cells are non-tumorigenic pluripotent-like stem cells residing in the bone marrow, peripheral blood, and organ connective tissues as pluripotent surface marker SSEA-3(+). They express pluripotency genes, differentiate into triploblastic-lineage cells, and self-renew at the single cell level. Muse cells do not express LIN28 but do express let-7 at higher levels than in iPSCs. In Muse cells, we demonstrated that let-7 inhibited the PI3K-AKT pathway, leading to sustainable expression of the key pluripotency regulator KLF4 as well as its downstream genes, POU5F1, SOX2, and NANOG. Let-7 also suppressed proliferation and glycolysis by inhibiting the PI3K-AKT pathway, suggesting its involvement in non-tumorigenicity. Furthermore, the MEK/ERK pathway is not controlled by let-7 and may have a pivotal role in maintaining self-renewal and suppression of senescence. The system found in Muse cells, in which the tumor suppressor let-7, but not LIN28, tunes the expression of pluripotency genes, might be a rational cell system conferring both pluripotency-like properties and a low risk for tumorigenicity.

OLIG2 is an in vivo bookmarking transcription factor in the developing neural tube in mouse.

Cells possess intrinsic features that are inheritable via epigenetic regulation, such as DNA methylation and histone modification. These inheritable features maintain a unique gene expression pattern, underlying cellular memory. Because of the degradation or displacement of mitotic chromosomes, most transcription factors do not contribute to cellular memory. However, accumulating in vitro evidence indicates that some transcription factors can be retained in mitotic chromosomes called as bookmarking. Such transcription factors may contribute to a novel third mechanism of cellular memory. Since most findings of transcription factor bookmarking have been reported in vitro, little is currently known in vivo. In the neural tube of mouse embryos, we discovered that OLIG2, a basic helix loop helix (bHLH) transcription factor that regulates proliferation of neural progenitors and the cell fate of motoneurons and oligodendrocytes, binds to chromatin through every cell cycle including M-phase. OLIG2 chromosomal localization coincides with mitotic cell features such as the phosphorylation of histone H3, KI67, and nuclear membrane breakdown. Chromosomal localization of OLIG2 is regulated by an N-terminus triple serine motif. Photobleaching analysis revealed slow OLIG2 mobility, suggesting a high affinity of OLIG2 to DNA. In Olig2 N-terminal deletion mutant mice, motoneurons and oligodendrocyte progenitor numbers are reduced in the neural tube, suggesting that the bookmarking regulatory domain is important for OLIG2 function. We conclude that OLIG2 is a de novo in vivo bookmarking transcription factor. Our results demonstrate the presence of in vivo bookmarking in a living organism and illustrate a novel function of transcription factors.

Establishment of neural stem cell culture from the central nervous system of the Iberian ribbed newt Pleurodeles waltl.

Urodele amphibians have exceptional regeneration ability in various organs. Among these, the Iberian ribbed newt (Pleurodeles waltl) has emerged as a useful model organism for investigating the mechanisms underlying regeneration. Neural stem cells (NSCs) are an important source of regeneration in the central nervous system (CNS) and their culture method in vitro has been well established. NSCs form spherical cell aggregates called neurospheres and their formation has been demonstrated in various vertebrates, including some urodele species, but not in P. waltl. In this study, we reported neurosphere formation in brain- and spinal cord-derived cells of post-metamorphic P. waltl. These neurospheres showed proliferative activity and similar expression of marker proteins. However, the surface morphology was found to vary according to their origin, implying that the characteristics of the neurospheres generated from the brain and spinal cord could be similar but not identical. Subsequent in vitro differentiation analysis demonstrated that spinal cord-derived neurospheres gave rise to neurons and glial cells. We also found that cells in neurospheres from P. waltl differentiated to oligodendrocytes, whereas those from axolotls were reported not to differentiate to this cell type under standard culture conditions. Based on our findings, implantation of genetically modified neurospheres together with associated technical advantages in P. waltl could reveal pivotal gene(s) and/or signaling pathway(s) essential for the complete spinal cord regeneration ability in the future.

Cytoplasmic Polyadenylation Element-Binding Protein 1 Post-transcriptionally Regulates Fragile X Mental Retardation 1 Expression Through 3' Untranslated Region in Central Nervous System Neurons.

Fragile X syndrome (FXS) is an inherited intellectual disability caused by a deficiency in Fragile X mental retardation 1 (Fmr1) gene expression. Recent studies have proposed the importance of cytoplasmic polyadenylation element-binding protein 1 (CPEB1) in FXS pathology; however, the molecular interaction between Fmr1 mRNA and CPEB1 has not been fully investigated. Here, we revealed that CPEB1 co-localized and interacted with Fmr1 mRNA in hippocampal and cerebellar neurons and culture cells. Furthermore, CPEB1 knockdown upregulated Fmr1 mRNA and protein levels and caused aberrant localization of Fragile X mental retardation protein in neurons. In an FXS cell model, CPEB1 knockdown upregulated the mRNA levels of several mitochondria-related genes and rescued the intracellular heat shock protein family A member 9 distribution. These findings suggest that CPEB1 post-transcriptionally regulated Fmr1 expression through the 3' untranslated region, and that CPEB1 knockdown might affect mitochondrial function.

A rare genetic variant in the cleavage site of prepro-orexin is associated with idiopathic hypersomnia.

Idiopathic hypersomnia (IH) is a rare, heterogeneous sleep disorder characterized by excessive daytime sleepiness. In contrast to narcolepsy type 1, which is a well-defined type of central disorders of hypersomnolence, the etiology of IH is poorly understood. No susceptibility loci associated with IH have been clearly identified, despite the tendency for familial aggregation of IH. We performed a variation screening of the prepro-orexin/hypocretin and orexin receptors genes and an association study for IH in a Japanese population, with replication (598 patients and 9826 controls). We identified a rare missense variant (g.42184347T>C; p.Lys68Arg; rs537376938) in the cleavage site of prepro-orexin that was associated with IH (minor allele frequency of 1.67% in cases versus 0.32% in controls, P = 2.7 × 10-8, odds ratio = 5.36). Two forms of orexin (orexin-A and -B) are generated from cleavage of one precursor peptide, prepro-orexin. The difference in cleavage efficiency between wild-type (Gly-Lys-Arg; GKR) and mutant (Gly-Arg-Arg; GRR) peptides was examined by assays using proprotein convertase subtilisin/kexin (PCSK) type 1 and PCSK type 2. In both PCSK1 and PCSK2 assays, the cleavage efficiency of the mutant peptide was lower than that of the wild-type peptide. We also confirmed that the prepro-orexin peptides themselves transmitted less signaling through orexin receptors than mature orexin-A and orexin-B peptides. These results indicate that a subgroup of IH is associated with decreased orexin signaling, which is believed to be a hallmark of narcolepsy type 1.

Sulfatide with ceramide composed of phytosphingosine (t18:0) and 2-hydroxy FAs in renal intercalated cells.

Diverse molecular species of sulfatide with differences in FA lengths, unsaturation degrees, and hydroxylation statuses are expressed in the kidneys. However, the physiological functions of specific sulfatide species in the kidneys are unclear. Here, we evaluated the distribution of specific sulfatide species in the kidneys and their physiological functions. Electron microscopic analysis of kidneys of Cst-deficient mice lacking sulfatide showed vacuolar accumulation in the cytoplasm of intercalated cells in the collecting duct, whereas the proximal and distal tubules were unchanged. Immunohistochemical analysis revealed that vacuolar H+-ATPase-positive vesicles were accumulated in intercalated cells in sulfatide-deficient kidneys. Seventeen sulfatide species were detected in the murine kidney by iMScope MALDI-MS analysis. The distribution of the specific sulfatide species was classified into four patterns. Although most sulfatide species were highly expressed in the outer medullary layer, two unique sulfatide species of m/z 896.6 (predicted ceramide structure: t18:0-C22:0h) and m/z 924.6 (predicted ceramide structure: t18:0-C24:0h) were dispersed along the collecting duct, implying expression in intercalated cells. In addition, the intercalated cell-enriched fraction was purified by fluorescence-activated cell sorting using the anti-vacuolar H+-ATPase subunit 6V0A4, which predominantly contained sulfatide species (m/z 896.6 and 924.6). The Degs2 and Fa2h genes, which are responsible for ceramide hydroxylation, were expressed in the purified intercalated cells. These results suggested that sulfatide molecular species with ceramide composed of phytosphingosine (t18:0) and 2-hydroxy FAs, which were characteristically expressed in intercalated cells, were involved in the excretion of NH3 and protons into the urine.

Cpeb1 expression is post-transcriptionally regulated by AUF1, CPEB1, and microRNAs.

Cytoplasmic polyadenylation element binding protein 1 (CPEB1) regulates the translation of numerous mRNAs. We previously showed that AU-rich binding factor 1 (AUF1) regulates Cpeb1 expression through the 3' untranslated region (3'UTR). To investigate the molecular basis of the regulatory potential of the Cpeb1 3'UTR, here we performed reporter analyses that examined expression levels of Gfp reporter mRNA containing the Cpeb1 3'UTR. Our findings indicate that CPEB1 represses the translation of Cpeb1 mRNA and that miR-145a-5p and let-7b-5p are involved in the reduction in Cpeb1 expression in the absence of AUF1. These results suggest that Cpeb1 expression is post-transcriptionally regulated by AUF1, CPEB1, and microRNAs.

Matrix Metalloproteinases in Human Decidualized Endometrial Stromal Cells.

Cyclic changes, such as growth, decidualization, shedding, and regeneration, in the human endometrium are regulated by the reciprocal action of female hormones, such as estradiol (E2), and progesterone (P4). Matrix metalloproteases (MMPs) and tissue inhibitors of MMPs (TIMPs) control the invasion of extravillous trophoblast cells after implantation. Several MMPs and TIMPs function in the decidua and endometrial stromal cells (ESCs). Here, we aimed to systematically investigate the changes in MMPs and TIMPs associated with ESC decidualization. We evaluated the expression of 23 MMPs, four TIMPs, and four anti-sense non-coding RNAs from MMP loci. Primary ESC cultures treated with E2 + medroxyprogesterone acetate (MPA), a potent P4 receptor agonist, showed significant down-regulation of MMP3, MMP10, MMP11, MMP12, MMP20, and MMP27 in decidualized ESCs, as assessed by quantitative reverse transcription PCR. Further, MMP15 and MMP19 were significantly upregulated in decidualized ESCs. siRNA-mediated silencing of Heart and Neural Crest Derivatives Expressed 2 (HAND2), a master transcriptional regulator in ESC decidualization, significantly increased MMP15 expression in untreated human ESCs. These results collectively indicate the importance of MMP15 and MMP19 in ESC decidualization and highlight the role of HAND2 in repressing MMP15 transcription, thereby regulating decidualization.

Transcriptional regulation of LGALS9 by HAND2 and FOXO1 in human endometrial stromal cells in women with regular cycles.

Uterine natural killer cells are regulated via surface inhibitory receptors for IL15 and galectin-9 (LGALS9) secreted by endometrial stromal cells (ESCs). However, the mechanism that regulates LGALS9 mRNA levels in ESCs is unclear. The aim of this study is to clarify the transcriptional regulation of LGALS9 in ESCs. Here, LGALS9 mRNA expression levels significantly decreased in the endometrial tissue in the early- to mid-secretory phase, and recovered in the mid- to late-secretory phase, compared to that in the proliferative phase. In ESCs, LGALS9 mRNA expression significantly decreased following estradiol + medroxyprogesterone acetate treatment for 1 day and increased after 12 days compared to that in the control. The transcriptional activity of the LGALS9 upstream region was upregulated by heart and neural crest derivatives expressed 2 (HAND2) and downregulated by forkhead box O1 (FOXO1). In ESCs, HAND2 expression significantly increased throughout the 12 days treatment with steroid hormones, whereas FOXO1 expression significantly increased on Day 1, reached a plateau, and significantly increased again after 6 days of treatment. Levels of FOXO1 phosphorylation (pFOXO1) remained unchanged after a 3-day treatment of ESCs with steroid hormones, but significantly increased following a 12-day treatment. pFOXO1 could not bind to the DNA and was thus unable to directly suppress LGALS9 transcription. Therefore, expression level of HAND2 and phosphorylation status of FOXO1 may determine LGALS9 mRNA expression. This study provides a novel molecular mechanism underlying the transcriptional regulation of LGALS9 mRNA in ESCs, which could be valuable in the treatment of diseases associated with decidualization failure.

Distribution, fine structure, and three-dimensional innervation of lamellar corpuscles in rat plantar skin.

Lamellar corpuscles function as mechanoreceptors in the skin, composed of axon terminals and lamellae constructed by terminal Schwann cells. They are classified into Pacinian, Meissner, and simple corpuscles based on histological criteria. Lamellar corpuscles in rat dermal papilla cells have been reported; however, the morphological aspects have yet to be thoroughly investigated. In the present study, we analyzed the enzyme activity, distribution, fine structure, and three-dimensional innervation of lamellar corpuscles in rat plantar skin. The lamellar corpuscles exhibiting non-specific cholinesterase were densely distributed in rat footpads, evident as notable skin elevations, especially at the apex, the highest portion of the ridges in each footpad. In contrast, only a few lamellar corpuscles were found in other plantar skin areas. Lamellar corpuscle was considered composed of a flat axon terminal Schwann cell lamellae, which were roughly concentrically arranged in the dermal papilla. These histological characteristics correspond to those of the simple corpuscle. Moreover, the axon tracing method revealed that one trunk axon innervated several simple corpuscles. The territory of the trunk axons overlapped with each other. Finally, the animals' footprints were analyzed. During the pausing and walking phases, footpads are often in contact with the floor. These results demonstrate that the type of lamellar corpuscles in the dermal papillae of rat plantar skin is a simple corpuscle and implies that their distribution pattern in the plantar skin is convenient for efficient sensing and transmission of mechanical stimuli from the ground.

Isolation and characterization of bone marrow-derived mesenchymal stem cells in Xenopus laevis.

Mesenchymal stem cells (MSCs) are multipotent cells that exist in mesenchymal tissues such as bone marrow and are able to differentiate into osteocytes, chondrocytes, and adipocytes. MSCs are generally collected as adherent cells on a plastic dish, and are positive for markers such as CD44, CD73, CD90, CD105 and CD166, and negative for CD11b, CD14, CD19, CD31, CD34, CD45, CD79a and HLA-DR. MSCs have been established from many kinds of mammals, but MSCs from amphibians have not yet been reported. We cultured adherent cells from the bone marrow of Xenopus laevis by modifying the protocol for culturing mammalian MSCs. The morphology of these cells was similar to that of mammalian MSCs. The amphibian MSCs were positive for cd44, cd73, cd90 and cd166, and negative for cd11b, cd14, cd19, cd31, cd34, cd45, cd79a and hla-dra. Moreover, they could be induced to differentiate into osteocyte-, chondrocyte-, and adipocyte-lineage cells by cytokine induction systems that were similar to those used for mammalian MSC differentiation. Thus, they are considered to be similar to mammalian MSCs. Unlike mammals, amphibians have high regenerative capacity. The findings from the present study will allow for future research to reveal how Xenopus MSCs are involved in the amphibian regenerative capacity and to elucidate the differences in the regenerative capacity between mammals and amphibians.

Keto form of curcumin derivatives strongly binds to Aß oligomers but not fibrils.

The accumulation of ß-amyloid (Aß) aggregates in the brain occurs early in the progression of Alzheimer's disease (AD), and non-fibrillar soluble Aß oligomers are particularly neurotoxic. During binding to Aß fibrils, curcumin, which can exist in an equilibrium state between its keto and enol tautomers, exists predominantly in the enol form, and binding activity of the keto form to Aß fibrils is much weaker. Here we described the strong binding activity the keto form of curcumin derivative Shiga-Y51 shows for Aß oligomers and its scant affinity for Aß fibrils. Furthermore, with imaging mass spectrometry we revealed the blood-brain barrier permeability of Shiga-Y51 and its accumulation in the cerebral cortex and the hippocampus, where Aß oligomers were mainly localized, in a mouse model of AD. The keto form of curcumin derivatives like Shiga-Y51 could be promising seed compounds to develop imaging probes and therapeutic agents targeting Aß oligomers in the brain.

Comparison of separation methods for tissue-derived extracellular vesicles in the liver, heart, and skeletal muscle.

Extracellular vesicles (EVs), which are nanosized vesicles released by cells as intracellular messengers, have high potential as biomarkers. EVs are usually collected from in vitro sources, such as cell culture media or biofluids, and not from tissues. Techniques enabling direct collection of EVs from tissues will extend the applications of EVs. We compared methods for separating EVs from solid liver, heart, and skeletal muscle. Compared with a precipitation method, an ultracentrifugation-based method for collection of EVs from solid tissues yielded a higher proportion of EVs positive for EV-related markers, with minimum levels of intracellular organelle-related markers. Some tissue-specific modifications, such as a sucrose cushion step, may improve the yield and purity of the collected EVs.

AUF1, an mRNA decay factor, has a discordant role in Cpeb1 expression.

Cytoplasmic polyadenylation element binding protein 1 (CPEB1) regulates polyadenylation and subsequent translation of CPE-containing mRNAs involved in various physiological and pathological phenomena. Although the significance of CPEB1-mediated translational regulation has recently been reported, the detailed regulatory mechanism of Cpeb1 expression remains unclear. To elucidate the post-transcriptional regulatory mechanisms of Cpeb1 expression, we constructed reporter plasmids containing various deletions or mutations in the Cpeb1 mRNA 3' untranslated region (3'UTR). We investigated their expression levels in Neuro2a neuroblastoma cells. We found that Cpeb1 expression is regulated through an AU-rich element in its 3'UTR. Furthermore, the mRNA decay factor AU-rich binding factor 1 (AUF1) regulates Cpeb1 expression, and knockdown of AUF1 upregulates Cpeb1 mRNA expression but results in a decrease in CPEB1 protein levels. These findings indicate that AUF1 has a discordant role in the expression of Cpeb1.

Downsizing effect of a modular radial head prosthesis on the lateral collateral ligament of the elbow: A cadaveric study.

BACKGROUND: It remains unclear how the head and stem diameters for the radial head prosthesis could affect mechanical properties of the lateral collateral ligament measured by strain changes during elbow and forearm motions. METHODS: Eight cadaveric specimens were secured to the device, which allows elbow flexion-extension and forearm pro-supination. Using six different implant combinations comprising 2 sizes for the head (long- and short-axis of the native head) and 3 sizes for the stem (press-fit, -1 mm, and -2 mm downsizing), prostheses were attached via the posterior approach. A differential variable reluctance transducer placed on the central portion of the radial collateral ligament were used for strain measurement with elbow flexion at 0°, 30°, 60°, and 90°. At each position, the strain patterns with the forearm in the neutral and 45° pro-supination positions were also assessed. FINDINGS: Specimens implanted with long-axis head component showed greater increases in the ligament strain during elbow flexion than intact specimens or those implanted with short-axis head. Compared to press-fit stem, implants with downsizing to -1 mm approximated strain patterns during pro-supination with elbow extension to intact condition. INTERPRETATION: Morphologic variation of the head and stem components in radial head prostheses led to altered strain patterns in the lateral collateral ligament during elbow and forearm motions. A short-axis head component can be used to prevent excessive strain changes after the prosthesis application. Downsizing of the stem component might be an option for approximating the biomechanics at the radiocapitellar joint during forearm rotation to the intact elbow.

A Novel Type of Stem Cells Double-Positive for SSEA-3 and CD45 in Human Peripheral Blood.

Peripheral blood (PB) contains several types of stem/progenitor cells, including hematopoietic stem and endothelial progenitor cells. We identified a population positive for both the pluripotent surface marker SSEA-3 and leukocyte common antigen CD45 that comprises 0.04% ± 0.003% of the mononuclear cells in human PB. The average size of the SSEA-3(+)/CD45(+) cells was 10.1 ± 0.3 µm and ∼22% were positive for CD105, a mesenchymal marker; ∼85% were positive for CD19, a B cell marker; and ∼94% were positive for HLA-DR, a major histocompatibility complex class II molecule relevant to antigen presentation. These SSEA-3(+)/CD45(+) cells expressed the pluripotency markers Nanog, Oct3/4, and Sox2, as well as sphingosine-1-phosphate (S1P) receptor 2, and migrated toward S1P, although their adherence and proliferative activities in vitro were low. They expressed NeuN at 7 d, Pax7 and desmin at 7 d, and alpha-fetoprotein and cytokeratin-19 at 3 d when supplied to mouse damaged tissues of the brain, skeletal muscle and liver, respectively, suggesting the ability to spontaneously differentiate into triploblastic lineages compatible to the tissue microenvironment. Multilineage-differentiating stress enduring (Muse) cells, identified as SSEA-3(+) in tissues such as the bone marrow and organ connective tissues, express pluripotency markers, migrate to sites of damage via the S1P-S1P receptor 2 system, and differentiate spontaneously into tissue-compatible cells after homing to the damaged tissue where they participate in tissue repair. After the onset of acute myocardial infarction and stroke, patients are reported to have an increase in the number of SSEA-3(+) cells in the PB. The SSEA-3(+)/CD45(+) cells in the PB showed similarity to tissue-Muse cells, although with difference in surface marker expression and cellular properties. Thus, these findings suggest that human PB contains a subset of cells that are distinct from known stem/progenitor cells, and that CD45(+)-mononuclear cells in the PB comprise a novel subpopulation of cells that express pluripotency markers.

The transcription factor HAND2 up-regulates transcription of the IL15 gene in human endometrial stromal cells.

Cyclic changes of the human endometrium, such as proliferation, secretion, and decidualization, occur during regular menstrual cycles. Heart- and neural crest derivatives-expressed transcript 2 (HAND2) is a key transcription factor in progestin-induced decidualization of human endometrial stromal cells (ESCs). It has been suggested that HAND2 regulates interleukin 15 (IL15), a key immune factor required for the activation and survival of uterine natural killer (uNK) cells. Activated uNK cells can promote spiral artery remodeling and secrete cytokines to induce immunotolerance. To date, no studies have evaluated the transcription factors that regulate IL15 expression in human ESCs. In the present study, we examined whether HAND2 controls IL15 transcriptional regulation in human ESCs. Quantitative RT-PCR and histological analyses revealed that HAND2 and IL15 levels increase considerably in the secretory phase of human endometrium tissues. Results from ChIP-quantitative PCR suggested that HAND2 binds to a putative HAND2 motif, which we identified in the upstream region of the human IL15 gene through in silico analysis. Using a luciferase reporter assay, we found that the upstream region of the human IL15 gene up-regulates reporter gene activities in response to estradiol and a progestin representative (medroxyprogesterone) in ESCs. The upstream region of the human IL15 gene also exhibited increasing responsiveness to transfection with a HAND2 expression vector. Of note, deletion and substitution variants of the putative HAND2 motif in the upstream region of IL15 did not respond to HAND2 transfection. These findings confirm that HAND2 directly up-regulates human IL15 transcription in ESCs.

GATA4/6 regulate DHH transcription in rat adrenocortical autografts.

Adrenal cortex autotransplantation with ACTH stimulation may be an alternative therapy for patients with bilateral adrenalectomy to avoid adrenal crisis, but its underlying mechanism has not been elucidated. Previously, we detected Dhh upregulation in rat adrenocortical autografts after transplantation. Here, we investigated potential regulators such as Gata4, Gata6, Sry and Sox9 which affect Dhh transcription in adrenocortical autografts with or without ACTH stimulation. In ACTH-stimulated autografts, Gata4 and Gata6 were downregulated compared to control autografts. This response was linked to rDhh repression. A reporter assay using the upstream region of rDhh and a GATA binding motif revealed that rDhh promoters were significantly upregulated by co-transfection with Gata4 or Gata6 or both. Sry and Sox9 expression in autografts with or without ACTH stimulation were verified by PCR and RNAscope analyses. The ovarian differentiation factors Foxl2 and Rspo1 were also upregulated in the autografts. Gata4 and Gata6 were found to be significant factors in the regulation of rDhh expression and could be associated with adrenocortical autograft maintenance. Gonadal primordia with bipotential testicular and ovarian functions may also be present in these autografts.

18-Oxocortisol Synthesis in Aldosterone-Producing Adrenocortical Adenoma and Significance of KCNJ5 Mutation Status.

Peripheral 18-oxocortisol (18oxoF) level could contribute to the detection of aldosterone-producing adenoma (APA) in patients with primary aldosteronism. However, peripheral 18oxoF varies among such patients, which is a big drawback concerning its clinical application. We studied 48 cases of APA, 35 harboring KCNJ5 mutation, to clarify the significance of clinical and pathological parameters about peripheral 18oxoF. Peripheral 18oxoF concentration ranged widely from 0.50 to 183.13 ng/dL and correlated positively with intratumoral areas stained positively for steroidogenic enzymes ( P<0.0001). The peripheral 18oxoF level also correlated significantly with that of circulating aldosterone ( P<0.0001) but not with that of cortisol, a precursor of 18oxoF. However, a significant correlation was detected between peripheral 18oxoF and intratumoral glucocorticoids ( P<0.05). In addition, peripheral 18oxoF correlated positively with the number of hybrid cells double positive for 11ß-hydroxylase and aldosterone synthase ( P<0.0001). Comparing between the cases with and those without KCNJ5 mutation, the KCNJ5-mutated group demonstrated a significantly higher concentration of peripheral 18oxoF (28.4±5.6 versus 3.0±0.9 ng/dL, P<0.0001) and a larger intratumoral environment including the hybrid cells ( P<0.001), possibly representing a deviation from normal aldosterone biosynthesis. After multivariate analysis, KCNJ5 mutation status turned out to be the most associated factor involved in 18oxoF synthesis in APA ( P<0.0001). Results of our present study first revealed that enhanced 18oxoF synthesis in APA could come from a functional deviation of aldosterone biosynthesis from the normal zona glomerulosa and the utility of peripheral 18oxoF measurement could be influenced by the prevalence of KCNJ5 mutation in an APA.

Direct conversion of adult human skin fibroblasts into functional Schwann cells that achieve robust recovery of the severed peripheral nerve in rats.

Direct conversion is considered a promising approach to obtain tissue-specific cells for cell therapies; however, this strategy depends on exogenous gene expression that may cause undesired adverse effects such as tumorigenesis. By optimizing the Schwann cell induction system, which was originally developed for trans-differentiation of bone marrow mesenchymal stem cells into Schwann cells, we established a system to directly convert adult human skin fibroblasts into cells comparable to authentic human Schwann cells without gene introduction. Serial treatments with beta-mercaptoethanol, retinoic acid, and finally a cocktail of basic fibroblast growth factor, forskolin, platelet-derived growth factor-AA, and heregulin-ß1 (EGF domain) converted fibroblasts into cells expressing authentic Schwann cell markers at an efficiency of approximately 75%. Genome-wide gene expression analysis suggested the conversion of fibroblasts into the Schwann cell-lineage. Transplantation of induced Schwann cells into severed peripheral nerve of rats facilitated axonal regeneration and robust functional recovery in sciatic function index comparable to those of authentic human Schwann cells. The contributions of induced Schwann cells to myelination of regenerated axons and re-formation of neuromuscular junctions were also demonstrated. Our data clearly demonstrated that cells comparable to functional Schwann cells feasible for the treatment of neural disease can be induced from adult human skin fibroblasts without gene introduction. This direct conversion system will be beneficial for clinical applications to peripheral and central nervous system injuries and demyelinating diseases.