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Dermal papilla cells (DPCs) are one type of mesenchymal cells; they play a key role on hair follicle induction. Their hair inductivity and proliferation abilities are rapidly lost during the 2-dimensional culture. Cell senescence is induced by inadequate culture conditions and telomere shortening. We previously reported that overexpression of TERT coding telomerase reverse transcriptase and BMI1 coding human B-cell-specific Moloney murine leukemia virus insertion region 1 (BMI1) avoided senescence of murine DPC and restored hair inductive activity.To evaluate the function of TERT and BMI1 in the human DPCs (hDPCs).Cultured hDPCs obtained from human scalp hair were transduced with TERT alone (hDP-T), BMI1 alone (hDP-B), both TERT and BMI1 (hDP-TB) and empty vector (hDP-E). The hair inductive activity of those cells was assessed by chamber assay in vivo. Gene expressions were analyzed by quantitative PCR (q-PCR).hDP-TB proliferated more than hDP-T and hDP-B in vitro and only hDP-TB showed hair inductivity in vivo. Moreover, the expressions of VCAN, CTNNB1, LEF1, FGF7 and VEGFA in hDP-TB were elevated compared to those in hDP-E.Overexpression of both TERT and BMI1 extends the life span of cultured hDPCs and ameliorates their hair inducing ability on mouse hair follicles.

The formation and deposition of immune complexes (IC) containing immunoglobulin (Ig)G antibodies induces an acute inflammatory response with tissue injury. One of the experimental models of IC-related vasculitis is the cutaneous reverse passive Arthus reaction, in which IgG antibodies are injected i.d., followed immediately by the i.v. application of the corresponding antigen. This reaction is characterized by edema, hemorrhage and neutrophil infiltration. To assess the role of the anti-inflammatory cytokine interleukin (IL)-10 in IC-related vasculitis, we investigated the cutaneous Arthus reaction using IL-10 knockout (IL-10KO) mice. Edema, which was quantified macroscopically by measuring the vascular leakage of Evans blue dye at 4 h after IC challenge, was significantly increased in IL-10KO mice compared with wild-type mice. In addition, hemorrhage, which was assessed by the average diameter of purpuric spots at 8 h after IC challenge, was enhanced significantly in IL-10KO mice compared with wild-type mice. Histological examination showed that the number of extravascular neutrophils was significantly increased in IL-10KO mice compared with wild-type mice at 4 and 8 h after IC challenge. Analysis of pro-inflammatory cytokine mRNA expression showed that IL-6 mRNA levels were significantly increased in IL-10KO mice compared with wild-type mice at 4 and 8 h after IC challenge. These results showed that IC-induced inflammation and vascular damage were significantly enhanced in the absence of IL-10. Thus, IL-10 may limit tissue disruption by suppressing the excessive infiltration of neutrophils and cytokine expression in a mouse model of type III vasculitis.

Lymphatic transport plays an important role in coordinating local immune responses. However, the biologic effects of impaired lymphatic flow in vivo are not fully understood. In this study, we investigated the roles of the lymphatic system in skin carcinogenesis and psoriasis-like inflammation using k-cyclin transgenic (kCYC+/−) mice, which demonstrate severe lymphatic dysfunction. kCYC+/− mice showed augmented tumor growth in the two-stage skin carcinogenesis model as well as severe clinical scores in imiquimod-induced psoriasis-like skin inflammation compared to wild-type mice. Although mRNA levels of inflammatory cytokines in skin after topical application of 12-O-tetradecanoylphorbol-13-acetate or imiquimod were comparable between kCYC+/– and wild-type mice, protein levels of inflammatory cytokines such as interleukin (IL)-17A, IL-22, and IL-23 were significantly upregulated in kCYC+/– mice in both models. Consistently, signal transducer and activator of transcription 3 pathway and nuclear factor-κB signaling were augmented in epidermal keratinocytes in kCYC+/– mice. These results suggest that lymphatic dysfunction in kCYC+/– mice caused accumulation of inflammatory cytokines, leading to the exacerbation of two-stage skin carcinogenesis and imiquimod-induced psoriasis-like skin inflammation. These findings add insight into the clinical problems of secondary malignancies and inflammatory dermatoses that may occur with extremity lymphedema.

Diabetes mellitus is caused by breakdown of blood glucose homeostasis, which is maintained by an exquisite balance between insulin and glucagon produced respectively by pancreatic beta cells and alpha cells. However, little is known about the mechanism of inducing glucagon secretion from human alpha cells. Many methods for generating pancreatic beta cells from human pluripotent stem cells (hPSCs) have been reported, but only two papers have reported generation of pancreatic alpha cells from hPSCs. Because NKX6.1 has been suggested as a very important gene for determining cell fate between pancreatic beta and alpha cells, we searched for the factors affecting expression of NKX6.1 in our beta cell differentiation protocols. We found that BMP antagonism and activation of retinoic acid signaling at stage 2 (from definitive endoderm to primitive gut tube) effectively suppressed NKX6.1 expression at later stages. Using two different hPSCs lines, treatment with BMP signaling inhibitor (LDN193189) and retinoic acid agonist (EC23) at Stage 2 reduced NKX6.1 expression and allowed differentiation of almost all cells into pancreatic alpha cells in vivo after transplantation under a kidney capsule. Our study demonstrated that the cell fate of pancreatic cells can be controlled by adjusting the expression level of NKX6.1 with proper timing of BMP antagonism and activation of retinoic acid signaling during the pancreatic differentiation process. Our method is useful for efficient induction of pancreatic alpha cells from hPSCs.

To identify factors involved in the earliest phase of the differentiation of human embryonic stem cells (hESCs) into brown adipocytes (BAs), we performed multi-time point microarray analyses. We found that growth/differentiation factor 15 (GDF15) expressions were specifically upregulated within three days of differentiation, when expressions of immature hESC markers were sustained. Although GDF15 expressions continued to increase in the subsequent differentiation phases, GDF15-deficient hESCs differentiated into mature BAs (Day 10) without apparent abnormalities. In addition, GDF15-deficient mice had normal brown adipose tissue (BAT) and were metabolically healthy. Unexpectedly, we found that interleukin-6 (IL6) expression was significantly lowered in the BAT of GDF15-/- mice. In addition, GDF15-/- hESCs showed abortive IL6 expressions in the later phase (&gt, Day 6) of the differentiation. Interestingly, GDF15 expression was markedly repressed throughout the whole course of the differentiation of IL6-/- hESCs into BAs, indicating IL6 is essential for the induction of GDF15 in the differentiation of hESCs. Finally, intraperitoneally transplanted BAT grafts of GDF15-/- donor mice, but not those of wild-type (WT) mice, failed in the long-term survival (12 weeks) in GDF15-/- recipient mice. Collectively, GDF15 is required for long-term survival of BAT grafts by creating a mutual gene induction loop with IL6.

Peribiliary glands (PBGs) are accessory glands with mucinous and serous acini in the biliary tree. The PBG is composed of a heterogeneous cell population, such as mucus- and pancreatic enzyme-producing epithelial cells, whereas it constitutes niches for multipotential stem/progenitor cells in the human extrahepatic bile duct (EHBD). By contrast, the nature of PBGs in the mouse EHBD remains unclear. Our aim was to establish a method for isolating and characterizing PBG-constituting cells in the mouse EHBD. We found that trophoblast cell surface protein 2 (Trop2) was expressed in the luminal epithelium of mouse EHBD exclusively, but not in the PBG. On the basis of the differential expression profile of Trop2, lumen-forming biliary epithelial cells (LBECs) and PBG-constituting epithelial cells (PBECs) were separately isolated for further characterization. Gene expression analysis revealed that the isolated mouse PBECs expressed several marker genes related to human PBGs. In the colony formation assay, PBECs showed significantly higher colony formation capacity than LBECs. In the organoid formation assay, PBECs formed cystic organoid with LBEC-like phenotype. Interestingly, PBECs proliferated, accompanied by reexpression of Trop2 in vivo after bile duct ligation. Furthermore, the unique expression profile of Trop2 was conserved in human EHBD. Our findings indicate that Trop2 is a useful marker in investigating the pathophysiological roles and characteristics of mouse and human PBGs in biliary diseases.

Fibrosis is an important wound-healing process in injured tissues, but excessive fibrosis is often observed in patients with chronic inflammation. Although oncostatin M (OSM) has been reported to play crucial roles for recovery from acute liver injury by inducing tissue inhibitor of metalloproteinase 1 (Timp1) expression, the role of OSM in chronic liver injury (CLI) is yet to be elucidated. Here, we show that OSM exerts powerful fibrogenic activity by regulating macrophage activation during CLI. Genetic ablation of the OSM gene alleviated fibrosis in a mouse model of chronic hepatitis. Conversely, continuous expression of OSM in a normal mouse liver by hydrodynamic tail vein injection (HTVi) induced severe fibrosis without necrotic damage of hepatocytes, indicating that OSM is involved in the fundamental process of liver fibrosis (LF) after hepatitis. In a primary coculture of hepatic stellate cells (HSCs) and hepatic macrophages (HMs), OSM up-regulated the expression of fibrogenic factors, such as transforming growth factor-β and platelet-derived growth factor in HMs, while inducing Timp1 expression in HSCs, suggesting the synergistic roles of OSM for collagen deposition in the liver. Fluorescence-activated cell sorting analyses using OSM-HTVi and OSM knockout mice have revealed that bone-marrow–derived monocyte/macrophage are responsive to OSM for profibrotic activation. Furthermore, depletion or blocking of HMs by administration of clodronate liposome or chemokine inhibitor prevented OSM-induced fibrosis. Conclusion: OSM plays a crucial role in LF by coordinating the phenotypic change of HMs and HSCs. Our data suggest that OSM is a promising therapeutic target for LF. (Hepatology 2018;67:296-312).

Common marmosets have attracted considerable attention as a small standard primate model in biomedical research. However, no marmoset diabetes model is available. Here, we established a marmoset diabetes model via the combination of partial pancreatectomy and intravenous streptozotocin (STZ). A partial pancreatectomy was performed in 11 common marmosets and multiple STZ doses were intravenously administered. Diabetes was diagnosed upon sustained hyperglycaemia (nonfasting blood glucose level >200 mg/dl). Blood glucose and biochemistry were periodically assessed, in addition to glucose tolerance testing, continual blood glucose determination using a continuous glucose monitoring system, urine testing and histological evaluation. In 8 of the 11 animals (73%), diabetes mellitus was induced. The diabetic marmosets also showed abnormal intravenous and oral glucose tolerance test results. Blood glucose levels decreased in response to human insulin administration. The hyperglycaemic state was irreversible and persisted for more than 3 months, and the animals’ condition was manageable via daily insulin administration. Thus, diabetes can be successfully induced and maintained in the common marmoset via partial pancreatectomy and STZ administration. This protocol effectively generates a valuable animal model for studying disease pathogenesis, risk factors and therapeutic interventions, including islet transplantation.

Nonalcoholic steatohepatitis (NASH) is a metabolic liver disease that progresses from simple steatosis to the disease state of inflammation and fibrosis. Previous studies suggest that apoptosis and necroptosis may contribute to the pathogenesis of NASH, based on several murine models. However, the mechanisms underlying the transition of simple steatosis to steatohepatitis remain unclear, because it is difficult to identify when and where such cell deaths begin to occur in the pathophysiological process of NASH. In the present study, our aim is to investigate which type of cell death plays a role as the trigger for initiating inflammation in fatty liver. By establishing a simple method of discriminating between apoptosis and necrosis in the liver, we found that necrosis occurred prior to apoptosis at the onset of steatohepatitis in the choline-deficient, ethionine-supplemented (CDE) diet model. To further investigate what type of necrosis is involved in the initial necrotic cell death, we examined the effect of necroptosis and ferroptosis inhibition by administering inhibitors to wild-type mice in the CDE diet model. In addition, necroptosis was evaluated using mixed lineage kinase domain-like protein (MLKL) knockout mice, which is lacking in a terminal executor of necroptosis. Consequently, necroptosis inhibition failed to block the onset of necrotic cell death, while ferroptosis inhibition protected hepatocytes from necrotic death almost completely, and suppressed the subsequent infiltration of immune cells and inflammatory reaction. Furthermore, the amount of oxidized phosphatidylethanolamine, which is involved in ferroptosis pathway, was increased in the liver sample of the CDE diet-fed mice. These findings suggest that hepatic ferroptosis plays an important role as the trigger for initiating inflammation in steatohepatitis and may be a therapeutic target for preventing the onset of steatohepatitis.

We developed a facile fabrication method for preparing poly(ethylene glycol)(PEG)-coated poly (lactic-co-glycolic acid) (PLGA) microspheres with homogeneous size distribution via a combination of mPEG-b-PLGA and Shirasu Porous Glass membrane emulsification. Subsequently, extracellular matrix (ECM) degrading enzymes, collagenase (COLase) or hyaluronidase (HAse) were loaded into the microspheres. The obtained microspheres exhibited a sustained release of COLase or HAse over 10 days. The degradation of ECM polymers by the released COLase and HAse was confirmed in vitro. Reversal of established dermal fibrosis via degradation of over-deposited ECM is a promising treatment for scleroderma. The therapeutic effects of COLase- and HAse-loaded PLGA microspheres on scleroderma were evaluated in vivo following their intradermal administration to a bleomycin-induced mice model of scleroderma. COLase- and HAse-loaded PLGA microspheres decreased scleroderma dermal thickness without altering the mechanical properties of skin, whereas the administration of free COLase and HAse solution induced overdecomposition of skin ECM and α-SMA expression. The facile one-pot synthesis of PEG-coated PLGA microspheres with high colloidal stability and narrow size distribution could be employed as a drug carrier for various diseases in future.

Under chronic or severe liver injury, liver progenitor cells (LPCs) of biliary origin are known to expand and contribute to the regeneration of hepatocytes and cholangiocytes. This regeneration process is called ductular reaction (DR), which is accompanied by dynamic remodeling of biliary tissue. Although the DR shows apparently distinct mode of biliary extension depending on the type of liver injury, the key regulatory mechanism remains poorly understood. Here, we show that Lutheran (Lu)/Basal cell adhesion molecule (BCAM) regulates the morphogenesis of DR depending on liver disease models. Lu+ and Lu- biliary cells isolated from injured liver exhibit opposite phenotypes in cell motility and duct formation capacities in vitro. By overexpression of Lu, Lu- biliary cells acquire the phenotype of Lu+ biliary cells. Lu-deficient mice showed severe defects in DR. Our findings reveal a critical role of Lu in the control of phenotypic heterogeneity of DR in distinct liver disease models., eLife digest Bile is a green to yellow liquid that the body uses to break down and digest fatty molecules. The substance is produced by the liver, and then it is collected and transported to the small bowel by a series of tubes known as the bile duct. When the liver is damaged, the ‘biliary’ cells that line the duct orchestrate the repair of the organ. In fact, the duct often reorganizes itself differently depending on the type of disease the liver is experiencing. For example, the biliary cells can form thin tube-like structures that deeply invade liver tissues, or they can grow into several robust pipes near the existing bile duct. However, it remains largely unknown which protein – or proteins – drive these different types of remodeling. Miura et al. find that, in mice, the biliary cells which invade an injured liver have a large amount of a protein called Lutheran at their surface, but that the cells that form robust ducts do not. This protein helps a cell attach to its surroundings. In addition, the biliary cells can adopt different types of repairing behaviors depending on the amount of Lutheran in their environment. Further experiments show that it is difficult for genetically modified mice without the protein to reshape their bile duct after liver injury. Finally, Miura et al. also detect Lutheran in the remodeling livers of patients with liver disease. Taken together, these results suggest that Lutheran plays an important role in tailoring the repairing roles of the biliary cells to a particular disease. The next step would be to clarify how different liver conditions coordinate the amount of Lutheran in biliary cells to create the right type of remodeling.

Background The dermal papilla is composed of a small clump of mesenchymal cells, called dermal papilla cells (DPCs). DPCs closely interact with epidermal cells to give rise to hair follicles and shafts during hair follicle development and the hair cycle. DPCs are promising cell sources for hair regeneration therapy for alopecia patients. However, once DPCs are put into conventional two-dimensional culture conditions, they quickly lose their capability to produce hair follicles. Objective We aimed to expand a sufficiently large population of DPCs that retain their hair inductive activity. Methods Murine DPCs were cultured in the presence of platelet-derived growth factor-AA (PDGF-AA) and fibroblast growth factor 2 (FGF2). Expressions of follicular-related genes were analyzed by real time PCR and hair inductive activity was determined by patch assay and chamber assay in vivo. Results FGF2 significantly increased the expression of platelet-derived growth factor receptor alpha (PDGFRα) in cultured vibrissal DPCs. PDGF-AA, a ligand of PDGFRα, promoted proliferation of DPCs synergistically when utilized with FGF2 and enhanced the expression of several follicular-related genes in DPCs. Hair reconstitution assays revealed that DPCs treated with both PDGF-AA and FGF-2 were able to maintain their hair inductive activity better than those treated with FGF2 alone. Conclusion Both cell proliferation and hair inductive activity in murine DPCs are maintained by the synergistic effect of FGF2 and PDGF-AA.

Sensitization and challenge using dinitrofluorobenzene (DNFB) induce contact hypersensitivity (CHS) with Th1 cell infiltration, whereas those using FITC generate CHS with Th2 cell infiltration. In this study, we attempted to determine the role of CXCR3, a chemokine receptor, in Th1- and Th2-type CHS induced by DNFB or FITC using CXCR3-deficient (CXCR3−/−) mice. Ear swelling was prolonged after DNFB challenge in CXCR3−/− mice, which was accompanied by increased Th1 cytokines and decreased TGF-β and IL-10 expression at a late time point of CHS, whereas there was no significant difference between wild-type and CXCR3−/− mice in FITC-induced CHS. In Th1-type CHS, the number of regulatory T cells (Tregs) was decreased in the challenged ear of CXCR3−/− mice compared with that of wild-type mice, suggesting that CXCR3 would be important in migration of Tregs into the site of inflammation. Moreover, we examined the characteristics of CXCR3+ Tregs both in vitro and in vivo, revealing that CXCR3+ Tregs expressed high levels of TGF-β and IL-10 as well as IFN-γ compared with CXCR3− Tregs. When CXCR3−/− mice were injected with CXCR3+ Tregs, the prolonged ear swelling induced by DNFB was normalized. Taken together, our results suggest that CXCR3+ Tregs play a key role for quenching Th1-type CHS.

CCL21 expression by lymphatic endothelial cells (LECs) is essential for migration of CCR7+ immune cells from skin to regional lymph nodes (LNs). We investigated the importance of mitogen-activated protein kinase (MAPK) signaling in CCL21 expression by ECs in vitro and in vivo. Normal human dermal lymphatic microvascular ECs (HMVEC-dLy) stimulated in vitro with oncostatin M (OSM) expressed high amounts of CCL21 mRNA. CCL21 protein expression by HMVEC-dLy was also markedly increased by OSM compared with unstimulated cultures. Marked phosphorylation of MAPK 44/42 was detected in HMVEC-dLy stimulated by OSM. CCL21 expression by HMVEC-dLy was blocked by a JAK inhibitor 1, JAK3 inhibitor, and U0126 (a MAPK kinase inhibitor) in vitro, all of which blocked phosphorylation of MAPK 44/42. In addition, injection of U0126 into murine skin significantly decreased CCL21 mRNA and protein expression. Moreover, injection of U0126 before sensitization decreased migration of dendritic cells to draining LNs and decreased contact hypersensitivity responses. In summary, these results suggest that the MAPK pathway is important for CCL21 expression by LECs in vitro and in vivo. Blocking MAPK signaling within skin may offer a novel approach to treatment of inflammatory skin diseases.

Induction of a desired cell type by defined transcription factors (TFs) using iPS technology can be used for cell replacement therapy. However, to overcome problems such as tumor formation, genomic insertional mutagenesis by viral transduction in the induction process needs to be avoided using alternative approaches. One approach could be the direct delivery of TF protein by a protein transduction system, whereby a protein transduction domain (PTD) is fused to facilitate the penetration of cell membrane. However, fusion proteins, including TFs, are reported to be biologically less active through the interference of PTD with proper protein folding. Here, we report a proof-of-concept study in which TF proteins fused with PTDs could be reactivated by removal of PTDs from cells. We demonstrated that Sox2 and Oct3/4 proteins fused with PTD were less active in mouse embryonic stem cells. Removal of PTD by a site-specific protease, derived from tobacco etch virus (TEV), substantially restored the functionality of these proteins, proved by enhanced rescue ability for differentiation induced by endogenous Sox2 and Oct3/4 repression. These results suggest that, by removing a PTD inside the cells, directly delivered TF proteins may exert substantially enhanced function than presently considered.

B cells play critical roles in the pathogenesis of lupus. To examine the influence of B cells on disease pathogenesis in a murine lupus model, New Zealand Black and New Zealand White F1 hybrid (NZB/W) mice were generated that were deficient for CD19 (CD19−/− NZB/W mice), a B cell-specific cell surface molecule that is essential for optimal B cell signal transduction. The emergence of anti-nuclear Abs was significantly delayed in CD19−/− NZB/W mice compared with wild type NZB/W mice. However, the pathologic manifestations of nephritis appeared significantly earlier, and survival was significantly reduced in CD19−/− NZB/W mice compared with wild type mice. These results demonstrate both disease-promoting and protective roles for B cells in lupus pathogenesis. Recent studies have identified a potent regulatory B cell subset (B10 cells) within the rare CD1dhiCD5+ B cell subset of the spleen that regulates acute inflammation and autoimmunity through the production of IL-10. In wild type NZB/W mice, the CD1dhiCD5+B220+ B cell subset that includes B10 cells was increased by 2.5-fold during the disease course, whereas CD19−/− NZB/W mice lacked this CD1dhiCD5+ regulatory B cell subset. However, the transfer of splenic CD1dhiCD5+ B cells from wild type NZB/W mice into CD19−/− NZB/W recipients significantly prolonged their survival. Furthermore, regulatory T cells were significantly decreased in CD19−/− NZB/W mice, but the transfer of wild type CD1dhiCD5+ B cells induced T regulatory cell expansion in CD19−/− NZB/W mice. These results demonstrate an important protective role for regulatory B10 cells in this systemic autoimmune disease.

Insulin-dependent diabetes mellitus (IDDM) is characterized by the rapid development of potentially severe metabolic abnormalities resulting from insulin deficiency. The transplantation of insulin-producing cells is a promising approach for the treatment of IDDM. The transcription factor pancreatic duodenal homeobox 1 (Pdx1) plays an important role in the differentiation of pancreatic beta cells. In this study, the human Pdx1 gene was transduced and expressed in murine adipose tissue-derived stem cells (ASCs). To evaluate pancreatic repair, we used a mouse model of pancreatic damage resulting in hyperglycemia, which involves injection of mice with streptozotocin (STZ). STZ-treated mice transplanted with Pdx1-transduced ASCs (Pdx1-ASCs) showed significantly decreased blood glucose levels and increased survival, when compared with control mice. While stable expression of Pdx1 in ASCs did not induce the pancreatic phenotype in vitro in our experiment, the transplanted stem cells became engrafted in the pancreas, wherein they expressed insulin and C-peptide, which is a marker of insulin-producing cells. These results suggest that Pdx1-ASCs are stably engrafted in the pancreas, acquire a functional beta-cell phenotype, and partially restore pancreatic function in vivo. The ease and safety associated with extirpating high numbers of cells from adipose tissues support the applicability of this system to developing a new cell therapy for IDDM.

Background and Aim: Multipotential mesenchymal stem cells (MSC), present in many organs and tissues, represent an attractive tool for the establishment of a successful stem cell-based therapy in the field of regeneration medicine. Adipose tissue mesenchymal stem cells (AT-MSC), known as adipose-derived stem cells (ASC) are especially attractive in the context of future clinical applications because of their high accessibility and minimal invasiveness during the procedure to obtain them. The goal of the present study was to induce human ASC into functional hepatocytes in vitro within a very short period of time and to check their therapeutic potential in vivo. Methods: In vitro generated ASC-derived hepatocytes were checked for hepatocyte-specific markers and functions. Afterwards, they were transplanted into nude mice with liver injury. Twenty-four hours after transplantation, biochemical parameters were evaluated in blood serum. Results: We have shown here that ASC can be differentiated into hepatocytes within 13 days and can reach the functional properties of primary human hepatocytes. After transplantation into mice with acute liver failure, ASC-derived hepatocytes can restore such liver functions as ammonia and purine metabolism. Markers of liver injury, alanine aminotransferase, aspartate aminotransferase, as well as ammonia, were decreased after ASC-derived hepatocyte transplantation. Conclusions: Our data highlight the properties of ASC as having a special affinity for hepatocyte differentiation in vitro and liver regeneration in vivo. Thus, ASC may be a superior choice for the establishment of a therapy for injured liver.

The pluripotency of embryonic stem (ES) cells is thought to be maintained by a few key transcription factors, including Oct3/4 and Sox2. The function of Oct3/4 in ES cells has been extensively characterized, but that of Sox2 has yet to be determined. Sox2 can act synergistically with Oct3/4 in vitro to activate Oct-Sox enhancers, which regulate the expression of pluripotent stem cell-specific genes, including Nanog, Oct3/4 and Sox2 itself. These findings suggest that Sox2 is required by ES cells for its Oct-Sox enhancer activity. Using inducible Sox2-null mouse ES cells, we show that Sox2 is dispensable for the activation of these Oct-Sox enhancers. In contrast, we demonstrate that Sox2 is necessary for regulating multiple transcription factors that affect Oct3/4 expression and that the forced expression of Oct3/4 rescues the pluripotency of Sox2-null ES cells. These results indicate that the essential function of Sox2 is to stabilize ES cells in a pluripotent state by maintaining the requisite level of Oct3/4 expression.

To analyze the functional differences of the insulin receptor substrate (IRS) family, the N-terminal fragments containing the pleckstrin homology (PH) domains and the phosphotyrosine-binding (PTB) domains of IRS (IRS-N) proteins, as well as intact IRS molecules, were expressed in Cos-1 cells, and insulin-induced tyrosine phosphorylation and subcellular distribution of IRS proteins were analyzed. In contrast to the distinct affinities toward phosphoinositides, these IRS-N fragments non-selectively inhibited insulin-induced tyrosine phosphorylation of IRS-1, IRS-2 and IRS-3, among which IRS3-N was most effective. The mutations of IRS-1 disrupting all the phosphoinositide-binding sites in both the PH and PTB domains significantly but not completely suppressed tyrosine phosphorylation of IRS-1, which was further inhibited by coexpression of all the IRS-N proteins examined. In contrast, the N-terminal PH domain-interacting region (PHIP-N) of PH-interacting protein (PHIP) did not impair tyrosine phosphorylation of either IRS molecule. The analysis using confocal microscopy also demonstrated that all the IRS-N proteins, but not PHIP-N, suppressed targeting of IRS-1 to the plasma membrane in response to insulin. Moreover, the phosphoinositide affinity-disrupting mutations of IRS-1 significantly impaired but did not completely abrogate the insulin-induced translocation of IRS-1 to the plasma membrane, which was further suppressed by IRS1-N overexpression. These findings suggest that both insulin-induced tyrosine phosphorylation and the cell surface targeting of IRS proteins may be regulated in a similar manner through a target molecule common to the members of the IRS family, and distinct from phosphoinositides or PHIP.

The in vitro formation of organs and/or tissues is a major goal for regenerative medicine that would also provide a powerful tool for analyzing both the mechanisms of development and disease processes for each target organ. Here, we present a method whereby pancreatic tissues can be formed in vitro from mouse embryonic stem (ES) cells. Embryoid body-like spheres (EBSs) induced from ES cell colonies were treated with retinoic acid (RA) and activin, which are candidate regulators of pancreatic development in vivo. These induced tissues had decreased expression of the sonic hedgehog (shh) gene and expressed several pancreatic marker genes. ES cell-derived pancreatic tissue was composed of exocrine cells, endocrine cells, and pancreatic duct-like structures. In addition, the ratio of exocrine to endocrine cells in the induced tissue was found to be sensitive to the concentrations of RA and activin in the present experiment.

Insulin-secreting cells have been generated from human embryonic or induced pluripotent stem cells (iPSCs) by mimicking developmental processes. However, these cells do not always secrete glucose-responsive insulin, one of the most important characteristics of pancreatic β-cells. We focused on the importance of endodermal differentiation from human iPSCs in order to obtain functional pancreatic β-cells.A six-stage protocol was established for the differentiation of human iPSCs to pancreatic β-cells using defined culture media without feeders or serum. The effects of CHIR99021, a selective glycogen synthase kinase-3β inhibitor, were examined in the presence of fibroblast growth factor 2, activin, and bone morphogenetic protein 4 (FAB) during definitive endodermal induction by immunostaining for SRY (sex determining region Y)-box 17 (SOX17) and Forkhead box protein A2 (FOXA2). Insulin secretion was compared between the last stage of monolayer culture and spheroid culture conditions. Cultured cells were transplanted under kidney capsules of streptozotocin-diabetic non-obese diabetic-severe combined immunodeficiency mice, and blood glucose levels were measured once a week. Immunohistochemical analyses were performed 4 and 12 weeks after transplantation.Addition of CHIR99021 (3 μmol/L) in the presence of FAB for 2 days improved endodermal cell viability, maintaining the high SOX17-positive rate. Spheroid formation after the endocrine progenitor stage showed more efficient insulin secretion than did monolayer culture. After cell transplantation, diabetic mice had lower blood glucose levels, and islet-like structures were detected in vivo.Functional pancreatic β-cells were generated from human iPSCs. Induction of definitive endoderm and spheroid formation may be key steps for producing these cells.

// Takayuki Kimura 1, 2 , Makoto Sugaya 1, 2 , Tomonori Oka 1, 2 , Andrew Blauvelt 3 , Hitoshi Okochi 2 , Shinichi Sato 1 1 Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan 2 Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan 3 Oregon Medical Research Center, Portland, Oregan, USA Correspondence to: Makoto Sugaya, e-mail: sugayam-der@h.u-tokyo.ac.jp Keywords: lymphatics, lymphedema, tumor immunity, cytotoxic T cells Received: March 18, 2015 Accepted: May 11, 2015 Published: May 27, 2015 ABSTRACT Tumor growth and metastasis of cancer involve autonomous tumor cell growth and host-tumor interactions. While tumor-specific immunity has been intensively studied in vitro , dynamic roles of lymphatic transport on tumor immunity in vivo have not been fully elucidated. In this study, we examined tumor growth and anti-tumor immune responses using kCYC mice, which demonstrate severe lymphatic dysfunction. Primary tumor growth was augmented in kCYC mice (compared to wild-type mice) when B16 melanoma or EL-4 lymphoma cells were subcutaneously injected. Expression of inflammatory cytokines such as IFN-γ, TNF-α, and IL-2 as well as IL-10 expression in draining lymph nodes (LNs) was significantly reduced in kCYC mice after tumor inoculation. Moreover, decreased levels of tumor-associated antigens were detected in draining LNs in kCYC mice, together with impaired antigen presentation. CD8 + T cells in draining LNs derived from kCYC mice bearing B16 melanoma also showed significantly decreased cytotoxic activity in vitro . Finally, tumor suppression activity of CD8 + T cells derived from kCYC mice bearing B16 melanoma was reduced when adoptively transferred to naive wild-type mice. In summary, these findings suggest that lymphatic transport is essential in generating optimal tumor-specific immune responses mediated by CD8 + T cells.

Cardiomyocytes are known to differentiate spontaneously from embryonic stem (ES) cells when they formed aggregates, so called "embryoid bodies", in the presence of serum. In this study, we explored the induction of cardiomyocytes from mouse ES cells in chemically defined serum-free medium by using a mesoderm-inducing factor, BMP4. Comparing the different inductive methods, we found a candidate cell surface marker, N-cadherin, for cardiomyocyte progenitors from ES cells. N-cadherin-positive cells highly expressed cardiogenic markers, Nkx2.5, Tbx5, and Isl1, and showed a high differentiation rate into cardiomyocyte lineage. These results indicate that N-cadherin can be a useful cell surface marker for the progenitors of cardiomyocyte differentiated from ES cells in the serum-free culture.

In the present study, we demonstrated that the mouse embryonic stem cells were differentiated into ciliated epithelial cells, with characteristics of normal ciliated cells. These cells expressed ciliary marker proteins, such as beta-tubulin IV and hepatocyte nuclear factor-3/forkhead homolog 4 (HFH-4), and processed microtubules were arranged in the 9 + 2 structure, which is the same specific alignment observed in normal ciliary microtubules. The cilia of these cells were beating at a frequency of 17-20 Hz. The differentiated embryoid bodies (EBs) containing these ciliated cells expressed respiratory marker genes such as thyroid transcription factor-1 and surfactant protein-C. For the induction of ciliated cells, culture of EBs in serum-free medium during the initial 2 days of the attachment was indispensable. When EBs were treated with bone morphogenetic proteins, the expression of HFH-4 was decreased, and the ciliated cells were scarcely differentiated. Previous methods for inducing ciliated cells in vitro from embryonic or adult tissues involved an air-liquid interface. The system used in this study more closely mimics the normal development of ciliated cells; thus, an added advantage of the system is as a tool for studying the differentiation mechanism of normal ciliated epithelial cells.

Cell surface molecules on lymphocytes positively or negatively modulate the Ag receptor signaling, and thus regulate the fate of the cell. CD22 is a B cell-specific cell surface protein that contains multiple ITIMs in the cytoplasmic tail, and critically regulates B cell activation and survival. CD22 regulation on B cell signaling is complex because CD22 can have both positive and negative roles in various contexts. We generated phosphospecific polyclonal Abs reacting four major CD22 tyrosine motifs (Y762, Y807, Y822, and Y842) and analyzed the pattern and intensity of phosphorylation of these tyrosine residues. The tyrosine motifs, Y762, Y822, and Y842, are considered as ITIM, whereas the other, Y807, is suggested to be important for Grb2 recruitment. Approximately 10% of the four tyrosine residues were constitutively phosphorylated. Upon anti-IgM ligation, CD22 Y762 underwent most rapid phosphorylation, whereas all four tyrosine residues were eventually phosphorylated equally at ∼35% of all CD22 molecules in the cell. By contrast, anti-CD40 stimulation specifically up-regulated anti-IgM-induced phosphorylation of tyrosines within two ITIM motifs, Y762 and Y842, which was consistent with in vivo finding of the negative role of CD22 in CD40 signaling. Thus, CD22 phosphorylation is not only quantitatively but also qualitatively regulated by different stimulations, which may determine the outcome of B cell signaling.

Summary Background Long-term cultures of epidermal cells from mouse skin have been notoriously difficult to establish. Objectives To develop a modified serum-free medium and technique for long-term culture of adult mouse epidermal keratinocytes. Methods Epidermal cells from trypsin-treated adult mouse dorsal and ventral skin were grown on type I collagen-coated dishes without feeder layers in a serum-free medium supplemented with only 10 ng mL−1 epidermal growth factor (EGF) and 10−10 mol L−1 cholera toxin (CT). Results After removing coexisting fibroblasts several times, we were able to obtain almost pure basal epidermal keratinocytes. Our technique supports sustained multiplication of mouse basal keratinocytes for more than 100 population doublings, and they retained the capacity to undergo terminal differentiation when given the appropriate stimulus. The epithelial nature of these cultivated cells was demonstrated both by phase-contrast microscopy and by immunostaining with antikeratin antibodies. EGF and CT, which have been reported to accelerate the cellular growth rate, were essential for successful long-term cultivation during multiple passages. Conclusions Our technique is very simple. It provides a useful and suitable model for investigations of growth, differentiation and skin remodelling in vitro.

Recently, the detection of side population (SP) cells, which have the ability to strongly efflux Hoechst 33342 fluorescence dye, has attracted attention as a method of stem cell isolation. We identified SP cells from mouse skin using the same method as from bone marrow. This population almost completely disappeared after treatment with the calcium channel blocker verapamil. SP cells were mainly localized in the epidermis, with a few in the dermis. The ratio of SP cells decreased as the mouse became older. Surface marker analysis revealed that the sorted SP cells expressed alpha6-integrin, beta1-integrin, Sca-1, keratin 14, and keratin 19, which are proliferating and progenitor cell markers, at levels higher than in non-SP cells, while they expressed E-cadherin, CD34, and CD71 at lower levels. The expression of breast cancer resistance protein 1 (BCRP1), which participates in dye efflux, was expressed at high levels at both the protein and mRNA level in sorted SP cells. Immunohistochemical analysis showed that BCRP1 was expressed in the basal layers and hair bulge regions of mouse skin. BCRP1 mRNA was found in basal layers and hair follicles of newborn skin by in situ hybridization. These results indicate that the localization of BCRP1-positive cells is compatible with that of keratinocyte stem cells. Based on the close relationship between BCRP1 and the SP cell phenotype, we conclude that keratinocyte stem cells are closely related to the SP- or BCRP1-positive cells.

To analyze the roles of insulin receptor substrate (IRS) proteins in insulin-stimulated cell cycle progression, we examined the functions of rat IRS-1 and IRS-3 in Chinese hamster ovary cells overexpressing the human insulin receptor. In this type of cell overexpressing IRS-1 or IRS-3, we showed that: 1) overexpression of IRS-3, but not IRS-1, suppressed the G1/S transition induced by insulin; 2) IRS-3 was more preferentially localized to the nucleus than IRS-1; 3) phosphorylation of glycogen synthase kinase 3 and MAPK/ERK was unaffected by IRS-3 overexpression, whereas that of protein kinase B was enhanced by either IRS; 4) overexpressed IRS-3 suppressed cyclin D1 expression in response to insulin; 5) among the signaling molecules regulating cyclin D1 expression, activation of the small G protein Ral was unchanged, whereas insulin-induced gene expression of c-myc, a critical component for growth control and cell cycle progression, was suppressed by overexpressed IRS-3; and 6) insulin-induced expression of p21, a cyclin-dependent kinase inhibitor, was decreased by overexpressed IRS-3. These findings imply that: 1) IRS-3 may play a unique role in mitogenesis by inhibiting insulin-stimulated cell cycle progression via a decrease in cyclin D1 and p21 expressions as well as suppression of c-myc mRNA induction in a manner independent of the activation of MAPK, protein kinase B, glycogen synthase kinase 3 and Ral; and 2) the interaction of IRS-3 with nuclear proteins may be involved in this process.

Systemic sclerosis (SSc) is characterized by fibrosis and autoimmmunity. Peripheral blood B cells from SSc patients specifically overexpress CD19, a critical cell-surface signal transduction molecule in B cells. CD19 deficiency in B cells also attenuates skin fibrosis in the tight-skin (TSK/+) mouse, a genetic model for SSc. Herein we analyzed two transgenic mouse lines that overexpress CD19. Remarkably, 20% increase of CD19 expression in mice spontaneously induced SSc-specific anti-DNA topoisomerase I (topo I) antibody (Ab) production, which was further augmented by 200% overexpression. In TSK/+ mice overexpressing CD19, skin thickness did not increase, although anti-topo I Ab levels were significantly augmented, indicating that abnormal CD19 signaling influences autoimmunity in TSK/+ mice and also that anti-topo I Ab does not have a pathogenic role. The molecular mechanisms for abnormal CD19 signaling were further assessed. B-cell antigen receptor crosslinking induced exaggerated calcium responses and augmented activation of extracellular signal-regulated kinase in TSK/+ B cells. CD22 function was specifically impaired in TSK/+ B cells. Consistently, CD19, a major target of CD22-negative regulation, was hyperphosphorylated in TSK/+ B cells. These findings indicate that reduced inhibitory signal provided by CD22 results in abnormal activation of signaling pathways including CD19 in TSK/+ mice and also suggest that this disrupted B cell signaling contribute to specific autoantibody production.

Recently, adult stem cells have been isolated from the skin and designated as skin-derived precursors (SKPs). These SKPs, cultured in vitro, can give rise to neurons, glia, smooth muscle cells, and adipocytes. In the current study, we confirmed the clonal expansion of SKPs using a sphere-forming culture system in a medium containing methylcellulose. Among the growth factors, only transforming growth factor-beta (TGF-beta) was revealed to uniquely facilitate the sphere formation and proliferation of the SKPs in combination with EGF and bFGF. In addition, TGF-beta did not alter phenotypical characteristics of the SKPs under sphere-forming conditions. The effect of TGF-beta on sphere formation was not observed in neural stem cells, which expressed a different set of cell surface markers from SKPs, suggesting that SKPs have distinct features. Although the number of SKPs decreased with age, TGF-beta increased the sphere colony formation and proliferation in all ages. These results suggest that SKPs maintained in the presence of TGF-beta during culture are of potential use in cell-replacement therapies employing adult tissue sources.

Skin wound healing is an interactive process involving soluble mediators, ECM, resident cells, and infiltrating cells. Little is known about wound healing in the presence of lymphedema. In this study, we investigated wound healing using kCYC+/− mice, which demonstrate severe lymphatic dysfunction. Wound healing was delayed significantly in kCYC+/− mice when compared with WT mice. In wounded skin of kCYC+/− mice, mast cell numbers were increased compared with WT mice, whereas macrophage numbers were decreased. Moreover, IL-10 expression by mast cells was increased, and expression of bFGF, mainly produced by macrophages, was decreased in wounded skin of kCYC+/− mice compared with WT mice. We next crossed kCYC+/− mice with IL-10−/− mice, which were reported to show accelerated wound closure. In kCYC+/−IL-10+/− mice, time course of wound healing, numbers of macrophages, and IL-10 mRNA expression levels in wounded skin were comparable with WT IL-10+/− mice. Similar results were obtained using a different lymphedema model, in which circumferential skin excision was performed on the tails of mice to remove the superficial lymphatics. In summary, these findings suggest that IL-10 plays an important role in delayed wound healing in the setting of lymphatic dysfunction.

CCR3 is a specific marker of anaplastic large cell lymphoma (ALCL) cells. ALCL cells also express CCL11, a ligand for CCR3, leading to the hypothesis that CCL11 may play an autocrine role in ALCL progression. In this study, we investigated a role of CCL11 in cell survival and growth of human Ki-JK cells, established from an ALCL patient, and murine EL-4 lymphoma cells. Both Ki-JK and EL-4 cells expressed cell surface CCR3. CCL11 increased cell survival rates of Ki-JK cells in a dose-dependent manner, whereas it promoted EL-4 cell proliferation. Furthermore, CCL11 induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in both Ki-JK cells and EL-4 cells. Cell survival and tumor proliferation promoted by CCL11 was completely blocked by inhibition of ERK phosphorylation. CCL11 induced expression of antiapoptotic proteins, Bcl-xL and survivin, in Ki-JK cells. CCL11 also enhanced tumor growth of EL-4 and Ki-JK cells in vivo. Consistent with these results, tumor cells of cutaneous ALCL expressed CCR3 and increased levels of phosphorylated ERK1/2, Bcl-xL, and survivin in situ. Thus, our findings prompt a novel therapeutic approach to treat relapses of an aggressive form of lymphoma based on the discovery that a cell surface marker of disease functions as a critical autocrine growth receptor. Cancer Res; 71(6); 2056–65. ©2011 AACR.

Adipose tissue-derived mesenchymal stem cells (ASCs) have been reported to be multipotent and to differentiate into various cell types, including osteocytes, adipocytes, chondrocytes, and neural cells. Recently, many authors have reported that ASCs are also able to differentiate into vascular endothelial cells (VECs) in vitro. However, these reports included the use of medium containing fetal bovine serum for endothelial differentiation. In the present study, we have developed a novel method for differentiating mouse ASCs into VECs under serum-free conditions. After the differentiation culture, over 80% of the cells expressed vascular endothelial-specific marker proteins and could take up low-density lipoprotein in vitro. This protocol should be helpful in clarifying the mechanisms of ASC differentiation into the VSC lineage.

Stem cells from various organs have been shown to regenerate muscle cells. Among them, skin-derived cells are promising because of their easy accessibility. We separated murine dermal cells into Sca-1 (+) and (−) fractions and investigated which of them could differentiate into muscle cells. After the cells were aggregated for 4 days and cultured on a collagen type I-coated plate for 7–10 days, the Sca-1 (−) fraction had expanded into many myoblastic cells, but the Sca-1 (+) fraction had not. Initial commitment to the myogenic lineage appeared to start during the aggregation. Sca-1 (−) cells proliferated exponentially and maintained their ability to differentiate into skeletal muscle cells within 7–10 days. About 60% of the cells showed positive staining for skeletal fast myosin heavy chain. Transplantation experiments revealed that the myoblastic cells arising after several passages were successfully engrafted into damaged host muscle. In conclusion, we have found that murine dermal Sca-1 (−) cells differentiate into muscle cells in vitro and in vivo after using an initial aggregation procedure. Their high differentiation efficiency and proliferation ability will offer substantial advantages for stem cell research.

Delineating the relative contributions of B lymphocytes during the course of autoimmune disease has been difficult. Therefore, the effects of depleting all mature B cells using a potent CD20 mAb, or of depleting circulating and marginal zone B cells using a ligand-blocking CD22 mAb, were compared in NZB/W F1 mice, a model for human systemic lupus erythematosus. Single low-dose mAb treatments depleted B cells efficiently in both NZB/W F1 and C57BL/6 mice. Prophylactic B cell depletion by repeated CD20 mAb treatments prolonged survival during pristane-accelerated lupus in NZB/W F1 mice, whereas CD22 mAb had little effect. Despite effective B cell depletion, neither mAb treatment prevented autoantibody generation. In addition, CD20, CD22, and control mAb-treated NZB/W F1 mice developed anti-mouse IgG autoantibodies in contrast to parental NZB and NZW strains, which may have reduced the effectiveness of B cell depletion. Despite this, low-dose CD20 mAb treatment initiated in 12–28-wk-old mice, and administered every 4 wk thereafter, significantly delayed spontaneous disease in NZB/W F1 mice. By contrast, B cell depletion initiated in 4-wk-old mice hastened disease onset, which paralleled depletion of the IL-10–producing regulatory B cell subset called B10 cells. B10 cells were phenotypically similar in NZB/W F1 and C57BL/6 mice, but were expanded significantly in young NZB/W F1 mice. Thus, B cell depletion had significant effects on NZB/W F1 mouse survival that were dependent on the timing of treatment initiation. Therefore, distinct B cell populations can have opposing protective and pathogenic roles during lupus progression.

Although contact hypersensitivity (CHS) has been considered a prototype of T cell-mediated immune reactions, recently a significant contribution of regulatory B cell subsets in the suppression of CHS has been demonstrated. CD22, one of the sialic acid-binding immunoglobulin-like lectins, is a B cell-specific molecule that negatively regulates BCR signaling. To clarify the roles of B cells in CHS, CHS in CD22−/− mice was investigated. CD22−/− mice showed delayed recovery from CHS reactions compared with that of wild-type mice. Transfer of wild-type peritoneal B-1a cells reversed the prolonged CHS reaction seen in CD22−/− mice, and this was blocked by the simultaneous injection with IL-10 receptor Ab. Although CD22−/− peritoneal B-1a cells were capable of producing IL-10 at wild-type levels, i.p. injection of differentially labeled wild-type/CD22−/− B cells demonstrated that a smaller number of CD22−/− B cells resided in lymphoid organs 5 d after CHS elicitation, suggesting a defect in survival or retention in activated CD22−/− peritoneal B-1 cells. Thus, our study reveals a regulatory role for peritoneal B-1a cells in CHS. Two distinct regulatory B cell subsets cooperatively inhibit CHS responses. Although splenic CD1dhiCD5+ B cells have a crucial role in suppressing the acute exacerbating phase of CHS, peritoneal B-1a cells are likely to suppress the late remission phase as “regulatory B cells.” CD22 deficiency results in disturbed CHS remission by impaired retention or survival of peritoneal B-1a cells that migrate into lymphoid organs.

Mesenchymal stem cells (MSCs), largely present in the adult human body, represent an attractive tool for the establishment of a stem cell-based therapy for liver diseases. Recently, the therapeutic potential and immunomodulatory activity of MSCs have been revealed. Adipose tissue-derived mesenchymal stem cells (AT-MSCs), so-called adipose-derived stem cells or adipose stromal cells, because of their high accessibility with minimal invasiveness, are especially attractive in the context of future clinical applications. The goal of the present study was to evaluate the therapeutic potential of AT-MSCs by their transplantation into nude mice with CCl4-caused liver injury. We observed that after transplantation, AT-MSCs can improve liver functions, which we verified by changes in the levels of biochemical parameters. Ammonia, uric acid, glutamic-pyruvic transaminase, and glutamic-oxaloacetic transaminase concentrations returned to a nearly normal level after AT-MSC transplantation. These results raised the question of how AT-MSCs can achieve this. To discover the possible mechanisms involved in this therapeutic ability of AT-MSCs, in vitro production of cytokines and growth factors was analyzed and compared with MSCs from bone marrow (BM-MSCs) and normal human dermal fibroblasts (NHDFs). As a result we observed that AT-MSCs secrete interleukin 1 receptor α (IL-1Rα), IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein 1, nerve growth factor, and hepatocyte growth factor in a volume higher than both BM-MSCs and NHDFs. Thus, our findings suggest that AT-MSCs may account for their broad therapeutic efficacy in animal models of liver diseases and in the clinical settings for liver disease treatment.Disclosure of potential conflicts of interest is found at the end of this article.

Contact hypersensitivity (CHS) is a cutaneous immune reaction mediated mainly by antigen-specific effector T cells and is regarded as a model for Th1/Tc1-mediated inflammation. However, recent reports have suggested pivotal roles of B cells in CHS. CD19 serves as a positive B-cell response regulator that defines signaling thresholds critical for B-cell responses. In the current study, we assessed the role of the B-cell-specific surface molecule CD19 on the development of CHS by examining CD19-deficient mice. Although CD19-deficient mice are hyposensitive to a variety of transmembrane signals, CD19 loss resulted in increased and prolonged reaction of CHS, suggesting an inhibitory role of CD19 expression in CHS. Sensitized lymph nodes and elicited ear lesions from CD19-deficient mice exhibited Th1/Tc1-shifted cytokine profile with increased interferon-gamma expression and decreased interleukin-10 expression. Adoptive transfer experiments revealed that CD19 expression in recipient mice was required for optimal suppression of CHS response, indicating its role in the elicitation phase. Furthermore, spleen B cells, especially marginal zone B cells, from wild-type mice were able to normalize exaggerated CHS reactions in CD19-deficient mice. Thus, CD19 expression in B cells is critical for termination of CHS responses, possibly through the function of regulatory B cells.

We have succeeded in culturing dermal papilla (DP) cells long term and developed new techniques that enhance their hair follicle-inducing efficiency in a patch assay. The outgrowing DP cells from mouse vibrissae were markedly stimulated by 10% fetal bovine serum-Dulbecco's modified essential medium that included fibroblast growth factor-2 (FGF-2). Moreover, the potency of proliferation was maintained during serial cultivations (more than 30 passages). We combined these established DP cells with epidermal cells and implanted them subcutaneously into athymic mice to examine their hair follicle-inducing ability. New hair follicles were induced by dissociated DP cells at earlier passages (under passage 4), but the cells from later passages could not induce follicles. We next aggregated the DP cells to form spheres and then injected them with epidermal cells. Unlike the dissociated DP cells, the spheres made from the later passaged cells (more than 10 passages) did induce new hair follicles. We examined several genes specific for DP of anagen follicles and confirmed that their expression level was elevated in the spheres compared with their expression level in adherent DP cells. These results suggest that FGF-2 is essential for dermal papilla cell culture and that sphere formation partially models the intact DP, resulting in hair follicle induction, even by later passaged cells.

We have developed a serum-free medium, designated ESF7, in which leukemia inhibitory factor (LIF) clearly stimulated murine embryonic stem (ES) cell proliferation accompanied by increased expression of nanog and Rex-1 and decreased FGF-5 expression. These effects were dependent on the concentration of LIF. The ES cells maintained in ESF7 medium for more than 2 yr retained an undifferentiated phenotype, as manifested by the expression of the transcription factor Oct-3/4, the stem cell marker SSEA-1, and alkaline phosphatase. Withdrawal of LIF from ESF7 medium resulted in ES cell apoptosis. Addition of serum to ESF7 medium promoted ES cell differentiation. Addition of BMP4 promoted ES cell differentiation into simple epithelial-like cells. In contrast, FGF-2 promoted ES cell differentiation into neuronal and glial-like cells. Under serum-free culture conditions, LIF was sufficient to stimulate cell proliferation, it inhibited cell differentiation, and it maintained self-renewal of ES cells. Because this simple serum-free adherent monoculture system supports the long-term propagation of pluripotent ES cells in vitro, it will allow the elucidation of ES cell responses to growth factors under defined conditions.

Recent studies have demonstrated that multipotent retinal stem or progenitor cells can be isolated from the ciliary epithelium (CE) of the eye using a neurosphere culture. In this study, we investigated the properties of growth and differentiation, and molecular profiles of rat adult ciliary epithelium (CE)-derived retinal progenitors and forebrain (FB) derived neurospheres. Under clonogenic culture conditions, we found that the CE-derived neurospheres contained fewer undifferentiated cells compared with the FB-derived neurospheres, and that CE-derived neurospheres initially expressed the set of Notch pathway molecules genes including Notch 1 and Delta 1, HES-1 and HES-5, but partially lose their expression after passaging. Furthermore, we found that the CE-derived neurospheres did not express several markers for in vivo embryonic retinal progenitors. Additionally, when the eye was divided into four subregions along its dorsoventral and nasotemporal axes and progenitor cells were obtained from the subregions, the progenitor cells did not express the subregion specific transcription factors, suggesting that subregional specificity is not maintained in vitro. Together, our results demonstrate that CE-derived progenitor cells may have intrinsic limitations in the production of cell types.

In lower vertebrates, multipotential retinal stem cells reside in a far peripheral retinal zone known as the ciliary marginal zone while more fate-restricted progenitor cells are located immediately adjacent to this retinal margin. To determine whether mammalian ciliary epithelium contains heterogenous stem cell and progenitor cell populations similar to lower vertebrates, we investigated the heterogeneity of the retinal progenitor (or stem) cells isolated from adult rabbit eyes, which are large and suited for precise identification of the region. Under clonogenic culture conditions for a neurosphere assay, spheres were generated from approximately 0.1% of the ciliary epithelial cells and expressed retinal selective markers when plated under conditions producing differentiation. Subsequently, we found that 13.7% of the primary spheres gave rise to a secondary sphere and that 77% of the primary sphere colonies were unipotential progenitors that generated either neurons or glial cells exclusively, while 23% were at least bipotential that could give rise to both cell types using double-labelled immunocytochemistry. Taken together, our results demonstrate that the ciliary epithelium of adult rabbits contains both (at least) bipotential retinal progenitor (or stem) cells and unipotential fate-restricted progenitor cells, similar to lower vertebrates.

Lipopolysaccharide (LPS) is a major gram-negative bacterial component that stimulates innate immune response and also induces B-lymphocyte activation. Recent studies have revealed that common molecular patterns of microorganisms such as LPS are recognized by toll-like receptors (TLRs). B cells have 2 known TLRs that mediate LPS signaling, TLR4 and RP105 (CD180). While TLR4 is expressed on immune cells of various types, RP105 is preferentially expressed on mature B cells. Here we demonstrate that CD19 plays a major role in regulating signal transduction through RP105. Anti-RP105 ligation induced normal proliferation of B cells from mice deficient for MyD88, an adaptor protein that mediates most TLR pathways. By contrast, the loss of CD19 resulted in modest B-cell proliferation against anti-RP105 stimulation as well as LPS stimulation. LPS induced tyrosine phosphorylation of CD19, which was RP105-dependent but TLR4-independent. CD19 formed a complex with Lyn and Vav following RP105 ligation, and CD19 expression was required for optimal Lyn activation and Vav phosphorylation. Consistently, B cells deficient for CD19 exhibited specific defect in the activation of c-Jun N-terminal kinases following RP105 ligation and LPS stimulation. In contrast, CD19 and phosphatidylinositol 3-kinase independently regulated intracellular calcium mobilization induced by anti-RP105 stimulation. Thus, signaling through the B-cell-specific LPS receptor RP105 is uniquely regulated by the B-cell-specific signaling component, Lyn/CD19/Vav complex.

The lymph transports tissue-resident dendritic cells (DCs) to regional lymph nodes (LNs), having important roles in immune function. The biological effects on tissue inflammation following lymphatic flow obstruction in vivo , however, are not fully known. In this study, we investigated the role of the lymphatic system in contact hypersensitivity (CHS) responses using k-cyclin transgenic (kCYC +/- ) mice, which demonstrate severe lymphatic dysfunction. kCYC +/- mice showed enhanced ear swelling to both DNFB and FITC, as well as stronger irritant responses to croton oil compared with wild-type littermates. Consistently, challenged ears of kCYC +/- mice exhibited massive infiltrates of inflammatory cells. In contrast, DC migration to regional LNs, drainage of cell-free antigen to LNs, antigen-specific IFN-γ production, and lymphocyte proliferation were impaired during the sensitization phase of CHS in kCYC +/- mice. Transfer experiments using lymphocytes from sensitized mice and real-time PCR analysis of cytokine expression using challenged ear revealed that ear swelling was enhanced because of impaired lymphatic flow. Collectively, we conclude that insufficient lymphatic drainage augments apparent inflammation to topically applied allergens and irritants. The findings add insight into the clinical problem of allergic and irritant contact dermatitis that commonly occurs in humans with peripheral edema of the lower legs.