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The hiPSC-derived spheroids we generated are more expensive to obtain than the human cell lines currently available for anti-SARS-CoV-2 drug evaluation, such as Calu-3 cells; however, the spheroids have better infection susceptibility than the existing human cell lines. Although we are cognizant that there are human lung (and colonic) organoid models for the study of SARS-CoV-2, the production of those organoids is greatly more costly and time consuming than the generation of human iPSC-derived spheroid cells.

Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are very attractive cell sources for the treatment of diabetes mellitus, because numerous cells can be obtained using their infinite proliferation potential to overcome the paucity of donor islets. Advances in differentiation protocols make it possible to generate glucose responsive hPSC-beta cells, which can ameliorate hyperglycemia in diabetic mice. These protocols have mainly been based on an adherent culture system. However, in clinical applications, suspension culture methods are more suitable for large-scale culture. There are reports that suspension culture and spheroid formation promote differentiation in various cell types, including hPSCs, but, to our knowledge, there are no reports comparing gene expression patterns between suspension and adherent cultured human iPSCs (hiPSCs) during definitive endoderm (DE) differentiation. In this study, we chose several stage marker genes, not only for DE but also for posterior epiblast and primitive streak, and we examined their time course expression in suspension and adherent cultures by quantitative PT-PCR (qPCR), western blot, flow cytometry and immunocytochemistry. Our results demonstrate that expressions of these marker genes are faster and more strongly induced in suspension culture than in adherent culture during the DE differentiation process, indicating that suspension culture favors DE differentiation.

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.

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.

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.

Maturity-onset diabetes of the young (MODY) is a heterozygous monogenic diabetes; more than 14 disease genes have been identified. However, the pathogenesis of MODY is not fully understood because the patients' pancreatic beta cells are inaccessible. To elucidate the pathology of MODY, we established MODY3 patient-derived iPS (MODY3-iPS) cells using non-integrating Sendai virus (SeV) vector and examined the mutant mRNA and protein of HNF1A (Hepatocyte Nuclear factor 1A) after pancreatic lineage differentiation. Our patient had a cytosine insertion in the HNF1A gene (P291fsinsC) causing frameshift and making a premature termination codon (PTC). We confirmed these MODY3-iPS cells possessed the characteristics of pluripotent stem cells. After we differentiated them into pancreatic beta cells, transcripts of HNF1A gene were cloned and sequenced. We found that P291fsinsC mutant transcripts were much less frequent than wild ones, but they increased after adding cycloheximide (CHX) to the medium. These results suggested that mutant mRNA was destroyed by nonsense-mediated mRNA decay (NMD). Moreover, we were not able to detect any band of mutant proteins in pancreatic lineage cells which were differentiated from MODY3-iPSCs by western blot (WB) analysis. A scarcity of the truncated form of mutant protein may indicate that MODY3 might be caused by a haplo-insufficiency effect rather than a dominant negative manner.

Background 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. Methods 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. Results 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. Conclusion Functional pancreatic β-cells were generated from human iPSCs. Induction of definitive endoderm and spheroid formation may be key steps for producing these cells.

Introduction To complement islet transplantation for type1 diabetic patients, cell-based therapy using pluripotent stem cells such as ES cells and iPS cells is promising. Many papers have already reported the induction of pancreatic β cells from these cell types, but a suspension culture system has not usually been employed. The aim of this study is to establish a suspension culture method for inducing functional islet-like cells from human iPS cells. Methods We used 30 ml spinner type culture vessels for human iPS cells throughout the differentiation process. Differentiated cells were analyzed by immunostaining and C-peptide secretion. Cell transplantation experiments were performed with STZ-induced diabetic NOD/SCID mice. Blood human C-peptide and glucagon levels were measured serially in mice, and grafts were analyzed histologically. Results We obtained spherical pancreatic beta-like cells from human iPS cells and detected verifiable amounts of C-peptide secretion in vitro. We demonstrated reversal of hyperglycemia in diabetic model mice after transplantation of these cells, maintaining non-fasting blood glucose levels along with the human glycemic set point. We confirmed the secretion of human insulin and glucagon dependent on the blood glucose level in vivo. Immunohistological analysis revealed that grafted cells became α, β and δ cells in vivo. Conclusions These results suggest that differentiated cells derived from human iPS cells grown in suspension culture mature and function like pancreatic islets in vivo., Highlights • Functional islet-like cells were induced from human iPS cells by suspension culture. • They ameliorated hyperglycemia in diabetic mice and secreted human insulin and glucagon. • Grafted cells became α, β and δ cells in vivo.

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.

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.

CD19 is a B-cell transmembrane molecule that is critical for B-cell activation. CD19 serves as a scaffold protein for key signal transduction molecules including Lyn, PI3K, and Vav, by providing docking sites for these molecules via phosphorylation of CD19-Y(513), CD19-Y(482), and CD19-Y(391). We investigated the process of CD19 tyrosine phophorylation during B-cell activation using Ab specific for each of these phosphorylated tyrosines. BCR engagement induced differential tyrosine phosphorylation, as CD19-Y(513) phophorylation occurred first, and CD19-Y(482) phosphorylation was delayed and transient. Different BCR isotypes exhibited distinct patterns of CD19 phosphorylation: IgG-BCR ligation resulted in faster phosphorylation of CD19-Y(513) and more intense phosphorylation of CD19-Y(391) than IgM-BCR ligation. This affected CD19-mediated downstream pathways involving Vav, PI3K, and Akt. Additionally, the phosphorylation profile of CD19 differed distinctly according to its plasma membrane location. CD19 phosphorylated at Y(513) was almost exclusively located within lipid rafts, whereas phosphorylated Y(482) and Y(391) were found both inside and outside of the rafts. Furthermore, the phosphorylation of all three tyrosines was remarkably enhanced and prolonged following the simultaneous stimulation of BCR and CD40. Thus, variations in phosphorylation patterns may contribute to the complexity of CD19-regulated signal transduction.

金沢大学大学院医学系研究科血管分子科学, The control of the stem cell compartment in epidermis is closely linked to the regulation of keratinocyte proliferation and differentiation. β1 integrins are expressed 2-fold higher by stem cells than transit-amplifying cells. Signaling from these β1 integrins is critical for the regulation of the epidermal stem cell compartment. To clarify the functional relevance of this differential expression of β1 integrins, we established HaCaT cells with high β1integrin expression by repeated flow cytometric sorting of this population from the parental cell line. In these obtained cells expressing β1 integrins by 5-fold, MAPK activation was markedly increased. Regarding the upstream of MAPK, Gab1 phosphorylation was also higher with high β1 integrin expression, while Shc phosphorylation was not altered. In addition, enhanced phosphatidylinositol 3-kinase activation was also observed. These observations suggest that Gab1 and phosphatidylinositol 3-kinase play pivotal roles in the β1 integrin-mediated regulation of the epidermal stem cell compartment. © 2007 Elsevier Inc. All rights reserved.

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.

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.

Mechanical stretching represents an important part of the signaling in skin. We have previously demonstrated that mechanical stretching induced proliferative phenotypes in human keratinocytes, as shown in increased 5-bromo-2'-deoxyuridine (BrdU) incorporation, ERK1/2 activation, and keratin K6 induction. Here we have further investigated the antiapoptotic signals in human keratinocytes provoked by mechanical stretching in vitro. Keratinocytes were plated on flexible silicone supports to transmit mechanical stretching to keratinocytes, involving continuous stretching by +20%. Stretching of these cells for 15-30 min caused the phosphorylation and activation of Akt. Inhibition of mitogen and extracellular signal-regulated kinase (MEK1/2) with U0126 and phosphoinositide 3-OH kinase (PI 3-K) with Wortmannin attenuated Akt activation. The epidermal growth factor (EGF) receptor kinase inhibitor, AG1478, and calcium channel inhibitor, gadolinium (Gd3+), also inhibited Akt activation. In summary, our results demonstrate the activation of the Akt signaling pathway by mechanical stretching, generating not only proliferative but also antiapoptotic signals in human keratinocytes.

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.

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.

Background: Mesenchymal stem cells (MSCs), including adipose tissue-derived mesenchymal stem cells (ADSCs), are multipotent and can differentiate into various cell types, including pancreatic β cells. Therefore, ADSCs present a potential cell source for the treatment of type 1 diabetes mellitus (T1DM). However, current in vitro protocols are insufficient to induce fully matured insulin-producing β cells. In this study, we assessed the effectiveness of overexpression of ΒETA2 (NeuroD1), a member of the basic helix–loop–helix transcription factor family, with murine insulinoma cell line-derived conditioned medium (MIN6-CM) to improve the differentiation capacity of ADSCs into insulin-producing cells. Method: Murine ADSCs were isolated from C57BL/6 mice, transduced with several transcriptional factors (TFs), and stable transfectants were established. MIN6-CM was prepared. Syngeneic recipient mice were rendered diabetic by a single injection of streptozotocin, and differentiated cells were transplanted under the kidney capsule of recipient mice. Next, blood glucose levels were monitored. Results: CM alone was sufficient to induce insulin mRNA expression in vitro. However, other TFs were not detected. ADSCs cultured with MIN6-CM induced insulin expressions in vitro, but other β cell-related TFs were been detected. However, BETA2 transduction in MIN6-CM resulted in robust expression of multiple β cell phenotypic markers. Moreover, insulin content analysis revealed insulin protein expression in vitro. Furthermore, in vivo transplant studies revealed the effectiveness of the simultaneous use of BETA2 transduction with the CM. Conclusion: These results suggest that the balance of cytokines and growth factors in addition to gene manipulation would benefit the efficient differentiation of ADSCs into pancreatic β cells. Our technology could provide a path to β cell differentiation and novel cell replacement-based therapies for T1DM.

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.

Despite a number of studies, the etiology of keloids remains unknown. We have investigated the response of fibroblasts derived from keloid tissue and normal adult skin to platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). Keloid fibroblasts were more responsive in both chemotactic and mitogenic assays to all three isoforms of PDGF than fibroblasts from normal skin. No enhanced response of the cells to either EGF or FGF was detected. The enhanced PDGF response of keloid fibroblasts appears to be mediated by elevated levels of PDGF alpha receptors, which are 4-5 times higher than those in normal human skin fibroblasts.

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.

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.

Background: Normal human epidermal keratinocytes usually proliferate in low-calcium and differentiate in high-calcium without a feeder layer, but they stop proliferating and differentiate at confluency even in low-calcium, serum-free medium. Objective: We speculated that this contact inhibition would be mediated in part by mechanical tension. To prove this, we created a new assay system. Methods: A 10 mm diameter cloning ring was put on the center of a 60 mm dish coated with type I collagen. Keratinocytes were plated in the ring and incubated for 4 h, then we had a circular epidermal monolayer sheet. We changed the mechanical tension by removing the ring and measured the diameter of the sheet under various conditions. Results: When we used keratinocyte-serum free medium (SFM) whose calcium concentration is below 0.1 mM as a medium, the keratinocytes in the perimeter migrated individually, and the keratinocytes in the center portion started differentiation. However, when we added calcium chloride to SFM (final concentration more than 0.5 mM), keratinocytes at the periphery showed marked lamellipodia without losing contact with the surrounding cells. These keratinocytes showed coordinate sheet-like outgrowth as a whole even in high concentrations of calcium. Conclusion: These results suggest that other than calcium concentration, change of the mechanical tension would be one of the factors that mediate proliferation or differentiation of keratinocytes and that this new assay can be useful in analyzing proliferation, differentiation, and migration of keratinocytes.

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.

Interleukin 15 (IL-15) is a potent stimulator of proliferation and an inhibitor of apoptosis in lymphocytes. We attempted to elucidate the mechanism of IL-15 function in HaCaT keratinocytes. We found that 5-bromo-2′-deoxyuridine incorporation increased in a dose-dependent manner with IL-15. This was blocked by MEK inhibitor U0126 or PI 3-K inhibitor LY294002. ERK1/2 and Akt phosphorylation by IL-15 were detected in a dose- and time-dependent manner. U0126 and LY294002 abolished ERK1/2 and Akt phosphorylation, respectively. DNA fragmentation and Annexin V binding accompanied by UVB-induced apoptosis were reduced by 30–50% with IL-15. Taken together, IL-15 induced cellular proliferation and had an anti-apoptotic effect on keratinocytes, in which ERK1/2 and Akt phosphorylation played crucial roles. The signal transduction pathways of IL-15 in keratinocytes were partially elucidated; they share a substantial part with growth signals induced by EGF. These results suggest a therapeutic approach to inflammatory skin diseases by controlling these signals.

The present invention provides a novel polypeptide, Connective Tissue Growth Factor (CTGF), polynucleotides encoding CTGF and polynucleotides regulating CTGF expression. The invention also provides agents that modulate CTGF and therapeutic methods for using the agents. Also provided are diagnostic methods for using CTGF and assays for identifying agents which affect the expression of CTGF polynucleotide.

Epidermal keratinocytes are continuously exposed to mechanical forces. The human skin surface can be thickened and enlarged by various stresses such as tissue expander or abrasive pressure. To investigate the mechanism of epidermal hyperproliferation by mechanical stress, keratinocytes were plated on flexible silicone dishes, which were continuously stretched by +20%. Stretching of cells for 24 h caused upregulation of 5-bromo-2'-deoxyuridine (BrdU)-positive cells to 200%-220% and activation of extracellular signal-regulated kinases (ERK)1/2. Inhibition of mitogen and ERK with U0126 and phosphoinositide 3-OH kinase attenuated BrdU incorporation and ERK1/2 activation. The EGF receptor kinase inhibitor and the calcium channel inhibitor also inhibited BrdU incorporation and the activation of ERK1/2. Twenty-four hours of stretching stimulated reporter activity driven by activator protein 1 (AP-1), induction of K6, and suppression of K10, which were inhibited by U0126. Our results indicate that mechanical stretching induces proliferative signals on human keratinocytes via induction of calcium influx, phosphorylation of epidermal growth factor receptor (EGFR), and ERK1/2. These mechanisms may contribute to the hyperproliferative nature of the epidermis, which is mechanically stretched by various stimuli.