Activation of cellular antioxidative stress and migration activities by purified components from immortalized stem cells from human exfoliated deciduous teeth.

Although stem cell-based regenerative medicine has been extensively studied, it remains difficult to reconstruct three dimensional tissues and organs in combination with vascular systems in vitro. One clinically successful therapy is transplantation of mesenchymal stem cells (MSC) into patients with graft versus host disease. However, transplanted cells are immediately damaged and destroyed because of innate immune reactions provoked by thrombogenic inflammation, and patients need to take immunosuppressive drugs for the immunological regulation of allogeneic cells. This reduces the benefits of stem cell transplantation. Therefore, alternative therapies are more realistic options for clinical use. In this study, we aimed to take advantage of the therapeutic efficacy of MSC and use multiple cytokines released from MSC, that is, stem cells from human exfoliated deciduous teeth (SHEDs). Here, we purified components from conditioned media of immortalized SHED (IM-SHED-CM) and evaluated the activities of intracellular dehydrogenase, cell migration, and antioxidative stress by studying the cells. The immortalization of SHED could make the stable supply of CM possible. We found that the fractionated component of 50-100 kD from IM-SHED-CM had higher efficacy than the original IM-SHED-CM in terms of intracellular dehydrogenase and cell migration in which intracellular signal transduction was activated via receptor tyrosine kinases, and the glutathione peroxidase and reductase system was highly active. Although antioxidative stress activities in the fractionated component of 50-100 kD had slightly lower than that of original IM-SHE-CM, the fraction still had the activity. Thus, the use of fractionated components of 50-100 kD from IM-SHED-CM could be an alternative choice for MSC transplantation because the purified components from CM could maintain the effect of cytokines from SHED.

Surface modulation of extracellular vesicles with cell-penetrating peptide-conjugated lipids for improvement of intracellular delivery to endothelial cells.

Exosomes (diameter 30-200 nm) are a subtype of extracellular vesicles secreted by cells containing DNA, microRNA (miRNA), and proteins. Exosomes are expected to be valuable as a means of delivering drugs or functional miRNAs in treatment of diseases. However, the delivery of exosomes is not sufficiently effective, even though exosomes have intrinsic delivery functions. Cell-penetrating peptides (CPPs) are short peptide families that facilitate cellular intake of molecules and vesicles. We previously reported that the modification of cells, and liposomes with CPP-conjugated-lipids, CPPs conjugated with poly (ethylene glycol)-conjugated phospholipids (PEG-lipid), that induce adhesion by CPPs, can be useful for cell-based assays and harvesting liposomes. In this study, we aimed to modulate the exosome surface using Tat peptide (YGRKKRRQRRR)-PEG-lipids to improve intracellular delivery to endothelial cells. We isolated and characterized exosomes from the medium of HEK 293 T cell cultures. Tat conjugated PEG-lipids with different spacer molecular weights and lipid types were incorporated into exosomes using fluorescein isothiocyanate labeling to optimize the number of Tat-PEG-lipids immobilized on the exosome surface. The exosomes modified with Tat-PEG-lipids were incubated with human umbilical vein endothelial cells (HUVECs) to study the interaction. Tat conjugated with 5 kDa PEG and C16 lipids incorporated on the exosome surface were highly detected inside HUVECs by flow cytometry. Fluorescence was negligible in HUVECs for control groups. Thus, Tat-PEG-lipids can be modified on the exosome surface, improving the intracellular delivery of exosomes.

Impact of spontaneous liposome modification with phospholipid polymer-lipid conjugates on protein interactions.

Liposome surface coating has been studied to avoid the immunological responses caused by the complement system, and alternative materials to poly(ethylene glycol) (PEG) have been explored recently since the production of anti-PEG IgM antibodies has been found in humans. We previously reported a liposome coating with poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC))-conjugated lipids (PMPC-lipids) and demonstrated its protective effect on blood protein interactions. Here, we attempted to modify the liposome surface by exogenously adding PMPC-lipids, which were spontaneously incorporated into the outer membrane via hydrophobic interactions. The polymerization degree of the PMPC segment was regulated from 10 to 100. The incorporated ratio of PMPC-lipid increased with a decrease in the degree of PMPC polymerization. Due to surface modification with PMPC-lipids, increase in the length of the PMPC-chain increased the size of the liposomes. The modified liposomes were kept stable for 14 d in terms of their size, polydispersity, and surface properties, where approximately 70% of PMPC-lipids were incorporated into the liposome surface. We demonstrated that liposome surface modification with PMPC-lipids can inhibit protein adsorption when exposed to serum, regardless of the degree of polymerization of PMPC. In addition, the PMPC-lipid modified surface was not recognized by the anti-PEG IgM antibody, whereas PEG-lipid was recognized by the antibody. Thus, we successfully fabricated an inert liposome surface via spontaneous modification with PMPC-lipids, where only the outer bilayer surface was modified. This technique can be available for full loading of water-soluble active pharmaceutical ingredient inside the modified liposome.

Hair cycle resting phase is regulated by cyclic epithelial FGF18 signaling.

Hair follicles repeatedly cycle through growth (anagen), regression (catagen), and resting (telogen) phases. Although the signaling molecules involved in the anagen and anagen-catagen transition have been studied extensively, the signaling that controls telogen is only partly understood. Here we show that fibroblast growth factor (Fgf)18 is expressed in a hair stem cell niche throughout telogen, and that it regulates the hair cycle through the non-growth phases. When the Fgf18 gene is conditionally knocked out in keratin 5-positive epithelial cells in mice, telogen becomes very short, giving rise to a strikingly rapid succession of hair cycles. In wild-type mice, hair follicle growth during anagen is strongly suppressed by local delivery of FGF18 protein. Our results demonstrate that epithelial FGF18 signaling and its reduction in the milieu of hair stem cells are crucial for the maintenance of resting and growth phase, respectively.

An FGF1:FGF2 chimeric growth factor exhibits universal FGF receptor specificity, enhanced stability and augmented activity useful for epithelial proliferation and radioprotection.

Structural instability of wild-type fibroblast growth factor (FGF)-1 and its dependence on exogenous heparin for optimal activity diminishes its potential utility as a therapeutic agent. Here we evaluated FGFC, an FGF1:FGF2 chimeric protein, for its receptor affinity, absolute heparin-dependence, stability and potential clinical applicability. Using BaF3 transfectants overexpressing each FGF receptor (FGFR) subtype, we found that, like FGF1, FGFC activates all of the FGFR subtypes (i.e., FGFR1c, FGFR1b, FGFR2c, FGFR2b, FGFR3c, FGFR3b and FGFR4) in the presence of heparin. Moreover, FGFC activates FGFRs even in the absence of heparin. FGFC stimulated keratinocytes proliferation much more strongly than FGF2, as would be expected from its ability to activate FGFR2b. FGFC showed greater structural stability, biological activity and resistance to trypsinization, and less loss in solution than FGF1 or FGF2. When FGFC was intraperitoneally administered to BALB/c mice prior to whole body gamma-irradiation, survival of small intestine crypts was significantly enhanced, as compared to control mice. These results suggest that FGFC could be useful in a variety of clinical applications, including promotion of wound healing and protection against radiation-induced damage.

betaKlotho is required for fibroblast growth factor (FGF) 21 signaling through FGF receptor (FGFR) 1c and FGFR3c.

Fibroblast growth factor (FGF) 21, a structural relative of FGF23 that regulates phosphate homeostasis, is a regulator of insulin-independent glucose transport in adipocytes and plays a role in the regulation of body weight. It also regulates ketogenesis and adaptive responses to starvation. We report that in a reconstituted receptor activation assay system using BaF3 cells, which do not endogenously express any type of FGF receptor (FGFR) or heparan sulfate proteoglycan, FGF21 alone does not activate FGFRs and that betaKlotho is required for FGF21 to activate two specific FGFR subtypes: FGFR1c and FGFR3c. Coexpression of betaKlotho and FGFR1c on BaF3 cells enabled FGF21, but not FGF23, to activate receptor signaling. Conversely, coexpression of FGFR1c and Klotho, a protein related to betaKlotho, enabled FGF23 but not FGF21 to activate receptor signaling, indicating that expression of betaKlotho/Klotho confers target cell specificity on FGF21/FGF23. In all of these cases, heparin enhanced the activation but was not essential. In 3T3-L1 adipocytes, up-regulation of glucose transporter (GLUT) expression by FGF21 was associated with expression of betaKlotho, which was absent in undifferentiated 3T3-L1 fibroblasts. It is thus suggested that betaKlotho expression is a crucial determinant of the FGF21 specificity of the target cells upon which it acts in an endocrine fashion.

Expression of fibroblast growth factors and their receptors during full-thickness skin wound healing in young and aged mice.

The highly ordered process of wound healing involves the coordinated regulation of cell proliferation and migration and tissue remodeling, predominantly by polypeptide growth factors. Consequently, the slowing of wound healing that occurs in the aged may be related to changes in the activity of these various regulatory factors. To gain additional insight into these issues, we quantified the absolute copy numbers of mRNAs encoding all the fibroblast growth factors (FGFs), their receptors (FGFRs) and two other growth factors in the dorsal skin of young and aged mice during the healing of full-thickness skin excisional wounds. In young adult mice (8 weeks old), FGF7, FGF10 and FGF22 mRNAs were all strongly expressed in healthy skin, and levels of FGF7 and 10 but not 22 increased 2- to 3.5-fold over differing time courses after wounding. The levels of FGF9, 16, 18 and especially 23 mRNAs were moderate or low in healthy skin but increased 2- to 33-fold after wounding. Among the four FGFRs, expression of only FGFR1 mRNA was augmented during wound healing. Expression of transforming growth factor-beta and hepatocyte growth factor was also high in healthy skin and was upregulated during healing. Notably, in aged mice (35 weeks old), where healing proceeded more slowly than in the young, both the basal and wound-induced mRNA expression of most of these genes was reduced. While these results confirm the established notion that FGFR2 IIIB ligands (FGF7 and FGF10) are important for wound healing, they also suggest that decreased expression of multiple FGF ligands contributes to the slowing of wound healing in aged mice and indicate the potential importance of further study of the involvement of FGF9, 16, 18 and 23 in the wound healing process.

Comprehensive analysis of FGF and FGFR expression in skin: FGF18 is highly expressed in hair follicles and capable of inducing anagen from telogen stage hair follicles.

We quantified the mRNA expression of all 22 fibroblast growth factor family members (FGF) and their four receptors (FGFR) in adult mouse full-thickness skin at various stages of the hair growth cycle. We found that in addition to mRNA encoding FGF previously identified in skin (FGF1, 2, 5, 7, 10, 13, and 22), FGF18 mRNA was also strongly expressed. Expression of these FGF varied throughout hair growth cycle: mRNA expression of FGF18 and 13 peaked at telogen; FGF7 and 10 at anagen V; and FGF5 and 22 at anagen VI. In situ hybridization revealed that FGF18 mRNA is mainly expressed in the anagen inner root sheath and telogen bulge of hair follicles. In culture, FGF18 stimulated DNA synthesis in human dermal fibroblasts, dermal papilla cells, epidermal keratinocytes and vascular endothelial cells. When FGF18 was administered subcutaneously to mice in a uniform telogen state, anagen hair growth was observed. Our findings suggest that FGF18 is important for the regulation of hair growth and the maintenance of skin in adult mice.