Mechanisms of Wharton's Jelly-derived MSCs in enhancing peripheral nerve regeneration.

Warton's jelly-derived Mesenchymal stem cells (WJ-MSCs) play key roles in improving nerve regeneration in acellular nerve grafts (ANGs); however, the mechanism of WJ-MSCs-related nerve regeneration remains unclear. This study investigated how WJ-MSCs contribute to peripheral nerve regeneration by examining immunomodulatory and paracrine effects, and differentiation potential. To this end, WJ-MSCs were isolated from umbilical cords, and ANGs (control) or WJ-MSCs-loaded ANGs (WJ-MSCs group) were transplanted in injury animal model. Functional recovery was evaluated by ankle angle and tetanic force measurements up to 16 weeks post-surgery. Tissue biopsies at 3, 7, and 14 days post-transplantation were used to analyze macrophage markers and interleukin (IL) levels, paracrine effects, and MSC differentiation potential by quantitative real-time polymerase chain reaction (RT-qPCR) and immunofluorescence staining. The WJ-MSCs group showed significantly higher ankle angle at 4 weeks and higher isometric tetanic force at 16 weeks, and increased expression of CD206 and IL10 at 7 or 14 days than the control group. Increased levels of neurotrophic and vascular growth factors were observed at 14 days. The WJ-MSCs group showed higher expression levels of S100ß; however, the co-staining of human nuclei was faint. This study demonstrates that WJ-MSCs' immunomodulation and paracrine actions contribute to peripheral nerve regeneration more than their differentiation potential.

Comparison of mesenchymal stem cell attachment efficiency in acellular neural graft for peripheral nerve regeneration.

Decellularized nerve allograft is an alternative to autologous nerve graft for nerve defects but has shown inferior clinical outcomes. Mesenchymal stem cells can play a key role in improving nerve regeneration of decellularized nerve allografts. The purpose of this study was to compare different mesenchymal stem cell seeding methods and to find the most efficient way to attach cells to nerve grafts for peripheral nerve regeneration. Wharton's jelly mesenchymal stem cells were collected from human umbilical cords and were seeded in the acellular nerve graft in five different ways as follows: PBS injection, fibrin glue drop, Matrigel drop, bioreactor, and Matrigel injection. A 6-mm sciatic nerve defect of Sprague-Dawley rats was bridged using mesenchymal stem cells-laden acellular nerve grafts according to the five seeding methods. Two days after implantation, the nerve tissue was biopsied and analyzed by the immunofluorescence staining of nuclei. The number of Wharton's jelly mesenchymal stem cells (+ h Nuclei) was counted in the inside, outside, and the total area of the graft sections under 200X magnification. The highest efficiency of mesenchymal stem cell attachment inside the graft and the highest total number of attached mesenchymal stem cells was observed in the group using Matrigel injection (p < 0.0001). This study showed mesenchymal stem cells can be more effectively attached to decellularized nerve graft using the injection method with Matrigel than other static or dynamic seeding methods in vivo.

Effect of glutaraldehyde-crosslinked cartilage acellular matrix film on anti-adhesion and nerve regeneration in a rat sciatic nerve injury model.

Decellularized extra-cellular matrix (ECM) has been studied as an alternative to anti-adhesive biomaterials and cartilage acellular matrix (CAM) has been shown to inhibit postoperative adhesion in several organs. This study aimed to evaluate the suitability of glutaraldehyde (GA) crosslinked CAM-films as anti-adhesion barriers for peripheral nerve injury. The films were successfully fabricated and showed improved physical properties such as mechanical strength, swelling ratio, and lengthened degradation period while maintaining the microstructure and chemical composition after GA crosslinking. In the in vitro study of CAM-film, the dsDNA content met the recommended limit of decellularization and more than 70% of the major ECM components were preserved after decellularization. The adhesion and proliferation of seeded human umbilical vein endothelial cells and fibroblasts were significantly lower in CAM-film than in control, but similar with Seprafilm. However, the CAM-film extract did not show cytotoxicity. In the in vivo study, the peri-neural fibrosis was thicker, adhesion score higher, and peri-neural collagen fibers more abundant in the control group than in the CAM-film group. The total number of myelinated axons was significantly higher in the CAM-film group than in the control group. The inflammatory marker decreased with time in the CAM-film group compared to that in the control group, whereas the nerve regenerative marker expression was maintained. Moreover, the ankle angles at contracture and toe-off were higher in the CAM film-treated rats than in the control rats. GA-crosslinked CAM films may be used during peripheral nerve surgery to prevent peri-neural adhesion and enhance nerve functional recovery.

Mesenchymal Stem Cells Derived from Wharton's Jelly Can Differentiate into Schwann Cell-Like Cells and Promote Peripheral Nerve Regeneration in Acellular Nerve Grafts.

BACKGROUND: Schwann cells (SCs) secrete neurotrophic factors and provide structural support and guidance during axonal regeneration. However, nearby nerves may be damaged to obtain primary SCs, and there is a lack of nervous tissue donors. We investigated the potential of Wharton's Jelly-derived mesenchymal stem cells (WJ-MSCs) in differentiating into Schwann cell-like cells (WJ-SCLCs) as an alternative to SCs. We also examined whether implantation of WJ-SCLCs-laden acellular nerve grafts (ANGs) are effective in inducing functional recovery and nerve regeneration in an animal model of peripheral nerve injury. METHODS: The differentiation of WJ-MSCs into WJ-SCLCs was determined by analyzing SC-specific markers. The secretion of neurotrophic factors was assessed by the Neuro Discovery antibody array. Neurite outgrowth and myelination of axons were found in a co-culture system involving motor neuron cell lines. The effects of ANGs on repairing sciatic nerves were evaluated using video gait angle test, isometric tetanic force analysis, and toluidine blue staining. RESULTS: Compared with undifferentiated WJ-MSCs, WJ-SCLCs showed higher expression levels of SC-specific markers such as S100ß, GFAP, KROX20, and NGFR. WJ-SCLCs also showed higher secreted amounts of brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and granulocyte-colony stimulating factor than did WJ-MSCs. WJ-SCLCs effectively promoted the outgrowth and myelination of neurites in motor neuron cells, and WJ-SCLCs laden ANGs significantly facilitated peripheral nerve regeneration in an animal model of sciatic nerve injury. CONCLUSION: WJ-MSCs were readily differentiated into WJ-SCLCs, which effectively promoted the regeneration of peripheral nerves. Transplantation of WJ-SCLCs with ANGs might be useful for assisting peripheral nerve regeneration.

Shikimic acid, a mannose bioisostere, promotes hair growth with the induction of anagen hair cycle.

Shikimic acid (SA) has recently been found to be a major component of plant stem cells. The exact effects of SA on human hair follicles (HFs) is unknown. The purpose of this study was to examine the effects of SA on hair growth. We investigated the effect of SA on an in vivo C57BL/6 mouse model. We examined the expression of mannose receptor (MR), which is a known receptor of SA, in human HFs and the effect of SA on human dermal papilla cells (hDPCs), outer root sheath cells (hORSCs), and on ex vivo human hair organ culture. SA significantly prolonged anagen hair growth in the in vivo mouse model. We confirmed expression of the MR in human HFs, and that SA increased the proliferation of hDPCs and hORSCs. It was found that SA enhanced hair shaft elongation in an ex vivo human hair organ culture. SA treatment of hDPCs led to increased c-myc, hepatocyte growth factor, keratinocyte growth factor and vascular endothelial growth factor levels and upregulation of p38 MAPK and cAMP response element-binding protein levels. Our results show that SA promotes hair growth and may serve as a new therapeutic agent in the treatment of alopecia.

Skin equivalent assay: An optimized method for testing for hair growth reconstitution capacity of epidermal and dermal cells.

Hair follicle reconstitution requires highly organized epithelial-mesenchymal interactions. Skin equivalents containing the epidermal and dermal cells with hair reconstitution capacity can reproduce these processes, but have not been established. This study was conducted to develop a hair follicle-producing three-dimensional (3D) skin equivalent assay using neonate mouse epidermal and dermal cells. A skin equivalent comprised of mouse dermal cells (MDCs) embedded in type I collagen and overlaid with mouse epidermal cells (MECs) was used. MDCs were mixed with type I collagen and cultured for 7 days. One day after adding MECs on top, the composites were grafted onto nude mice. MDCs cultured on a two-dimensional (2D) plate for 7 days and mixed with MECs as a negative control, and freshly isolated MDCs and MECs mixture (chamber assay) as a positive control were also grafted. Six weeks after grafting, regenerated hair follicles were analysed. Our 3D skin equivalent culture assay reproducibly regenerated hair follicles, while MDCs precultured in the 2D model with MECs did not. Compared to the chamber assay, which produced randomly oriented hair follicles, nearly all regenerated hair follicles in our assay extruded through the skin and numerous regenerated hair follicles were higher than those in the chamber assay. Several representative genes associated with hair induction showed higher expression in our assay than in the 2D model. When Wnt3a was added, the number of regenerated hairs increased. Organized hair follicle regeneration was accomplished using our assay. This approach can be applied to assess a test agent with hair growth-promoting effects.

The effect of cilostazol, a phosphodiesterase 3 (PDE3) inhibitor, on human hair growth with the dual promoting mechanisms.

BACKGROUND: Cilostazol, a phosphodiesterase 3 (PDE3) inhibitor, increases the intracellular level of cyclic adenosine monophosphate to cause vasodilation. Topical application of cilostazol is reported to improve local blood flow and enhance wound healing; however, its effect on human hair follicles is unknown. OBJECTIVE: The purpose of this study was to determine the effect of cilostazol on hair growth. METHODS: We investigated the expression of PDE3 in human dermal papilla cells (DPCs), outer root sheath cells (ORSCs), and hair follicles. The effects of cilostazol on DPC and ORSC proliferation were evaluated using BrdU and WST-1 assays. The expression of various growth factors in DPCs was investigated by growth factor antibody array. Additionally, hair shaft elongation was measured using ex vivo hair follicle organ cultures, and anagen induction was evaluated in C57BL/6 mice. Finally, the effects of cilostazol on vessel formation and activation of the mitogen-activated protein kinase pathway were evaluated. RESULTS: We confirmed high mRNA and protein expression of PDE3 in human DPCs. Cilostazol not only enhanced the proliferation of human DPCs but also regulated the secretion of several growth factors responsible for hair growth. Furthermore, it promoted hair shaft elongation ex vivo, with increased proliferation of matrix keratinocytes. Cilostazol also accelerated anagen induction by stimulating vessel formation and upregulating the levels of phosphorylated extracellular signal-regulated kinase, c-Jun N-terminal kinase, and P38 after its topical application in C57BL/6 mice. CONCLUSION: Our results show that cilostazol promotes hair growth and may serve as a therapeutic agent for the treatment of alopecia.

Acute Stress-Induced Changes in Follicular Dermal Papilla Cells and Mobilization of Mast Cells: Implications for Hair Growth.

BACKGROUND: Stress is a known cause of hair loss in many species. OBJECTIVE: In this study, we investigated the role of acute stress on hair growth using a rat model. METHODS: Rats were immobilized for 24 hours and blood samples, and skin biopsies were taken. The effect of stress-serum on the in vitro proliferation of rat and human dermal papilla cells (hDPCs), as well as serum cortisol and corticotropin-releasing hormone levels, were measured. Mast cell staining was performed on the biopsied tissue. In addition, Western blot and quantitative real time polymerase chain reaction were used to assess mast cell tryptase and cytokine expression, respectively in rat skin biopsies. RESULTS: Stress-serum treatment reduced significantly the number of viable hDPCs and arrested the cell cycle in the G1 phase, compared to serum from unrestrained rats (p<0.05, respectively). Moreover, restrained rats had significantly higher levels of cortisol in serum than unrestrained rats (p<0.01). Acute stress serum increased mast cell numbers and mast cell tryptase expression, as well as inducing interleukin (IL)-6 and IL-1ß up-regulation. CONCLUSION: These results suggest that acute stress also has an inhibitory effect on hair growth via cortisol release in addition to substance P-mast cell pathway.

Enhancement of Human Hair Growth Using Ecklonia cava Polyphenols.

BACKGROUND: Ecklonia cava is a brown alga that contains various compounds, including carotenoids, fucoidans, and phlorotannins. E. cava polyphenols (ECPs) are known to increase fibroblast survival. The human dermal papilla cell (hDPC) has the properties of mesenchymal-origin fibroblasts. OBJECTIVE: This study aims to investigate the effect of ECPs on human hair growth promotion in vitro and ex vivo. METHODS: MTT assays were conducted to examine the effect of ECPs on hDPC proliferation. Hair growth was measured using ex-vivo hair follicle cultures. Real-time polymerase chain reaction was performed to evaluate the mRNA expression of various growth factors in ECP-treated hDPCs. RESULTS: Treatment with 10 µg/ml purified polyphenols from E. cava (PPE) enhanced the proliferation of hDPCs 30.3% more than in the negative control (p<0.001). Furthermore, 0.1 µg/ml PPE extended the human hair shaft 30.8% longer than the negative control over 9 days (p<0.05). Insulin-like growth factor-1 (IGF-1) mRNA expression increased 3.2-fold in hDPCs following treatment with 6 µg/ml PPE (p<0.05). Vascular endothelial growth factor (VEGF) mRNA expression was also increased 2.0-fold by 3 µg/ml PPE (p<0.05). Treatment with 10 µg/ml PPE reduced oxidative stress in hDPCs (p<0.05). CONCLUSION: These results suggest that PPE could enhance human hair growth. This can be explained by hDPC proliferation coupled with increases in growth factors such as IGF-1 and VEGF. Reducing oxidative stress is also thought to help increase hDPCs. These favorable results suggest that PPE is a promising therapeutic candidate for hair loss.

Role of Arachidonic Acid in Promoting Hair Growth.

BACKGROUND: Arachidonic acid (AA) is an omega-6 polyunsaturated fatty acid present in all mammalian cell membranes, and involved in the regulation of many cellular processes, including cell survival, angiogenesis, and mitogenesis. The dermal papilla, composed of specialized fibroblasts located in the bulb of the hair follicle, contributes to the control of hair growth and the hair cycle. OBJECTIVE: This study investigated the effect of AA on hair growth by using in vivo and in vitro models. METHODS: The effect of AA on human dermal papilla cells (hDPCs) and hair shaft elongation was evaluated by MTT assay and hair follicle organ culture, respectively. The expression of various growth and survival factors in hDPCs were investigated by western blot or immunohistochemistry. The ability of AA to induce and prolong anagen phase in C57BL/6 mice was analyzed. RESULTS: AA was found to enhance the viability of hDPCs and promote the expression of several factors responsible for hair growth, including fibroblast growth factor-7 (FGF-7) and FGF-10. Western blotting identified the role of AA in the phosphorylation of various transcription factors (ERK, CREB, and AKT) and increased expression of Bcl-2 in hDPCs. In addition, AA significantly promoted hair shaft elongation, with increased proliferation of matrix keratinocytes, during ex vivo hair follicle culture. It was also found to promote hair growth by induction and prolongation of anagen phase in telogen-stage C57BL/6 mice. CONCLUSION: This study concludes that AA plays a role in promoting hair growth by increasing the expression of growth factors in hDPCs and enhancing follicle proliferation and survival.

Comparative Analysis of Human Epidermal and Peripheral Blood γδ T Cell Cytokine Profiles.

BACKGROUND: Human epidermal γδ T cells are known to play crucial roles in the defense and homeostasis of the skin. However, their precise mechanism of action in skin inflammation remains less clear. OBJECTIVE: In this study, we analyzed the cytokine expression profile of human epidermal γδ T cells and compared it to that of peripheral blood γδ T cells to investigate the specific activity of epidermal γδ T cells in modulating skin inflammation. METHODS: We isolated γδ T cells from epidermal tissue or peripheral blood obtained from healthy volunteers. Isolated γδ T cells were stimulated using immobilized anti-CD3 antibody and interleukin-2 plus phytohaemagglutinin, and were then analyzed using a cytokine array kit. RESULTS: Both epidermal and peripheral blood γδ T cells produced comparable levels of granulocyte-macrophage colony-stimulating factor, I-309, interferon-γ, macrophage migration inhibitory factor, macrophage inflammatory protein-1α, and chemokine (C-C) ligand 5. The epidermal γδ T cells produced significantly higher levels of interleukin-4, -8, -13, and macrophage inflammatory protein-1ß than the peripheral blood γδ T cells did. Notably, the epidermal γδ T cells produced several hundred-fold higher levels of interleukin-13 than interleukin-4. CONCLUSION: These results suggest that the epidermal γδ T cells have a stronger potential to participate in the Th2-type response than the peripheral blood γδ T cells do. Furthermore, epidermal γδ T cells might play an important role in the pathogenesis of Th2-dominant skin diseases because of their active production of interleukin-13.

Valproic acid promotes human hair growth in in vitro culture model.

BACKGROUND: ß-Catenin, the transducer of Wnt signaling, is critical for the development and growth of hair follicles. In the absence of Wnt signals, cytoplasmic ß-catenin is phosphorylated by glycogen synthase kinase (GSK)-3 and then degraded. Therefore, inhibition of GSK-3 may enhance hair growth via ß-catenin stabilization. Valproic acid is an anticonvulsant and a mood-stabilizing drug that has been used for decades. Recently, valproic acid was reported to inhibit GSK-3ß in neuronal cells, but its effect on human hair follicles remains unknown. OBJECTIVES: To determine the effect of VPA on human hair growth. METHODS: We investigated the effect of VPA on cultured human dermal papilla cells and outer root sheath cells and on an in vitro culture of human hair follicles, which were obtained from scalp skin samples of healthy volunteers. Anagen induction by valproic acid was evaluated using C57BL/6 mice model. RESULTS: Valproic acid not only enhanced the viability of human dermal papilla cells and outer root sheath cells but also promoted elongation of the hair shaft and reduced catagen transition of human hair follicles in organ culture model. Valproic acid treatment of human dermal papilla cells led to increased ß-catenin levels and nuclear accumulation and inhibition of GSK-3ß by phosphorylation. In addition, valproic acid treatment accelerated the induction of anagen hair in 7-week-old female C57BL/6 mice. CONCLUSIONS: Valproic acid enhanced human hair growth by increasing ß-catenin and therefore may serve as an alternative therapeutic option for alopecia.

Effects of glucocorticoid on human dermal papilla cells in vitro.

Glucocorticoid (GC) is synthesized mostly in the adrenal gland and is secreted in response to stressful conditions. The stress-induced increase in systemic GC may mediate diverse types of cellular damage. However, the specific effects of GC on the dermal papilla cells (DPCs) of hair follicles remain unknown, although stress-related hair loss has increased significantly in recent years. The objective of this study was to determine the effect of a synthetic GC, dexamethasone (Dex), on human DPCs in vitro. We evaluated the effects of Dex on cell proliferation, survival, and the expression of growth factors in DPCs. Dex treatment (1µM) significantly reduced the number of viable cells and the expression of the Ki-67 protein, VEGF and HGF were downregulated following treatment of DPCs with Dex. Taken together, we concluded that Dex inhibits human hair growth by inhibiting both the proliferation of, and growth factors expression by, DPCs.