scholarly journals In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics

2018 ◽  
Author(s):  
Chee-Wai Wong ◽  
Beverley F. Kinnear ◽  
Radoslaw M. Sobota ◽  
Rajkumar Ramalingam ◽  
Catherine F. LeGrand ◽  
...  
Keyword(s):  
Cell Fate ◽  
Dermal Fibroblast ◽  
Human Keratinocytes ◽  
Human Dermal Fibroblast ◽  
Dermal Fibroblasts ◽  
Primary Human Keratinocytes ◽  
Matrix Deposition ◽  
Forming Efficiency

SummaryThe long-term expansion of keratinocytes under serum- and feeder free conditions generally results in diminished proliferation and an increased commitment to terminal differentiation. Here we present a serum and xenogeneic feeder free culture system that retains the self-renewal capacity of primary human keratinocytes. In vivo, the tissue microenvironment is a major contributor to determining cell fate and a key component of the microenvironment is the extracellular matrix (ECM). Accordingly, acellular ECMs derived from human dermal fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, were used as the basis for a xenogeneic-free keratinocyte expansion protocol. A phospholipase A2 decellularisation procedure produced matrices which, by proteomics analysis, resembled in composition the core matrix proteins of skin dermis. On these ECMs keratinocytes proliferated rapidly, retained their small size, expressed p63, did not express keratin 10 and rarely expressed keratin 16. Moreover, the colony forming efficiency of keratinocytes cultured on these acellular matrices was markedly enhanced. Collectively these data indicate that the dermal fibroblast-derived matrices support the in vitro expansion of keratinocytes that maintained stem-like characteristics under serum free conditions.

scholarly journals In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Chee-Wai Wong ◽  
Catherine F. LeGrand ◽  
Beverley F. Kinnear ◽  
Radoslaw M. Sobota ◽  
Rajkumar Ramalingam ◽  
...  
Keyword(s):  
Dermal Fibroblast ◽  
Human Keratinocytes ◽  
Dermal Fibroblasts ◽  
Collagen I ◽  
Matrix Composition ◽  
Major Influence ◽  
Primary Human Keratinocytes ◽  
Matrix Deposition

AbstractThe long-term expansion of keratinocytes under conditions that avoid xenogeneic components (i.e. animal serum- and feeder cell-free) generally causes diminished proliferation and increased terminal differentiation. Here we present a culture system free of xenogeneic components that retains the self-renewal capacity of primary human keratinocytes. In vivo the extracellular matrix (ECM) of the tissue microenvironment has a major influence on a cell’s fate. We used ECM from human dermal fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, in a xenogeneic-free keratinocyte expansion protocol. Phospholipase A2 decellularisation produced ECM whose components resembled the core matrix composition of natural dermis by proteome analyses. Keratinocytes proliferated rapidly on these matrices, retained their small size, expressed p63, lacked keratin 10 and rarely expressed keratin 16. The colony forming efficiency of these keratinocytes was enhanced over that of keratinocytes grown on collagen I, indicating that dermal fibroblast-derived matrices maintain the in vitro expansion of keratinocytes in a stem-like state. Keratinocyte sheets formed on such matrices were multi-layered with superior strength and stability compared to the single-layered sheets formed on collagen I. Thus, keratinocytes expanded using our xenogeneic-free protocol retained a stem-like state, but when triggered by confluence and calcium concentration, they stratified to produce epidermal sheets with a potential clinical use.

scholarly journals In VivoApplicability ofNeosartorya fischeriAntifungal Protein 2 (NFAP2) in Treatment of Vulvovaginal Candidiasis

2018 ◽  
Vol 63 (2) ◽  
pp. e01777-18 ◽  
Author(s):  
Renátó Kovács ◽  
Jeanett Holzknecht ◽  
Zoltán Hargitai ◽  
Csaba Papp ◽  
Attila Farkas ◽  
...  
Keyword(s):  
Human Keratinocytes ◽  
Dermal Fibroblasts ◽  
Vulvovaginal Candidiasis ◽  
Antifungal Protein ◽  
Primary Human Keratinocytes ◽  
Fungal Cell ◽  
Efficient Treatment ◽  
Content Type

ABSTRACTAs a consequence of emerging numbers of vulvovaginitis cases caused by azole-resistant and biofilm-formingCandidaspecies, fast and efficient treatment of this infection has become challenging. The problem is further exacerbated by the severe side effects of azoles as long-term-use medications in the recurrent form. There is therefore an increasing demand for novel and safely applicable effective antifungal therapeutic strategies. The small, cysteine-rich, and cationic antifungal proteins from filamentous ascomycetes are potential candidates, as they inhibit the growth of severalCandidaspp.in vitro; however, no information is available about theirin vivoantifungal potency against yeasts. In the present study, we investigated the possible therapeutic application of one of their representatives in the treatment of vulvovaginal candidiasis,Neosartorya fischeriantifungal protein 2 (NFAP2). NFAP2 inhibited the growth of a fluconazole (FLC)-resistantCandida albicansstrain isolated from a vulvovaginal infection, and it was effective against both planktonic cells and biofilmin vitro. We observed that the fungal cell-killing activity of NFAP2 is connected to its pore-forming ability in the cell membrane. NFAP2 did not exert cytotoxic effects on primary human keratinocytes and dermal fibroblasts at the MICin vitro. In vivomurine vulvovaginitis model experiments showed that NFAP2 significantly decreases the number of FLC-resistantC. albicanscells, and combined application with FLC enhances the efficacy. These results suggest that NFAP2 provides a feasible base for the development of a fundamental new, safely applicable mono- or polytherapeutic topical agent for the treatment of superficial candidiasis.

A novel design of wound bandage using heparin-polyvinylpyrrolidone/TiO2 nanocomposite to improved antibacterial treatment and burn wound healing effect: In vitro and in vivo evaluation

2021 ◽  
Vol 11 (11) ◽  
pp. 1808-1818
Author(s):  
Xiuli Li ◽  
Jigang Wang ◽  
Xin Li ◽  
Xiaoqian Hou ◽  
Hao Wang ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Dermal Fibroblast ◽  
Synergistic Effects ◽  
Bacterial Species ◽  
Human Dermal Fibroblast ◽  
Burn Wound ◽  
In Vivo Evaluation ◽  
Closure Rate

In our current study, porous heparin-polyvinylpyrrolidone/TiO2 nanocomposite (HpPVP/TiO2) bandage were prepared via the incorporation of TiO2 into HpPVP hydrogels for biomedical applications such as burn infection. The effect of the HpPVP hydrogels and the nanoparticles of TiO2 composition on the functional group and the surface properties of the as-fabricated bandages were characterized by Fourier transform infrared spectroscopy (FTIR) and X-ray diffractometry (XRD). The presence of TiO2 nanoparticles created the internal structure of the HpPVP hydrogel that aids in a homogeneous porous structure, as indicated by the scanning electron microscope (SEM). The size distribution of the TiO2 nanoparticles was measured using a transmission electron microscope (TEM). The studies on the mechanical properties of the HpPVP hydrogel indicate that the addition of TiO2 nanoparticles increases its strength. The prepared HpPVP/TiO2 nanocomposite dressing has excellent antimicrobial activity were tested against bacterial species (Staphylococcus aureus and Escherichia coli) and has good biocompatibility against human dermal fibroblast cells (HFFF2) for biological applications. In addition, in vivo evaluations in Kunming mice exposed that the as-fabricated HpPVP/TiO2 nanocomposite bandages increased the wound curing and facilitated accelerate skin cell construction along with collagen development. The synergistic effects of the HpPVP/TiO2 nanocomposite hydrogel dressing material, such as its excellent hydrophilic nature, good bactericidal activity, biocompatibility and wound closure rate through in vivo test makes it a suitable candidate for burn infections.

scholarly journals Inhibition of Calcineurin/NFAT Signaling Blocks Oncogenic H-Ras Induced Autophagy in Primary Human Keratinocytes

2021 ◽  
Vol 9 ◽  
Author(s):  
Shuangshuang Wang ◽  
Hua Qian ◽  
Liwei Zhang ◽  
Panpan Liu ◽  
Dexuan Zhuang ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Human Keratinocytes ◽  
Primary Human Keratinocytes ◽  
Activated T Cells ◽  
Ras Pathway ◽  
Ras Family ◽  
Interfering Rna

Mutations of H-Ras, a member of the RAS family, are preferentially found in cutaneous squamous cell carcinomas (SCCs). H-Ras has been reported to induce autophagy, which plays an essential role in tissue homeostasis in multiple types of cancer cells and in fibroblasts, however, the potential role of H-Ras in regulating autophagy in human keratinocytes has not been reported. In this study, we found that the stable expression of the G12V mutant of H-RAS (H-RasG12V) induced autophagy in human keratinocytes, and interestingly, the induction of autophagy was strongly blocked by inhibiting the calcineurin/nuclear factor of activated T cells (NFAT) pathway with either a calcineurin inhibitor (Cyclosporin A) or a NFAT inhibitor (VIVIT), or by the small interfering RNA (siRNA) mediated knockdown of calcineurin B1 or NFATc1 in vitro, as well as in vivo. To characterize the role of the calcineurin/NFAT pathway in H-Ras induced autophagy, we found that H-RasG12V promoted the nuclear translocation of NFATc1, an indication of the activation of the calcineurin/NFAT pathway, in human keratinocytes. However, activation of NFATc1 either by the forced expression of NFATc1 or by treatment with phenformin, an AMPK activator, did not increase the formation of autophagy in human keratinocytes. Further study revealed that inhibiting the calcineurin/NFAT pathway actually suppressed H-Ras expression in H-RasG12V overexpressing cells. Finally, chromatin immunoprecipitation (ChIP) assays showed that NFATc1 potentially binds the promoter region of H-Ras and the binding efficiency was significantly enhanced by the overexpression of H-RasG12V, which was abolished by treatment with the calcineurin/NFAT pathway inhibitors cyclosporine A (CsA) or VIVIT. Taking these data together, the present study demonstrates that the calcineurin/NFAT signaling pathway controls H-Ras expression and interacts with the H-Ras pathway, involving the regulation of H-Ras induced autophagy in human keratinocytes.

scholarly journals A protein phosphatase network controls the temporal and spatial dynamics of differentiation commitment in human epidermis

2017 ◽  
Author(s):  
Ajay Mishra ◽  
Angela Oliveira Pisco ◽  
Benedicte Oules ◽  
Tony Ly ◽  
Kifayathullah Liakath-Ali ◽  
...  
Keyword(s):  
Stem Cell ◽  
Terminal Differentiation ◽  
Human Keratinocytes ◽  
Cell Renewal ◽  
Stable States ◽  
Primary Human Keratinocytes ◽  
Network Modelling ◽  
Epidermal Homeostasis

AbstractEpidermal homeostasis depends on a balance between stem cell renewal and terminal differentiation1,2. While progress has been made in characterising the stem and differentiated cell compartments3, the transition between the two cell states, termed commitment4, is poorly understood. Here we characterise commitment by integrating transcriptomic and proteomic data from disaggregated primary human keratinocytes held in suspension for up to 12h. We have previously shown that commitment begins at approximately 4h and differentiation is initiated by 8h5. We find that cell detachment induces a network of protein phosphatases. The pro-commitment phosphatases – including DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA – promote terminal differentiation by negatively regulating ERK MAPK and positively regulating key API transcription factors. Their activity is antagonised by concomitant upregulation of the anti-commitment phosphatase DUSP10. The phosphatases form a dynamic network of transient positive and negative interactions, with DUSP6 predominating at commitment. Boolean network modelling identifies a mandatory switch between two stable states (stem cell and differentiated cell) via an unstable (committed) state. In addition phosphatase expression is spatially regulated relative to the location of stem cells, both in vivo and in response to topographical cues in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment.

scholarly journals In Vitro and In Vivo Photoprotective Effects of (-)-Loliode Isolated from the Brown Seaweed, Sargassum horneri

Molecules ◽  
2021 ◽  
Vol 26 (22) ◽  
pp. 6898
Author(s):  
Lei Wang ◽  
Hyun-Soo Kim ◽  
Jun-Geon Je ◽  
Xiaoting Fu ◽  
Caoxing Huang ◽  
...  
Keyword(s):  
Brown Seaweed ◽  
Human Keratinocytes ◽  
Dermal Fibroblasts ◽  
Sargassum Horneri ◽  
Skin Damage ◽  
In Vivo Models ◽  
In Vivo Test ◽  
Skin Cells

Skin is the largest organ of humans. Overexposure to ultraviolet (UV) is the primary environmental factor that causes skin damage. The compound, (-)-loliode, isolated from the brown seaweed Sargassum horneri, showed strong antioxidant and anti-inflammatory activities in in vitro and in vivo models. To further explore the potential of (-)-loliode in cosmetics, in the present study, we investigated the photoprotective effect of (-)-loliode in vitro in skin cells and in vivo in zebrafish. The results indicated that (-)-loliode significantly reduced intracellular reactive oxygen species (ROS) level, improved cell viability, and suppressed apoptosis of UVB-irradiated human keratinocytes. In addition, (-)-loliode remarkably attenuated oxidative damage, improved collagen synthesis, and inhibited matrix metalloproteinases expression in UVB-irradiated human dermal fibroblasts. Furthermore, the in vivo test demonstrated that (-)-loliode effectively and dose-dependently suppressed UVB-induced zebrafish damage displayed in decreasing the levels of ROS, nitric oxide, lipid peroxidation, and cell death in UVB-irradiated zebrafish. These results indicate that (-)-loliode possesses strong photoprotective activities and suggest (-)-loliode may an ideal ingredient in the pharmaceutical and cosmeceutical industries.

Ultrastructural and metabolic effects of products of activated mononuclear leucocytes on cultured human dermal fibroblasts

1984 ◽  
Vol 42 ◽  
pp. 682-683
Author(s):  
I. Stachura ◽  
Matias Pardo ◽  
Jennifer Worrall ◽  
Theresa L. Whiteside
Keyword(s):  
Plasma Membranes ◽  
Dermal Fibroblast ◽  
Human Dermal Fibroblast ◽  
Dermal Fibroblasts ◽  
Metabolic Effects ◽  
Normal Human Skin ◽  
Normal Human ◽  
Mononuclear Leucocytes ◽  
Distinctive Phenotype

Products of antigen- or mitogen-activated mononuclear leucocytes (ML) are known to modulate fibroblast proliferation and collagen production in vitro. In tissue, ML accumulate at sites of inflammation and are probably involved in the process of fibrosis. We have established that supernatants (SN) of concanavalin A-activated ML increase synthesis of glycosaminoglycan (GAG) in human dermal fibroblast (DF) cultures). When explants of normal human skin were cultured in CMRL 1066 medium supplemented with 15% pooled human serum and containing MLSN the outgrowing DF acquired a distinctive phenotype. In comparison to control DF, the cells treated with MLSN exhibited a marked increase in the number of intracytoplasmic organelles especially dilated cisternae of RER filled with electrondense material, abundant lysosomes, prominent Golgi apparatus and bundles of microfilaments often extending beyond the cell boundaries. Cell surfaces were shaggy and floccular material accumulated in patches along the plasma membranes.

Effects Of (1→3), (1→6)-β-D-Glucan Behavior in Human Dermal Fibroblast Cells under Serum Starvation

2007 ◽  
Vol 342-343 ◽  
pp. 401-404 ◽  
Author(s):  
Yeon I Woo ◽  
Hyun Joo Son ◽  
Hye Ryeon Lim ◽  
Mi Hee Lee ◽  
Hyun Sook Baek ◽  
...  
Keyword(s):  
Wound Healing ◽  
Dermal Fibroblast ◽  
Human Dermal Fibroblast ◽  
Positive Influence ◽  
Dermal Fibroblasts ◽  
Serum Starvation ◽  
Healing Agent ◽  
And Migration ◽  
Free Serum

Glucans have been reported to stimulate immunity and to promote wound healing. Adult human dermal fibroblast (aHDF) cultured in serum free (serum-starvation). Proliferation of aHDF was measured at various concentrations of β-glucan by MTT assay, and migration was observed for 36h on microscope. The result of fibroblast bioassay, β-glucan had positive influence. In this study, the direct effects of β-glucan on proliferation and migration of human dermal fibroblasts were examined in vitro. That means β-D-glucan has the effect to enhance proliferation and aHDF migration speed, and has the potential as a wound healing agent.

scholarly journals Differential Efficacy of Novel Antiviral Substances in 3D and Monolayer Cell Culture

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1294
Author(s):  
Robert Koban ◽  
Markus Neumann ◽  
Philipp P. Nelson ◽  
Heinz Ellerbrok
Keyword(s):  
Cell Culture ◽  
Antiviral Activity ◽  
Virus Replication ◽  
3D Culture ◽  
Human Keratinocytes ◽  
Infection Model ◽  
Cowpox Virus ◽  
Primary Human Keratinocytes

Repurposing of approved drugs that target host functions also important for virus replication promises to overcome the shortage of antiviral therapeutics. Mostly, virus biology including initial screening of antivirals is studied in conventional monolayer cells. The biology of these cells differs considerably from infected tissues. 3D culture models with characteristics of human tissues may reflect more realistically the in vivo events during infection. We screened first, second, and third generation epidermal growth factor receptor (EGFR)-inhibitors with different modes of action and the EGFR-blocking monoclonal antibody cetuximab in a 3D cell culture infection model with primary human keratinocytes and cowpox virus (CPXV) for antiviral activity. Antiviral activity of erlotinib and osimertinib was nearly unaffected by the cultivation method similar to the virus-directed antivirals tecovirimat and cidofovir. In contrast, the host-directed inhibitors afatinib and cetuximab were approx. 100-fold more efficient against CPXV in the 3D infection model, similar to previous results with gefitinib. In summary, inhibition of EGFR-signaling downregulates virus replication comparable to established virus-directed antivirals. However, in contrast to virus-directed inhibitors, in vitro efficacy of host-directed antivirals might be seriously affected by cell cultivation. Results obtained for afatinib and cetuximab suggest that screening of such drugs in standard monolayer culture might underestimate their potential as antivirals.

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