Keratinocyte Progenitor Cells Research Articles

The lncRNA SNHG26 drives the inflammatory-to-proliferative state transition of keratinocyte progenitor cells during wound healing

The cell transition from an inflammatory phase to a subsequent proliferative phase is crucial for wound healing, yet the driving mechanism remains unclear. By profiling lncRNA expression changes during human skin wound healing and screening lncRNA functions, we identify SNHG26 as a pivotal regulator in keratinocyte progenitors underpinning this phase transition. Snhg26-deficient mice exhibit impaired wound repair characterized by delayed re-epithelization accompanied by exacerbated inflammation. Single-cell transcriptome analysis combined with gain-of-function and loss-of-function of SNHG26 in vitro and ex vivo reveals its specific role in facilitating inflammatory-to-proliferative state transition of keratinocyte progenitors. A mechanistic study unravels that SNHG26 interacts with and relocates the transcription factor ILF2 from inflammatory genomic loci, such as JUN, IL6, IL8, and CCL20, to the genomic locus of LAMB3. Collectively, our findings suggest that lncRNAs play cardinal roles in expediting tissue repair and regeneration and may constitute an invaluable reservoir of therapeutic targets in reparative medicine.

Antimicrobial Peptide Reduces Cytotoxicity and Inflammation in Canine Epidermal Keratinocyte Progenitor Cells Induced by Pseudomonas aeruginosa Infection.

The direct effects and antimicrobial activity of synthetic antimicrobial peptides (AMPs) obtained from dogs, including cBD, cBD103, and cCath, against P. aeruginosa wild-type strain PAO1 and canine keratinocytes were analyzed. Antibacterial effects on planktonic bacteria were assessed by determining the minimum bactericidal concentrations (MBCs) of AMPs and by a time-kill assay. Antibiofilm effects were assessed using the microtiter plate assay. We also evaluated the effects of AMPs on cell cytotoxicity and host immune response induced by stimulating canine epidermal keratinocyte progenitor (CPEK) cells with PAO1 and its LPS. cBD, cBD103, and cCath all exhibited dose-dependent antimicrobial and antibiofilm effects. In particular, 25 μg/mL cBD103 showed rapid bactericidal activity within 60 min and inhibited biofilm formation. In addition, pretreatment with cBD103 (25 µg/mL) and cCath (50 µg/mL) 1 h before stimulation significantly reduced the cytotoxicity of the CPEK cells by PAO1 and LPS-induced IL-6 and TNF-a expressions. cBD had little effect on the response to PAO1 and LPS in the cells. These results indicate the therapeutic potential of AMPs in P. aeruginosa skin infections. However, further studies on the mechanism of action of AMPs in keratinocytes and clinical trials are needed.

Nutritional supplementation of an apple callus extract to target epidermal aging

The global increase in life expectancy has led to a growing elderly population, which places a significant burden on healthcare systems. As a result, there is a growing interest to improve healthspan and promote healthy aging with accessible means, such as nutrition and lifestyle interventions. The skin, the body's largest organ and first line of defense against environmental stressors, is particularly vulnerable to aging due to both intrinsic and extrinsic factors. Various molecular and biochemical changes can drive skin aging, including the depletion of stem cells, which can lead to impaired skin properties. Nutrition plays a crucial role in skin aging, and dietary supplements could be a viable intervention to support a healthy skin. In this study, a plant callus extract from a specific apple variety (Malus Domestica Borkh), the Uttwiler Spätlauber, was evaluated as a dietary supplement to combat epidermal aging. The extract was found to maintain the function of keratinocyte progenitor cells during aging in vitro. Furthermore, an open-label pilot clinical study involving 31 female volunteers showed that the intake of apple callus extract led to improvements in skin elasticity, density, and firmness after 56 days of supplementation. This study highlights the potential of plant callus extracts as natural dietary supplements to support healthy skin aging. The use of plant callus cultures provides a sustainable and reproducible source of bioactive molecules, which is gaining acceptance in the nutraceutical market. These findings suggest that dietary interventions can play a role in promoting healthy skin aging and contribute to a holistic approach to improving healthspan during the aging process.

Comparative Analysis of Cx31 and Cx43 in Differentiation-Competent Rodent Keratinocytes

When considering connexin expression and regulation, the epidermis of the skin is one of the most complex tissues found in mammals even though it largely contains a single cell type, the keratinocyte. In the rodent epidermis, up to 9 connexin family members have been detected at the mRNA level. Many of these connexins are temporally and spatially regulated in coordination with keratinocyte progenitor cell differentiation and migration from the stratum basale to form the stratum spinosum and stratum granulosum layers before finally forming the stratum corneum. Cx43 is the principal connexin found in basal keratinocytes and to a lesser degree found in keratinocytes that have begun to differentiate where Cx26, Cx30 and Cx31 become prevalent. Here we show that the CRISPR-Cas9 ablation of Cx43 reduces overall gap junction coupling in monolayer cultures of rat epidermal keratinocytes (REKs) and dysregulates the differentiation of REKs when grown in organotypic cultures. Natively found in differentiated keratinocytes, Cx31 readily assembles into gap junctions when expressed in REKs where it can extensively co-assemble into the same gap junctions with co-expressed Cx30. Time-lapse imaging indicated that many Cx31 gap junctions are mobile within the plasma membrane undergoing both fusion and fission events. Finally, the persistence of pre-existing Cx31 gap junctions in the presence of the protein trafficking blocker, brefeldin A, is longer than that found for Cx43 gap junctions indicating that it has a distinctly different life expectancy in REKs. Collectively, this study highlights the importance of Cx43 in rodent keratinocyte differentiation and suggests that Cx31 acquires life-cycle properties that are distinct from Cx43.

Deriving Keratinocyte Progenitor Cells and Keratinocytes from Human-Induced Pluripotent Stem Cells.

Skin or hair loss (alopecia) may occur due to a wide variety of causes ranging from trauma to pathological processes including acquired or congenital causes. It would be ideal to replace them with immunologically compatible cells to avoid potentially exacerbating the condition. Deriving the replacement cells from human-induced pluripotent stem cells (hiPSCs) allows for sufficient scale up and using hiPSCs as the choice of human pluripotent stem cells (hPSC) will ensure immunocompatibility. Here we offer a protocol for differentiating hiPSCs into keratinocyte progenitor cells (KPC) and keratinocytes employing all-trans retinoic acid (ATRA) and L-ascorbic acid, (L-AA), bone morphogenic protein-4 (BMP4), and epidermal growth factor (EGF). We observed that the hiPSC-derived KPCs express the same panel of markers as primary hair follicle bulge stem cells (HFBSCs), including CD200, integrin α-6 (ITGA6), integrin β-1 (ITGB1), the transcription factor P63, keratin 15 (KRT15), and keratin 19 (KRT19). If permitted to differentiate further, the hiPSC-derived KPC lose CD200 expression and rather come to express keratin 14 (KRT14) indicating emergence of more mature terminally-differentiated keratinocytes. The HFBSCs are transplantable for hair follicle (HF) restoration, and the keratinocytes may be transplantable for therapy for large burns or ulcers. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Reprogramming of normal human skin fibroblasts into normal hiPSCs using episomal DNA cocktail Basic Protocol 2: Differentiation of hiPSCs into KPCs and keratinocytes Alternate Protocol 2: EBS formation protocol using AggreWell™ plates (Antonchuk, 2013) Support Protocol 1: Passage hiPSC-KPC Support Protocol 2: Immunocytochemistry (ICC) Support Protocol 3: Immunofluorescence staining of cells for flow cytometry (FC).

Chemically defined and xenogeneic-free culture method for human epidermal keratinocytes on laminin-based matrices.

The basal keratinocyte progenitor cells in cultured epithelial autografts (CEAs) regenerate human epidermis after transplantation, a curative therapy for severe burns and, recently, diseases with epidermal loss, such as junctional epidermolysis bullosa (EB). Although a culturing technique for skin keratinocytes was developed four decades ago, the xenogeneic nature of that conventional CEA culture system restricts its use to the treatment of critical and life-threatening cases, such as severe burns on >30% of total body surface area and EB. In the present protocol, we describe how to implement a defined, xeno-free culture system that supports long-term ex vivo expansion of functional human epidermal keratinocytes. Skin-specific basement membrane proteins called laminins play important roles in the maintenance of phenotypic integrity and in supporting the survival of keratinocytes that are adhered to them. This fully human keratinocyte culture system is 'regulatory friendly' and increases the potential of epithelial cellular therapy, which can be expanded to treat less severe burns and other skin defects, such as chronic diabetic wounds. It takes between 7 and 14 d to obtain an initial culture. Conservatively, a secondary culture from the primary culture can be expanded up to 20-fold within 4-5 d once cells reach confluency.

Metabolic activity and intracellular pH in induced pluripotent stem cells differentiating in dermal and epidermal directions

Induced pluripotent stem cells (iPSC) are a promising tool for personalized cell therapy, in particular, in the field of dermatology. Metabolic plasticity of iPSC are not completely understood due to the fact that iPSC have a mixed mitochondrial phenotype, which still resembles that of somatic cells. In this study we investigated the metabolic changes in iPSC undergoing differentiation in two directions, dermal and epidermal, using two-photon fluorescence microscopy combined with FLIM. Directed differentiation of iPSC into dermal fibroblasts and keratinocyte progenitor cells was induced. Cellular metabolism was examined on the basis of the fluorescence of the metabolic cofactors NAD(P)H and FAD. The optical redox ratio (FAD/NAD(P)H) and the fluorescence lifetimes of NAD(P)H and FAD were traced using two-photon fluorescence microscopy combined with FLIM. Evaluation of the intracellular pH was carried out with the fluorescent pH sensor SypHer-2 and fluorescence microscopy. In this study, evaluation of the metabolic status of iPSC during dermal and epidermal differentiation was accomplished for the first time with the use of optical metabolic imaging. Based on the data on the FAD/NAD(P)H redox ratio and on the fluorescence lifetimes of protein-bound form of NAD(P)H and closed form of FAD, we registered a metabolic shift toward a more oxidative status in the process of iPSC differentiation into dermal fibroblasts and keratinocyte progenitor cells. Biosynthetic processes occurring in dermal fibroblasts associated with the synthesis of fibronectin and versican, that stimulate increased energy metabolism and lower the intracellular pH. No intracellular pH shift is observed in the culture of keratinocyte progenitor cells, which reflects the incomplete process of differentiation in this type of cells. Presented results provide the basis for further understanding the metabolic features of iPSC during differentiation process, which is essential for developing new treatment strategies in cell therapy and tissue engineering.

Engineering human cell spheroids to model embryonic tissue fusion in vitro.

Epithelial-mesenchymal interactions drive embryonic fusion events during development, and perturbations of these interactions can result in birth defects. Cleft palate and neural tube defects can result from genetic defects or environmental exposures during development, yet very little is known about the effect of chemical exposures on fusion events during human development because of a lack of relevant and robust human in vitro assays of developmental fusion behavior. Given the etiology and prevalence of cleft palate and the relatively simple architecture and composition of the embryonic palate, we sought to develop a three-dimensional culture system that mimics the embryonic palate and could be used to study fusion behavior in vitro using human cells. We engineered size-controlled human Wharton’s Jelly stromal cell (HWJSC) spheroids and established that 7 days of culture in osteogenesis differentiation medium was sufficient to promote an osteogenic phenotype consistent with embryonic palatal mesenchyme. HWJSC spheroids supported the attachment of human epidermal keratinocyte progenitor cells (HPEKp) on the outer spheroid surface likely through deposition of collagens I and IV, fibronectin, and laminin by mesenchymal spheroids. HWJSC spheroids coated in HPEKp cells exhibited fusion behavior in culture, as indicated by the removal of epithelial cells from the seams between spheroids, that was dependent on epidermal growth factor signaling and fibroblast growth factor signaling in agreement with palate fusion literature. The method described here may broadly apply to the generation of three-dimensional epithelial-mesenchymal co-cultures to study developmental fusion events in a format that is amenable to predictive toxicology applications.

Tu1312 - Hydrotalcite Protects Human Esophageal Keratinocyte Progenitor Cells from Nsaid-Induced Injury By Upregulating Expression of Anti-Apoptosis Protein Survivin, IGF-1 and Cox2. Direct Actions Independent of Neural and Vascular Factors

Tu1312 - Hydrotalcite Protects Human Esophageal Keratinocyte Progenitor Cells from Nsaid-Induced Injury By Upregulating Expression of Anti-Apoptosis Protein Survivin, IGF-1 and Cox2. Direct Actions Independent of Neural and Vascular Factors

Keratinocyte progenitor cells in human subcutaneous adipose tissue

Keratinocyte progenitor cells in human subcutaneous adipose tissue

Evaluation of oral keratinocyte progenitor and T-lymphocite cells response during early healing after augmentation of keratinized gingiva with a 3D collagen matrix - a pilot study

BackgroundThe aim of the present study is to analyze the behavior of selected populations of oral keratinocytes and T-lymphocytes, responsible for re-constructing and maintaining the oral epithelial tissue architecture, following augmentation of the keratinized oral mucosa using a 3D-collagen matrix.MethodsDifferent groups of oral keratinocytes were isolated from biopsies harvested from 3 patients before the surgical procedure, as well as 7 and 14 days after the augmentation procedure. T-lymphocytes were isolated from peripheral blood at same timepoints. Keratinocytes were characterized for stem and differentiation markers, such as p63, cytokeratin 10 and 14, and in vitro parameters, such as cell viability, cell size and colony-forming efficiency. T-lymphocytes were analyzed for viability and the expression of various cluster of differentiation markers. The methods included magnetic separation of cell populations, immunofluorescence, flow cytometry, and histology of oral biopsies.ResultsBoth at 7 and 14 days, the majority of cells that repopulate the matrix were actively proliferating/progenitor oral keratinocytes with the phenotype integrin alfa6beta4 + CD71+. These cells display in vitro characteristics similar to the progenitor cells analyzed before the matrix placement. T-lymphocytes expressed CD8 and CD69 markers, while CD25 was absent.ConclusionThe study shows that two weeks after the collagen membrane placement, the healing process appeared to be histologically complete, with no abnormal immune response induced by the matrix, however, with a higher than usual content of active proliferating cells, the majority of keratinocytes being characterized as transit amplifying cells.

Breaking dogma for future therapy using stem cell - Where we have reached?

Breaking dogma for future therapy using stem cell - Where we have reached?

Correction of Hair Shaft Defects through Allele-Specific Silencing of Mutant Krt75

Dominant mutations in keratin genes can cause a number of inheritable skin disorders characterized by intraepidermal blistering, epidermal hyperkeratosis, or abnormalities in skin appendages, such as nail plate dystrophy and structural defects in hair. Allele-specific silencing of mutant keratins through RNA interference is a promising therapeutic approach for suppressing the expression of mutant keratins and related phenotypes in the epidermis. However, its effectiveness on skin appendages remains to be confirmed in vivo. In this study, we developed allele specific siRNAs capable of selectively suppressing the expression of a mutant Krt75, which causes hair shaft structural defects characterized by the development of blebs along the hair shaft in mice. Hair regenerated from epidermal keratinocyte progenitor cells isolated from mutant Krt75 mouse models reproduced the blebbing phenotype when grafted in vivo. In contrast, mutant cells manipulated with a lentiviral vector expressing mutant Krt75-specific shRNA persistently suppressed this phenotype. The phenotypic correction was associated with significant reduction of mutant Krt75 mRNA in the skin grafts. Thus, data obtained from this study demonstrated the feasibility of utilizing RNA interference to achieve durable correction of hair structural phenotypes through allele-specific silencing of the mutant keratin genes.

The Photoprotective Effect of S-Methylmethionine Sulfonium in Skin.

S-Methylmethionine sulfonium (SMMS) was reported to have wound-healing effects; we therefore have investigated the photoprotective effect of SMMS in the present study. SMMS increased the viability of keratinocyte progenitor cells (KPCs) and human dermal fibroblasts (hDFs) following ultraviolet B (UVB) irradiation, and reduced the UVB-induced apoptosis in these cells. SMMS increased the phosphorylation of extracellular signal-regulated kinases (ERK), and the inhibitor of the mitogen-activated protein kinase pathway significantly decreased the SMMS-induced viability of KPCs and hDFs. In addition, SMMS attenuated the UVB-induced reactive oxygen species (ROS) generation in KPCs and hDFs. SMMS induced the collagen synthesis and reduced the matrix metalloproteinase-1 expression in UVB-irradiated hDFs. In animal studies, application of 5% and 10% SMMS before and after UVB-irradiation significantly decreased the UVB-induced erythema index and depletion of Langerhans cells. In summary, SMMS protects KPCs and hDFs from UVB irradiation, and reduces UVB-induced skin erythema and immune suppression. Therefore, SMMS can be used as a cosmetic raw material, and protect skin from UVB.

Post-transcriptional Regulation of Keratinocyte Progenitor Cell Expansion, Differentiation and Hair Follicle Regression by miR-22.

Hair follicles (HF) undergo precisely regulated recurrent cycles of growth, cessation, and rest. The transitions from anagen (growth), to catagen (regression), to telogen (rest) involve a physiological involution of the HF. This process is likely coordinated by a variety of mechanisms including apoptosis and loss of growth factor signaling. However, the precise molecular mechanisms underlying follicle involution after hair keratinocyte differentiation and hair shaft assembly remain poorly understood. Here we demonstrate that a highly conserved microRNA, miR-22 is markedly upregulated during catagen and peaks in telogen. Using gain- and loss-of-function approaches in vivo, we find that miR-22 overexpression leads to hair loss by promoting anagen-to-catagen transition of the HF, and that deletion of miR-22 delays entry to catagen and accelerates the transition from telogen to anagen. Ectopic activation of miR-22 results in hair loss due to the repression a hair keratinocyte differentiation program and keratinocyte progenitor expansion, as well as promotion of apoptosis. At the molecular level, we demonstrate that miR-22 directly represses numerous transcription factors upstream of phenotypic keratin genes, including Dlx3, Foxn1, and Hoxc13. We conclude that miR-22 is a critical post-transcriptional regulator of the hair cycle and may represent a novel target for therapeutic modulation of hair growth.

Breast cancer resistance protein identifies clonogenic keratinocytes in human interfollicular epidermis.

IntroductionThere is a practical need for the identification of robust cell-surface markers that can be used to enrich for living keratinocyte progenitor cells. Breast cancer resistance protein (ABCG2), a member of the ATP binding cassette (ABC) transporter family, is known to be a marker for stem/progenitor cells in many tissues and organs.MethodsWe investigated the expression of ABCG2 protein in normal human epidermis to evaluate its potential as a cell surface marker for identifying and enriching for clonogenic epidermal keratinocytes outside the pilosebaceous tract.ResultsImmunofluorescence and immunoblotting studies of human skin showed that ABCG2 is expressed in a subset of basal layer cells in the epidermis. Flow cytometry analysis showed approximately 2-3% of keratinocytes in non-hair-bearing epidermis expressing ABCG2; this population also expresses p63, β1 and α6 integrins and keratin 14, but not CD34, CD71, C-kit or involucrin. The ABCG2-positive keratinocytes showed significantly higher colony forming efficiency when co-cultured with mouse 3T3 feeder cells, and more extensive long-term proliferation capacity in vitro, than did ABCG2-negative keratinocytes. Upon clonal analysis, most of the freshly isolated ABCG2-positive keratinocytes formed holoclones and were capable of generating a stratified differentiating epidermis in organotypic culture models.ConclusionsThese data indicate that in skin, expression of the ABCG2 transporter is a characteristic of interfollicular keratinocyte progentior cells and suggest that ABCG2 may be useful for enriching keratinocyte stem cells in human interfollicular epidermis.

Keratinocyte progenitor cells reside in human subcutaneous adipose tissue.

The differentiation of adipose-derived stem cells (ASCs) towards epithelial lineages has yet to be demonstrated using a standardized method. This study investigated whether keratinocyte progenitor cells are present in the ASC population. ASCs isolated from subcutaneous adipose tissue were cultured and examined for the expression of the keratinocyte progenitor markers p63 and desmoglein 3 (DSG3) by immunofluorescence microscopy and flow cytometry. In addition, p63 and DSG3 expression levels were assessed before and after differentiation of ASCs into adipocytes by real-time PCR and western blot analysis, as well as in subcutaneous adipose tissue by real-time reverse transcriptase polymerase chain reaction. Both markers were expressed in ASCs, but were downregulated after the differentiation of ASCs into adipocytes; p63-positive cells were also detected in subcutaneous adipose tissue. ASCs co-cultured with human fibroblasts and incubated with all-trans retinoic acid and bone morphologic protein 4 showed an upregulation in DSG3 level, which was also increased in the presence of type IV collagen. They also showed an upregulation in cytokeratin-5 level only in the presence of type IV collagen. These results provide the demonstration that keratinocyte progenitor cells reside in subcutaneous adipose tissue.

278 Novel Mechanisms and Signaling Pathways of Esophageal Keratinocyte Progenitor Cells: Interactions of COX2/prostaglandin E, c-AMP/PKA and KGF. Implications for Esophageal Epithelial Renewal, Healing and the Therapeutic Actions of Hydrotalcite.

278 Novel Mechanisms and Signaling Pathways of Esophageal Keratinocyte Progenitor Cells: Interactions of COX2/prostaglandin E, c-AMP/PKA and KGF. Implications for Esophageal Epithelial Renewal, Healing and the Therapeutic Actions of Hydrotalcite.

Efficient Generation of Functional Epithelial and Epidermal Cells from Human Pluripotent Stem Cells Under Defined Conditions

Human pluripotent stem cells (hPSCs) have an unparalleled potential to generate limitless quantities of any somatic cell type. However, current methods for producing populations of various somatic cell types from hPSCs are generally not standardized and typically incorporate undefined cell culture components often resulting in variable differentiation efficiencies and poor reproducibility. To address this, we have developed a defined approach for generating epithelial progenitor and epidermal cells from hPSCs. In doing so, we have identified an optimal starting cell density to maximize yield and maintain high purity of K18+/p63+ simple epithelial progenitors. In addition, we have shown that the use of synthetic, defined substrates in lieu of Matrigel and gelatin can successfully facilitate efficient epithelial differentiation, maintaining a high (>75%) purity of K14+/p63+ keratinocyte progenitor cells and at a two to threefold higher yield than a previously reported undefined differentiation method. These K14+/p63+ cells also exhibited a higher expansion potential compared to cells generated using an undefined differentiation protocol and were able to terminally differentiate and recapitulate an epidermal tissue architecture in vitro. In summary, we have demonstrated the production of populations of functional epithelial and epidermal cells from multiple hPSC lines using a new, completely defined differentiation strategy.

35 Novel Insight Into Esophageal Mucosal Defense and Its Enhancement by Hydrotalcite. Direct Evidence That VEGF, Its Receptor 2 and KGFR Are Expressed in Esophageal Keratinocyte Progenitor Cells and Regulate Their Growth and Epithelial Cell Renewal

35 Novel Insight Into Esophageal Mucosal Defense and Its Enhancement by Hydrotalcite. Direct Evidence That VEGF, Its Receptor 2 and KGFR Are Expressed in Esophageal Keratinocyte Progenitor Cells and Regulate Their Growth and Epithelial Cell Renewal