Differentiation Of Epidermal Stem Cells Research Articles

P75 neurotrophin receptor activates epidermal stem cell differentiation via ERK, JNK and AKT pathways

P75 neurotrophin receptor activates epidermal stem cell differentiation via ERK, JNK and AKT pathways

Effects of porcine acellular dermal matrix treatment on wound healing and scar formation: Role of Jag1 expression in epidermal stem cells

ABSTRACTSkin wound healing involves Notch/Jagged1 signaling. However, little is known how Jag1 expression level in epidermal stem cells (ESCs) contributes to wound healing and scar formation. We applied multiple cellular and molecular techniques to examine how Jag1 expression in ESCs modulates ESCs differentiation to myofibroblasts (MFB) in vitro, interpret how Jag1 expression in ESCs is involved in wound healing and scar formation in mice, and evaluate the effects of porcine acellular dermal matrix (ADM) treatment on wound healing and scar formation. We found that Jag1, Notch1 and Hes1 expression was up-regulated in the wound tissue during the period of wound healing. Furthermore, Jag1 expression level in the ESCs was positively associated with the level of differentiation to MFB. ESC-specific knockout of Jag1 delayed wound healing and promoted scar formation in vivo. In addition, we reported that porcine ADM treatment after skin incision could accelerate wound closure and reduce scar formation in vivo. This effect was associated with decreased expression of MFB markers, including α-SMA Col-1 and Col-III in wound tissues. Finally, we confirmed that porcine ADM treatment could increase Jag1, Notch1 and Hesl expression in wound tissues. Taken together, our results suggested that ESC-specific Jag1 expression levels are critical for wound healing and scar formation, and porcine ADM treatment would be beneficial in promoting wound healing and preventing scar formation by enhancing Notch/Jagged1 signaling pathway in ESCs.

MicroRNA-203 Modulates Wound Healing and Scar Formation via Suppressing Hes1 Expression in Epidermal Stem Cells

Background/Aims: Little is known how miR-203 is involved in epidermal stem cells (ESCs) differentiation and scar formation. Methods: We first used luciferase assay to determine the interaction of miR-203 with the 3’-UTR in regulation of Hes1 expression. We then used flow cytometry to analyze the effects of miR-203 expression on the differentiation of ESCs to MFB by determination of CK15 ratio and α-SMA. To confirm the results of flow cytometry analysis, we used Western blot to examine the expression of α-SMA, Collagen I (Col I), and Collagen III (Col III), as well as the expression of Notch1, Jagged1, and Hes1 in ESCs after the treatment of pre-miR-203 or anti-miR-203. Finally, we examined the effects local anti-miR-203 treatment on would closure and scar formation using a mouse skin wound model. Results: Pre-miR-203 treatment increased ESCs differentiation to MFB cells, as indicated by decreased CK15 ratio and increased MFB biomarkers. This phenomenon was reversed by overexpression of Hes1 in ESCs. In addition, skin incision increased expression of miR-203 in wound tissue. Local treatment of anti-miR-203 could accelerate wound closure and reduce scar formation in vivo, which was associated with increased re-epithelialization, skin attachment regeneration, and collagen reassignment. Finally, we confirmed that anti-miR-203 treatment could inhibit ESCs differentiation in vivo via increasing Hesl expression. Conclusion: Taken together, our results suggested that overexpression of miR-203 in ESCs after skin wound may be a critical mechanism underlying the scar formation.

Circular RNAs are abundantly expressed and upregulated during human epidermal stem cell differentiation

ABSTRACTThe expression patterns of endogenous circular RNA (circRNA) molecules during epidermal stem cell (EpSC) differentiation have not previously been explored. Here, we show that circRNAs are abundantly expressed in EpSCs and that their expression change dramatically during differentiation in a coordinated manner. Overall, circRNAs are expressed at higher levels in the differentiated cells, and many upregulated circRNAs are derived from developmental genes, including four different circRNAs from DLG1. The observed changes in circRNA expression were largely independent of host gene expression, and circRNAs independently upregulated upon differentiation are more prone to AGO2 binding and have more predicted miRNA binding sites compared to stably expressed circRNAs. In particular, upregulated circRNAs from the HECTD1 and ZNF91 genes have exceptionally high numbers of AGO2 binding sites and predicted miRNA target sites, and circZNF91 contains 24 target sites for miR-23b-3p, which is known to play important roles in keratinocyte differentiation. We also observed that upregulated circRNAs are less likely to be flanked by homologues inverted Alu repeats compared to stably expressed circRNAs. This coincide with DHX9 being upregulated in the differentiated keratinocytes. Finally, none of the circRNAs upregulated upon differentiation were also upregulated upon DNMT3A or DNMT3B knockdown, making it unlikely that epigenetic mechanisms are governing the observed circRNA expression changes. Together, we provide a map of circRNA expression in EpSCs and their differentiated counterparts and shed light on potential function and regulation of differentially expressed circRNAs.

Cd271 mediates proliferation and differentiation of epidermal stem cells to support cutaneous burn wound healing.

Burn wounds can significantly reduce the quality of life of patients with respect to their physiology and psychology and can even threaten their lives. Many treatments have been proposed, including stem cell therapy but no effective method can as yet cure such damage. Our study highlights the role of Cd271 in epidermal stem cells (eSC) during the healing of burn wounds. The expression of Cd271 increases together with burn wound healing. Injection of Cd271-over-expressing eSC into wounds promotes the healing rate in a mouse burn model. Over-expression of Cd271 enhances the abilities of eSC with regard to their differentiation, proliferation and migration and even their resistance to apoptosis in vitro. These results are in accordance with a hypothesis suggesting that Cd271 promotes the healing of skin burn wounds by improving the potential of eSC for differentiation, proliferation and migration. Our findings shed light on the role of Cd271 in wound healing and may provide new therapeutic approaches for curing burn wounds of the skin.

Basic fibroblast growth factor reduces scar by inhibiting the differentiation of epidermal stem cells to myofibroblasts via the Notch1/Jagged1 pathway

BackgroundBasic fibroblast growth factor (bFGF) plays an important role in promoting wound healing and reducing scar, but the possible molecular mechanisms are still unclear. Our previous studies have found that activating the Notch1/Jagged1 pathway can inhibit the differentiation of epidermal stem cells (ESCs) to myofibroblasts (MFB). Herein, we document that bFGF reduces scar by inhibiting the differentiation of ESCs to MFB via activating the Notch1/Jagged1 pathway.MethodsIn in-vitro study, ESCs were isolated from 10 neonatal SD rats (1–3 days old), cultured in keratinocyte serum-free medium, and divided into six groups: bFGF group, bFGF + SU5402 group, bFGF + DAPT group, siJagged1 group, bFGF + siJagged1 group, and control group. Jagged1 of the ESCs in the siJagged1 group and bFGF + siJagged1 group was knocked down by small-interfering RNA transfection. Expression of ESC markers (CK15/CK10), MFB markers (α-SMA, Collagen I, Collagen III), and Notch1/Jagged1 components (Jagged1, Notch1, Hes1) was detected by FCM, qRT-PCR, and western blot analysis to study the relationships of bFGF, ESCs, and Notch1/Jagged1 pathway. In in-vivo study, the wound healing time and scar hyperplasia were observed on rabbit ear scar models. The quality of wound healing was estimated by hematoxylin and eosin staining and Masson staining. Expression of ESC markers, MFB markers and Notch1/Jagged1 components was elucidated by immunohistochemistry, immunofluorescence, and western blot analysis.ResultsThe in-vitro study showed that bFGF could significantly upregulate the expression of ESC markers and Notch1/Jagged1 components, while downregulating the expression of MFB markers at the same time. However, these effects could be obviously decreased when we knocked down Jagged1 or added DAPT. Similarly, in in-vivo study, bFGF also exhibited its functions in inhibiting the differentiation of rabbit ESCs to MFB by activating the Notch1/Jagged1 pathway, which improved the wound healing quality and alleviated scar significantly.ConclusionThese results provide evidence that bFGF can reduce scar by inhibiting the differentiation of ESCs to MFB via the Notch1/Jagged1 pathway, and present a new promising potential direction for the treatment of scar.

Pluripotency factor Nanog is tumorigenic by deregulating DNA damage response in somatic cells.

The pluripotency gene Nanog is not expressed in normal adult tissues but is overexpressed in some human cancers. However, the tumorigenic roles of Nanog remain unclear. The ectopic expression of Nanog is not sufficient to induce spontaneous tumors in mice but can promote metastasis of established tumors by activating the expression of metastatic genes. The expression of Nanog in mouse skin activates tumor suppressor p53, leading to the differentiation of epidermal stem cells. In the absence of p53, Nanog induces spontaneous squamous cell carcinoma, identifying a novel role of Nanog in tumorigenesis. Therefore, the induction of p53 and differentiation in Nanog-expressing skin suppresses the tumorigenic activities of Nanog, which include the induction of DNA double-stranded break damage. Notably, Nanog interacts with the KRAB-associated protein 1 (KAP1) and inhibits its sumoylation activity, impairing KAP1-mediated chromatin remodeling, which is important for efficiently activating DNA damage response. In summary, Nanog is an oncogene with multiple roles in promoting tumorigenesis and metastasis.

An Essential Role for Parathyroid Hormone in Gill Formation and Differentiation of Ion-Transporting Cells in Developing Zebrafish.

In vertebrates, parathyroid hormone (PTH) is important for skeletogenesis and Ca(2+) homeostasis. However, little is known about the molecular mechanisms by which PTH regulates skeleton formation and Ca(2+) balance during early development. Using larval zebrafish as an in vivo model system, we determined that PTH1 regulates the differentiation of epithelial cells and the development of craniofacial cartilage. We demonstrated that translational gene knockdown of PTH1 decreased Ca(2+) uptake at 4 days after fertilization. We also observed that PTH1-deficient fish exhibited reduced numbers of epithelial Ca(2+) channel (ecac)-expressing cells, Na(+)/K(+)-ATPase-rich cells, and H(+)-ATPase-rich cells. Additionally, the density of epidermal stem cells was decreased substantially in the fish experiencing PTH1 knockdown. Knockdown of PTH1 caused a shortening of the jaw and impeded the development of branchial arches. Results from in situ hybridization suggested that the expression of collagen 2a1a (marker for proliferating chondrocytes) was substantially reduced in the cartilage that forms the jaw and branchial aches. Disorganization of chondrocytes in craniofacial cartilage also was observed in PTH1-deficient fish. The results of real-time PCR demonstrated that PTH1 morphants failed to express the transcription factor glial cell missing 2 (gcm2). Coinjection of PTH1 morpholino with gcm2 capped RNA rescued the phenotypes observed in the PTH1 morphants, suggesting that the defects in PTH1-deficient fish were caused, at least in part, by the suppression of gcm2. Taken together, the results of the present study reveal critical roles for PTH1 in promoting the differentiation of epidermal stem cells into mature ionocytes and cartilage formation during development.

Nanog down-regulates the Wnt signaling pathway via β-catenin phosphorylation during epidermal stem cell proliferation and differentiation.

BackgroundSkin tissue homeostasis is maintained by a balance between the proliferation and differentiation of epidermal stem cells (EpSCs). EpSC proliferation and differentiation are complex processes regulated by many factors and signaling pathways. This study aimed to explore the connection between the Nanog and the Wnt/β-catenin pathway in the proliferation and differentiation of EpSCs.ResultsOur results demonstrated that during the study period, EpSC underwent differentiation when incubated in the presence neuropeptide substance P (SP), there was an opposing expression trend of Nanog and β-catenin after SP treatment, which could be antagonized by the Wnt antagonist, Dkk-1. The transduced EpSCs had a greater proliferative ability than the SP treatment group and they did not undergo differentiation upon SP treatment. More important, β-catenin expression was down-regulated but phosphorylated β-catenin expression and phosphorylated GSK-3β expression was up-regulated upon Nanog overexpression.ConclusionsThese results strongly suggest that Nanog plays an important role in maintaining the proliferation and differentiation homeostasis of EpSCs by promoting β-catenin phosphorylation via GSK-3β to inhibit the activity of the Wnt/β-catenin signaling pathway. This is important for precise regulation of proliferation and differentiation of EpSC in the application of tissue engineering.

The nanoscale geometrical maturation of focal adhesions controls stem cell differentiation and mechanotransduction.

We show that the nanoscale adhesion geometry controls the spreading and differentiation of epidermal stem cells. We find that cells respond to such hard nanopatterns similarly to their behavior on soft hydrogels. Cellular responses were seen to stem from local changes in diffusion dynamics of the adapter protein vinculin and associated impaired mechanotransduction rather than impaired recruitment of proteins involved in focal adhesion formation.

Low-intensity pulsed electromagnetic fields and the differentiation of epidermal stem cells

Objective To observe the effect of a low-intensity pulsed electromagnetic field (LIPEMF) on the differentiation of epidermal stem cells (EpSCs),and to explain the regulation of any observed effect on the gene level.Methods EpSCs were isolated from the back skin of neonatal Sprague-Dawley rats,and divided into three experimental groups and a control group.The three experimental aliquots received 5 mT magnetic field irradiation pulsed at 15 Hz,50 Hz or 75 Hz.After 10 days of continuous stimulation,immunofluorescence was used to observe any differentiation,RT-PCRs tested for c-myc and β-catenin mRNA,and immunohistochemical methods were applied to test for cytokeratin 18 (CK18).Results The EpSCs of the three stimulation groups all differentiated,and differentiation-related CK 18 showed significant expression in the 50 Hz group.Expression of c-myc mRNA increased significantly in all three experimental samples compared with the controls.Expression of β-catenin mRNA decreased significantly in all three samples compared with the controls.Conclusion LIPEMFs can induce the differentiation of EpSCs.Irradiation at 50 Hz and 5 mT is the most effective.The effect may be related to enhanced expression of c-myc and inhibited expression of β-catenin. Key words: Epidermis stem cells; Electromagnetic fields; Differentiation

MicroRNA-203 inhibits the progression of esophageal squamous cell carcinoma with restored epithelial tissue architecture in vivo

MicroRNA (miR)-203 has been shown to induce squamous differentiation of epidermal stem cells through the suppression of p63. The aim of this study was to assess the tumor suppressor effect of miR-203 in esophageal squamous cell carcinoma (ESCC) with focus on the regulation of the cell fate decisions and organization of tumor tissue architecture invivo. Our investigation establishing stable clones from ESCC cell lines with induced miR-203 expression resulted in significant growth inhibition in a mouse xenograft model. Small foci were observed in xenograft tumors with stratified squamous differentiation in conjunction with restored baso-apical polarity. The expression of the basement membrane protein laminine was localized at the center of the foci and the basal cell marker p75NTR was expressed in the innermost layer. The expression of ki67 and p63 was co-localized at the center layers, while involucrin was expressed in the outer layers. Flow cytometry revealed that the p75NTR-positive cells expressing p63 and Bmi1 were well maintained, while the expression of p63 was suppressed in the p75NTR-negative cells. Our cDNA microarray analysis demonstrated the upregulation of genes involved in regulating tissue architecture, such as BMP-4 and ZO-1 in the mir-203 transfectant. Investigation using surgically removed ESCC specimens revealed that the expression of miR-203 significantly correlated with a favorable prognosis. These results demonstrated that miR-203 regulated both basal and supra-basal cell components to induce differentiation with restored epithelial tissue architecture, leading to significant tumor growth inhibition invivo. Those results suggest the use of miR-203 as a novel therapeutic and diagnostic target in patients with ESCC.

Helium-Neon Laser Irradiation Promotes the Proliferation and Migration of Human Epidermal Stem Cells In Vitro: Proposed Mechanism for Enhanced Wound Re-epithelialization

The present study was conducted to investigate the effects of helium-neon (He-Ne) laser irradiation on the proliferation, migration, and differentiation of cultured human epidermal stem cells (ESCs). A He-Ne laser with a wavelength of 632.8 nm is known to have photobiological effects, and is widely used for accelerating wound healing; however, the cellular mechanisms involved have not been completely understood. The ESCs were prepared from human foreskin, and irradiated by using He-Ne laser at 632.8 nm with 2 J/cm(2). The ESC proliferation, migration, and differentiation were examined by using XTT assay, scratch assay, and flow cytometry technology, respectively. The phosphorylation of extracellular signal-regulated kinases (ERK) was analyzed by using Western blotting. He-Ne laser irradiation markedly promoted cell proliferation and migration accompanied by an increase in the phosphorylation of ERK, but did not significantly influence cell differentiation. Our data indicated that photostimulation with a He-Ne laser resulted in a significant increase in human ESC proliferation and migration in vitro, which might contribute, at least partially, to accelerated wound re-epithelialization by low-level laser therapy.

Decoupling geometrical and chemical cues directing epidermal stem cell fate on polymer brush-based cell micro-patterns

The intricacy of the different parameters involved in cell adhesion to biomaterials and fate decision (e.g. proliferation, differentiation, apoptosis) makes the decoupling of the respective effects of surface properties, extra-cellular matrix protein adsorption and ultimately cell behaviour difficult. This work presents a micro-patterned polymer brush platform to control the adsorption of extra-cellular matrix (ECM) proteins to well defined micron-size areas and consequently control cell adhesion, spreading and shape independently of other chemical and physical surface properties. Protein patterns can be readily generated with brushes presenting a range of hydrophilicity and surface charge density. The surface properties of the selected brushes are fully characterised using a combination of FTIR, XPS, ellipsometry, atomic force microscopy, water contact goniometry, dynamic light scattering and ζ-potential measurements. Interactions of proteins relevant to cell patterning and culture with these brushes are studied by surface plasmon resonance, dynamic light scattering, ellipsometry and immuno-fluorescence microscopy. Finally this platform is used in an assay investigating the relative contributions of matrix geometry and surface chemistry on epidermal stem cell differentiation. It is found that moderate hydrophobicity does not impact stem cell commitment, whereas strongly negative surface potential increases the incidence of differentiation. This correlates with a marked decrease in the formation of focal adhesions (but not cell spreading).

Stochastic simulation of structured skin cell population dynamics

The epidermis is the outmost skin tissue. It operates as a first defense system to process inflammatory signals and responds by producing inflammatory mediators that promote the recruitment of immune cells. Various skin diseases such as atopic dermatitis occur as a result of the defect of proper skin barrier function and successive impaired inflammatory responses. The onset of such a skin disease links to the disturbed epidermal homeostasis regulated by appropriate self-renewal and differentiation of epidermal stem cells. The theory of physiologically structured population models provides a versatile framework to formulate mathematical models which describe the growth dynamics of a cell population such as the epidermis. In this paper, we develop an algorithm to implement stochastic simulation for a class of physiologically structured population models. We demonstrate that the developed algorithm is applicable to several cell population models and typical age-structured population models. On the basis of the developed algorithm, we investigate stochastic dynamics of skin cell populations and spread of inflammation. It is revealed that demographic stochasticity can bring considerable impact on the outcome of inflammation spread at the tissue level.

Microenvironment‐dependent homeostasis and differentiation of epidermal basal undifferentiated keratinocytes and their clinical applications in skin repair

Skin homeostasis is maintained by controlling the balance between proliferation and differentiation of epidermal stem cells. The microenvironment, including extrinsic stresses, growth factors, soluble molecules, cell-ECM and cell-cell communications, plays an important role in cell fate determination in vivo and in vitro. In response to external signals, keratinocytes cooperate with other cell types to modulate and facilitate the wound microenvironment during wound healing; however, the aberrant signals or conjunctions in the environment will lead to pathologic abnormalities. In addition, despite some drawbacks, the epidermal stem-cellbased bioengineered skin substitutes have greatly improved the quality of cutaneous repair. Thus, exploring the characteristics and regulation mechanisms of microenvironment-dependent homeostasis and differentiation of epidermal basal undifferentiated keratinocytes is necessary to understand skin development and wound repair and to design novel therapeutic strategies for skin wound healing.

Mechanisms regulating epidermal stem cells

The skin epidermis contains different appendages such as the hair follicle and the sebaceous glands. Recent studies demonstrated that several types of stem cells (SCs) exist in different niches within the epidermis and maintain discrete epidermal compartments, but the exact contribution of each SC populations under physiological conditions is still unclear. In addition, the precise mechanisms controlling the balance between proliferation and differentiation of epidermal SC still remain elusive. Recent studies provide new insights into these important questions by showing the contribution of hair follicle SC to the sebaceous lineage and the importance of chromatin modifications and micro-RNAs (miRs) in regulating epidermal SCs renewal and differentiation. In this review, we will discuss the importance of these papers to our understanding of the mechanisms that control epidermal SC functions.

Nanog and β-catenin: A new convergence point in EpSC proliferation and differentiation

Skin tissue homeostasis is maintained by the balanced proliferation and differentiation of certain types of proliferating cells such as epidermal stem cells (EpSCs). The proliferation and differentiation of EpSCs are complex processes which are not well understood. This study aimed to find the internal relationship between the Nanog pathway and the Wnt/β-catenin pathway in the proliferation and differentiation process of EpSCs. In brief, EpSCs were isolated from rat epidermis and cultured. The MTT assay, western blotting, polymerase chain reaction (PCR) and immunocytochemistry were performed during the proliferation and differentiation process of EpSCs. Our results showed that 10−7 M neuropeptide substance P could effectively stimulate proliferation of EpSCs and that a possible link exists between the Nanog pathway and the Wnt/β-catenin pathway.

Neural differentiation of epidermal stem cells

The bulge of hair follicles contains a mixed population of stem cells,including epidermal stem cells,neural crest stem cells,as well as another type of stem cells which can be induced into melanocytes.Bulge stem cells have the ability to divide,proliferate and are pluripotent as other adult stem cells.The neural crest stem cells can differentiate in vitro into neurons,glia and melanocytes under proper induction.Meanwhile,it was proved that implanting these cells into the gap region of damaged nerves could greatly enhance the regeneration rate of nerve and restoration of nerve function.In this review,distribution of the stated stem cells in hair follicle bulge and their characteristics of differentiating into neurons were discussed,as well as their possible application in regenerative medicine. Key words: Epidermal stem cells; Differentiate into neurons; Regenerative medicine

Kin-19/casein kinase Iα has dual functions in regulating asymmetric division and terminal differentiation in C. elegans epidermal stem cells

Casein Kinase I (CKI) is a conserved component of the Wnt signaling pathway, which regulates cell fate determination in metazoans. We show that post-embryonic asymmetric division and fate specification of C. elegans epidermal stem cells are controlled by a non-canonical Wnt/b-catenin signaling pathway, involving the b-catenins WRM-1 and SYS-1, and that C. elegans kin-19/CKIa functions in this pathway. Furthermore, we find that kin-19 is the only member of the Wnt asymmetry pathway that functions with, or in parallel to, the heterochronic temporal patterning pathway to control withdrawal from self-renewal and subsequent terminal differentiation of epidermal stem cells. We show that, except in the case of kin-19, the Wnt asymmetry pathway and the heterochronic pathway function separately and in parallel to control different aspects of epidermal stem cell fate specification. However, given the function of kin-19/CKIa in both pathways, and that CKI, Wnt signaling pathway and heterochronic pathway genes are widely conserved in animals, our findings suggest that CKIa may function as a regulatory hub through which asymmetric division and terminal differentiation are coordinated in adult stem cells of vertebrates.