Stagewise keratinocyte differentiation from human embryonic stem cells by defined signal transduction modulators
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE144241 | GEO | 2020/01/25
REPOSITORIES: GEO
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Project description:Keratinocyte is the predominant cell type in the epidermis of skin, and it provides the protective barrier function for the body. Various signaling pathways have been implicated in keratinocyte differentiation in animal models; However, their temporal regulation and interactions are still to be explored in pluripotent stem cell models. In this report, we use human embryonic stem cells to demonstrate that epidermal ectoderm and subsequent keratinocyte cell fate can be determined step by step under the regulation of defined factors. The inhibition of TGF? initiates ectodermal lineage differentiation, and the activation of BMP pathway drives epidermal TP63 expression. Meanwhile, the timely activation of WNT pathway suppresses extraembryonic lineage, and promotes epidermal cell fate. With further specification by NOTCH inhibition, more than 90% of cells become TP63-positive stage ? keratinocytes. Finally, stage ? keratinocytes are produced under defined hypo-calcium keratinocyte culture conditions, and are further matured in mouse xenograft model. This study not only establishes an in vitro platform to study keratinocyte cell fate determination, but also provides an efficient protocol to produce keratinocytes for disease models and clinical applications.
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Project description:Stagewise keratinocyte differentiation from human embryonic stem cells by defined signal transduction modulators
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Project description:Histamines suppress epidermal keratinocyte differentiation. Previously, we reported that konjac ceramide (kCer) suppresses histamine-stimulated cell migration of HaCaT keratinocytes. kCer specifically binds to Nrp1 and does not interact with histamine receptors. The signaling mechanism of kCer in HaCaT cells is also controlled by an intracellular signaling cascade activated by the Sema3A-Nrp1 pathway. In the present study, we demonstrated that kCer treatment induced HaCaT keratinocyte differentiation after migration of immature cells. kCer-induced HaCaT cell differentiation was accompanied by some features of keratinocyte differentiation markers. kCer induced activating phosphorylation of p38MAPK and c-Fos, which increased the protein levels of involucrin that was the latter differentiation marker. In addition, we demonstrated that the effects of both kCer and histamines are regulated by an intracellular mechanism of Rac1 activation/RhoA inhibition downstream of the Sema3A/Nrp1 receptor and histamine/GPCR pathways. In summary, the effects of kCer on cell migration and cell differentiation are regulated by cascade crosstalk between downstream Nrp1 and histamine-GPCR pathways in HaCaT cells.
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Project description:Many medicinal plants have been used to treat wounds. Here, we revealed the potential wound healing effects of Curcuma amarissima (CA). Our cell viability assay showed that CA extract increased the viability of HaCaT cells that were cultured in the absence of serum. This increase in cell viability was proved to be associated with the pharmacological activities of CA extract in inducing cell proliferation. To further define possible molecular mechanisms of action, we performed Western blot analysis and immunofluorescence study, and our data demonstrated that CA extract rapidly induced ERK1/2 and Akt activation. Consistently, CA extract accelerated cell migration, resulting in rapid healing of wounded human keratinocyte monolayer. Specifically, the CA-induced increase of cell monolayer wound healing was blocked by the MEK inhibitor (U0126) or the PI3K inhibitor (LY294002). Moreover, CA extract induced the expression of Mcl-1, which is an anti-apoptotic protein, supporting that CA extract enhances human keratinocyte survival. Taken together, our study provided convincing evidence that Curcuma amarissima can promote proliferation and survival of human keratinocyte through stimulating the MAPK and PI3K/Akt signaling cascades. These promising data emphasize the possibility to develop this plant as a wound healing agent for the potential application in regenerative medicine.
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Project description:Characterization at the global transcriptome level of the gene networks ensuring the regulation of the immature status of cutaneous stem- and precursor-cells is a necessary step to further understand the concept of ‘stemness’ in this tissue. Moreover, considering possible clinical applications, the search for molecular targets to control stem cell characteristics and regenerative capacity is a necessary step to improve therapeutic approaches. Cutaneous cell therapy is concerned by this objective, as skin graft bioengineering requires a phase of ex vivo expansion of keratinocytes from donors, during which the preservation of a sufficient stem cell pool is critical for graft take and long-term skin regeneration. We have investigated the role of transcription factor KLF4 in native keratinocyte precursors from adult human skin. A stable lentiviral-based shRNA-mediated KLF4 knock‑down (KD) approach was developed and used to study the properties of KLF4-deficient [KLF4KD] versus control [KLF4WT] native keratinocyte precursors. Using long-term cultures and clonal assays, we found that decreased KLF4 expression increased keratinocyte precursor immaturity and clonogenic potential, thus promoting self-renewal. Importantly, [KLF4KD] keratinocyte precursors exhibited an improved grafting capacity in an in vivo skin xenograft model and in serial grafting. To analyze the biological impact of KLF4 knock‑down at the molecular level, comparative transcriptome profiling of [KLF4WT] and [KLF4KD] cells was performed using next-generation RNA sequencing (RNA-seq). This set of data provides a material that permits the dissection of genetic networks modulated by KLF4 in human keratinocyte precursor cells, and controlling their immature status and epidermis regeneration capacity.
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Project description:To characterize the global transcriptome of human keratinocyte stem cells (KSC) and keratinocyte progenitors (KP), primary basal keratinocyte subpopulations enriched in quiescent stem cells [Itg-?6bright / Trf-Rdim] or in cycling progenitors [Itg-?6bright / Trf-Rbright] were purified by flow cytometry from human skin samples. Sorted keratinocytes were then seeded in culture plates and maintained in culture medium overnight (15 hours), and genome-wide transcriptome analysis of these two subpopulations was directly performed, in order to globally maintain the initial phenotypes. After RNA preparation, gene profiling was performed using oligonucleotide microarrays (26068 probes). LOWESS normalisation was applied. Keywords: epidermis, keratinocyte stem cells, progenitors, transcriptome, stemness Dye-swap hybridization were performed. Slides were scanned with a Genepix 4000 microarray scanner (Axon Instruments, Molecular devices, Sunnyvale, CA). For each hybridized spot, the Cy3 and Cy5 fluorescence values were obtained by using Genepix Pro 4.0 software (Axon Instruments) and were saved as a result file
2015-05-06 | E-GEOD-68583 | biostudies-arrayexpress
Project description:Comparative analysis of gene expression in cultured primary keratinocytes isolated from newborn control (K14-cre; GPx4fl/+) and knockout (K14-cre; GPx4fl/fl) mice. Selenoproteins are essential for skin function, as targeted abolition of selenoproteins in epidermal tissue results in newborn mice manifesting gross abnormalities of skin and hair, accompanied by retarded growth and premature death. To investigate whether lack of a single selenoprotein could induce similar phenotypic effect in mice, we generated keratinocyte-specific knockout mice lacking glutathione peroxidase 4 (GPx4), an essential selenoprotein in skin, to examine phenotypic changes resulting from the lack of GPx4 in skin. Ablation of GPx4 results in focal alopecia and disturbed hair follicle morphogenesis, with GPx4 being essential during early stages of hair follicle morphogenesis as well as for keratinocyte adhesion and proliferation in culture.
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Project description:Characterization at the global transcriptome level of the gene networks ensuring the regulation of the immature status of cutaneous stem- and precursor-cells is a necessary step to further understand the concept of ‘stemness’ in this tissue. Moreover, considering possible clinical applications, the search for molecular targets to control stem cell characteristics and regenerative capacity is a necessary step to improve therapeutic approaches. Cutaneous cell therapy is concerned by this objective, as skin graft bioengineering requires a phase of ex vivo expansion of keratinocytes from donors, during which the preservation of a sufficient stem cell pool is critical for graft take and long-term skin regeneration. We have investigated the role of transcription factor MXD4/MAD4 in native keratinocyte precursors from adult human skin. A stable lentiviral-based shRNA-mediated MXD4 knock‑down (KD) approach was developed and used to study the properties of MXD4-deficient [MXD4KD] versus control [MXD4WT] native keratinocyte precursors. Using long-term cultures and clonal assays, we found that decreased MXD4 expression increased keratinocyte precursor immaturity and clonogenic potential, thus promoting self-renewal. Importantly, [MXD4KD] keratinocyte precursors exhibited an improved capacity for three-dimensional epidermis reconstruction, which was preserved at a late ex vivo expansion stage where this potential was lost in [MXD4WT] keratinocytes. To analyze the biological impact of MXD4 knock‑down at the molecular level, comparative transcriptome profiling of [MXD4WT] and [MXD4KD] cells was performed using next-generation RNA sequencing (RNA-seq). This set of data provides a material that permits the dissection of genetic networks modulated by MXD4 in human keratinocyte precursor cells, and controlling their immature status and epidermis regeneration capacity.
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