Differential effects of the extracellular microenvironment on human embryonic stem cell differentiation into keratinocytes and their subsequent replicative life span.

Culture microenvironment plays a critical role in the propagation and differentiation of human embryonic stem cells (hESCs) and their differentiated progenies. Although high efficiency of hESC differentiation to keratinocytes (hESC-Kert) has been achieved, little is known regarding the effects of early culture microenvironment and pertinent extracellular matrix (ECM) interactions during epidermal commitment on subsequent proliferative capacity of hESC-Kert. The aim of this study is to evaluate the effects of the different ECM microenvironments during hESC differentiation on subsequent replicative life span of hESC-Kert. In doing so, H1-hESCs were differentiated to keratinocytes (H1-Kert) in two differentiation systems. The first system employed autologous fibroblast feeder support, in which keratinocytes (H1-Kert(ACC)) were derived by coculture of hESCs with hESC-derived fibroblasts (H1-ebFs). The second system employed a novel decellularized matrix from H1-ebFs to create a dermoepidermal junction-like (DEJ) matrix. H1-Kert(AFF) were derived by differentiation of hESCs on the feeder-free system employing the DEJ matrix. Our study indicated that the feeder-free system with the use of DEJ matrix was more efficient in differentiation of hESCs toward epidermal progenitors. However, the feeder-free system was not sufficient to support the subsequent replicative capacity of differentiated keratinocytes. Of note, H1-Kert(AFF) showed limited replicative capacity with reduced telomere length and early cellular senescence. We further showed that the lack of cell-cell interactions during epidermal commitment led to heightened production of TGF-ß1 by hESC-Kert during extended culture, which in turn was responsible for resulting in the limited replicative life span with cellular senescence of hESC-Kert derived under the feeder-free culture system. This study highlights for the first time the importance of the culture microenvironment and cell-ECM interactions during differentiation of hESCs on subsequent replicative life span and cellular senescence of the differentiated keratinocytes, with implications for use of these cells for applications in tissue engineering and regenerative medicine.

Investigation of human embryonic stem cell-derived keratinocytes as an in vitro research model for mechanical stress dynamic response.

The epidermis is mainly composed of keratinocytes forming a protective barrier. It is perpetually subjected to mechanical stress and strain during development, homeostasis and disease. Perturbation of the normal strain with alteration of its biological response may lead to severe diseases such as psoriasis and epidermolysis bullosa. To date, most of the studies about skin response to mechanical stress used immortalized cell lines (i.e. HaCaT) or primary cells from donors, which suffer issues of limited physiological relevance and inter-donor variability. It is therefore necessary to develop a new human model for the study of normal skin physiology and response to mechanical stress. In this study, we investigated the use of keratinocytes derived from human embryonic stem cells (hESCs) as a reliable alternative model to HaCaT for study of the effects of mechanical tension. With comparison to HaCaT, hESC-derived keratinocytes (hESC-Kert) were exposed to up to 3 days of cyclic mechanical stress, and gene expression changes were analyzed. Dynamic expression of several key mechanical stress related-genes was studied at mRNA level using qPCR. The expression of matrix-metallopeptidase9 was studied at protein level using ELISA. The two cell types displayed similar gene expression kinetics for most of the genes including E-cadherin, cateninß1, connexin43, desmoglein1, endothelin1, integrinα6, interleukinα1, keratin1, 6, and 10, keratinocyte growth-factor-receptor and lamininα5. Unlike HaCaT, hESC-Kert displayed early gene and protein expression of matrix metallopeptidase 9 following mechanical stimulation, suggesting that these cells have remodeling capacity that resembles that of normal human skin. Our study confirmed the use of hESC-Kert as a good model for study of skin response to mechanical stress.

Human embryonic stem cells derived keratinocyte as an in vitro research model for the study of immune response.

BACKGROUND: The innate immune response (IMR) is critical for the oral mucosa due to their continuous exposure to various oral pathogens. Keratinocytes play important role in IMR. Therefore, to date, keratinocytes from different sources have been used as in vitro research model for the study of IMR. However, current keratinocyte research models are hampered by the limited supply, patients' dependency and batch to batch variation. Therefore, in this study, we demonstrated the use of human embryonic stem cells (hESCs) derived keratinocytes (H9-Kert) as an alternative research model for the study of IMR. METHODS: The expression kinetics of toll-like receptor (TLR) 2, TLR 4, interleukin (IL) -6, IL-8, inducible nitric oxide synthase (iNOS) and tumour necrosis factor-alpha (TNF-α), in H9-Kert and immortalized human keratinocyte cell line (HaCaT) were analysed at mRNA levels by both reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. The activation of the inflammatory transcription factor nuclear factor kappa-b (NFĸB) was assayed in these cells by transiently transfecting the cells with NFĸB reporter plasmid. Activation of NFĸB following treatment with heat-killed Porphyromonas gingivalis (P. gingivalis), an oral pathogen, was determined by assaying for the reporter, secreted alkaline phosphatase activity. RESULTS: The expression of TLRs, cytokines and activation of NFĸB following bacterial stimulation showed in both H9-Kert and the widely used HaCaT keratinocyte cell line was similar. CONCLUSION: Overall, our results support the potential application of hESCs as an alternative limitless cell source for primary keratinocytes which can be used as consistent and dependable research tool with minimum variations and no donor's dependency.

Removal of chlorophenolic derivatives by soil isolated ascomycete of Paraconiothyrium variabile and studying the role of its extracellular laccase.

The ability of Paraconiothyrium variabile, a laccase producing ascomycete recently isolated from soil, was studied to eliminate chlorophenol derivatives in submerged culture medium. Among the tested compounds, ρ-chlorophenol (ρ-CP) and pentachlorophenol (PCP) were found to have minimum and maximum toxic effects, respectively, on the growth of the microorganism and at the same time high and low bioelimination percentages. The fungal strain was able to remove 86% of ρ-CP (with initial concentration of 40 mg l(-1)) and 56% of 2,4-dichlorophenol (2,4-DCP; with same concentration as ρ-CP) after 9 days of incubation while no elimination was observed in the presence of 2,4,6-trichlorophenol (2,4,6-TCP) and PCP. Monitoring of laccase production level in the fermentation broth together with pollutant removal confirmed the key role of this copper-containing oxidase in chlorophenol derivatives elimination. The type of laccase inducer (guaiacol) and its final concentration (250 µM) and also initial pH of the fermentation broth (pH=5.5) in the elimination of ρ-CP increased the final removal yield from 86% to 94.3%.