Epigenetic regulation and signalling pathways in Merkel cell development.

Merkel cells are specialized epithelial cells connected to afferent nerve endings responsible for light-touch sensations, formed at specific locations in touch-sensitive regions of the mammalian skin. Although Merkel cells are descendants of the epidermal lineage, little is known about the mechanisms responsible for the development of these unique mechanosensory cells. Recent studies have highlighted that the Polycomb group (PcG) of proteins play a significant role in spatiotemporal regulation of Merkel cell formation. In addition, several of the major signalling pathways involved in skin development have been shown to regulate Merkel cell development as well. Here, we summarize the current understandings of the role of developmental regulators in Merkel cell formation, including the interplay between the epigenetic machinery and key signalling pathways, and the lineage-specific transcription factors involved in the regulation of Merkel cell development.

ZNF750 Regulates Skin Barrier Function by Driving Cornified Envelope and Lipid Processing Pathways.

The epidermis is a constantly renewing stratified epithelial tissue that provides essential protective barrier functions. The major barrier is located at the outermost layers of the epidermis, formed by terminally differentiated keratinocytes reinforced by proteins of their cornified envelope and sequestered intercellular lipids. Disruptions to epidermal differentiation characterize various skin disorders. ZNF750 is an epithelial transcription factor essential for in vitro keratinocyte differentiation, whose truncating mutation in humans causes autosomal dominant psoriasis-like skin disease. In this study, we utilized an epidermal-specific Znf750 conditional knockout mouse model to uncover the role ZNF750 plays in epidermal development. We show that deletion of Znf750 in the developing skin does not block epidermal differentiation completely, suggesting in vivo compensatory feedback mechanisms, although it does result in impaired barrier function and perinatal lethality. Molecular dissection revealed ultrastructural defects in the differentiated layers of the epidermis, accompanied by alterations in the expression of ZNF750-dependent genes encoding key cornified envelope precursor proteins and lipid-processing enzymes, including gene subsets known to be mutated in human skin diseases involving impaired barrier function. Together, our findings provide molecular insights into the pathogenesis of human skin disease by linking ZNF750 to a subset of epidermal differentiation genes involved in barrier formation pathways.

Histone Mono-Ubiquitination in Transcriptional Regulation and Its Mark on Life: Emerging Roles in Tissue Development and Disease.

Epigenetic regulation plays an essential role in driving precise transcriptional programs during development and homeostasis. Among epigenetic mechanisms, histone mono-ubiquitination has emerged as an important post-transcriptional modification. Two major histone mono-ubiquitination events are the mono-ubiquitination of histone H2A at lysine 119 (H2AK119ub), placed by Polycomb repressive complex 1 (PRC1), and histone H2B lysine 120 mono-ubiquitination (H2BK120ub), placed by the heteromeric RNF20/RNF40 complex. Both of these events play fundamental roles in shaping the chromatin epigenetic landscape and cellular identity. In this review we summarize the current understandings of molecular concepts behind histone mono-ubiquitination, focusing on their recently identified roles in tissue development and pathologies.

Enhanced Induction of Definitive Endoderm Differentiation of Mouse Embryonic Stem Cells in Simulated Microgravity.

Directed in vitro differentiation of pluripotent stem cells toward definitive endoderm (DE) offers great research and therapeutic potential since these cells can further differentiate into cells of the respiratory and gastrointestinal tracts, as well as associated organs such as pancreas, liver, and thyroid. We hypothesized that culturing mouse embryonic stem cells (mESCs) under simulated microgravity (SMG) conditions in rotary bioreactors (BRs) will enhance the induction of directed DE differentiation. To test our hypothesis, we cultured the cells for 6 days in two-dimensional monolayer colony cultures or as embryoid bodies (EBs) in either static conditions or, dynamically, in the rotary BRs. We used flow cytometry and quantitative polymerase chain reaction to analyze the expression of marker proteins and genes, respectively, for pluripotency (Oct3/4) and mesendodermal (Brachyury T), endodermal (FoxA2, Sox17, CxCr4), and mesodermal (Vimentin, Meox1) lineages. Culture in the form of EBs in maintenance media in the presence of leukemia inhibitory factor, in static or SMG conditions, induced expression of some of the differentiation markers, suggesting heterogeneity of the cells. This is in line with previous studies showing that differentiation is initiated as cells are aggregated into EBs even without supplementing differentiation factors to the media. Culturing EBs in static conditions in differentiation media (DM) in the presence of activin A reduced Oct3/4 expression and significantly increased Brachyury T and CxCr4 expression, but downregulated FoxA2 and Sox17. However, culturing in SMG BRs in DM upregulated Brachyury T and all of the DE markers and reduced Oct3/4 expression, indicating the advantage of dynamic cultures in BRs to specifically enhance directed DE differentiation. Given the potential discrepancies between the SMG conditions on earth and actual microgravity conditions, as observed in other studies, future experiments in space flight are required to validate the effects of reduced gravity on mESC differentiation.

Protein-polymer conjugates for forming photopolymerizable biomimetic hydrogels for tissue engineering.

Collagen, fibrin and albumin are popular proteins for making biological scaffolds for tissue engineering because of their biocompatibility, biodegradability, and availability. A major drawback of biological protein-based biomaterials is the limited control over their physical and biodegradation properties. Our laboratory has been developing new protein-based biomaterials with tunable properties without the use of cytotoxic protein cross-linking techniques. We describe the formation and assembly of photopolymerizable biomimetic hydrogel scaffolds made from protein-polymer conjugates of poly(ethylene glycol) (PEG) and collagen, fibrin or albumin. The conjugation of PEG to these proteins (PEGylation) was verified by SDS-PAGE and the polymerization reaction into a hydrogel network was confirmed by shear rheometry. The differences in rheology and swelling characteristics of the three hydrogel materials underscore the importance of the molecular relationship between the PEG and the protein constituent in this protein-polymer arrangement. The biofunctionality of the PEGylated collagen and fibrinogen hydrogels sustained both cell adhesion and proteolytic degradation that enabled 3-D cell spreading and migration within the hydrogel network. PEG-albumin hydrogels exhibited poor cell spreading and migration by virtue of the fact that the albumin backbone lacks any known cell adhesion sites. Despite differences in the biological and structural composition of the PEGylated fibrinogen and collagen hydrogels, the rate of cellular migration within each material was not significantly different.

Polymer-conjugated albumin and fibrinogen composite hydrogels as cell scaffolds designed for affinity-based drug delivery.

Serum albumin was conjugated to poly-(ethylene glycol) (PEG) and cross-linked to form mono-PEGylated albumin hydrogels. These hydrogels were used as a basis for drug carrying tissue engineering scaffold materials, based on the natural affinity of various drugs and compounds for the tethered albumin in the polymer network. The results of the drug release validation experiments showed that the release kinetics of the drugs from the mono-PEGylated albumin hydrogels were controlled by the molecular weight (MW) of PEG conjugated to the albumin protein, the drug MW and its inherent affinity for albumin. Composite hydrogels containing both mono-PEGylated albumin and PEGylated fibrinogen were used specifically for three-dimensional (3D) cell culture scaffolds, with inherent bioactivity, proteolytic biodegradability and controlled drug release properties. The specific characteristics of these complex hydrogels were governed by the ratio between the concentrations of each protein, the addition of free PEG diacrylate (PEG DA) molecules to the hydrogel matrix and the MW of the PEG conjugated to each protein. Comprehensive characterization of the drug release and degradation properties, as well as 3D cell culture experiments using these composite materials, demonstrated the effectiveness of this combined approach in creating a tissue engineering scaffold material with controlled drug release features.