Chin Yan Lim

The hair follicle is a specialized appendage of the skin that is critical for multiple functions, including thermoregulation, immune surveillance, and sebum production. Mammals are born with a fixed number of hair follicles that develop embryonically. Postnatally, these hair follicles undergo regenerative cycles of regression and growth that recapitulate many of the embryonic signaling pathways. Furthermore, hair cycles have a direct impact on skin regeneration in homeostasis, cutaneous wound healing, and disease conditions such as alopecia. Here, we review the current knowledge of hair follicle formation during embryonic development and the post-natal hair cycle, with an emphasis on the molecular signaling pathways underlying these processes. We then discuss efforts to capitalize on the field’s understanding of in vivo mechanisms to bioengineer hair follicles or hair-bearing skin in vitro and how such models may be further improved to develop strategies for hair regeneration.

The mammalian epidermis undergoes constant renewal, replenished by a pool of stem cells and terminal differentiation of their progeny. This is accompanied by changes in gene expression and morphology that are orchestrated, in part, by epigenetic modifiers. Here, we define the role of the histone acetyltransferase KAT2A in epidermal homeostasis and provide a comparative analysis that reveals key functional divergence with its paralog KAT2B. In contrast to the reported function of KAT2B in epidermal differentiation, KAT2A supports the undifferentiated state in keratinocytes. RNA-seq analysis of KAT2A- and KAT2B- depleted keratinocytes revealed dysregulated epidermal differentiation. Depletion of KAT2A led to premature expression of epidermal differentiation genes in the absence of inductive signals, whereas loss of KAT2B delayed differentiation. KAT2A acetyltransferase activity was indispensable in regulating epidermal differentiation gene expression. The metazoan-specific N terminus of KAT2A was also required to support its function in keratinocytes. We further showed that the interplay between KAT2A- and KAT2B-mediated regulation was important for normal cutaneous wound healing in vivo. Overall, these findings reveal a distinct mechanism in which keratinocytes use a pair of highly homologous histone acetyltransferases to support divergent functions in self-renewal and differentiation processes.

Pathogenic variants in KMT5B , a lysine methyltransferase, are associated with global developmental delay, macrocephaly, autism, and congenital anomalies (OMIM # 617788). Given the relatively recent discovery of this disorder, it has not been fully characterized. Deep phenotyping of the largest ( n = 43) patient cohort to date identified that hypotonia and congenital heart defects are prominent features that were previously not associated with this syndrome. Both missense variants and putative loss-of-function variants resulted in slow growth in patient-derived cell lines. KMT5B homozygous knockout mice were smaller in size than their wild-type littermates but did not have significantly smaller brains, suggesting relative macrocephaly, also noted as a prominent clinical feature. RNA sequencing of patient lymphoblasts and Kmt5b haploinsufficient mouse brains identified differentially expressed pathways associated with nervous system development and function including axon guidance signaling. Overall, we identified additional pathogenic variants and clinical features in KMT5B -related neurodevelopmental disorder and provide insights into the molecular mechanisms of the disorder using multiple model systems.

Skin is a complex organ made up of different cell layers, appendages, connective tissues, and immune repertoires. These different components interact extensively to maintain the overall functions of the integumentary system. In particular, appendages such as hair follicles critically contribute to the skin’s function in thermoregulation, sensory perception, and homeostatic regeneration. Despite a strong need for better skin regenerative therapeutics, efforts to bio-engineer highly functional appendage-containing human reconstituted skin in vitro have not yielded much success. Here, we report methods in generating and incorporating hair follicle-primed heterotypic spheroids into epidermal-dermal skin constructs that induced invaginating outgrowths with follicle-like organization and lineage gene expression. By co-culturing epithelial keratinocytes (KCs) with dermal papilla (DP) cells in low attachment plates, we established the media and culture conditions that best supported the viability, signalling and remodelling of the cell aggregates to form 3D KC-DP spheroids with the expression of both DP inductiveness and hair follicle lineage genes. We show that long-term growth and maturation of KC cells in these spheroids was supported by incorporation into epidermal-dermal constructs but not in scaffold-less media. When cultured, the bio-fabricated constructs developed invaginations from the integrated spheroids with follicle-forming potential. The generation of these constructs is a step towards the development of functional hair-bearing skin mimetics.

Ageing-related delays and dysregulated inflammation in wound healing are well-documented in both human and animal models. However, cellular and molecular changes underlying this impairment in healing progression are not fully understood. In this study, we characterised ageing-associated changes to macrophages in wounds of young and aged mice and investigated transcriptomic differences that may impact the progression of wound healing. Full-thickness wounds created on the dorsum of C57BL/6J young and aged mice were excised on Days 3 and 7 post-wounding for analysis by immunohistochemistry, flow cytometry, and RNA sequencing. Our data revealed that macrophages were significantly reduced in aged wounds in comparison to young. Functional transcriptomic analyses showed that macrophages from aged wounds exhibited significantly reduced expression of cell cycle, DNA replication, and repair pathway genes. Furthermore, we uncovered an elevated pro-inflammatory gene expression program in the aged macrophages correlated with poor inflammation resolution and excessive tissue damage observed in aged wounds. Altogether, our work provides insights into how poorly healing aged wounds are phenotypically defined by the presence of macrophages with reduced proliferative capacity and an exacerbated inflammatory response, both of which are pathways that can be targeted to improve healing in the elderly.

Dynamic shifts in chromatin states occur during embryonic epidermal development to support diverse epigenetic pathways that regulate skin formation and differentiation. However, it is not known whether the epigenomes established during embryonic development are maintained into adulthood or how these epigenetic mechanisms may be altered upon physiological ageing of the tissue. Here, we systematically profiled the nuclear enrichment of five key histone modifications in young and aged mouse epidermis and identified distinct chromatin states that are tightly correlated with cellular differentiation, as well as chromatin alterations that accompanied epidermal ageing. Our data showed that histone modifications, which become differentially enriched in undifferentiated basal or differentiated suprabasal cells during embryonic development, retained their distinct cell-type specific enrichment patterns in both young and aged adult tissues. Specifically, high levels of H3K4me3, H4K20me1 and H4K16ac marked the proliferative basal cells, while differentiated suprabasal cells accumulated H3K27me3 and H4K20me3 heterochromatin with a concomitant deacetylation of H4K16. We further identified shifts in the chromatin in the aged basal epidermis, which exhibited markedly reduced levels of H4K16ac, absence of high H4K20me1 staining and increased cell-to-cell variability in total histone H3 and H4 content. Changes in the chromatin profiles in aged tissues paralleled the altered expression of their corresponding histone modifiers in the basal keratinocytes. These results thus reveal the key histone signatures of epidermal differentiation that are conserved from embryonic development to adult homoeostasis, and provide insights into the epigenetic pathways underlying physiological skin ageing.

Diabetes mellitus (DM) is a chronic metabolic disease with increasing prevalence worldwide. Diabetic foot ulcers (DFUs) are a serious complication of DM. It is estimated that 15–25% of DM patients develop DFU at least once in their lifetime. The lack of effective wound dressings and targeted therapy for DFUs often results in prolonged hospitalization and amputations. As the incidence of DM is projected to rise, the demand for specialized DFU wound management will continue to increase. Hence, it is of great interest to improve and develop effective DFU-specific wound dressings and therapies. In the last decade, 3D bioprinting technology has made a great contribution to the healthcare sector, with the development of personalized prosthetics, implants, and bioengineered tissues. In this review, we discuss the challenges faced in DFU wound management and how 3D bioprinting technology can be applied to advance current treatment methods, such as biomanufacturing of composite 3D human skin substitutes for skin grafting and the development of DFU-appropriate wound dressings. Future co-development of 3D bioprinting technologies with novel treatment approaches to mitigate DFU-specific pathophysiological challenges will be key to limiting the healthcare burden associated with the increasing prevalence of DM.

Post-translational modifications on nucleosomal histones represent a key epigenetic regulatory mechanism to mediate the complex gene expression, DNA replication, and cell cycle changes that occur in embryonic cells undergoing lineage specification, maturation, and differentiation during development. Here, we investigated the dynamics of 13 key histone marks in epidermal cells at three distinct stages of embryonic skin development and identified significant changes that corresponded with the maturation of the proliferative basal epidermal cells and terminally differentiated cells in the stratified layers. In particular, H3K4me3 and H3K27ac were accumulated and became more prominent in the basal cells at later stages of epidermal development, while H3K27me3 was found to be low in the basal cells but highly enriched in the differentiated suprabasal cell types. Constitutive heterochromatin marked by H4K20me3 was also significantly elevated in differentiated epidermal cells at late gestation stages, which exhibited a concomitant loss of H4K16 acetylation. These differential chromatin profiles were established in the embryonic skin by gestation day 15 and further amplified at E18 and in postnatal skin. Our results reveal the dynamic chromatin states that occur as epidermal progenitor cells commit to the lineage and differentiate into the different cells of the stratified epidermis and provide insight to the underlying epigenetic pathways that support normal epidermal development and homoeostasis.

There are considerable challenges in directly targeting the mutant p53 protein, given the large heterogeneity of p53 mutations in the clinic. An alternative approach is to exploit the altered fitness of cells imposed by loss-of-wild-type p53. Here we identify niclosamide through a HTS screen for compounds selectively killing p53-deficient cells. Niclosamide impairs the growth of p53-deficient cells and of p53 mutant patient-derived ovarian xenografts. Metabolome profiling reveals that niclosamide induces mitochondrial uncoupling, which renders mutant p53 cells susceptible to mitochondrial-dependent apoptosis through preferential accumulation of arachidonic acid (AA), and represents a first-in-class inhibitor of p53 mutant tumors. Wild-type p53 evades the cytotoxicity by promoting the transcriptional induction of two key lipid oxygenation genes, ALOX5 and ALOX12B, which catalyzes the dioxygenation and breakdown of AA. Therefore, we propose a new paradigm for targeting cancers defective in the p53 pathway, by exploiting their vulnerability to niclosamide-induced mitochondrial uncoupling.

Embryonic stem cells have the ability to self-renew or differentiate and these processes are under tight control. We previously reported that the polyamine regulator AMD1 is critical for embryonic stem cell self-renewal. The polyamines putrescine, spermidine, and spermine are essential organic cations that play a role in a wide array of cellular processes. Here, we explore the essential role of the polyamines in the promotion of self-renewal and identify a new stem cell regulator that acts downstream of the polyamines: MINDY1. MINDY1 protein levels are high in embryonic stem cells (ESCs) and are dependent on high polyamine levels. Overexpression of MINDY1 can promote ESC self-renewal in the absence of the usually essential cytokine Leukemia Inhibitory Factor (LIF). MINDY1 protein is prenylated and this modification is required for its ability to promote self-renewal. We go on to show that Mindy1 RNA is targeted for repression by mir-710 during Neural Precursor cell differentiation. Taken together, these data demonstrate that high polyamine levels are required for ESC self-renewal and that they function, in part, through promotion of high MINDY1 levels. Stem Cells 2018;36:1170–1178