Publications by authors named "Fiona M Watt"

210 Publications

Fibrotic enzymes modulate wound-induced skin tumorigenesis.

EMBO Rep 2021 Mar 29:e51573. Epub 2021 Mar 29.

VIB Center for Inflammation Research, Ghent, Belgium.

Fibroblasts are a major component of the microenvironment of most solid tumours. Recent research elucidated a large heterogeneity and plasticity of activated fibroblasts, indicating that their role in cancer initiation, growth and metastasis is complex and context-dependent. Here, we performed genome-wide expression analysis comparing fibroblasts in normal, inflammatory and tumour-associated skin. Cancer-associated fibroblasts (CAFs) exhibit a fibrotic gene signature in wound-induced tumours, demonstrating persistent extracellular matrix (ECM) remodelling within these tumours. A top upregulated gene in mouse CAFs encodes for PRSS35, a protease capable of collagen remodelling. In human skin, we observed PRSS35 expression uniquely in the stroma of high-grade squamous cell carcinomas. Ablation of PRSS35 in mouse models of wound- or chemically-induced tumorigenesis resulted in aberrant collagen composition in the ECM and increased tumour incidence. Our results indicate that fibrotic enzymes expressed by CAFs can regulate squamous tumour initiation by remodelling the ECM.
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http://dx.doi.org/10.15252/embr.202051573DOI Listing
March 2021

Developmental cell programs are co-opted in inflammatory skin disease.

Science 2021 01;371(6527)

Biosciences Institute, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.

The skin confers biophysical and immunological protection through a complex cellular network established early in embryonic development. We profiled the transcriptomes of more than 500,000 single cells from developing human fetal skin, healthy adult skin, and adult skin with atopic dermatitis and psoriasis. We leveraged these datasets to compare cell states across development, homeostasis, and disease. Our analysis revealed an enrichment of innate immune cells in skin during the first trimester and clonal expansion of disease-associated lymphocytes in atopic dermatitis and psoriasis. We uncovered and validated in situ a reemergence of prenatal vascular endothelial cell and macrophage cellular programs in atopic dermatitis and psoriasis lesional skin. These data illustrate the dynamism of cutaneous immunity and provide opportunities for targeting pathological developmental programs in inflammatory skin diseases.
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http://dx.doi.org/10.1126/science.aba6500DOI Listing
January 2021

A blueprint for translational regenerative medicine.

Sci Transl Med 2020 12;12(572)

Department of Materials, Imperial College London, London SW7 2AZ, UK.

The past few decades have produced a large number of proof-of-concept studies in regenerative medicine. However, the route to clinical adoption is fraught with technical and translational obstacles that frequently consign promising academic solutions to the so-called "valley of death." Here, we present a proposed blueprint for translational regenerative medicine. We offer principles to help guide the selection of cells and materials, present key in vivo imaging modalities, and argue that the host immune response should be considered throughout design and development. Last, we suggest a pathway to navigate the often complex regulatory and manufacturing landscape of translational regenerative medicine.
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http://dx.doi.org/10.1126/scitranslmed.aaz2253DOI Listing
December 2020

Genomic landscape and clonal architecture of mouse oral squamous cell carcinomas dictate tumour ecology.

Nat Commun 2020 11 9;11(1):5671. Epub 2020 Nov 9.

Centre for Stem Cells & Regenerative Medicine, King's College London, Guy's Hospital, Great Maze Pond, London, SE1 9RT, UK.

To establish whether 4-nitroquinoline N-oxide-induced carcinogenesis mirrors the heterogeneity of human oral squamous cell carcinoma (OSCC), we have performed genomic analysis of mouse tongue lesions. The mutational signatures of human and mouse OSCC overlap extensively. Mutational burden is higher in moderate dysplasias and invasive SCCs than in hyperplasias and mild dysplasias, although mutations in p53, Notch1 and Fat1 occur in early lesions. Laminin-α3 mutations are associated with tumour invasiveness and Notch1 mutant tumours have an increased immune infiltrate. Computational modelling of clonal dynamics indicates that high genetic heterogeneity may be a feature of those mild dysplasias that are likely to progress to more aggressive tumours. These studies provide a foundation for exploring OSCC evolution, heterogeneity and progression.
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http://dx.doi.org/10.1038/s41467-020-19401-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652942PMC
November 2020

Contribution of GATA6 to homeostasis of the human upper pilosebaceous unit and acne pathogenesis.

Nat Commun 2020 10 20;11(1):5067. Epub 2020 Oct 20.

Centre for Stem Cells and Regenerative Medicine, King's College London, Floor 28, Tower Wing, Guy's Hospital, Great Maze Pond, London, SE1 9RT, UK.

Although acne is the most common human inflammatory skin disease, its pathogenic mechanisms remain incompletely understood. Here we show that GATA6, which is expressed in the upper pilosebaceous unit of normal human skin, is down-regulated in acne. GATA6 controls keratinocyte proliferation and differentiation to prevent hyperkeratinisation of the infundibulum, which is the primary pathological event in acne. When overexpressed in immortalised human sebocytes, GATA6 triggers a junctional zone and sebaceous differentiation program whilst limiting lipid production and cell proliferation. It modulates the immunological repertoire of sebocytes, notably by upregulating PD-L1 and IL10. GATA6 expression contributes to the therapeutic effect of retinoic acid, the main treatment for acne. In a human sebaceous organoid model GATA6-mediated down-regulation of the infundibular differentiation program is mediated by induction of TGFβ signalling. We conclude that GATA6 is involved in regulation of the upper pilosebaceous unit and may be an actionable target in the treatment of acne.
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http://dx.doi.org/10.1038/s41467-020-18784-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7575575PMC
October 2020

Clinically Relevant Vulnerabilities of Deep Machine Learning Systems for Skin Cancer Diagnosis.

J Invest Dermatol 2021 Apr 12;141(4):916-920. Epub 2020 Sep 12.

Centre for Stem Cells and Regenerative Medicine, King's College London, London, United Kingdom; St John's Institute of Dermatology, Guys Hospital, London, United Kingdom.

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http://dx.doi.org/10.1016/j.jid.2020.07.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990050PMC
April 2021

Human epidermal stem cell differentiation is modulated by specific lipid subspecies.

Proc Natl Acad Sci U S A 2020 09 25;117(36):22173-22182. Epub 2020 Aug 25.

Centre for Stem Cells and Regenerative Medicine, King's College London, SE1 9RT London, United Kingdom;

While the lipids of the outer layers of mammalian epidermis and their contribution to barrier formation have been extensively described, the role of individual lipid species in the onset of keratinocyte differentiation remains unknown. A lipidomic analysis of primary human keratinocytes revealed accumulation of numerous lipid species during suspension-induced differentiation. A small interfering RNA screen of 258 lipid-modifying enzymes identified two genes that on knockdown induced epidermal differentiation: , encoding elongation of very long-chain fatty acids protein 1, and , encoding fatty acid transport protein 1. By intersecting lipidomic datasets from suspension-induced differentiation and knockdown keratinocytes, we pinpointed candidate bioactive lipid subspecies as differentiation regulators. Several of these-ceramides and glucosylceramides-induced differentiation when added to primary keratinocytes in culture. Our results reveal the potential of lipid subspecies to regulate exit from the epidermal stem cell compartment.
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http://dx.doi.org/10.1073/pnas.2011310117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486749PMC
September 2020

Regulation of ERK basal and pulsatile activity control proliferation and exit from the stem cell compartment in mammalian epidermis.

Proc Natl Acad Sci U S A 2020 07 10;117(30):17796-17807. Epub 2020 Jul 10.

Centre for Stem Cells and Regenerative Medicine, King's College London, SE1 9RT London, United Kingdom;

Fluctuation in signal transduction pathways is frequently observed during mammalian development. However, its role in regulating stem cells has not been explored. Here we tracked spatiotemporal ERK MAPK dynamics in human epidermal stem cells. While stem cells and differentiated cells were distinguished by high and low stable basal ERK activity, respectively, we also found cells with pulsatile ERK activity. Transitions from Basal-Pulse (stem) to Basal-Pulse, Basal-Pulse, and Basal-Pulse (differentiated) cells occurred in expanding keratinocyte colonies and in response to differentiation stimuli. Pharmacological inhibition of ERK induced differentiation only when cells were in the Basal-Pulse state. Basal ERK activity and pulses were differentially regulated by DUSP10 and DUSP6, leading us to speculate that DUSP6-mediated ERK pulse down-regulation promotes initiation of differentiation, whereas DUSP10-mediated down-regulation of mean ERK activity promotes and stabilizes postcommitment differentiation. Levels of MAPK1/MAPK3 transcripts correlated with DUSP6 and DUSP10 transcripts in individual cells, suggesting that ERK activity is negatively regulated by transcriptional and posttranslational mechanisms. When cells were cultured on a topography that mimics the epidermal-dermal interface, spatial segregation of mean ERK activity and pulses was observed. In vivo imaging of mouse epidermis revealed a patterned distribution of basal cells with pulsatile ERK activity, and down-regulation was linked to the onset of differentiation. Our findings demonstrate that ERK MAPK signal fluctuations link kinase activity to stem cell dynamics.
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http://dx.doi.org/10.1073/pnas.2006965117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395546PMC
July 2020

A framework for advancing our understanding of cancer-associated fibroblasts.

Nat Rev Cancer 2020 03 24;20(3):174-186. Epub 2020 Jan 24.

Department of Anatomy, University of California, San Francisco, San Francisco, CA, USA.

Cancer-associated fibroblasts (CAFs) are a key component of the tumour microenvironment with diverse functions, including matrix deposition and remodelling, extensive reciprocal signalling interactions with cancer cells and crosstalk with infiltrating leukocytes. As such, they are a potential target for optimizing therapeutic strategies against cancer. However, many challenges are present in ongoing attempts to modulate CAFs for therapeutic benefit. These include limitations in our understanding of the origin of CAFs and heterogeneity in CAF function, with it being desirable to retain some antitumorigenic functions. On the basis of a meeting of experts in the field of CAF biology, we summarize in this Consensus Statement our current knowledge and present a framework for advancing our understanding of this critical cell type within the tumour microenvironment.
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http://dx.doi.org/10.1038/s41568-019-0238-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046529PMC
March 2020

NOTCH1 signaling in oral squamous cell carcinoma via a TEL2/SERPINE1 axis.

Oncotarget 2019 Nov 26;10(63):6791-6804. Epub 2019 Nov 26.

Centre for Stem Cells and Regenerative Medicine, King's College London, Tower Wing, Guy's Hospital, London, UK.

Inactivating mutations in the EGF-like ligand binding domain of are a prominent feature of the mutational landscape of oral squamous cell carcinoma (OSCC). In this study, we investigated mutations in keratinocyte lines derived from OSCC biopsies that had been subjected to whole exome sequencing. One line, SJG6, was found to have truncating mutations in both alleles, resulting in loss of NOTCH1 expression. Overexpression of the NOTCH1 intracellular domain (NICD) in SJG6 cells promoted cell adhesion and differentiation, while suppressing proliferation, migration and clonal growth, consistent with the previously reported tumour suppressive function of NOTCH1 in OSCC. Comparative gene expression profiling identified as being downregulated on NICD overexpression and predicted an interaction between and genes involved in cell proliferation and migration. Mechanistically, overexpression of NICD resulted in upregulation of /TEL2, which negatively regulates expression. Knockdown of phenocopied the effects of NICD overexpression in culture. Consistent with previous studies and our findings, there were inverse correlations between and expression and survival in OSCC primary tumours. Our results suggest that the tumour suppressive role of in OSCC is mediated, at least in part, by inhibition of via .
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http://dx.doi.org/10.18632/oncotarget.27306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6887571PMC
November 2019

Heterogeneity within Stratified Epithelial Stem Cell Populations Maintains the Oral Mucosa in Response to Physiological Stress.

Cell Stem Cell 2019 12;25(6):814-829.e6

Department of Pathology & Laboratory Medicine, the University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA; Lineberger Comprehensive Cancer Center, the University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address:

Stem cells in stratified epithelia are generally believed to adhere to a non-hierarchical single-progenitor model. Using lineage tracing and genetic label-retention assays, we show that the hard palatal epithelium of the oral cavity is unique in displaying marked proliferative heterogeneity. We identify a previously uncharacterized, infrequently-dividing stem cell population that resides within a candidate niche, the junctional zone (JZ). JZ stem cells tend to self-renew by planar symmetric divisions, respond to masticatory stresses, and promote wound healing, whereas frequently-dividing cells reside outside the JZ, preferentially renew through perpendicular asymmetric divisions, and are less responsive to injury. LRIG1 is enriched in the infrequently-dividing population in homeostasis, dynamically changes expression in response to tissue stresses, and promotes quiescence, whereas Igfbp5 preferentially labels a rapidly-growing, differentiation-prone population. These studies establish the oral mucosa as an important model system to study epithelial stem cell populations and how they respond to tissue stresses.
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http://dx.doi.org/10.1016/j.stem.2019.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925542PMC
December 2019

High-throughput micropatterning platform reveals Nodal-dependent bisection of peri-gastrulation-associated versus preneurulation-associated fate patterning.

PLoS Biol 2019 10 21;17(10):e3000081. Epub 2019 Oct 21.

Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, Toronto, Ontario, Canada.

In vitro models of postimplantation human development are valuable to the fields of regenerative medicine and developmental biology. Here, we report characterization of a robust in vitro platform that enabled high-content screening of multiple human pluripotent stem cell (hPSC) lines for their ability to undergo peri-gastrulation-like fate patterning upon bone morphogenetic protein 4 (BMP4) treatment of geometrically confined colonies and observed significant heterogeneity in their differentiation propensities along a gastrulation associable and neuralization associable axis. This cell line-associated heterogeneity was found to be attributable to endogenous Nodal expression, with up-regulation of Nodal correlated with expression of a gastrulation-associated gene profile, and Nodal down-regulation correlated with a preneurulation-associated gene profile expression. We harness this knowledge to establish a platform of preneurulation-like fate patterning in geometrically confined hPSC colonies in which fates arise because of a BMPs signalling gradient conveying positional information. Our work identifies a Nodal signalling-dependent switch in peri-gastrulation versus preneurulation-associated fate patterning in hPSC cells, provides a technology to robustly assay hPSC differentiation outcomes, and suggests conserved mechanisms of organized fate specification in differentiating epiblast and ectodermal tissues.
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http://dx.doi.org/10.1371/journal.pbio.3000081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6822778PMC
October 2019

Delta-like 1-mediated cis-inhibition of Jagged1/2 signalling inhibits differentiation of human epidermal cells in culture.

Sci Rep 2019 07 25;9(1):10825. Epub 2019 Jul 25.

Centre for Stem Cells and Regenerative Medicine, Faculty of Life Sciences & Medicine, King's College London, 28th Floor, Tower Wing, Guy's Hospital, SE1 9RT, London, UK.

Epidermal homeostasis depends on a balance between self-renewal of stem cells and terminal differentiation of their progeny. Notch signalling is known to play a role in epidermal  stem cell patterning and differentiation. However, the molecular mechanisms are incompletely understood. Here we demonstrate dynamic patterns of Notch ligand and receptor expression in cultured human epidermis. Notch2 and 3 act together to promote differentiation, while Notch1 decreases stem cell proliferation. The Notch ligand Jagged1 triggers differentiation when presented on an adhesive substrate or on polystyrene beads and over-rides the differentiation inhibitory effect of cell spreading. In contrast, Delta-like 1 (Dll1) overexpression abrogates the pro-differentiation effect of Jagged1 in a cell autonomous fashion. We conclude that Dll1 expression by stem cells not only stimulates differentiation of neighbouring cells in trans, but also inhibits differentiation cell autonomously. These results highlight the distinct roles of different Notch receptors and ligands in controlling epidermal homeostasis.
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http://dx.doi.org/10.1038/s41598-019-47232-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658703PMC
July 2019

The role of keratins in modulating carcinogenesis via communication with cells of the immune system.

Cell Stress 2019 Mar 23;3(4):136-138. Epub 2019 Mar 23.

Centre for Stem Cells & Regenerative Medicine, King's College London, Guy's Hospital, Great Maze Pond, London SE1 9RT, UK.

Keratins are intermediate filament proteins expressed by epithelial cells and provide mechanical support for diverse epithelia. In our recent study (Sequeira Nat Comm 9(1):3437), we analysed the role of keratin 76 (Krt76) in inflammation and cancer. Krt76 is expressed throughout embryonic development in the differentiated epithelial layers of a subset of stratified epithelia including tongue, palate and stomach. It is significantly downregulated in human oral squamous cell carcinoma (OSCC), correlating strongly with poor prognosis. We have shown that Krt76 mice exhibit systemic inflammation with increased levels of circulating B cells, regulatory T cells and effector T cells. When mice are given a chemical carcinogen in the drinking water, tongue and gastric cancer formation is accelerated in Krt76 mutant mice. Our data suggest that the increased tumour susceptibility of Krt76 mice is in part due to the enhanced accumulation of regulatory T cells in the tumour microenvironment. Our results support the notion that keratins, in addition to their function as cytoskeletal components, regulate immunity and affect tumour susceptibility of epithelial cells.
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http://dx.doi.org/10.15698/cst2019.04.184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6551831PMC
March 2019

Mechanisms, Hallmarks, and Implications of Stem Cell Quiescence.

Stem Cell Reports 2019 06;12(6):1190-1200

Centre for Stem Cells and Regenerative Medicine, King's College London, Guy's Hospital, Floor 28, Tower Wing, Great Maze Pond, London SE1 9RT, UK. Electronic address:

Cellular quiescence is a dormant but reversible cellular state in which cell-cycle entry and proliferation are prevented. Recent studies both in vivo and in vitro demonstrate that quiescence is actively maintained through synergistic interactions between intrinsic and extrinsic signals. Subtypes of adult mammalian stem cells can be maintained in this poised, quiescent state, and subsequently reactivated upon tissue injury to restore homeostasis. However, quiescence can become deregulated in pathological settings. In this review, we discuss the recent advances uncovering intracellular signaling pathways, transcriptional changes, and extracellular cues within the stem cell niche that control induction and exit from quiescence in tissue stem cells. We discuss the implications of quiescence as well as the pharmacological and genetic approaches that are being explored to either induce or prevent quiescence as a therapeutic strategy.
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http://dx.doi.org/10.1016/j.stemcr.2019.05.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6565921PMC
June 2019

Mutant Lef1 controls Gata6 in sebaceous gland development and cancer.

EMBO J 2019 05 18;38(9). Epub 2019 Mar 18.

Centre for Stem Cells and Regenerative Medicine, King's College London, London, UK

Mutations in Lef1 occur in human and mouse sebaceous gland (SG) tumors, but their contribution to carcinogenesis remains unclear. Since Gata6 controls lineage identity in SG, we investigated the link between these two transcription factors. Here, we show that Gata6 is a β-catenin-independent transcriptional target of mutant Lef1. During epidermal development, Gata6 is expressed in a subset of Sox9-positive Lef1-negative hair follicle progenitors that give rise to the upper SG Overexpression of Gata6 by lentiviral injection is sufficient to induce ectopic sebaceous gland elements. In mice overexpressing mutant Lef1, Gata6 ablation increases the total number of skin tumors yet decreases the proportion of SG tumors. The increased tumor burden correlates with impaired DNA mismatch repair and decreased expression of Mlh1 and Msh2 genes, defects frequently observed in human sebaceous neoplasia. Gata6 specifically marks human SG tumors and also defines tumors with elements of sebaceous differentiation, including a subset of basal cell carcinomas. Our findings reveal that Gata6 controls sebaceous gland development and cancer.
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http://dx.doi.org/10.15252/embj.2018100526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484415PMC
May 2019

Genome-wide association study in frontal fibrosing alopecia identifies four susceptibility loci including HLA-B*07:02.

Nat Commun 2019 03 8;10(1):1150. Epub 2019 Mar 8.

St. John's Institute of Dermatology, King's College London, London, Guy's Hospital, London, SE1 9RT, UK.

Frontal fibrosing alopecia (FFA) is a recently described inflammatory and scarring type of hair loss affecting almost exclusively women. Despite a dramatic recent increase in incidence the aetiopathogenesis of FFA remains unknown. We undertake genome-wide association studies in females from a UK cohort, comprising 844 cases and 3,760 controls, a Spanish cohort of 172 cases and 385 controls, and perform statistical meta-analysis. We observe genome-wide significant association with FFA at four genomic loci: 2p22.2, 6p21.1, 8q24.22 and 15q2.1. Within the 6p21.1 locus, fine-mapping indicates that the association is driven by the HLA-B*07:02 allele. At 2p22.1, we implicate a putative causal missense variant in CYP1B1, encoding the homonymous xenobiotic- and hormone-processing enzyme. Transcriptomic analysis of affected scalp tissue highlights overrepresentation of transcripts encoding components of innate and adaptive immune response pathways. These findings provide insight into disease pathogenesis and characterise FFA as a genetically predisposed immuno-inflammatory disorder driven by HLA-B*07:02.
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http://dx.doi.org/10.1038/s41467-019-09117-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6408457PMC
March 2019

Identifying Extrinsic versus Intrinsic Drivers of Variation in Cell Behavior in Human iPSC Lines from Healthy Donors.

Cell Rep 2019 02;26(8):2078-2087.e3

Centre for Stem Cells and Regenerative Medicine, King's College London, Floor 28, Tower Wing, Guy's Hospital, Great Maze Pond, London SE1 9RT, UK. Electronic address:

Large cohorts of human induced pluripotent stem cells (iPSCs) from healthy donors are a potentially powerful tool for investigating the relationship between genetic variants and cellular behavior. Here, we integrate high content imaging of cell shape, proliferation, and other phenotypes with gene expression and DNA sequence datasets from over 100 human iPSC lines. By applying a dimensionality reduction approach, Probabilistic Estimation of Expression Residuals (PEER), we extracted factors that captured the effects of intrinsic (genetic concordance between different cell lines from the same donor) and extrinsic (cell responses to different fibronectin concentrations) conditions. We identify genes that correlate in expression with intrinsic and extrinsic PEER factors and associate outlier cell behavior with genes containing rare deleterious non-synonymous SNVs. Our study, thus, establishes a strategy for examining the genetic basis of inter-individual variability in cell behavior.
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http://dx.doi.org/10.1016/j.celrep.2019.01.094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381787PMC
February 2019

Patterning of human epidermal stem cells on undulating elastomer substrates reflects differences in cell stiffness.

Acta Biomater 2019 03 30;87:256-264. Epub 2019 Jan 30.

Centre for Stem Cells and Regenerative Medicine, King's College London, 28th Floor, Tower Wing, Guy's Hospital, Great Maze Pond, London SE1 9RT, United Kingdom. Electronic address:

In human skin the junction between epidermis and dermis undulates, the width and depth of the undulations varying with age and disease. When primary human epidermal keratinocytes are seeded on collagen-coated polydimethylsiloxane (PDMS) elastomer substrates that mimic the epidermal-dermal interface, the stem cells become patterned by 24 h, resembling their organisation in living skin. We found that cell density and nuclear height were higher at the base than the tips of the PDMS features. Cells on the tips not only expressed higher levels of the stem cell marker β1 integrin but also had elevated E-cadherin, Desmoglein 3 and F-actin than cells at the base. In contrast, levels of the transcriptional cofactor MAL were higher at the base. AFM measurements established that the Young's modulus of cells on the tips was lower than on the base or cells on flat substrates. The differences in cell stiffness were dependent on Rho kinase activity and intercellular adhesion. On flat substrates the Young's modulus of calcium-dependent intercellular junctions was higher than that of the cell body, again dependent on Rho kinase. Cell patterning was influenced by the angle of the slope on undulating substrates. Our observations are consistent with the concept that epidermal stem cell patterning is dependent on mechanical forces exerted at intercellular junctions in response to undulations in the epidermal-dermal interface. STATEMENT OF SIGNIFICANCE: In human skin the epidermal-dermal junction undulates and epidermal stem cells are patterned according to their position. We previously created collagen-coated polydimethylsiloxane (PDMS) elastomer substrates that mimic the undulations and provide sufficient topographical information for stem cells to cluster on the tips. Here we show that the stiffness of cells on the tips is lower than cells on the base. The differences in cell stiffness depend on Rho kinase activity and intercellular adhesion. We propose that epidermal stem cell patterning is determined by mechanical forces exerted at intercellular junctions in response to the slope of the undulations.
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http://dx.doi.org/10.1016/j.actbio.2019.01.063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401207PMC
March 2019

Micro-scaled topographies direct differentiation of human epidermal stem cells.

Acta Biomater 2019 01 5;84:133-145. Epub 2018 Dec 5.

Centre for Stem Cells and Regenerative Medicine, King's College London, 28th Floor, Tower Wing, Guy's Hospital, Great Maze Pond, London SE1 9RT, United Kingdom. Electronic address:

Human epidermal stem cells initiate terminal differentiation when spreading is restricted on ECM-coated micropatterned islands, soft hydrogels or hydrogel-nanoparticle composites with high nanoparticle spacing. The effect of substrate topography, however, is incompletely understood. To explore this, primary human keratinocytes enriched for stem cells were seeded on a topographical library with over 2000 different topographies in the micrometre range. Twenty-four hours later the proportion of cells expressing the differentiation marker transglutaminase-1 was determined by high content imaging. As predicted, topographies that prevented spreading promoted differentiation. However, we also identified topographies that supported differentiation of highly spread cells. Topographies supporting differentiation of spread cells were more irregular than those supporting differentiation of round cells. Low topography coverage promoted differentiation of spread cells, whereas high coverage promoted differentiation of round cells. Based on these observations we fabricated a topography in 6-well plate format that supported differentiation of spread cells, enabling us to examine cell responses at higher resolution. We found that differentiated spread cells did not assemble significant numbers of hemidesmosomes, focal adhesions, adherens junctions, desmosomes or tight junctions. They did, however, organise the actin cytoskeleton in response to the topographies. Rho kinase inhibition and blebbistatin treatment blocked the differentiation of spread cells, whereas SRF inhibition did not. These observations suggest a potential role for actin polymerization and actomyosin contraction in the topography-induced differentiation of spread cells. STATEMENT OF SIGNIFICANCE: The epidermis is the outer covering of the skin. It is formed by layers of cells called keratinocytes. The basal cell layer contains stem cells, which divide to replace cells in the outermost layers that are lost through a process known as differentiation. In this manuscript we have developed surfaces that promote the differentiation of epidermal stem cells in order to understand the signals that control differentiation. The experimental tools we have developed have the potential to help us to devise new treatments that control diseases such as psoriasis and eczema in which epidermal stem cell proliferation and differentiation are disturbed.
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http://dx.doi.org/10.1016/j.actbio.2018.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6336537PMC
January 2019

Hair follicle epidermal stem cells define a niche for tactile sensation.

Elife 2018 10 25;7. Epub 2018 Oct 25.

Laboratory for Tissue Microenvironment, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan.

The heterogeneity and compartmentalization of stem cells is a common principle in many epithelia, and is known to function in epithelial maintenance, but its other physiological roles remain elusive. Here we show transcriptional and anatomical contributions of compartmentalized epidermal stem cells in tactile sensory unit formation in the mouse hair follicle. Epidermal stem cells in the follicle upper-bulge, where mechanosensory lanceolate complexes innervate, express a unique set of extracellular matrix (ECM) and neurogenesis-related genes. These epidermal stem cells deposit an ECM protein called EGFL6 into the collar matrix, a novel ECM that tightly ensheathes lanceolate complexes. EGFL6 is required for the proper patterning, touch responses, and αv integrin-enrichment of lanceolate complexes. By maintaining a quiescent original epidermal stem cell niche, the old bulge, epidermal stem cells provide anatomically stable follicle-lanceolate complex interfaces, irrespective of the stage of follicle regeneration cycle. Thus, compartmentalized epidermal stem cells provide a niche linking the hair follicle and the nervous system throughout the hair cycle.
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http://dx.doi.org/10.7554/eLife.38883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6226291PMC
October 2018

Lrig1 marks a population of gastric epithelial cells capable of long-term tissue maintenance and growth in vitro.

Sci Rep 2018 10 15;8(1):15255. Epub 2018 Oct 15.

BRIC - Biotech Research & Innovation Centre, University of Copenhagen, DK-2200, Copenhagen N, Denmark.

The processes involved in renewal of the epithelium that lines the mouse stomach remain unclear. Apart from the cells in the isthmus, several other populations located deeper in the gastric glands have been suggested to contribute to the maintenance of the gastric epithelium. Here, we reveal that Lrig1 is expressed in the basal layer of the forestomach and the lower part of glands in the corpus and pylorus. In the glandular epithelium of the stomach, Lrig1 marks a heterogeneous population comprising mainly non-proliferative cells. Yet, fate-mapping experiments using a knock-in mouse line expressing Cre specifically in Lrig1 cells demonstrate that these cells are able to contribute to the long-term maintenance of the gastric epithelium. Moreover, when cultured in vitro, cells expressing high level of Lrig1 have much higher organoid forming potential than the corresponding cellular populations expressing lower levels of Lrig1. Taken together, these observations show that Lrig1 is expressed primarily by differentiated cells, but that these cells can be recruited to contribute to the maintenance of the gastric epithelium. This confirms previous observations that cells located in the lower segments of gastric glands can participate in tissue replenishment.
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http://dx.doi.org/10.1038/s41598-018-33578-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6189208PMC
October 2018

Dynamic Culture Substrates That Mimic the Topography of the Epidermal-Dermal Junction.

Tissue Eng Part A 2019 02 28;25(3-4):214-223. Epub 2018 Dec 28.

1 Center for Stem Cells and Regenerative Medicine, King's College London, Guy's Hospital, London, United Kingdom.

Impact Statement: In human skin the junction between the epidermis and dermis undulates. Epidermal stem cells pattern according to their position relative to those undulations. Here we describe a rig in which epidermal cells are cultured on a collagen-coated poly(d,l-lactide-co-glycolide) (PLGA) membrane. When a vacuum is applied the membrane is induced to undulate. Stem cells cluster in response to the vacuum, whereas differentiating cells do not. Rho kinase inhibition results in loss of clustering, suggesting a role for Rho family members in the process. This dynamic platform is a new tool for investigating changes in the skin with age and disease.
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http://dx.doi.org/10.1089/ten.TEA.2018.0125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6388717PMC
February 2019

Fibroblast state switching orchestrates dermal maturation and wound healing.

Mol Syst Biol 2018 08 29;14(8):e8174. Epub 2018 Aug 29.

Centre for Stem Cells and Regenerative Medicine, King's College London, London, UK

Murine dermis contains functionally and spatially distinct fibroblast lineages that cease to proliferate in early postnatal life. Here, we propose a model in which a negative feedback loop between extracellular matrix (ECM) deposition and fibroblast proliferation determines dermal architecture. Virtual-tissue simulations of our model faithfully recapitulate dermal maturation, predicting a loss of spatial segregation of fibroblast lineages and dictating that fibroblast migration is only required for wound healing. To test this, we performed live imaging of dermal fibroblasts, which revealed that homeostatic tissue architecture is achieved without active cell migration. In contrast, both fibroblast proliferation and migration are key determinants of tissue repair following wounding. The results show that tissue-scale coordination is driven by the interdependence of cell proliferation and ECM deposition, paving the way for identifying new therapeutic strategies to enhance skin regeneration.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113774PMC
http://dx.doi.org/10.15252/msb.20178174DOI Listing
August 2018

The reward of great collaborations.

Authors:
Fiona M Watt

Nat Cell Biol 2018 Sep;20(9):1011

King's College London Centre for Stem Cells and Regenerative Medicine, London, UK.

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http://dx.doi.org/10.1038/s41556-018-0157-5DOI Listing
September 2018

Immunomodulatory role of Keratin 76 in oral and gastric cancer.

Nat Commun 2018 08 24;9(1):3437. Epub 2018 Aug 24.

Centre for Stem Cells & Regenerative Medicine, King's College London, Guy's Hospital, Great Maze Pond, London, SE1 9RT, UK.

Keratin 76 (Krt76) is expressed in the differentiated epithelial layers of skin, oral cavity and squamous stomach. Krt76 downregulation in human oral squamous cell carcinomas (OSCC) correlates with poor prognosis. We show that genetic ablation of Krt76 in mice leads to spleen and lymph node enlargement, an increase in regulatory T cells (Tregs) and high levels of pro-inflammatory cytokines. Krt76 Tregs have increased suppressive ability correlated with increased CD39 and CD73 expression, while their effector T cells are less proliferative than controls. Loss of Krt76 increases carcinogen-induced tumours in tongue and squamous stomach. Carcinogenesis is further increased when Treg levels are elevated experimentally. The carcinogenesis response includes upregulation of pro-inflammatory cytokines and enhanced accumulation of Tregs in the tumour microenvironment. Tregs also accumulate in human OSCC exhibiting Krt76 loss. Our study highlights the role of epithelial cells in modulating carcinogenesis via communication with cells of the immune system.
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http://dx.doi.org/10.1038/s41467-018-05872-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6109110PMC
August 2018

Bench to bedside: Current advances in regenerative medicine.

Curr Opin Cell Biol 2018 12 11;55:59-66. Epub 2018 Jul 11.

Centre for Stem Cells and Regenerative Medicine, King's College London, Floor 28. Tower Wing, Guy's Hospital, Great Maze Pond, London SE1 9RT, UK. Electronic address:

Regenerative medicine is a diverse and rapidly evolving field, employing core expertise from biologists, engineers, and clinicians. Recently the field has made significant progress towards regenerating or replacing tissues lost to age, disease or injury. Current strategies include transplantation of adult or pluripotent stem cells to replace tissue or support tissue healing. Promising approaches for the future of regenerative medicine include stimulating endogenous stem cells for in situ repair, transplantation of organoids to repair minor tissue injury, and the use of interspecies chimerism to produce functional metabolic organs for transplantation. In our review we focus on these emerging strategies, paying particular attention to their current and prospective translational impacts and challenges.
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http://dx.doi.org/10.1016/j.ceb.2018.05.006DOI Listing
December 2018

Loxl2 is dispensable for dermal development, homeostasis and tumour stroma formation.

PLoS One 2018 28;13(6):e0199679. Epub 2018 Jun 28.

Centre for Stem Cells and Regenerative Medicine, King's College London, Guy's Hospital, London, United Kingdom.

Lysyl oxidase-like 2 (LOXL2) is a copper-dependent monoamine oxidase that contributes to the remodelling of the extracellular matrix (ECM) by cross linkage of collagen and elastin fibres and has emerged as a potential therapeutic target in cancer and fibrosis. In the skin, LOXL2 is essential for epidermal cell polarity and differentiation. However, its role in the dermis has not been evaluated. We found that Loxl2 is dispensable for mouse dermal development, maturation and homeostasis, yet affects dermal stiffness. Neither loss of Loxl2 nor increased Loxl2 expression affected dermal architecture following treatment with the phorbol ester TPA. Furthermore, Loxl2 expression did not alter the stroma of DMBA-TPA-induced tumours. We conclude that, although Loxl2 is expressed in both dermis and epidermis, its function appears largely confined to the epidermis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0199679PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023175PMC
December 2018