Simon L.I.J. Denil

Lgr5-expressing chief cells drive epithelial regeneration and cancer in the oxyntic stomach

The daily renewal of the corpus epithelium is fuelled by adult stem cells residing within tubular glands, but the identity of these stem cells remains controversial. Lgr5 marks homeostatic stem cells and ‘reserve’ stem cells in multiple tissues. Here, we report Lgr5 expression in a subpopulation of chief cells in mouse and human corpus glands. Using a non-variegated Lgr5-2A-CreERT2 mouse model, we show by lineage tracing that Lgr5-expressing chief cells do not behave as corpus stem cells during homeostasis, but are recruited to function as stem cells to effect epithelial renewal following injury by activating Wnt signalling. Ablation of Lgr5+ cells severely impairs epithelial homeostasis in the corpus, indicating an essential role for these Lgr5+ cells in maintaining the homeostatic stem cell pool. We additionally define Lgr5+ chief cells as a major cell-of-origin of gastric cancer. These findings reveal clinically relevant insights into homeostasis, repair and cancer in the corpus.

Long-Term Culture of Self-renewing Pancreatic Progenitors Derived from Human Pluripotent Stem Cells

Summary Pluripotent stem cells have been proposed as an unlimited source of pancreatic β cells for studying and treating diabetes. However, the long, multi-step differentiation protocols used to generate functional β cells inevitably exhibit considerable variability, particularly when applied to pluripotent cells from diverse genetic backgrounds. We have developed culture conditions that support long-term self-renewal of human multipotent pancreatic progenitors, which are developmentally more proximal to the specialized cells of the adult pancreas. These cultured pancreatic progenitor (cPP) cells express key pancreatic transcription factors, including PDX1 and SOX9, and exhibit transcriptomes closely related to their in vivo counterparts. Upon exposure to differentiation cues, cPP cells give rise to pancreatic endocrine, acinar, and ductal lineages, indicating multilineage potency. Furthermore, cPP cells generate insulin+ β-like cells in vitro and in vivo, suggesting that they offer a convenient alternative to pluripotent cells as a source of adult cell types for modeling pancreatic development and diabetes.

Array-based sequencing of filaggrin gene for comprehensive detection of disease-associated variants

4. Meyaard L. The inhibitory collagen receptor LAIR-1 (CD305). J Leukoc Biol 2008;83:799-803. 5. Funchal GA, Jaeger N, Czepielewski RS, Machado MS, Muraro SP, Stein RT, et al. Respiratory syncytial virus fusion protein promotes TLR-4-dependent neutrophil extracellular trap formation by human neutrophils. PLoS One 2015; 10:e0124082. 6. Van Avondt K, van der Linden M, Naccache PH, Egan DA, Meyaard L. Signal inhibitory receptor on leukocytes-1 limits the formation of neutrophil extracellular traps, but preserves intracellular bacterial killing. J Immunol 2016; 196:3686-94. 7. Sangaletti S, Tripodo C, Vitali C, Portararo P, Guarnotta C, Casalini P, et al. Defective stromal remodeling and neutrophil extracellular traps in lymphoid tissues favor the transition from autoimmunity to lymphoma. Cancer Discov 2014;4:110-29. 8. DeVincenzo JP, McClure MW, Symons JA, Fathi H, Westland C, Chanda S, et al. Activity of oral ALS-008176 in a respiratory syncytial virus challenge study. N Engl J Med 2015;373:2048-58. 9. Lukens MV, van de Pol AC, Coenjaerts FE, Jansen NJ, Kamp VM, Kimpen JL, et al. A systemic neutrophil response precedes robust CD8(1) T-cell activation during natural respiratory syncytial virus infection in infants. J Virol 2010;84: 2374-83.

Low-dose calcipotriol can elicit wound closure, anti-microbial, and anti-neoplastic effects in epidermolysis bullosa keratinocytes

Recessive dystrophic epidermolysis bullosa (RDEB) patients suffer from chronic and repeatedly infected wounds predisposing them to the development of aggressive and life-threatening skin cancer in these areas. Vitamin D3 is an often neglected but critical factor for wound healing. Intact skin possesses the entire enzymatic machinery required to produce active 1-alpha,25-dihydroxyvitamin D3 (calcitriol), underscoring its significance to proper skin function. Injury enhances calcitriol production, inducing the expression of calcitriol target genes including the antimicrobial peptide cathelicidin (hCAP18), an essential component of the innate immune system and an important wound healing factor. We found significantly reduced hCAP18 expression in a subset of RDEB keratinocytes which could be restored by calcipotriol treatment. Reduced scratch closure in RDEB cell monolayers was enhanced up to 2-fold by calcipotriol treatment, and the secretome of calcipotriol-treated cells additionally showed increased antimicrobial activity. Calcipotriol exhibited anti-neoplastic effects, suppressing the clonogenicity and proliferation of RDEB tumor cells. The combined wound healing, anti-microbial, and anti-neoplastic effects indicate that calcipotriol may represent a vital therapeutic option for RDEB patients which we could demonstrate in a single-patient observation study.

Tiled array-based sequencing identifies enrichment of loss-of-function variants in the highly homologous filaggrin gene in African-American children with severe atopic dermatitis

Filaggrin (FLG) loss‐of‐function (LOF) variants are a major risk factor for the common inflammatory skin disease, atopic dermatitis (AD) and are often population‐specific. African‐American (AA) children are disproportionately affected with AD, often later developing asthma and/or allergic rhinitis and comprise an atopy health disparity group for which the role of FLG LOF is not well known. Discovery of FLG LOF using exome sequencing is challenging given the known difficulties for accurate short‐read alignment to FLGs high homology repeat variation. Here, we employed an array‐based sequencing approach to tile across each FLG repeat and discover FLG LOF in a well‐characterized cohort of AA children with moderate‐to‐severe AD. Five FLG LOF were identified in 23% of our cohort. Two novel FLG LOF singletons, c.488delG and p.S3101*, were discovered as well as p.R501*, p.R826* and p.S3316* previously reported for AD. p.S3316* (rs149484917) is likely an African ancestral FLG LOF, reported in African individuals in ExAC (Exome Aggregation Consortium), Exome Variant Server (ESP), and 4 African 1000G population databases (ESN, MSL, ASW, and ACB). The proportion of FLG LOF (11.5%) among the total FLG alleles in our cohort was significantly higher in comparisons with FLG LOF reported for African individuals in ExAC (2.5%; P = 4.3 × 10−4) and ESP (1.7%; P = 3.5 × 10−5) suggesting a disease‐enrichment effect for FLG LOF. Our results demonstrate the utility of array‐based sequencing in discovering FLG LOF, including novel and population‐specific, which are of higher prevalence in our AA severe AD group than previously reported.

ENPP1 Mutation Causes Recessive Cole Disease by Altering Melanogenesis

Cole disease is a genodermatosis of pigmentation following a strict dominant mode of inheritance. In this study, we investigated eight patients affected with an overlapping genodermatosis after recessive inheritance. The patients presented with hypo- and hyperpigmented macules over the body, resembling dyschromatosis universalis hereditaria in addition to punctuate palmoplantar keratosis. By homozygosity mapping and whole-exome sequencing, a biallelic p.Cys120Arg mutation in ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) was identified in all patients. We found that this mutation, like those causing dominant Cole disease, impairs homodimerization of the ENPP1 enzyme that is mediated by its two somatomedin-B-like domains. Histological analysis revealed structural and molecular changes in affected skin that were likely to originate from defective melanocytes because keratinocytes do not express ENPP1. Consistently, RNA-sequencing analysis of patient-derived primary melanocytes revealed alterations in melanocyte development and in pigmentation signaling pathways. We therefore conclude that germline ENPP1 cysteine-specific mutations, primarily affecting the melanocyte lineage, cause a clinical spectrum of dyschromatosis, in which the p.Cys120Arg allele represents a recessive and more severe form of Cole disease.