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Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrinα6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Major shifts in intestinal commensal bacteria often result in changes in CD4+ T lymphocyte populations, leading to an influx of Th17 cells, chronic inflammation, and eventually cancer. Consequently, the inappropriate propagation of certain commensal species in the gut has been associated with mucosal inflammatory diseases and cancer development. Recent experiments investigating the relationships between food-borne pathogens, enteric bacteria, and cancer have exposed the ability of certain bacterial species to significantly reduce tumor size and tumor progression in mice. In similar studies, pro-inflammatory Th17 and Th1 cells were at times found present along with anti-inflammatory Treg populations in the intestinal mucosa. This antitumor response was mediated by a balanced production of pro- and anti-inflammatory cytokines, resulting in a controlled threshold of mucosal immunity largely moderated by CD4+ T lymphocyte populations, through a dendritic cell-dependent pathway. These findings provide new evidence that certain species of bacteria can help manage subcutaneous tumor development by calibrating mucosal and, in some instances, systemic thresholds of innate and adaptive immunity.

Relative to conventional two-dimensional (2-D) culture, three-dimensional (3-D) suspension culture of epithelial cells more closely mimics the in vivo cell microenvironment regarding cell architecture, cell to matrix interaction, and osmosis exchange. However, primary normal human keratinocytes (NHKc) rapidly undergo terminal differentiation and detachment-induced cell death (anoikis) upon disconnection from the basement membrane, thus greatly constraining their use in 3-D suspension culture models. Here, we examined the 3-D anchorage-free growth potential of NHKc isolated from neonatal skin explants of 59 different individuals. We found that 40% of all isolates naturally self-assembled into multicellular spheroids within 24 h in anchorage-free culture, while 60% did not. Placing a single spheroid back into 2-D monolayer culture yielded proliferating cells that expressed elevated levels of nuclear P63 and basal cytokeratin 14. These cells also displayed prolonged keratinocyte renewal and a gene expression profile corresponding to cellular heterogeneity, quiescence, and de-differentiation. Notably, spheroid-derived (SD) NHKc were enriched for a P63/K14 double-positive population that formed holoclonal colonies and reassembled into multicellular spheroids during 3-D suspension subculture. This study reveals marked phenotypic differences in neonatal keratinocyte suspension cultures isolated from different individuals and present a model system that can be readily employed to study epithelial cell behavior, along with a variety of dermatological diseases.

Epigenetic regulation has a profound influence on stem cell fate during normal development in maintenance of physiologic tissue homeostasis. Here we report diminished ten-eleven translocation (TET) methylcytosine dioxygenase expression and loss of the DNA hydroxymethylation mark 5-hydroxymethylcytosine (5-hmC) in keratinocyte stem cells and transit amplifying cells in human psoriasis and in imiquimod-induced murine psoriasis. Loss of 5-hmC was associated with dysregulated keratinocyte stem cell kinetics, resulting in accumulation of nestin and FABP5-expressing transit amplifying cells to produce classic psoriatic epidermal architecture. Moreover, 5-hmC loss was accompanied by diminished TET1 and TET2 mRNA expression. Genome-wide mapping of epidermal 5-hmC in murine psoriasis revealed loci-specific loss of 5-hmC in genes regulating stem cell homeostasis, including MBD1, RTN1, STRN4, PRKD2, AKT1, and MAPKAP2, as well as those associated with RAR and Wnt/β-catenin signaling pathways. In vitro restoration of TET expression by ascorbic acid was accomplished in cultured human keratinocyte stem cells to show similar Ca++-induced differentiation, resulting in increased 5-hmC levels and reduced nestin expression. To our knowledge, an epigenetic deficiency in psoriasis with relevance to stem cell dysregulation has not been previously reported. This observation raises the possibility that epigenetic modifiers that impact on the TET–5-hmC pathway may be a relevant approach of heretofore unappreciated therapeutic utility.

Human papillomavirus (HPV) infection of the genital tract is common; however, only about 10 to 15% of infections persist, and approximately 10 to 15% of these persistent infections result in cancer. Basal epidermal stem cells are the presumed target cells for HPV infection, providing a reservoir of latently infected cells that persist over time and initiate lesions. However, it is not known whether stem cell density has any influence on transformation of human keratinocytes by HPV. We explored the relationship between stem cell properties of normal human keratinocytes and their susceptibility to transformation by HPV16 DNA. Normal human keratinocyte isolates (NHKc) derived from different donors were cultured in three-dimensional anchorage-free suspension to assess their spheroid-forming ability. NHKc spheroids were then plated back into plastic monolayer culture and transfected with full-length HPV16 DNA, which we have previously shown to integrate into the host cell genome upon transfection. Spheroid-derived NHKc (SD-NHKc) and fluorescence-activated cell sorting-purified populations of basal stem-like keratinocytes, expressing low levels of epidermal growth factor receptor and high levels of integrin alpha 6 (EGFR lo /ITGα6 hi ), responded to transfection with HPV16 DNA with more vigorous proliferation, greater immortalization efficiency, and faster progression to differentiation resistance than autologous mass-cultured cells. Conversely, cells committed to terminal differentiation (EGFR hi /ITGα6 lo ) grew slowly after transfection with HPV16 and failed to generate immortalized or DR clones. HPV16 DNA induced stem cell properties in mass-cultured NHKc. We conclude that HPV16 preferentially immortalizes basal keratinocytes with stem cell properties and that these cells readily achieve a differentiation-resistant phenotype upon immortalization by HPV16. IMPORTANCE This paper explores the relationship between the stem cell properties of normal human epidermal cells in culture and these cells' susceptibility to transformation by HPV16 DNA, the HPV type present in about 50% of cervical cancers. We report variable susceptibilities to HPV16-mediated transformation among different keratinocyte isolates derived from neonatal foreskin. Our findings provide strong experimental evidence that HPV16 preferentially transforms basal keratinocytes with stem cell properties. Insights gained from these studies increase our understanding of the host cell-specific factors influencing individual susceptibility to HPV-driven transformation and the contributing factors leading to preneoplastic and neoplastic progression of HPV-positive lesions.

Aims: To identify novel antibiotic-producing microbial strains with unprecedented pertinence. We hypothesize that site-specific soil samples will contain a variety of antibioticproducing species (APS) with diverse specificity of molecular elements. Place and Duration of Study: Laboratory of Microbiology, Division of Biological and Health Sciences, University of Pittsburgh, Bradford, PA-16701, USA, between August 2010 and May 2011. Methodology: The environmental soil samples were collected from residential and recreational sites in Southern, PA, USA at longitude: -76 42 21.7116, latitude: 39 56 35.7252; approximately 201 meters above sea level. Over 70 natural antibiotic-producing soil bacteria were screened against 19 pathogenic microorganisms. Agar-plug assay was established to identify the antibiotics’ potency and pathogenic inhibitory index calculations were employed to measure the inhibitory potential of each isolate; 16S rRNA sequencing was used for microbial classification. Results: A total of 71 microorganisms from residential soil demonstrated zones of inhibition (ZOI), followed by 9 organisms from recreational soil sample. A total of 15 bioactive strains demonstrated convincing growth inhibitory properties against 16 clinically relevant pathogens; 40% revealed pDNA presence, of which 67% exhibited stringent potencies against S. aureus. We observed a highly bioactive residential soil microbiota compared to recreational soil. Conclusion: 16S rRNA sequence analysis corroborated several of the species belonging to Enterobacteriaceae, Xanthomonadaceae, and Bacillaceae. These findings may indicate a co-evolutionary biosynthesis of novel antibiotics driven by the increase of bioactive microbiota in residential environments.

The field of nanotechnology has recently seen vast advancements in its applications for therapeutic strategy. This technological revolution has led way to nanomedicine, which spurred the development of clever drug delivery designs and ingenious nanovehicles for the monitoring of cellular events in vivo. The clinical implementations of this technology are innumerable and have demonstrated utility as diagnostic tools and fortifying machineries for the mammalian immune system. Recently engineered viral vectors and multi-subunit packaging RNAs have verified stable enough for long-term existence in the physiological environment and therefore reveal unique potential as artificial immunosurveillance devices. Physiological and pathological events recorded by nanodevices could help develop "biocatalogs" of patients' infection history, frequency of disease, and much more. In this article, we introduce a novel design concept for a multilayer synthetic immune network parallel to the natural immune system; an artificial network of continuously patrolling nanodevices incorporated in the blood and lymphatic systems, and adapted for molecular event recording, anomaly detection, drug delivery, and gene silencing. We also aim to discuss the approaches and advances recently reported in nanomedicine, especially as it pertains to promising viral and RNA-based nanovehicles and their prospective applications for the development of a synthetic immunosurveillance system (SIS). Alternative suggestions and limitations of these technologies are also discussed.

Human papillomavirus (HPV) infection is the primary cause of cervical cancer. While HPV infection is very common, cervical cancer is a rare outcome. Why some women develop cervical cancer and others do not is not well understood, but likely involves host cell-specific factors and a woman's ability to get rid of HPV-infected cells. We have previously reported marked variability of epidermal growth factor receptor (EGFR) expression in normal human epidermal tissue from different individuals and demonstrated that increased immortalization by HPV16 E6 and E7 oncogenes correlated with lower basal levels of EGFR. We now report a significantly greater transformation response in stem cell-like keratinocytes after transfection with the full length HPV16 genome. Primary normal human keratinocyte strains (NHKc) from 59 individuals were cultured in 3-D anchorage-free suspension to assess spheroid forming ability and to determine resistance to anchorage-independent cell death. Only about 40% of NHKc strains survived and formed spheroids in 3-D culture and grew when plated back in monolayers as spheroid-derived NHKc (SD-NHKc). Fluorescence-activated cell sorting analysis found that SD-NHKc were enriched in cells expressing known keratinocyte stem cell markers: low levels of EGFR and high levels of integrin α6, while spheroid-non forming strains (SNF-NHKc) had dramatically reduced numbers of EGFRlo/ITGα6hi cells. Isolation and cultivation of EGFRlo/ITGα6hi keratinocytes isolated from primary monolayer NHKc cultures revealed this cell population to behave like SD-NHKc with respect to anchorage-independent survival, proliferative potential, cell morphology, and clonogenicity. Upon transfection with HPV16 DNA, most NHKc strains acquired anchorage-independence and exhibited increased expression of EGFR. Importantly, EGFRlo/ITGα6hi keratinocytes and SD-NHKc displayed exuberant growth responses after transfection with HPV16 DNA and significantly higher transformation efficiency by HPV16. In contrast, SNF-NHKc grew more sluggishly and were relatively resistant to HPV16-mediated immortalization. Our study indicates that stem cell-like keratinocytes are preferentially transformed by HPV16 DNA and that stemness is a phenotypic determinant of susceptibility to HPV16-mediated transformation in primary human keratinocytes. Citation Format: Yvon Woappi, Kim E. Creek, Lucia Pirisi. Stem cell properties of normal human keratinocytes influence transformation responses to HPV16. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4048.