Know your enemy: Unexpected, pervasive and persistent viral and bacterial contamination of primary cell cultures.

In biomedical research, cell culture contamination is one of the main culprits of experimental failure. Contamination sources and concomitant remedies are numerous and challenging to manage. We herein describe two cases of uncommon contamination of cell cultures that we encountered, and the successful determination and eradication strategies. The first case describes the infection with human adenovirus C that originated from pharyngeal tonsils used for isolation of primary tonsillar epithelial cells. It is known that viral contamination of in vitro cell cultures can occur symptomless and is therefore difficult to identify. The contamination was pervasive and persistent, as it was widely spread in flow cabinets and apparatus, and has caused a serious delay to our research projects and the inevitable loss of valuable (patient-derived) cell sources. Eradication was successful by formalin gas sterilization of the flow cabinet and elimination of all infected cell lines from our biobank after PCR-guided determination. Secondly, we encountered a spore-forming bacterium, namely Brevibacillus brevis, in our cell culture facility. This bacterium originated from contaminated tap water pipes and spread via regular aseptic culture techniques due to survival of the bacterial spores in 70% ethanol. B brevis overgrew the cultures within a few days after seeding of the primary cells. Chlorine solution effectively killed this spore-forming bacterium. Both cases of contamination were identified using DNA sequencing which enabled the deployment of targeted aseptic techniques for the elimination of the persistent contamination.

Psoriasis-Associated Late Cornified Envelope (LCE) Proteins Have Antibacterial Activity.

Terminally differentiating epidermal keratinocytes express a large number of structural and antimicrobial proteins that are involved in the physical barrier function of the stratum corneum and provide innate cutaneous host defense. Late cornified envelope (LCE) genes, located in the epidermal differentiation complex on chromosome 1, encode a family of 18 proteins of unknown function, whose expression is largely restricted to epidermis. Deletion of two members, LCE3B and LCE3C (LCE3B/C-del), is a widely-replicated psoriasis risk factor that interacts with the major psoriasis-psoriasis risk gene HLA-C*06. Here we performed quantitative trait locus analysis, utilizing RNA-seq data from human skin and found that LCE3B/C-del was associated with a markedly increased expression of LCE3A, a gene directly adjacent to LCE3B/C-del. We confirmed these findings in a 3-dimensional skin model using primary keratinocytes from LCE3B/C-del genotyped donors. Functional analysis revealed that LCE3 proteins, and LCE3A in particular, have defensin-like antimicrobial activity against a variety of bacterial taxa at low micromolar concentrations. No genotype-dependent effect was observed for the inside-out or outside-in physical skin barrier function. Our findings identify an unknown biological function for LCE3 proteins and suggest a role in epidermal host defense and LCE3B/C-del-mediated psoriasis risk.

Transcription factor p63 bookmarks and regulates dynamic enhancers during epidermal differentiation.

The transcription factor p63 plays a pivotal role in keratinocyte proliferation and differentiation in the epidermis. However, how p63 regulates epidermal genes during differentiation is not yet clear. Using epigenome profiling of differentiating human primary epidermal keratinocytes, we characterized a catalog of dynamically regulated genes and p63-bound regulatory elements that are relevant for epithelial development and related diseases. p63-bound regulatory elements occur as single or clustered enhancers, and remarkably, only a subset is active as defined by the co-presence of the active enhancer mark histone modification H3K27ac in epidermal keratinocytes. We show that the dynamics of gene expression correlates with the activity of p63-bound enhancers rather than with p63 binding itself. The activity of p63-bound enhancers is likely determined by other transcription factors that cooperate with p63. Our data show that inactive p63-bound enhancers in epidermal keratinocytes may be active during the development of other epithelial-related structures such as limbs and suggest that p63 bookmarks genomic loci during the commitment of the epithelial lineage and regulates genes through temporal- and spatial-specific active enhancers.