Reprogramming of human pancreatic exocrine cells to ß-like cells.

Rodent acinar cells exhibit a remarkable plasticity as they can transdifferentiate to duct-, hepatocyte- and islet ß-like cells. We evaluated whether exocrine cells from adult human pancreas can similarly respond to proendocrine stimuli. Exocrine cells from adult human pancreas were transduced directly with lentiviruses expressing activated MAPK (mitogen-activated protein kinase) and STAT3 (signal transducer and activator of transcription 3) and cultured as monolayers or as 3D structures. Expression of STAT3 and MAPK in human exocrine cells activated expression of the proendocrine factor neurogenin 3 in 50% to 80% of transduced exocrine cells. However, the number of insulin-positive cells increased only in the exocrine cells grown initially in suspension before 3D culture. Lineage tracing identified human acinar cells as the source of Ngn3- and insulin-expressing cells. Long-term engraftment into immunocompromised mice increased the efficiency of reprogramming to insulin-positive cells. Our data demonstrate that exocrine cells from human pancreas can be reprogrammed to transplantable insulin-producing cells that acquire functionality. Given the large number of exocrine cells in a donor pancreas, this approach presents a novel strategy to expand cell therapy in type 1 diabetes.

Practical problems encountered during the cultivation of an open-source reconstructed human epidermis model on a polycarbonate membrane and protein quantification.

During recent years, the importance of in vitro technology in skin research has increased significantly. A variety of skin culture models have been developed and commercialized. In this respect, the availability of reconstructed human epidermis (RHE) equivalents represents a significant improvement compared to the use of monolayer cultures. However, when an in-house RHE model is being developed, researchers might encounter some difficulties during cultivation. The scope of this paper is to report our experiences and practical problems with the development of a three-dimensional RHE model cultured on a polycarbonate membrane. Some important issues including cell density, the use of lysing enzymes, culture media, cell storage and viability, cell confluency and protein extraction are reported and optional solutions are given.

Current status of healthy human skin models: can histone deacetylase inhibitors potentially improve the present replacement models?

Histone deacetylase inhibitors (HDACi), a relatively new group of epigenetic agents, are being investigated as powerful chemotherapeutics because of their antiproliferative and prodifferentiation effects both in vitro and in vivo, in various tumor cell lines. Only little is known with respect to their effects on normal cells. Yet, to understand tissue pathology and evaluate potential effects of new chemical entities in tissue homeostasis, insight into the physiology of healthy tissue is necessary. Therefore, this review addresses the effects of HDACi on healthy human primary skin cell cultures and three-dimensional epidermal models. In general, HDACi exert an effect on both the epidermal morphology and differentiation process of human skin. The latter is manifested through cell cycle arrest, disorganization of the basal layer, thinning of the stratum spinosum and thickening of the stratum corneum, reorganization of the cytoskeleton and increased formation of cornified envelopes. This overview shows that, although only a limited number of reports exist, these molecules might be an interesting tool for the development and study of new human skin models.

Unique patterns of mitochondrial genes, transcripts and proteins in different male-sterile cytoplasms of Daucus carota.

Restriction fragment analysis of mitochondrial and chloroplast DNAs from a brown anther and a petaloid cytoplasmic male-sterile (cms) line revealed unique patterns for each cms line distinct from those of normal fertile cytoplasms, but identical restriction fragments for all chloroplast DNAs. The restauration of fertility through the introduction of nuclear restorer genes had no effect on the overall mitochondrial DNA (mtDNA) structure. The genomic environment and transcription patterns of several mitochondrial genes differ between cms and normal cytoplasms, while no difference has so far been detected between cms and the corresponding fertility-restored lines in mitochondrial DNAs and mRNAs. Mitochondrial translation products analysed by in-organello synthesized proteins revealed a number of polypeptides unique to each cytoplasm. Most prominent is a 17-kDa polypeptide that is present in the brown anther cms line but not in fertile mitochondria. Synthesis of this protein was not visibly affected by fertility restauration. The different cms phenotypes in carrot are thus associated with extensive and unique mtDNA rearrangements and distinct alterations in transcription and translation patterns.