Xeno-free workflow exhibits comparable efficiency and quality of keratinocytes isolated from human skin biopsies.

INTRODUCTION: Regenerative solutions of the skin represent a hope for burn victims with extensive skin loss and chronic wound patients. The development of xeno-free workflow is crucial for clinical application in compliance with the directives of the European Medicines Agency. This study aimed at evaluating the outcome of the xeno-free isolation workflow of keratinocytes from human skin biopsy. METHODS: Skin biopsies were obtained from volunteers. The epidermis was digested with TrypLE™ Select, which was deactivated by dilution or with trypsin, deactivated by media with fetal bovine serum. Freshly isolated cells were compared for total cell number, viability, activity of caspase 3, gene expression and the presence of the keratinocyte surface markers cytokeratin 14. The cells were cultured in xeno-free conditions for one week and characterized regarding the number and viability as well as the metalloproteinase secretion. RESULTS: The number of obtained cells was similar in both workflows. The cell viability was less in the TrypLE group, with slight reduction of the cell surface marker cytokeratin 14. Caspase 3 activity was comparable as well as the gene expression of the apoptotic markers BAX, BCL2 and SLUG, as well as the keratinocyte markers cytokeratin 14, stratifin and filaggrin. Upon culture, the number of keratinocytes, their viability and secretion of matrix metalloproteinases 1 and 10 were equal in both groups. CONCLUSION: This study reports the possibility of isolating functioning and viable keratinocytes through a xeno-free workflow for clinical application.

TET2 as a tumor suppressor and therapeutic target in T-cell acute lymphoblastic leukemia.

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy resulting from overproduction of immature T-cells in the thymus and is typified by widespread alterations in DNA methylation. As survival rates for relapsed T-ALL remain dismal (10 to 25%), development of targeted therapies to prevent relapse is key to improving prognosis. Whereas mutations in the DNA demethylating enzyme TET2 are frequent in adult T-cell malignancies, TET2 mutations in T-ALL are rare. Here, we analyzed RNA-sequencing data of 321 primary T-ALLs, 20 T-ALL cell lines, and 25 normal human tissues, revealing that TET2 is transcriptionally repressed or silenced in 71% and 17% of T-ALL, respectively. Furthermore, we show that TET2 silencing is often associated with hypermethylation of the TET2 promoter in primary T-ALL. Importantly, treatment with the DNA demethylating agent, 5-azacytidine (5-aza), was significantly more toxic to TET2-silenced T-ALL cells and resulted in stable re-expression of the TET2 gene. Additionally, 5-aza led to up-regulation of methylated genes and human endogenous retroviruses (HERVs), which was further enhanced by the addition of physiological levels of vitamin C, a potent enhancer of TET activity. Together, our results clearly identify 5-aza as a potential targeted therapy for TET2-silenced T-ALL.

A reassessment of DNA-immunoprecipitation-based genomic profiling.

DNA immunoprecipitation followed by sequencing (DIP-seq) is a common enrichment method for profiling DNA modifications in mammalian genomes. However, the results of independent DIP-seq studies often show considerable variation between profiles of the same genome and between profiles obtained by alternative methods. Here we show that these differences are primarily due to the intrinsic affinity of IgG for short unmodified DNA repeats. This pervasive experimental error accounts for 50-99% of regions identified as 'enriched' for DNA modifications in DIP-seq data. Correction of this error profoundly altered DNA-modification profiles for numerous cell types, including mouse embryonic stem cells, and subsequently revealed novel associations among DNA modifications, chromatin modifications and biological processes. We conclude that both matched input and IgG controls are essential in order for the results of DIP-based assays to be interpreted correctly, and that complementary, non-antibody-based techniques should be used to validate DIP-based findings to avoid further misinterpretation of genome-wide profiling data.