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 TrypLEâ\u84¢ 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.Funding agencies: Centre for Advanced Medical Product, Sweden; Hand and Plastic Surgery Department, Linköping University Hospital, Region Östergötland, Sweden.</p

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

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is an aggres-sive malignancy resulting from overproduction of immature T-cells in the thymus and is typified by widespread alterations in DNA methyl-ation. As survival rates for relapsed T-ALL remain dismal (10 to 25%), development of targeted therapies to prevent relapse is key to improv-ing 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, respec-tively. Furthermore, we show that TET2 silencing is often associated with hypermethylation of the TET2 promoter in primary T-ALL. Impor-tantly, 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 ret-roviruses (HERVs), which was further enhanced by the addition of phys-iological 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