Ex vivo generated natural killer cells acquire typical natural killer receptors and display a cytotoxic gene expression profile similar to peripheral blood natural killer cells

Ex vivo differentiation systems of natural killer (NK) cells from CD34+ hematopoietic stem cells are of potential importance for adjuvant immunotherapy of cancer. Here, we analyzed ex vivo differentiation of NK cells from cord blood-derived CD34+ stem cells by gene expression profiling, real-time RT-PCR, flow cytometry, and functional analysis. Additionally, we compared the identified characteristics to peripheral blood (PB) CD56(bright) and CD56(dim) NK cells. The data show sequential expression of CD56 and the CD94 and NKG2 receptor chains during ex vivo NK cell development, resulting finally in the expression of a range of genes with partial characteristics of CD56(bright) and CD56(dim) NK cells from PB. Expression of characteristic NK cell receptors and cytotoxic genes was mainly found within the predominant ex vivo generated population of NKG2A+ NK cells, indicating the importance of NKG2A expression during NK cell differentiation and maturation. Furthermore, despite distinct phenotypic characteristics, the detailed analysis of cytolytic genes expressed within the ex vivo differentiated NK cells revealed a pattern close to CD56(dim) NK cells. In line with this finding, ex vivo generated NK cells displayed potent cytotoxicity. This supports that the ex vivo differentiation system faithfully reproduces major steps of the differentiation of NK cells from their progenitors, constitutes an excellent model to study NK cell differentiation, and is valuable to generate large-scale NK cells appropriate for immunotherapy

Safety and Optimization of Metabolic Labeling of Endothelial Progenitor Cells for Tracking

Metabolic labeling is one of the most powerful methods to label the live cell for in vitro and in vivo tracking. However, the cellular mechanisms by modified glycosylation due to metabolic agents are not fully understood. Therefore, metabolic labeling has not yet been widely used in EPC tracking and labeling. In this study, cell functional properties such as proliferation, migration and permeability and gene expression patterns of metabolic labeling agent-treated hUCB-EPCs were analyzed to demonstrate cellular effects of metabolic labeling agents. As the results, 10 mu M Ac4ManNAz treatment had no effects on cellular function or gene regulations, however, higher concentration of Ac4ManNAz (> 20 mu M) led to the inhibition of functional properties (proliferation rate, viability and rate of endocytosis) and down-regulation of genes related to cell adhesion, PI3K/AKT, FGF and EGFR signaling pathways. Interestingly, the new blood vessel formation and angiogenic potential of hUCB-EPCs were not affected by Ac4ManNAz concentration. Based on our results, we suggest 10 mu M as the optimal concentration of Ac4ManNAz for in vivo hUCB-EPC labeling and tracking. Additionally, we expect that our approach can be used for understanding the efficacy and safety of stem cell-based therapy in vivo