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 CD56dim 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.

Summary Despite many advances in cardiovascular medicine, heart failure (HF) remains the leading cause of death in developed countries affecting at least 10 million people in Western Europe alone. The poor long‐term prognosis of HF patients, and immense public health implications has fuelled interest in finding new therapeutic modalities. Recent observations of the beneficial effect of stem cells on the damaged heart in animal experiments have generated tremendous excitement and stimulated clinical studies suggesting that this approach is feasible, safe, and potentially effective in humans. Cell‐based myocardial regeneration is currently explored for a wide range of cardiac disease states, including acute and chronic ischemic myocardial damage, cardiomyopathy and as biological heart pacemakers. The aim of the present manuscript is to review the work that has been done to establish the role of stem cells in cardiac repair, give an update on the clinical trials performed so far, as well as to discuss critically the controversies, challenges and future surrounding this novel therapeutic concept.

We identified ZNF683/HOBIT as the most highly upregulated transcription factor gene during ex vivo differentiation of human CD34+ cord blood progenitor cells to CD56+ natural killer (NK) cells. ZNF683/HOBIT mRNA was preferentially expressed in NK cells compared to other human peripheral blood lymphocytes and monocytes. During ex vivo differentiation, ZNF683/HOBIT mRNA started to increase shortly after addition of IL-15 and further accumulated in parallel to the generation of CD56+ NK cells. shRNA-mediated knockdown of ZNF683/HOBIT resulted in a substantial reduction of CD56−CD14− NK-cell progenitors and the following generation of CD56+ NK cells was largely abrogated. The few CD56+ NK cells, which escaped the developmental inhibition in the ZNF683/HOBIT knockdown cultures, displayed normal levels of NKG2A and KIR receptors. Functional analyses of these cells showed no differences in degranulation capacity from control cultures. However, the proportion of IFN-γ-producing cells appeared to be increased upon ZNF683/HOBIT knockdown. These results indicate a key role of ZNF683/HOBIT for the differentiation of the human NK-cell lineage and further suggest a potential negative control on IFN-γ production in more mature human NK cells.

No abstract

The possibility to modulate ex vivo human NK cell differentiation towards specific phenotypes will contribute to a better understanding of NK cell differentiation and facilitate tailored production of NK cells for immunotherapy. In this study, we show that addition of a specific low dose of IL-12 to an ex vivo NK cell differentiation system from cord blood CD34+ stem cells will result in significantly increased proportions of cells with expression of CD62L as well as KIRs and CD16 which are preferentially expressed on mature CD56dim peripheral blood NK cells. In addition, the cells displayed decreased expression of receptors such as CCR6 and CXCR3, which are typically expressed to a lower extent by CD56dim than CD56bright peripheral blood NK cells. The increased number of CD62L and KIR positive cells prevailed in a population of CD33+NKG2A+ NK cells, supporting that maturation occurs via this subtype. Among a series of transcription factors tested we found Gata3 and TOX to be significantly downregulated, whereas ID3 was upregulated in the IL-12-modulated ex vivo NK cells, implicating these factors in the observed changes. Importantly, the cells differentiated in the presence of IL-12 showed enhanced cytokine production and cytolytic activity against MHC class I negative and positive targets. Moreover, in line with the enhanced CD16 expression, these cells exhibited improved antibody-dependent cellular cytotoxicity for B-cell leukemia target cells in the presence of the clinically applied antibody rituximab. Altogether, these data provide evidence that IL-12 directs human ex vivo NK cell differentiation towards more mature NK cells with improved properties for potential cancer therapies.