Subsequently these expanded effector cells were

Subsequently, these expanded effector D609 were examined for their cytotoxicity activity (PI% and LDH release) against targets, where they also showed strong killing of the K562 leukemia cell line. However, the observation of the slight cytotoxicity induced by fresh effector cells suggests proper activation of CD3depleted UCB-MNCs during expansion and differentiation. This finding is in accordance with our above mentioned data that showed increasing levels of CD2, as an activation marker for NK cell during expansion. In a somewhat similar experimental setup, Boissel et al. also showed the high cytolytic potential against K562 for NK cell expanded on wharton\'s jelly fibroblasts with no cytokine treatment (Boissel et al., 2008). However, we found more effective cytolytic activity, which may be due to cytokine supplementation in our experiments. To further understand whether expanded NK cells were activated or not, we also examined the expression of the CD107a as a marker of NK cell degranulation (Alter et al., 2004; Winchester, 2001). Our results showed CD107a up regulation on expanded NK cells after stimulation with the K562 cell line. This finding suggests that higher release of NK cell granule contents can induce apoptosis and death of targets. On the other hand, a significant decrease in IFN-γ and TNF-α release during the culture period suggests that cytokine release may not be critically involved in the observed increase in cytotoxicity. These observations possibly highlight the more prominent role of granule-released perforin/granzyme than cytockine release in the cytolytic activity induced by NK cells.
Conclusion Overall, we demonstrated here a method that provides optimized differentiation and proliferation of NK cells from CD3depleted UCB-MNCs using a feeder layer of BMSCs in the presence of cytokines. We introduce this method as an appropriate and cost-effective approach to provide sufficient functional NK cells that could be applicable for cellular therapies. The following are the supplementary data related to this article.
Acknowledgments
Introduction MicroRNAs are known to have the capability to modulate multiple cell signaling pathways simultaneously (Hashimoto et al., 2013; Shalgi et al., 2009; Subramanyam et al., 2011). Many of these small molecule regulators of gene expression have been described to modulate somatic cell reprogramming to pluripotency (Anokye-Danso et al., 2011; Li and He, 2012; Subramanyam et al., 2011). For example, the miR-302/367 cluster is highly upregulated during cellular reprogramming to induced pluripotent stem cells (iPSCs) and highly expressed in embryonic stem cells (ESC) (Card et al., 2008; Lee et al., 2013; Lipchina et al., 2012). It is known that OCT4, SOX2 and NANOG upregulate transcription of the miR-302/367 cluster (Card et al., 2008; Sandmaier and Telugu, 2015), which in turn targets components of TGF-β (Lipchina et al., 2011; Subramanyam et al., 2011), and RHOC pathways (Subramanyam et al., 2011). Alongside, miRs of the miR-290/295 cluster (Parchem et al., 2014) have been associated with targeting components of the cell cycle\'s G1-S barrier (Wang et al., 2008), as well as the WNT signaling inhibitor Dkk1 (Zovoilis et al., 2009) and the pro-apoptotic genes Caspase 2 and EiI24 (Zheng et al., 2011) and were found to increase reprogramming efficiency (Parchem et al., 2014). On the other hand, the miR-143/145 cluster was described as a major inhibitor of somatic cell reprogramming (Barta et al., 2015). This cluster has increased expression levels during ESC differentiation exerting its function by targeting OCT4, SOX2 and KLF4 (Xu et al., 2009), but also genes related to cell proliferation pathway (Ding et al., 2015) and c-Myc expression (Sachdeva et al., 2009). The miR-29 family of microRNAs is highly conserved amongst mammalian species and its members have as well been investigated for their roles in modulation of somatic cell fate reprogramming from fibroblasts to iPSCs (Guo et al., 2013; Pfaff et al., 2011; Yang et al., 2011). The family comprises of three variants where miR-29b-1 and miR-29a are located in the same cluster on chromosome 7q32.3, while miR-29b-2 and miR-29c are located on chromosome 1q32.2 in human cells (Mott et al., 2010). In mice the respective miR-29 clusters are located at chromosomes 6qA3.3 and 1qH6. Importantly, the mature sequence of all miR-29 variants is conserved amongst human and mouse species (Slusarz and Pulakat, 2015). During reprogramming of mouse embryonic fibroblasts (MEFs) into iPSCs using separate retroviral vectors encoding for the four transcription factors Oct4, Sox2, Klf4 or c-Myc, it was found that c-Myc is an inhibitor of miR-29 clusters transcription, whereas Sox2 and Klf4 were reported to induce miR-29 expression levels (Yang et al., 2011). Moreover, blocking of miR-29a by anti-miRs resulted in enhanced reprogramming efficiencies and the effect of miR-29a inhibition was partially attributed to indirect down-regulation of p53, orchestrated by high levels of p85α and CDC42 proteins, both of which are targets of miR-29 (Yang et al., 2011). In our own previous work we analyzed the effects of a set of 379 microRNAs in OG2-MEFs from Oct4-GFP reporter mice undergoing nuclear reprogramming after transduction with OKS lentiviral vector. In this work, we confirmed that transfection of miR-29 family members at the early stage of somatic cell reprogramming rather decreases the total number of Oct4-GFP+ colonies compared to scramble controls (Pfaff et al., 2011).