Expansion of NK cells by engineered K562 cells co-expressing 4-1BBL and mMICA, combined with soluble IL-21.

NK cells hold promise for protecting hosts from cancer and pathogen infection through direct killing and expressing immune-regulatory cytokines. In our study, a genetically modified K562 cell line with surface expression of 4-1BBL and MICA was constructed to expand functional NK cells in vitro for further adoptive immunotherapy against cancer. After a long-term up to 21 day co-culture with newly isolated peripheral blood mononuclear cells (PBMCs) in the presence of soluble IL-21 (sIL-21), notable increase in proportion of expanded NK cells was observed, especially the CD56(bright)CD16(+) subset. Apparent up-regulation of activating receptors CD38, CD69 and NKG2D was detected on expanded NK cells, so did inhibitory receptor CD94; the cytotoxicity of expanded NK cells against target tumor cells exceeded that of NK cells within fresh PBMCs. The intracellular staining showed expanded NK cells produced immune-regulatory IFN-γ. Taken together, we expanded NK cells with significant up-regulation of activating NKG2D and moderate enhancement of cytotoxicity, with IFN-γ producing ability and a more heterogeneous population of NK cells. These findings provide a novel perspective on expanding NK cells in vitro for further biology study and adoptive immunotherapy of NK cells against cancer.

Enhanced differentiation of human amniotic fluid-derived stem cells into insulin-producing cells in vitro.

AIMS/INTRODUCTION: To investigate the ability of human amniotic fluid stem cells (hAFSCs) to differentiate into insulin-producing cells. MATERIALS AND METHODS: hAFSCs were induced to differentiate into pancreatic cells by a multistep protocol. The expressions of pancreas-related genes and proteins, including pancreatic and duodenal homeobox-1, insulin, and glucose transporter 2, were detected by polymerase chain reaction and immunofluorescence. Insulin secreted from differentiated cells was tested by enzyme-linked immunosorbent assay. RESULTS: hAFSCs were successfully isolated from amniotic fluid that expressed the pluripotent markers of embryonic stem cells, such as Oct3/4, and mesenchymal stem cells, such as integrin ß-1 and ecto-5'-nucleotidase. Here, we first obtained the hAFSCs that expressed pluripotent marker stage-specific embryonic antigen 1. Real-time polymerase chain reaction analysis showed that pancreatic and duodenal homeobox-1, paired box gene 4 and paired box gene 6 were expressed in the early phase of induction, and then stably expressed in the differentiated cells. The pancreas-related genes, such as insulin, glucokinase, glucose transporter 2 and Nkx6.1, were expressed in the differentiated cells. Immunofluorescence showed that these differentiated cells co-expressed insulin, C-peptide, and pancreatic and duodenal homeobox-1. Insulin was released in response to glucose stimulation in a manner similar to that of adult human islets. CONCLUSIONS: The present study showed that hAFSCs, under selective culture conditions, could differentiate into islet-like insulin-producing cells, which might be used as a potential source for transplantation in patients with type 1 diabetes mellitus.