CAR-T-cell therapy against MM currently shows promising results, but usually with serious toxicities. CAR-NK cells may exert less toxicity when redirected against resistant myeloma cells. CARs can be designed through the use of receptors, such as NKG2D, which recognizes a wide range of ligands to provide broad target specificity. Here, we test this approach by analyzing the antitumor activity of activated and expanded NK cells (NKAE) and CD45RA− T cells from MM patients that were engineered to express an NKG2D-based CAR. NKAE cells were cultured with irradiated Clone9.mbIL21 cells. Then, cells were transduced with an NKG2D-4-1BB-CD3z-CAR. CAR-NKAE cells exhibited no evidence of genetic abnormalities. Although memory T cells were more stably transduced, CAR-NKAE cells exhibited greater in vitro cytotoxicity against MM cells, while showing minimal activity against healthy cells. In vivo, CAR-NKAE cells mediated highly efficient abrogation of MM growth, and 25% of the treated mice remained disease free. Overall, these results demonstrate that it is feasible to modify autologous NKAE cells from MM patients to safely express a NKG2D-CAR. Additionally, autologous CAR-NKAE cells display enhanced antimyeloma activity demonstrating that they could be an effective strategy against MM supporting the development of NKG2D-CAR-NK-cell therapy for MM.

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Regenerative medicine has increasingly made use of adult stem cells in the last years (1, 2). Micro-engineering a biomimetic three dimensional structure provides a realistic approach for stem cell niche studies, and further translational applications. A promising approach for engineering artificial stem cell niches is provided by high-throughput microfluidic technologies. In this work, a droplet-based microfluidic-assisted encapsulation device for the generation of multi-layered cellular structures on demand using alginate and Puramatrix is presented. This novel technology is based on gravity-driven flows, passive mixing principle and a gelation system where the use of a double laminar oil flow where only one contains the cross-linking agent allows both the uniform gelation of the inner core and the continuous generation of a stream of cross-linked hydrogel beads. The soft consecutive coating of the inner core with a second and a third layer without exposing the encapsulated cells to external forces that might reduce their viability represents a promising technology towards 3D stem cell encapsulation. Furthermore, we demonstrate the suitability of the presented technology for encapsulation of stem cells by using human Mesenchymal Stem cells (hMScs) and human Hematopoietic stem cells (hHScs). Preliminary results demonstrate a niche model capable of mid-term culture of primitive hHScs in a microfluidic environment. Therefore, the presented method could apply for the artificial reconstruction of the stem cell niche components as an efficient approach to study stem cell behaviour in vitro under controlled conditions, opening a wide field of potential applications within uTAS for 3D cell culture and tissue engineering applications.

Studying the dysregulation of expression of glutamate receptors is crucial to better understand the mechanisms associated with cognitive disabilities in Down syndrome (DS) patients. By using data of microarray experiments previously deposited in GEO Dataset, we studied the expression of 26 glutamate receptor genes in DS brain samples since prenatal to adult age in several brain structures. Overall, our results showed a complexity in the expression of the genes which were dependent mainly on the brain structure analyzed; especially, the hippocampus showed a different expression pattern. While in the general brain analysis the overexpressed genes were GRIN3A and GRIN2C, higher expression levels of GRM1, GRID2, and GRIK1 gene receptors were recorded in hippocampus. Our results suggest that the glutamatergic system in association with other neurotransmitter systems in the human brain would associate with glutamatergic receptor alterations to bring upon synaptic changes and cognitive deficits in DS models.