Elimination of proliferating cells from CNS grafts using a Ki67 promoter-driven thymidine kinase.

Pluripotent stem cell (PSC)-based cell therapy is an attractive concept for neurodegenerative diseases, but can lead to tumor formation. This is particularly relevant as proliferating neural precursors rather than postmitotic mature neurons need to be transplanted. Thus, safety mechanisms to eliminate proliferating cells are needed. Here, we propose a suicide gene approach, based on cell cycle-dependent promoter Ki67-driven expression of herpes simplex virus thymidine kinase (HSV-TK). We generated a PSC line expressing this construct and induced neural differentiation. In vitro, proliferating PSC and early neural precursor cells (NPC) were killed by exposure to ganciclovir. In vivo, transplantation of PSC led to tumor formation, which was prevented by early ganciclovir treatment. Transplanted NPC did not lead to tumor formation and their survival and neural maturation were not affected by ganciclovir. In conclusion, the cell cycle promoter-driven suicide gene approach described in this study allows killing of proliferating undifferentiated precursor cells without expression of the suicide gene in mature neurons. This approach could also be of use for other stem cell-based therapies where the final target consists of postmitotic cells.

A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem Cells (hNSC).

We present here a microfluidic device that generates sub-millimetric hollow hydrogel spheres, encapsulating cells and coated internally with a layer of reconstituted extracellular matrix (ECM) of a few microns thick. The spherical capsules, composed of alginate hydrogel, originate from the spontaneous instability of a multi-layered jet formed by co-extrusion using a coaxial flow device. We provide a simple design to manufacture this device using a DLP (digital light processing) 3D printer. Then, we demonstrate how the inner wall of the capsules can be decorated with a continuous ECM layer that is anchored to the alginate gel and mimics the basal membrane of a cellular niche. Finally, we used this approach to encapsulate human Neural Stem Cells (hNSC) derived from human Induced Pluripotent Stem Cells (hIPSC), which were further differentiated into neurons within the capsules with negligible loss of viability. Altogether, we show that these capsules may serve as cell micro-containers compatible with complex cell culture conditions and applications. These developments widen the field of research and biomedical applications of the cell encapsulation technology.