Genome Editing in Neuroepithelial Stem Cells to Generate Human Neurons with High Adenosine-Releasing Capacity

Abstract

As a powerful regulator of cellular homeostasis and metabolism, adenosine is involved in diverse neurological processes including pain, cognition, and memory. Altered adenosine homeostasis has also been associated with several diseases such as depression, schizophrenia, or epilepsy. Based on its protective properties, adenosine has been considered as a potential therapeutic agent for various brain disorders. Since systemic application of adenosine is hampered by serious side effects such as vasodilatation and cardiac suppression, recent studies aim at improving local delivery by depots, pumps, or cell‐based applications. Here, we report on the characterization of adenosine‐releasing human embryonic stem cell‐derived neuroepithelial stem cells (long‐term self‐renewing neuroepithelial stem [lt‐NES] cells) generated by zinc finger nuclease (ZFN)‐mediated knockout of the adenosine kinase (ADK) gene. ADK‐deficient lt‐NES cells and their differentiated neuronal and astroglial progeny exhibit substantially elevated release of adenosine compared to control cells. Importantly, extensive adenosine release could be triggered by excitation of differentiated neuronal cultures, suggesting a potential activity‐dependent regulation of adenosine supply. Thus, ZFN‐modified neural stem cells might serve as a useful vehicle for the activity‐dependent local therapeutic delivery of adenosine into the central nervous system. stem cells translational medicine 2018;7:477–486

Acquisition of chromosome 1q duplication in parental and genome‐edited human‐induced pluripotent stem cell‐derived neural stem cells results in their higher proliferation rate in vitro and in vivo

“…NSCs derived from human ESCs and iPSCs are important vehicles for genetic and molecular therapies in the central nervous system. Genetic modifications of these cells allow for monitoring their differentiation progress, 36 improve our understanding of neural development and disease, 37 and may increase their potential for regenerative therapies 38 . To overcome the disadvantages of genetic modification methods based on random integration, we used ZFN technology for targeted genome modification in hiPSC‐NSCs.…”

Emerging Role of Purine Metabolizing Enzymes in Brain Function and Tumors

“…In order to improve local delivery and raise the level of adenosine in CNS, adenosine releasing polymers and cell-mediated cell therapy have been proposed. Indeed, human neuroepithelial stem cells with ADK gene knockout were able to differentiate in cells exhibiting higher release of adenosine compared to control cells [ 70 ].…”

Sulfated Polysaccharides from Macroalgae Are Potent Dual Inhibitors of Human ATP-Hydrolyzing Ectonucleotidases NPP1 and CD39

“…Therefore, we used this cancer cell line to determine the hydrolysis of extracellular ATP, added to the cells, resulting in the formation of adenosine, which is known to produce antiproliferative, antiangiogenic, metastasis-promoting and immunosuppressive effects when released into the tumor microenvironment. Dipyridamole was added to block the cellular uptake of adenosine via the SLC29 transporter family [ 45 ], and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was present to inhibit metabolism of adenosine to inosine [ 46 ]. Cells treated with ATP, dipyridamole and EHNA were studied in the absence and in the presence of sulfopolysaccharide 7 , and extracellular adenosine accumulation was quantified by capillary electrophoresis coupled to UV detection.…”

“…Human NTPDases (subtypes 1, 2, 3 and 8) were recombinantly expressed as previously described and cell membrane preparations overexpressing one of the NTPDase subtypes were used for the experiments according to described procedures [ 46 ]. For monitoring the inhibitory activity of the compounds at the NTPDases two different assay system were utilized.…”