Cryopreservation of GABAergic Neuronal Precursors for Cell-Based Therapy

Abstract

Cryopreservation protocols are essential for stem cells storage in order to apply them in the clinic. Here we describe a new standardized cryopreservation protocol for GABAergic neural precursors derived from the medial glanglionic eminence (MGE), a promising source of GABAergic neuronal progenitors for cell therapy against interneuron-related pathologies. We used 10% Me2SO as cryoprotectant and assessed the effects of cell culture amplification and cellular organization, as in toto explants, neurospheres, or individualized cells, on post-thaw cell viability and retrieval. We confirmed that in toto cryopreservation of MGE explants is an optimal preservation system to keep intact the interneuron precursor properties for cell transplantation, together with a high cell viability (>80%) and yield (>70%). Post-thaw proliferation and self-renewal of the cryopreserved precursors were tested in vitro. In addition, their migration capacity, acquisition of mature neuronal morphology, and potency to differentiate into multiple interneuron subtypes were also confirmed in vivo after transplantation. The results show that the cryopreserved precursor features remained intact and were similar to those immediately transplanted after their dissection from the MGE. We hope this protocol will facilitate the generation of biobanks to obtain a permanent and reliable source of GABAergic precursors for clinical application in cell-based therapies against interneuronopathies.

Application of Medial Ganglionic Eminence Cell Transplantation in Diseases Associated With Interneuron Disorders

“…MGE cells were transplanted into the brain and differentiated into interneurons, expressing GABA and specific interneuron markers, such as PV, SST, NPY, and calretinin (CR). Their ratios were similar to those typically produced by the MGE during development and conform to their intrinsically defined differentiation program ( Rodriguez-Martinez et al, 2017 ). All of these properties make MGE cells the most promising neuronal progenitor cells for cell-based therapies for interneuron disorder-related diseases.…”

Persistent seizure control in epileptic mice transplanted with gamma‐aminobutyric acid progenitors

“…Henderson et al (2014) 18 used bilateral transplantation of MGE cells (transfected with a neural stem cell nucleofector kit and pre-incubated in media containing growth factors) into a single hilar site in the dentate gyrus. Freshly harvested or in toto cryopreserved MGE progenitors have significantly better cell viability and migratory properties than growth factor treated preparations 47 and this protocol difference, combined with a single injection site strategy, may contribute to the relatively small area of new interneuron integration observed in Henderson et al (2014) 18 . Given the widespread hippocampal damage and circuit re-organization characteristic of the systemic pilocarpine model 29,32–34 it would be difficult to achieve a significant therapeutic effect with such a limited distribution of MGE-derived interneurons.…”

Clustered gamma-protocadherins regulate cortical interneuron programmed cell death

“…For these experiments, dissected MGEs from each embryo were collected in 500 uL L15 and kept on ice until cryopreserved. MGEs were resuspended in 10% DMSO in L15 and cryopreserved as previously described ( Rodríguez-Martínez et al, 2017 ). Vials were cooled to −80°C at a rate of −1 °C/minute in a Nalgene Mr. Frosty Freezing Container and then transferred to liquid nitrogen for long term storage.…”