Cryopreservation of GABAergic Neuronal Precursors for Cell-Based Therapy

RESEARCH ARTICLE Cryopreservation of GABAergic Neuronal Precursors for Cell-Based Therapy Daniel Rodrı́guez-Martı́nez☯, Marı́a Magdalena Martı́nez-Losa☯, Manuel Alvarez-Dolado* Laboratory of Cell-based Therapy for Neuropathologies, Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), CSIC, Seville, Spain ☯ These authors contributed equally to this work. * a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Rodrı́guez-Martı́nez D, Martı́nez-Losa MM, Alvarez-Dolado M (2017) Cryopreservation of GABAergic Neuronal Precursors for Cell-Based Therapy. PLoS ONE 12(1): e0170776. doi:10.1371/ journal.pone.0170776 Editor: Carlos E. Ambrósio, Faculty of Animal Sciences and Food Engineering, University of São Paulo, BRAZIL Received: October 7, 2016 Accepted: January 10, 2017 Published: January 25, 2017 Copyright: © 2017 Rodrı́guez-Martı́nez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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%). Postthaw 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. Data Availability Statement: All relevant data are within the paper and its figures. Introduction Funding: This work was supported by the Ministerio de Economı́a y Competitividad (SAF 0907746 y 12- 36853), Junta de Andalucı́a (CTS2563), and Ramón Areces Fundation. DR and MML were respectively recipients of a Formative and a Sara Borrell fellowship from the Carlos III Health Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Interneuron-related pathologies (interneuronopathies) comprise a wide and relevant group of diseases, including epilepsy, schizophrenia, infantile encephalopathies, autism spectrum disorder, or Alzheimer’s disease [1–5]. In the last years, different groups have been working in innovative cell-based therapeutic approaches to treat this group of neuropathologies [6–9]. Very promising results have been achieved grafting GABAergic interneuron precursors derived from the MGE, the subpallial region of the embryonic brain where most of the cortical interneurons are generated during development [10,11]. This therapeutic strategy has led to reversion of symptomatology in multiple animal models of the above mentioned diseases [6–9]. PLOS ONE | DOI:10.1371/journal.pone.0170776 January 25, 2017 1 / 17 MGE in Toto Cryopreservation Competing Interests: The authors have declared that no competing interests exist. Hitherto, the transplants were performed isolating the MGE-derived precursors from E12-E14 mouse embryos, following on mechanical dissociation, and immediate grafting into the neonatal or adult brain, with no culture or any further manipulation. After transplantation, these precursors spread out and cover wide areas of the cortex, striatum and hippocampus [12]. They can migrate several mm during the first week, to later stop and acquire the morphology of mature interneurons. Four weeks after transplantation they have fully differentiated, expressing GABA and specific interneuron subtype markers such as, parvalbumin, somatostatin, calretining, or NP-Y. Their proportions are similar to those normally generated by the MGE during development [12,13], in concordance with their intrinsically determined differentiation program [14]. It has been shown they integrate in the host circuitry and are able to modify the cortical and hippocampal inhibitory tone [12,13]. Moreover, electron microscopy, electrophysiological analysis of spontaneous and evoked synaptic currents, and simultaneous electrode recordings of transplanted interneurons and host projection neurons have shown they form functional inhibitory synaptic connections [12,15,16]. Finally, these precursors present a good long term survival rate (around 15% a year and a half after transplantation in the mouse brain) with no side effects such as gliosis, or tumor formation, what points to their high safety standard [6]. All these properties make the MGE-derived GABAergic precursors the most promising neuronal progenitor for cell-based therapies against interneuronopathies. To apply these precursors in the clinical setting it would be necessary a permanent source of cells ready for transplantation. The establishment of biobanks, in where to store MGEderived cells from altruist donations, should facilitate the provision of cells ready for their immediate use. An alternative would be the generation of GABAergic precursors from induced pluripotent stem cells (iPSC). Several groups have reported driven differentiation and transplantation of iPSC-derived interneurons in animal models of epilepsy with promising results [17,18]. In any case, these cell cultures would need somehow to be stored as well. To our knowledge, currently there is no description of a specific preservation system for this type of interneuron precursors. Therefore, it is crucial to set up an efficient cryopreservation protocol to properly collect these GABAergic precursors, preserving always their unique intrinsic features, and so facilitating their clinical application. Cryopreservation is the process by which cells or tissues are frozen at very low temperatures, generally between -80˚C and -196˚C, to reduce cellular functions and keep life suspended [19]. It is a controlled process of reversible cellular dehydration and enzyme activity suspension that allows cell storage fo (...truncated)