Cryopreservation of GABAergic neuronal precursors for cell-based therapy

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% Me2 SO 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.Ministerio de Economía y Competitividad 09-07746 y 12-36853Junta de Andalucía CTS- 256

GABAergic deficits in absence of LPA1 receptor, associated anxiety-like and coping behaviors, and amelioration by interneuron precursor transplants into the dorsal hippocampus

Defects in GABAergic function can cause anxiety- and depression-like behaviors among other neuropsychiatric disorders. Therapeutic strategies using the transplantation of GABAergic interneuron progenitors derived from the medial ganglionic eminence (MGE) into the adult hippocampus reversed the symptomatology in multiple rodent models of interneuron-related pathologies. In turn, the lysophosphatidic acid receptor LPA has been reported to be essential for hippocampal function. Converging evidence suggests that deficits in LPA receptor signaling represent a core feature underlying comparable hippocampal dysfunction and behaviors manifested in common neuropsychiatric conditions. Here, we first analyzed the GABAergic interneurons in the hippocampus of wild-type and maLPA-null mice, lacking the LPA receptor. Our data revealed a reduction in the number of neurons expressing GABA, calcium-binding proteins, and neuropeptides such as somatostatin and neuropeptide Y in the hippocampus of maLPA-null mice. Then, we used interneuron precursor transplants to test links between hippocampal GABAergic interneuron deficit, cell-based therapy, and LPA receptor-dependent psychiatric disease-like phenotypes. For this purpose, we transplanted MGE-derived interneuron precursors into the adult hippocampus of maLPA-null mice, to test their effects on GABAergic deficit and behavioral symptoms associated with the absence of the LPA receptor. Transplant studies in maLPA-null mice showed that grafted cells were able to restore the hippocampal host environment, decrease the anxiety-like behaviors and neutralize passive coping, with no abnormal effects on motor activity. Furthermore, grafted MGE-derived cells maintained their normal differentiation program. These findings reinforce the use of cell-based strategies for brain disorders and suggest that the LPA receptor represents a potential target for interneuron-related neuropsychiatric disorders.This work was supported by grants from the Spanish Ministry of Science, Innovation and Universities, co-funded by the European Regional Development Fund (ERDF, EU), (PSI2017-82604R, to LJS; PSI2017-83408-P to CP; SAF09-07746, to MAD; PI16/01510, to GET) and Andalusian Regional Ministries of Economy, Knowledge, Business and University (SEJ-4515 -to LJS; SEJ1863 to CP) and of Health and Families (Nicolas Monardes Programme to GET)

Cryopreservation of GABAergic Neuronal Precursors for Cell-Based Therapy.

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

Inhibitory interneuron transplants restore brain functions in an Alzheimer mouse model

Trabajo presentado en 44th Annual Meeting Society for Neuroscience, celebrado en Washington, DC (USA) del 15 al 19 de noviembre de 2014Alzheimer’s disease (AD) and other cognitive disorders result in neuronal hypersynchrony and altered oscillatory rhythmic activity, but the underlying mechanisms are poorly understood. The brain relies on inhibitory interneurons to regulate network excitability and oscillatory rhythms required for cognitive functions. We previously showed that impaired Nav1.1-expressing inhibitory interneurons contribute to memory deficits in a mouse model of familial AD (hAPPJ20 mice). Here, we address whether inhibitory interneuron transplants from the medial ganglionic eminence (MGE) can restore brain functions in hAPPJ20 mice. Nav1.1-overexpressing, but not wild-type, MGE interneuron transplants reduced network hypersynchrony, enhanced gamma oscillatory activity, and improved cognitive functions in hAPPJ20 mice. We conclude that interneuron transplants restore network stability and brain rhythms that support cognitive function in a familial AD mouse model.Support by Alzheimer's Association and NIHPeer Reviewe

Hypoxia Promotes Efficient Differentiation of Human Embryonic Stem Cells to Functional Endothelium

12 páginas, 5 figuras.-- et al.Early development of mammalian embryos occurs in an environment of relative hypoxia. Nevertheless, human embryonic stem cells (hESC), which are derived from the inner cell mass of blastocyst, are routinely cultured under the same atmospheric conditions (21% O2) as somatic cells. We hypothesized that O2 levels modulate gene expression and differentiation potential of hESC, and thus, we performed gene profiling of hESC maintained under normoxic or hypoxic (1% or 5% O2) conditions. Our analysis revealed that hypoxia downregulates expression of pluripotency markers in hESC but increases significantly the expression of genes associated with angio- and vasculogenesis including vascular endothelial growth factor and angiopoitein-like proteins. Consequently, we were able to efficiently differentiate hESC to functional endothelial cells (EC) by varying O2 levels; after 24 hours at 5% O2, more than 50% of cells were CD34+. Transplantation of resulting endothelial-like cells improved both systolic function and fractional shortening in a rodent model of myocardial infarction. Moreover, analysis of the infarcted zone revealed that transplanted EC reduced the area of fibrous scar tissue by 50%. Thus, use of hypoxic conditions to specify the endothelial lineage suggests a novel strategy for cellular therapies aimed at repair of damaged vasculature in pathologies such as cerebral ischemia and myocardial infarction.This project was supported by the Regenerative Medicine Program of Valencia with funding from the Instituto Carlos III and the Valencian Health Ministry. Other sources of funding include Spanish Ministry for Science and Innovation (MICINN) project SAF2008-00011, Red de Terápia Celular (TerCell), CIBERDEM (ISCIII), and ISCIII project EMER07/012.Peer reviewe

GABAergic precursors grafts in animal models of epilepsy

Trabajo presentado en II EMBO Workshop on Cortical Interneurons in Health and Disease, celebrado en Costa d’en Blanes, Mallorca (España), del 24 al 27 de junio de 2012Introduction: Refractory epilepsy is present in about 30% of TLE patients, despite carefully optimized drug treatment. These patients have no treatment other than major resective surgery. In the last years, cell-based therapies have emerged as a promising alternative. Recently, we have demonstrated that MGE-derived precursors transplanted into the normal adult and neonatal telencephalon migrate, differentiate and incorporate as fully functional GABAergic interneurons. Moreover, grafting in a mouse model of hyperactivity, generated by partial elimination of GABAergic interneurons, replaced the deficit in interneurons and restored the normal levels of inhibition in the ablated hippocampus. To further analyze the use of these precursors as an antiepileptic therapy for refractory TLE epilepsy, we have performed transplants in the pilocarpine mouse model of epilepsy. Results: Intrahippocampal bilateral transplants of GFP+ cells from the MGE were performed 5 days after pilocarpine induction of status epilepticus. We monitorized the control and transplanted mice daily from 2 weeks to 4 months after the transplant to asses the possible antiepileptic effects. We observed a significant delay in the latency time to develop the spontaneous recurrent seizures (SRS) in the transplanted group. In addition, the percentage of transplanted mice that fully developed the SRS was significantly reduced with respect their controls and this small group suffered the SRS four times less frequently. Immohistochemical analysis reveled that GFP+ cells spread widely through CA and DG areas. They differentiated into normal interneuron subtypes, with mature morphology and expressing specific markers such as parvalbumin, somatostatin, NP-Y, and calretinin. Interestingly, we observed a protective effect on the hippocampal sclerosis, correlated with reduced levels of cell death. Grafted cells did not affect the sprouting of mossy fibers, although we cannot discard a direct interaction with the grafted cells. In fact, patch clamp analysis of sIPSC in the grafted area confirmed a modulation of the inhibitory synaptic function. Conclusion: MGE-derived precursor grafts into the hippocampus protects against the development of TLE and elicit a reduction in hippocampal sclerosis. This cell-based therapy could be extremely beneficial for the treatment of refractory epilepsy.Financial Support: Ministerio de Ciencia y Tecnología (SAF 2009- 07746)Peer Reviewe

Distribution and differentiation of <i>in toto</i> cryopreserved MGE-derived precursors after transplantation.

<p>(A) Detection of grafted cells by immunohistochemistry against GFP in the neocortex (Ctx) and hippocampus four weeks after the transplant. (B) Note the wide distribution of cryopreserved grafted cells in multiple cortical layers and the dark background due to dendrite concentration in layer IV. (C-F) MGE-derived cells in the cortex differentiated into neurons presenting typical morphology of interneuron subtypes e.g., bitufted (C), multipolar (D), chandelier cells with synaptic boutons resembling candlesticks (E), or basket cells (F). In hippocampus, grafted cells accumulated in CA1 and dentate gyrus (DG). Corpus callosum (cc); cortical layers (III-VI); striatum (St); subiculum (Sb). Scale bars for A and B: 200μm; in C for D-F: 25μm.</p

Interneuron sub-type differentiation of MGE-derived <i>in toto</i> cryopreserved cells after transplantation.

<p>Immunohistochemical colocalization of MGE <i>in toto</i> cryopreserved GFP⁺ cells with the interneuron subtype markers PV (A, C) and SOM (B, D) in the cerebral cortex (A-B) and hippocampus (C-D). Arrowheads show double-positive cells. Scale bar 100μm.</p

Morphological analysis of MGE-derived cells after transplantation.

<p>Morphological analysis of MGE-derived cells after transplantation.</p

Cell recovery yield after cryopreservation.

<p>Representation of the retrieved cell percentage for each cryopreservation protocol immediately after thawing. Values expressed as mean + SEM (n = 5). Statistical analysis: One-way ANOVA with Tukey's multiple comparison post-hoc test (* = p<0.05).</p