Transplant of GABAergic Precursors Restores Hippocampal Inhibitory Function in a Mouse Model of Seizure Susceptibility

Defects in GABAergic function can cause epilepsy. in the last years, cell-based therapies have attempted to correct these defects with disparate success on animal models of epilepsy. Recently, we demonstrated that medial ganglionic eminence (MGE)-derived cells grafted into the neonatal normal brain migrate and differentiate into functional mature GABAergic interneurons. These cells are able to modulate the local level of GABA-mediated synaptic inhibition, which suggests their suitability for cell-based therapies. However, it is unclear whether they can integrate in the host circuitry and rescue the loss of inhibition in pathological conditions. Thus, as proof of principle, we grafted MGE-derived cells into a mouse model of seizure susceptibility caused by specific elimination of GABAergic interneuron subpopulations in the mouse hippocampus after injection of the neurotoxic saporin conjugated to substance P (SSP-Sap). This ablation was associated with significant decrease in inhibitory postsynaptic currents (IPSC) on CA1 pyramidal cells and increased seizure susceptibility induced by pentylenetetrazol (PTZ). Grafting of GFP(+) MGE-derived cells in SSP-Sap-treated mice repopulates the hippocampal ablated zone with cells expressing molecular markers of mature interneurons. Interestingly, IPSC kinetics on CA1 pyramidal cells of ablated hippocampus significantly increased after transplantation, reaching levels similar to the normal mice. More importantly, this was associated with reduction in seizure severity and decrease in postseizure mortality induced by PTZ. Our data show that MGE-derived cells fulfill most of the requirements for an appropriate cell-based therapy, and indicate their suitability for neurological conditions where a modulation of synaptic inhibition is needed, such as epilepsy.Spanish Ministry of Science and InnovationCIPFCarlos III Institute (Spanish Ministry of Science and Innovation)Generalitat ValencianaAndalusian Ctr Mol Biol & Regenerat Med CABIMER, Dept Cell Therapy & Regenerat Med, Seville, SpainCIPF, Valencia, SpainUniversidade Federal de São Paulo, Dept Physiol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Physiol, São Paulo, BrazilSpanish Ministry of Science and Innovation: SAF 07/61880Spanish Ministry of Science and Innovation: FIS 07/0079Web of Scienc

Intrinsically determined cell death of developing cortical interneurons

Cortical inhibitory circuits are formed by GABAergic interneurons, a cell population that originates far from the cerebral cortex in the embryonic ventral forebrain. Given their distant developmental origins, it is intriguing how the number of cortical interneurons is ultimately determined. One possibility, suggested by the neurotrophic hypothesis1-5, is that cortical interneurons are overproduced, and then following their migration into cortex, excess interneurons are eliminated through a competition for extrinsically derived trophic signals. Here we have characterized the developmental cell death of mouse cortical interneurons in vivo, in vitro, and following transplantation. We found that 40% of developing cortical interneurons were eliminated through Bax- (Bcl-2 associated X-) dependent apoptosis during postnatal life. When cultured in vitro or transplanted into the cortex, interneuron precursors died at a cellular age similar to that at which endogenous interneurons died during normal development. Remarkably, over transplant sizes that varied 200-fold, a constant fraction of the transplanted population underwent cell death. The death of transplanted neurons was not affected by the cell-autonomous disruption of TrkB (tropomyosin kinase receptor B), the main neurotrophin receptor expressed by central nervous system (CNS) neurons6-8. Transplantation expanded the cortical interneuron population by up to 35%, but the frequency of inhibitory synaptic events did not scale with the number of transplanted interneurons. Together, our findings indicate that interneuron cell death is intrinsically determined, either cell-autonomously, or through a population-autonomous competition for survival signals derived from other interneurons

Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes

Recent studies have suggested that bone marrow cells possess a broad differentiation potential, being able to form new liver cells, cardiomyocytes and neurons(1,2). Several groups have attributed this apparent plasticity to 'transdifferentiation'(3-5). Others, however, have suggested that cell fusion could explain these results(6-9). Using a simple method based on Cre/lox recombination to detect cell fusion events, we demonstrate that bone-marrow-derived cells (BMDCs) fuse spontaneously with neural progenitors in vitro. Furthermore, bone marrow transplantation demonstrates that BMDCs fuse in vivo with hepatocytes in liver, Purkinje neurons in the brain and cardiac muscle in the heart, resulting in the formation of multinucleated cells. No evidence of transdifferentiation without fusion was observed in these tissues. These observations provide the first in vivo evidence for cell fusion of BMDCs with neurons and cardiomyocytes, raising the possibility that cell fusion may contribute to the development or maintenance of these key cell types.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62789/1/nature02069.pd

Mesenchymal stem cells rescue cardiomyoblasts from cell death in an in vitro ischemia model via direct cell-to-cell connections

<p>Abstract</p> <p>Background</p> <p>Bone marrow derived mesenchymal stem cells (MSCs) are promising candidates for cell based therapies in myocardial infarction. However, the exact underlying cellular mechanisms are still not fully understood. Our aim was to explore the possible role of direct cell-to-cell interaction between ischemic H9c2 cardiomyoblasts and normal MSCs. Using an in vitro ischemia model of 150 minutes of oxygen glucose deprivation we investigated cell viability and cell interactions with confocal microscopy and flow cytometry.</p> <p>Results</p> <p>Our model revealed that adding normal MSCs to the ischemic cell population significantly decreased the ratio of dead H9c2 cells (H9c2 only: 0.85 ± 0.086 vs. H9c2+MSCs: 0.16 ± 0.035). This effect was dependent on direct cell-to-cell contact since co-cultivation with MSCs cultured in cell inserts did not exert the same beneficial effect (ratio of dead H9c2 cells: 0.90 ± 0.055). Confocal microscopy revealed that cardiomyoblasts and MSCs frequently formed 200-500 nm wide intercellular connections and cell fusion rarely occurred between these cells.</p> <p>Conclusion</p> <p>Based on these results we hypothesize that mesenchymal stem cells may reduce the number of dead cardiomyoblasts after ischemic damage via direct cell-to-cell interactions and intercellular tubular connections may play an important role in these processes.</p

Quantum Dots Do Not Affect the Behaviour of Mouse Embryonic Stem Cells and Kidney Stem Cells and Are Suitable for Short-Term Tracking

Quantum dots (QDs) are small nanocrystals widely used for labelling cells in order to enable cell tracking in complex environments in vitro, ex vivo and in vivo. They present many advantages over traditional fluorescent markers as they are resistant to photobleaching and have narrow emission spectra. Although QDs have been used effectively in cell tracking applications, their suitability has been questioned by reports showing they can affect stem cell behaviour and can be transferred to neighbouring cells. Using a variety of cellular and molecular biology techniques, we have investigated the effect of QDs on the proliferation and differentiation potential of two stem cell types: mouse embryonic stem cells and tissue-specific stem cells derived from mouse kidney. We have also tested if QDs released from living or dead cells can be taken up by neighbouring cells, and we have determined if QDs affect the degree of cell-cell fusion; this information is critical in order to assess the suitability of QDs for stem cell tracking. We show here that QDs have no effect on the viability, proliferation or differentiation potential of the two stem cell types. Furthermore, we show that the extent of transfer of QDs to neighbouring cells is <4%, and that QDs do not increase the degree of cell-cell fusion. However, although the QDs have a high labelling efficiency (>85%), they are rapidly depleted from both stem cell populations. Taken together, our results suggest that QDs are effective cell labelling probes that are suitable for short-term stem cell tracking

Rapid and Sensitive Lentivirus Vector-Based Conditional Gene Expression Assay to Monitor and Quantify Cell Fusion Activity

Cell-to-cell fusion is involved in multiple fundamental biological processes. Prominent examples include osteoclast and giant cell formation, fertilization and skeletal myogenesis which involve macrophage, sperm-egg and myoblast fusion, respectively. Indeed, the importance of cell fusion is underscored by the wide range of homeostatic as well as pathologic processes in which it plays a key role. Therefore, rapid and sensitive systems to trace and measure cell fusion events in various experimental systems are in demand. Here, we introduce a bipartite cell fusion monitoring system based on a genetic switch responsive to the site-specific recombinase FLP. To allow flexible deployment in both dividing as well as non-dividing cell populations, inducer and reporter modules were incorporated in lentivirus vector particles. Moreover, the recombinase-inducible transcription units were designed in such a way as to minimize basal activity and chromosomal position effects in the “off” and “on” states, respectively. The lentivirus vector-based conditional gene expression assay was validated in primary human mesenchymal stem cells and in a differentiation model based on muscle progenitor cells from a Duchenne muscular dystrophy patient using reporter genes compatible with live- and single-cell imaging and with whole population measurements. Using the skeletal muscle cell differentiation model, we showed that the new assay displays low background activity, a 2-log dynamic range, high sensitivity and is amenable to the investigation of cell fusion kinetics. The utility of the bipartite cell fusion monitoring system was underscored by a study on the impact of drug- and RNAi-mediated p38 MAPK inhibition on human myocyte differentiation. Finally, building on the capacity of lentivirus vectors to readily generate transgenic animals the present FLP-inducible system should be adaptable, alone or together with Cre/loxP-based assays, to cell lineage tracing and conditional gene manipulation studies in vivo

Study of Muscle Cell Dedifferentiation after Skeletal Muscle Injury of Mice with a Cre-Lox System

Background: Dedifferentiation of muscle cells in the tissue of mammals has yet to be observed. One of the challenges facing the study of skeletal muscle cell dedifferentiation is the availability of a reliable model that can confidentially distinguish differentiated cell populations of myotubes and non-fused mononuclear cells, including stem cells that can coexist within the population of cells being studied. Methodology/Principal Findings: In the current study, we created a Cre/Lox-β-galactosidase system, which can specifically tag differentiated multinuclear myotubes and myotube-generated mononuclear cells based on the activation of the marker gene, β-galactosidase. By using this system in an adult mouse model, we found that β-galactosidase positive mononuclear cells were generated from β-galactosidase positive multinuclear myofibers upon muscle injury. We also demonstrated that these mononuclear cells can develop into a variety of different muscle cell lineages, i.e., myoblasts, satellite cells, and muscle derived stem cells. Conclusions/Significance: These novel findings demonstrated, for the first time, that cellular dedifferentiation of skeletal muscle cells actually occurs in mammalian skeletal muscle following traumatic injury in vivo. © 2011 Mu et al

Granulocyte-colony stimulating factor for stroke treatment: mechanisms of action and efficacy in preclinical studies

G-CSF is widely employed for the treatment of chemotherapy-induced neutropenia. Recently, neuroprotective effects of G-CSF in animal stroke models were discovered including infarct size reduction and enhancement of functional recovery. The underlying mechanisms of action of G-CSF in ischemia appear to be a direct anti-apoptotic activity in neurons and a neurogenesis inducing capacity. Additional effects may be based on the stimulation of new blood-vessel formation, the stimulation of immunocompetence and -modulation as well as on bone marrow mobilization. In addition to a discussion of these mechanisms, we will review the available preclinical studies and analyze their impact on the overall efficacy of G-CSF in experimental stroke

Mesenchymal Stem Cells Transfer Mitochondria to the Cells with Virtually No Mitochondrial Function but Not with Pathogenic mtDNA Mutations

It has been reported that human mesenchymal stem cells (MSCs) can transfer mitochondria to the cells with severely compromised mitochondrial function. We tested whether the reported intercellular mitochondrial transfer could be replicated in different types of cells or under different experimental conditions, and tried to elucidate possible mechanism. Using biochemical selection methods, we found exponentially growing cells in restrictive media (uridine− and bromodeoxyuridine [BrdU]+) during the coculture of MSCs (uridine-independent and BrdU-sensitive) and 143B-derived cells with severe mitochondrial dysfunction induced by either long-term ethidium bromide treatment or short-term rhodamine 6G (R6G) treatment (uridine-dependent but BrdU-resistant). The exponentially growing cells had nuclear DNA fingerprint patterns identical to 143B, and a sequence of mitochondrial DNA (mtDNA) identical to the MSCs. Since R6G causes rapid and irreversible damage to mitochondria without the removal of mtDNA, the mitochondrial function appears to be restored through a direct transfer of mitochondria rather than mtDNA alone. Conditioned media, which were prepared by treating mtDNA-less 143B ρ0 cells under uridine-free condition, induced increased chemotaxis in MSC, which was also supported by transcriptome analysis. Cytochalasin B, an inhibitor of chemotaxis and cytoskeletal assembly, blocked mitochondrial transfer phenomenon in the above condition. However, we could not find any evidence of mitochondrial transfer to the cells harboring human pathogenic mtDNA mutations (A3243G mutation or 4,977 bp deletion). Thus, the mitochondrial transfer is limited to the condition of a near total absence of mitochondrial function. Elucidation of the mechanism of mitochondrial transfer will help us create a potential cell therapy-based mitochondrial restoration or mitochondrial gene therapy for human diseases caused by mitochondrial dysfunction

Bone Marrow Transplantation Results in Human Donor Blood Cells Acquiring and Displaying Mouse Recipient Class I MHC and CD45 Antigens on Their Surface

Background: Mouse models of human disease are invaluable for determining the differentiation ability and functional capacity of stem cells. The best example is bone marrow transplants for studies of hematopoietic stem cells. For organ studies, the interpretation of the data can be difficult as transdifferentiation, cell fusion or surface antigen transfer (trogocytosis) can be misinterpreted as differentiation. These events have not been investigated in hematopoietic stem cell transplant models. Methodology/Principal Findings: In this study we investigated fusion and trogocytosis involving blood cells during bone marrow transplantation using a xenograft model. We report that using a standard SCID repopulating assay almost 100 % of the human donor cells appear as hybrid blood cells containing both mouse and human surface antigens. Conclusion/Significance: Hybrid cells are not the result of cell-cell fusion events but appear to be due to efficient surface antigen transfer, a process referred to as trogocytosis. Antigen transfer appears to be non-random and includes all donor cells regardless of sub-type. We also demonstrate that irradiation preconditioning enhances the frequency of hybrid cell