Stefania Corti

Identification of a Primitive Brain–Derived Neural Stem Cell Population Based on Aldehyde Dehydrogenase Activity

Stem cells are undifferentiated cells defined by their ability to self‐renew and differentiate to progenitors and terminally differentiated cells. Stem cells have been isolated from almost all tissues, and an emerging idea is that they share common characteristics such as the presence of ATP‐binding cassette transporter G2 and high telomerase and aldehyde dehydrogenase (ALDH) activity, raising the hypothesis of a set of universal stem cell markers. In the present study, we describe the isolation of primitive neural stem cells (NSCs) from adult and embryonic murine neurospheres and dissociated tissue, based on the expression of high levels of ALDH activity. Single‐cell suspension was stained with a fluorescent ALDH substrate termed Aldefluor and then analyzed by flow cytometry. A population of cells with low side scatter (SSClo) and bright ALDH (ALDHbr) activity was isolated. SSCloALDHbr cells are capable of self‐renewal and are able to generate new neurospheres and neuroepithelial stem‐like cells. Furthermore, these cells are multipotent, differentiating both in neurons and macroglia, as determined by immunocytochemistry and real‐time reverse transcription–polymerase chain reaction analysis. To evaluate the engraftment potential of SSCloALDHbr cells in vivo, we transplanted them into mouse brain. Donor‐derived neurons with mature morphology were detected in the cortex and subcortical areas, demonstrating the capacity of this cell population to differentiate appropriately in vivo. The ALDH expression assay is an effective method for direct identification of NSCs, and improvement of the stem cell isolation protocol may be useful in the development of a cell‐mediated therapeutic strategy for neurodegenerative diseases.

Genetic Correction of Human Induced Pluripotent Stem Cells from Patients with Spinal Muscular Atrophy

Motor neurons generated from genetically corrected iPSCs derived from patients with spinal muscular atrophy show rescue of the disease phenotype. Engineering iPSC-Derived Motor Neurons for Cell Therapy Spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by mutations in the gene encoding the survival motor neuron 1 (SMN1) protein. The mutant protein causes loss of spinal cord motor neurons, and there is no effective therapy. Humans have a paralogous gene, SMN2, that differs from SMN1 by a single nucleotide variant within exon 7 that results in the production of an incomplete and nonfunctional protein. Now, Corti et al. investigate the feasibility of genetically engineering induced pluripotent stem cells (iPSCs) derived from SMA patients to generate motor neurons that do not show the disease phenotype. The authors generated human SMA-iPSCs using nonviral, nonintegrating episomal vectors and then performed genetic editing with oligonucleotides to modify SMN2 to produce a functional SMN1-like protein. Uncorrected SMA-iPSC–derived motor neurons reproduced disease-specific features, whereas motor neurons derived from genetically corrected SMA-iPSCs showed rescue of the disease phenotype. Upon direct transplantation into a severe SMA mouse model, corrected SMA-iPSC–derived motor neurons engrafted in the spinal cord and improved the disease phenotype. This study demonstrates the feasibility of generating patient-specific iPSCs and their motor neuron progeny that are genetically corrected and free of exogenous sequences and suggests the potential of this approach for clinical translation. Spinal muscular atrophy (SMA) is among the most common genetic neurological diseases that cause infant mortality. Induced pluripotent stem cells (iPSCs) generated from skin fibroblasts from SMA patients and genetically corrected have been proposed to be useful for autologous cell therapy. We generated iPSCs from SMA patients (SMA-iPSCs) using nonviral, nonintegrating episomal vectors and used a targeted gene correction approach based on single-stranded oligonucleotides to convert the survival motor neuron 2 (SMN2) gene into an SMN1-like gene. Corrected iPSC lines contained no exogenous sequences. Motor neurons formed by differentiation of uncorrected SMA-iPSCs reproduced disease-specific features. These features were ameliorated in motor neurons derived from genetically corrected SMA-iPSCs. The different gene splicing profile in SMA-iPSC motor neurons was rescued after genetic correction. The transplantation of corrected motor neurons derived from SMA-iPSCs into an SMA mouse model extended the life span of the animals and improved the disease phenotype. These results suggest that generating genetically corrected SMA-iPSCs and differentiating them into motor neurons may provide a source of motor neurons for therapeutic transplantation for SMA.

Stem cell therapy in stroke

Abstract.Recent work has focused on cell transplantation as a therapeutic option following ischemic stroke, based on animal studies showing that cells transplanted to the brain not only survive, but also lead to functional improvement. Neural degeneration after ischemia is not selective but involves different neuronal populations, as well as glial and endothelial cell types. In models of stroke, the principal mechanism by which any improvement has been observed, has been attributed to the release of trophic factors, possibly promoting endogenous repair mechanisms, reducing cell death and stimulating neurogenesis and angiogenesis. Initial human studies indicate that stem cell therapy may be technically feasible in stroke patients, however, issues still need to be addressed for use in human subjects.

Neural stem cell transplantation can ameliorate the phenotype of a mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA), a motor neuron disease (MND) and one of the most common genetic causes of infant mortality, currently has no cure. Patients with SMA exhibit muscle weakness and hypotonia. Stem cell transplantation is a potential therapeutic strategy for SMA and other MNDs. In this study, we isolated spinal cord neural stem cells (NSCs) from mice expressing green fluorescent protein only in motor neurons and assessed their therapeutic effects on the phenotype of SMA mice. Intrathecally grafted NSCs migrated into the parenchyma and generated a small proportion of motor neurons. Treated SMA mice exhibited improved neuromuscular function, increased life span, and improved motor unit pathology. Global gene expression analysis of laser-capture-microdissected motor neurons from treated mice showed that the major effect of NSC transplantation was modification of the SMA phenotype toward the wild-type pattern, including changes in RNA metabolism proteins, cell cycle proteins, and actin-binding proteins. NSC transplantation positively affected the SMA disease phenotype, indicating that transplantation of NSCs may be a possible treatment for SMA.

Isolation and characterization of murine neural stem/progenitor cells based on Prominin-1 expression.

The identification of strategies for the isolation of neural stem cells (NSCs) has important implications for the understanding of their biology and the development of therapeutic applications. It has been previously described that human neural stem and progenitor cells (NSPCs) can be isolated from the central nervous system (CNS) using antibodies to prominin (CD133) and fluorescence-activated cell sorting (FACS). Although this antigen displayed an identical membrane topology in several human and murine tissues there was uncertainty as to the relationship between human and mouse prominin because of the low level of amino acid identity. Here we show that prominin expression can be used to identify and isolate also murine NSPCs from the developing or adult brain. Prominin is co-expressed with known neural stem markers like SOX 1-2, Musashi and Nestin. Moreover, neurosphere-forming cells with multipotency and self-renewal capacity reside within the prominin-positive fraction. Transplantation experiments show that CD133-positive cells give rise to neurons and glial cells in vivo, and that many neurons display appropriate phenotypic characteristics of the recipient tissues. The demonstration that CD133 is a stem cell antigen for murine NSPCs as it is for human NSPCs is useful for the investigation of mammal neurogenesis and development of preclinical tests of NSPCs transplantation in mouse analogues of human diseases.

Neuroectodermal and microglial differentiation of bone marrow cells in the mouse spinal cord and sensory ganglia.

There is now evidence that bone marrow (BM) can generate cells expressing neuronal antigens in adult mouse brain. In the present study, we examined the spinal cord and dorsal root ganglia (DRG) of adult mice 3 months after BM cell transplantation from transgenic donor mice expressing the enhanced green fluorescent protein (GFP). To determine whether GFP+ cells acquire neuroectodermal phenotypes, we tested, by immunocytochemistry followed by confocal analysis, the coexpression of the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal markers NeuN, neurofilament (NF), and class III β‐tubulin (TuJ1). Rare GFP+ cells coexpressing TuJ1, NF, and NeuN were found both in spinal cord and in sensory ganglia. These cells have small dimensions and short cytoplasmic processes, probably reflecting an immature phenotype. Double GFP and GFAP positivity was found only in spinal cord. To determine whether cell fusion with endogenous cells occurred, we investigated the nuclear content of cells coexpressing GFP and neuronal or astrocytic markers, demonstrating that these cells have only one nucleus and a DNA ploidy that it is not different from that of surrounding neurons and astrocytes. Large numbers of GFP+ cells are also positively stained for F4/80, a microglial‐recognizing antibody, and present a characteristic microglial‐like morphology both in spinal cord and, with a higher frequency, in sensory ganglia. These data support a potential role for BM‐derived stem cells in spinal cord neuroneogenesis. They also confirm that the microglial compartment within the CNS and in DRG undergoes a relatively fast turnover, with the contribution of hematopoietic stem cells. Both these findings might prove useful for the development of treatments for spinal cord neurodegenerative and acquired disorders.

The Mitochondrial Disulfide Relay System Protein GFER Is Mutated in Autosomal-Recessive Myopathy with Cataract and Combined Respiratory-Chain Deficiency

A disulfide relay system (DRS) was recently identified in the yeast mitochondrial intermembrane space (IMS) that consists of two essential components: the sulfhydryl oxidase Erv1 and the redox-regulated import receptor Mia40. The DRS drives the import of cysteine-rich proteins into the IMS via an oxidative folding mechanism. Erv1p is reoxidized within this system, transferring its electrons to molecular oxygen through interactions with cytochrome c and cytochrome c oxidase (COX), thereby linking the DRS to the respiratory chain. The role of the human Erv1 ortholog, GFER, in the DRS has been poorly explored. Using homozygosity mapping, we discovered that a mutation in the GFER gene causes an infantile mitochondrial disorder. Three children born to healthy consanguineous parents presented with progressive myopathy and partial combined respiratory-chain deficiency, congenital cataract, sensorineural hearing loss, and developmental delay. The consequences of the mutation at the level of the patients muscle tissue and fibroblasts were 1) a reduction in complex I, II, and IV activity; 2) a lower cysteine-rich protein content; 3) abnormal ultrastructural morphology of the mitochondria, with enlargement of the IMS space; and 4) accelerated time-dependent accumulation of multiple mtDNA deletions. Moreover, the Saccharomyces cerevisiae erv1(R182H) mutant strain reproduced the complex IV activity defect and exhibited genetic instability of the mtDNA and mitochondrial morphological defects. These findings shed light on the mechanisms of mitochondrial biogenesis, establish the role of GFER in the human DRS, and promote an understanding of the pathogenesis of a new mitochondrial disease.

Direct reprogramming of human astrocytes into neural stem cells and neurons

Generating neural stem cells and neurons from reprogrammed human astrocytes is a potential strategy for neurological repair. Here we show dedifferentiation of human cortical astrocytes into the neural stem/progenitor phenotype to obtain progenitor and mature cells with a neural fate. Ectopic expression of the reprogramming factors OCT4, SOX2, or NANOG into astrocytes in specific cytokine/culture conditions activated the neural stem gene program and induced generation of cells expressing neural stem/precursor markers. Pure CD44 + mature astrocytes also exhibited this lineage commitment change and did not require passing through a pluripotent state. These astrocyte-derived neural stem cells gave rise to neurons, astrocytes, and oligodendrocytes and showed in vivo engraftment properties. ASCL1 expression further promoted neuronal phenotype acquisition in vitro and in vivo. Methylation analysis showed that epigenetic modifications underlie this process. The restoration of multipotency from human astrocytes has potential in cellular reprogramming of endogenous central nervous system cells in neurological disorders.

Embryonic stem cell-derived neural stem cells improve spinal muscular atrophy phenotype in mice.

Spinal muscular atrophy, characterized by selective loss of lower motor neurons, is an incurable genetic neurological disease leading to infant mortality. We previously showed that primary neural stem cells derived from spinal cord can ameliorate the spinal muscular atrophy phenotype in mice, but this primary source has limited translational value. Here, we illustrate that pluripotent stem cells from embryonic stem cells show the same potential therapeutic effects as those derived from spinal cord and offer great promise as an unlimited source of neural stem cells for transplantation. We found that embryonic stem cell-derived neural stem cells can differentiate into motor neurons in vitro and in vivo. In addition, following their intrathecal transplantation into spinal muscular atrophy mice, the neural stem cells, like those derived from spinal cord, survived and migrated to appropriate areas, ameliorated behavioural endpoints and lifespan, and exhibited neuroprotective capability. Neural stem cells obtained using a drug-selectable embryonic stem cell line yielded the greatest improvements. As with cells originating from primary tissue, the embryonic stem cell-derived neural stem cells integrated appropriately into the parenchyma, expressing neuron- and motor neuron-specific markers. Our results suggest translational potential for the use of pluripotent cells in neural stem cell-mediated therapies and highlight potential safety improvements and benefits of drug selection for neuroepithelial cells.

Modulated Generation of Neuronal Cells from Bone Marrow by Expansion and Mobilization of Circulating Stem Cells with in Vivo Cytokine Treatment

The aim of the present study is to determine whether the expansion and mobilization of circulating bone marrow (BM) stem cells by in vivo treatment with granulocyte-colony stimulating factor (G-CSF) and stem cell factor (SCF) increase the amount of BM-derived neuronal cells in mouse brain. The presence of BM-derived cells in the brain was traced by transplanting into lethally irradiated adults and newborns adult BM from transgenic mice that ubiquitously expressed enhanced green fluorescent protein (GFP). GFP+ and Y-chromosome+ donor-derived cells were present in several brain areas of all treated mice (cortical and subcortical areas, cerebellum, olfactory bulb). The presence of GFP+ cells expressing nuclear neural specific antigen (NeuN), neurofilament, and beta-III tubulin in cortical forebrain and olfactory bulb (OB) was higher in G-CSF-SCF treated groups (P < 0.05, analysis of variance, Fisher post hoc). We observed that overall the amount of double positive cells was higher in animals treated at birth than in adults and in OB than in forebrain areas (P < 0.05). Temporal cortical areas of cytokine-treated adult animals revealed a mean threefold increase in the number of GFP+ cells expressing the nuclear neural specific antigen (211 +/- 86 GFP+NeuN+/mm(3) in G-CSF + SCF treated mice and 66 +/- 33 GFP+NeuN+/mm(3) in control animals). GFP+ cells coexpressing neuronal markers contain only one nucleus and have a DNA index (a measure of DNA ploidy) identical to that of surrounding neurons, thus excluding donor cell fusion with endogenous cells as a relevant phenomenon under these experimental conditions. Our results indicate that G-CSF and SCF administration modulates the availability of GFP+ cells in the brain and enhances their capacity to acquire neuronal characteristics. Cytokine stimulation of autologous stem cells might be seen as a new strategy for neuronal repair in neurodegenerative diseases.