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.

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease without any effective therapy. To evaluate the potential of wild-type bone marrow (BM)-derived stem cells to modify the ALS phenotype, we generated BM chimeric Cu/Zn superoxide dismutase (SOD1) mice by transplantation of BM cells derived from mice expressing green fluorescent protein (GFP) in all tissues and from Thy1-YFP mice that express a spectral variant of GFP (yellow fluorescent protein) in neurons only. In the recipient cerebral cortex, we observed rare GFP+ and YFP+ neurons, which were probably generated by cell fusion, as demonstrated by fluorescence in situ hybridization (FISH) analysis, suggesting that this phenomenon is not limited to Purkinje cells. GFP-positive microglial cells were extensively present in both the brain and spinal cord of the affected animals. Completely differentiated and immature GFP+ myofibres were also present in the heart and skeletal muscles of SOD1 mice, confirming that BM cells can participate in striated muscle tissue regeneration. Moreover, wild-type BM chimeric SOD1 mice showed a significantly delayed disease onset and an increased life span, probably due to a positive ‘non-neuronal environmental’ effect rather than to neuronogenesis. This improvement in SOD1-G93A mouse survival is comparable with that previously obtained using some safer pharmacological agents. BM transplantation-related complications in humans preclude its clinical application for ALS treatment. However, our data suggest that further studies aimed at improving the degree of tissue chimerism by BM-derived cells may provide valuable insights into strategies to slow ALS progression.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by the degeneration of the motor neurons. We tested whether treatment of superoxide dismutase (SOD1)-G93A transgenic mouse, a model of ALS, with a neural stem cell subpopulation double positive for Lewis X and the chemokine receptor CXCR4 (LeX+CXCR4+) can modify the disease's progression. In vitro, after exposure to morphogenetic stimuli, LeX+CXCR4+ cells generate cholinergic motor neuron-like cells upon differentiation. LeX+CXCR4+ cells deriving from mice expressing Green Fluorescent Protein in all tissues or only in motor neurons, after a period of priming in vitro, were grafted into spinal cord of SOD1-G93A mice. Transplanted transgenic mice exhibited a delayed disease onset and progression, and survived significantly longer than non-treated animals by 23 days. Examination of the spinal cord revealed integration of donor-derived cells that differentiated mostly in neurons and in a lower proportion in motor neuron-like cells. Quantification of motor neurons of the spinal cord suggests a significant neuroprotection by LeX+CXCR4+ cells. Both VEGF- and IGF1-dependent pathways were significantly modulated in transplanted animals compared to controls, suggesting a role of these neurotrophins in MN protection. Our results support the therapeutic potential of neural stem cell fractions through both neurogenesis and growth factors release in motor neuron disorders.

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.

Objective. The minimum clinically important difference (MCID) is a standard way of measuring clinical relevance. The objective of this work was to establish the MCID for the 6‐minute walking test (6minWT) and the Gross Motor Function Measure (GMFM‐88) in pediatric gait disorders. Methods. A cohort, pretest‐posttest study was conducted in a hospitalized care setting. A total of 182 patients with acquired brain injury (ABI) or cerebral palsy (CP) performed 20 robot‐assisted gait training sessions complemented with 20 sessions of physical therapy over 4 weeks. Separate MCIDs were calculated using 5 distribution‐based approaches, complemented with an anonymized survey completed by clinical professionals. Results. The MCID range for the 6minWT was 20‐38 m in the ABI cohort, with subgroup ranges of 20‐36 m for GMFCS I‐II, 23‐46 m for GMFCS III, and 24‐46 m for GMFCS IV. MCIDs for the CP population were 6‐23 m, with subgroup ranges of 4‐28 m for GMFCS I‐II, 9‐19 m for GMFCS III, and 10‐27 m for GMFCS IV. For GMFM‐88 total score, MCID values were 1.1%‐5.3% for the ABI cohort and 0.1%‐3.0% for the CP population. For dimension “D” of the GMFM, MCID ranges were 2.3%‐6.5% and 0.8%‐5.2% for ABI and CP populations, respectively. For dimension “E,” MCID ranges were 2.8%‐6.5% and 0.3%‐4.9% for ABI and CP cohorts, respectively. The survey showed a large interquartile range, but the results well mimicked the distribution‐based methods. Conclusions. This study identified for the first time MCID ranges for 6minWT and GMFM‐88 in pediatric patients with neurological impairments, offering useful insights for clinicians to evaluate the impact of treatments. Distribution‐based methods should be used with caution: methods based on pre‐post correlation may underestimate MCID when applied to patients with small improvements over the treatment period. Our results should be complemented with estimates obtained using consensus‐ and anchor‐based approaches.