Highly Efficient Differentiation and Enrichment of Spinal Motor Neurons Derived from Human and Monkey Embryonic Stem Cells

Background

There are no cures or efficacious treatments for severe motor neuron diseases. It is extremely difficult to obtain naïve spinal motor neurons (sMNs) from human tissues for research due to both technical and ethical reasons. Human embryonic stem cells (hESCs) are alternative sources. Several methods for MN differentiation have been reported. However, efficient production of naïve sMNs and culture cost were not taken into consideration in most of the methods.

Methods/Principal Findings

We aimed to establish protocols for efficient production and enrichment of sMNs derived from pluripotent stem cells. Nestin+ neural stem cell (NSC) clusters were induced by Noggin or a small molecule inhibitor of BMP signaling. After dissociation of NSC clusters, neurospheres were formed in a floating culture containing FGF2. The number of NSCs in neurospheres could be expanded more than 30-fold via several passages. More than 33% of HB9+ sMN progenitor cells were observed after differentiation of dissociated neurospheres by all-trans retinoic acid (ATRA) and a Shh agonist for another week on monolayer culture. HB9+ sMN progenitor cells were enriched by gradient centrifugation up to 80% purity. These HB9+ cells differentiated into electrophysiologically functional cells and formed synapses with myotubes during a few weeks after ATRA/SAG treatment.

Conclusions and Significance

The series of procedures we established here, namely neural induction, NSC expansion, sMN differentiation and sMN purification, can provide large quantities of naïve sMNs derived from human and monkey pluripotent stem cells. Using small molecule reagents, reduction of culture cost could be achieved.     

Role of miRNA-146?in proliferation and differentiation of mouse neural stem cells

Neural stem cells (NSCs) have been defined as neural cells with the potential to self-renew and eventually generate all cell types of the nervous system. NSCs serve as an ideal cell type for nervous system repair. In the present study, miR-146 overexpression and predicted target (notch 1) were used to study proliferation and differentiation of mouse NSCs. shRNA were used to demonstrate the function of Notch 1 in proliferation of mouse NSCs and luciferase reporter assay was used to assess and confirm the binding sequence of 3′-UTR between Notch 1 and miR-146. Results showed that miR-146 overexpression and knockdown of notch 1 inhibited proliferation of mouse NSCs under serum-free cultural conditions and promoted spontaneous differentiation of mouse NSCs under contained serum cultural conditions respectively. Mouse NSCs spontaneously underwent differentiation into neurogenic cells with contained serum medium. However, when miR-146 was overexpressed, differentiation efficiency of glial cells from NSCs was increased, suggesting that Notch1 promoted NSC proliferation and repressed spontaneous differentiation of NSC in serum-free medium. In conclusion, our results demonstrate that miR-146 promoted spontaneous differentiation of NSCs, and this mechanism was influenced by miR-146, as well as its target (notch 1) and downstream gene.     

Similar variation in carbon storage between deciduous and evergreen treeline species across elevational gradients

Background and Aims

The most plausible explanation for treeline formation so far is provided by the growth limitation hypothesis (GLH), which proposes that carbon sinks are more restricted by low temperatures than by carbon sources. Evidence supporting the GLH has been strong in evergreen, but less and weaker in deciduous treeline species. Here a test is made of the GLH in deciduous–evergreen mixed species forests across elevational gradients, with the hypothesis that deciduous treeline species show a different carbon storage trend from that shown by evergreen species across elevations.

Methods

Tree growth and concentrations of non-structural carbohydrates (NSCs) in foliage, branch sapwood and stem sapwood tissues were measured at four elevations in six deciduous–evergreen treeline ecotones (including treeline) in the southern Andes of Chile (40°S, Nothofagus pumilio and Nothofagus betuloides; 46°S, Nothofagus pumilio and Pinus sylvestris) and in the Swiss Alps (46°N, Larix decidua and Pinus cembra).

Key Results

Tree growth (basal area increment) decreased with elevation for all species. Regardless of foliar habit, NSCs did not deplete across elevations, indicating no shortage of carbon storage in any of the investigated tissues. Rather, NSCs increased significantly with elevation in leaves (P < 0·001) and branch sapwood (P = 0·012) tissues. Deciduous species showed significantly higher NSCs than evergreens for all tissues; on average, the former had 11 % (leaves), 158 % (branch) and 103 % (sapwood) significantly (P < 0·001) higher NSCs than the latter. Finally, deciduous species had higher NSC (particularly starch) increases with elevation than evergreens for stem sapwood, but the opposite was true for leaves and branch sapwood.

Conclusions

Considering the observed decrease in tree growth and increase in NSCs with elevation, it is concluded that both deciduous and evergreen treeline species are sink limited when faced with decreasing temperatures. Despite the overall higher requirements of deciduous tree species for carbon storage, no indication was found of carbon limitation in deciduous species in the alpine treeline ecotone.     

The Effects of Controlled Release of Neurotrophin-3 from PCLA Scaffolds on the Survival and Neuronal Differentiation of Transplanted Neural Stem Cells in a Rat Spinal Cord Injury Model

Neural stem cells (NSCs) have emerged as a potential source for cell replacement therapy following spinal cord injury (SCI). However, poor survival and low neuronal differentiation remain major obstacles to the use of NSCs. Biomaterials with neurotrophic factors are promising strategies for promoting the proliferation and differentiation of NSCs. Silk fibroin (SF) matrices were demonstrated to successfully deliver growth factors and preserve their potency. In this study, by incorporating NT-3 into a SF coating, we successfully developed NT-3-immobilized scaffolds (membranes and conduits). Sustained release of bioactive NT-3 from the conduits for up to 8 weeks was achieved. Cell viability was confirmed using live/dead staining after 14 days in culture. The efficacy of the immobilized NT-3 was confirmed by assessing NSC neuronal differentiation in vitro. NSC neuronal differentiation was 55.2±4.1% on the NT-3-immobilized membranes, which was significantly higher than that on the NT-3 free membrane. Furthermore, 8 weeks after the NSCs were seeded into conduits and implanted in rats with a transected SCI, the conduit+NT-3+NSCs group achieved higher NSC survival (75.8±15.1%) and neuronal differentiation (21.5±5.2%) compared with the conduit+NSCs group. The animals that received the conduit+NT-3+NSCs treatment also showed improved functional outcomes, as well as increased axonal regeneration. These results indicate the feasibility of fabricating NT-3-immobilized scaffolds using the adsorption of NT-3/SF coating method, as well as the potential of these scaffolds to induce SCI repair by promoting survival and neuronal differentiation of transplanted NSCs.     

Mild Hypoxia Enhances Proliferation and Multipotency of Human Neural Stem Cells

Background

Neural stem cells (NSCs) represent an optimal tool for studies and therapy of neurodegenerative diseases. We recently established a v-myc immortalized human NSC (IhNSC) line, which retains stem properties comparable to parental cells. Oxygen concentration is one of the most crucial environmental conditions for cell proliferation and differentiation both in vitro and in vivo. In the central nervous system, physiological concentrations of oxygen range from 0.55 to 8% oxygen. In particular, in the in the subventricular zone niche area, it''s estimated to be 2.5 to 3%.

Methodology/Principal Findings

We investigated in vitro the effects of 1, 2.5, 5, and 20% oxygen concentrations on IhNSCs both during proliferation and differentiation. The highest proliferation rate, evaluated through neurosphere formation assay, was obtained at 2.5 and 5% oxygen, while 1% oxygen was most noxious for cell survival. The differentiation assays showed that the percentages of β-tubIII+ or MAP2+ neuronal cells and of GalC+ oligodendrocytes were significantly higher at 2.5% compared with 1, 5, or 20% oxygen at 17 days in vitro. Mild hypoxia (2.5 to 5% oxygen) promoted differentiation into neuro-oligodendroglial progenitors as revealed by the higher percentage of MAP2+/Ki67+ and GalC+/Ki67+ residual proliferating progenitors, and enhanced the yield of GABAergic and slightly of glutamatergic neurons compared to 1% and 20% oxygen where a significant percentage of GFAP+/nestin+ cells were still present at 17 days of differentiation.

Conclusions/Significance

These findings raise the possibility that reduced oxygen levels occurring in neuronal disorders like cerebral ischemia transiently lead to NSC remaining in a state of quiescence. Conversely, mild hypoxia favors NSC proliferation and neuronal and oligodendroglial differentiation, thus providing an important advance and a useful tool for NSC-mediated therapy of ischemic stroke and neurodegenerative diseases like Parkinson''s disease, multiple sclerosis, and Alzheimer''s disease.     

CD24 Expression Identifies Teratogen-Sensitive Fetal Neural Stem Cell Subpopulations: Evidence from Developmental Ethanol Exposure and Orthotopic Cell Transfer Models

Background

Ethanol is a potent teratogen. Its adverse neural effects are partly mediated by disrupting fetal neurogenesis. The teratogenic process is poorly understood, and vulnerable neurogenic stages have not been identified. Identifying these is a prerequisite for therapeutic interventions to mitigate effects of teratogen exposures.

Methods

We used flow cytometry and qRT-PCR to screen fetal mouse-derived neurosphere cultures for ethanol-sensitive neural stem cell (NSC) subpopulations, to study NSC renewal and differentiation. The identity of vulnerable NSC populations was validated in vivo, using a maternal ethanol exposure model. Finally, the effect of ethanol exposure on the ability of vulnerable NSC subpopulations to integrate into the fetal neurogenic environment was assessed following ultrasound guided, adoptive transfer.

Results

Ethanol decreased NSC mRNAs for c-kit, Musashi-1and GFAP. The CD24⁺ NSC population, specifically the CD24⁺CD15⁺ double-positive subpopulation, was selectively decreased by ethanol. Maternal ethanol exposure also resulted in decreased fetal forebrain CD24 expression. Ethanol pre-exposed CD24⁺ cells exhibited increased proliferation, and deficits in cell-autonomous and cue-directed neuronal differentiation, and following orthotopic transplantation into naïve fetuses, were unable to integrate into neurogenic niches. CD24^depleted cells retained neurosphere regeneration capacity, but following ethanol exposure, generated increased numbers of CD24⁺ cells relative to controls.

Conclusions

Neuronal lineage committed CD24⁺ cells exhibit specific vulnerability, and ethanol exposure persistently impairs this population’s cell-autonomous differentiation capacity. CD24⁺ cells may additionally serve as quorum sensors within neurogenic niches; their loss, leading to compensatory NSC activation, perhaps depleting renewal capacity. These data collectively advance a mechanistic hypothesis for teratogenesis leading to microencephaly.     

Lamin A/C Haploinsufficiency Modulates the Differentiation Potential of Mouse Embryonic Stem Cells

Background

Lamins are structural proteins that are the major determinants of nuclear architecture and play important roles in various nuclear functions including gene regulation and cell differentiation. Mutations in the human lamin A gene cause a spectrum of genetic diseases that affect specific tissues. Most available mouse models for laminopathies recapitulate disease symptoms for muscle diseases and progerias. However, loss of human lamin A/C also has highly deleterious effects on fetal development. Hence it is important to understand the impact of lamin A/C expression levels on embryonic differentiation pathways.

Methodology and Principal Findings

We have investigated the differentiation potential of mouse embryonic stem cells containing reduced levels of lamin A/C by detailed lineage analysis of embryoid bodies derived from these cells by in vitro culture. We initially carried out a targeted disruption of one allele of the mouse lamin A/C gene (Lmna). Undifferentiated wild-type and Lmna^+/− embryonic stem cells showed similar expression of pluripotency markers and cell cycle profiles. Upon spontaneous differentiation into embryoid bodies, markers for visceral endoderm such as α-fetoprotein were highly upregulated in haploinsufficient cells. However, neuronal markers such as β-III tubulin and nestin were downregulated. Furthermore, we observed a reduction in the commitment of Lmna^+/− cells into the myogenic lineage, but no discernible effects on cardiac, adipocyte or osteocyte lineages. In the next series of experiments, we derived embryonic stem cell clones expressing lamin A/C short hairpin RNA and examined their differentiation potential. These cells expressed pluripotency markers and, upon differentiation, the expression of lineage-specific markers was altered as observed with Lmna^+/− embryonic stem cells.

Conclusions

We have observed significant effects on embryonic stem cell differentiation to visceral endoderm, neuronal and myogenic lineages upon depletion of lamin A/C. Hence our results implicate lamin A/C level as an important determinant of lineage-specific differentiation during embryonic development.     

Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells

Background

Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS).

Methodology/Principal Findings

We performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC) and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC, glia and neurons. We isolated a population of NSC that was CD184⁺/CD271⁻/CD44⁻/CD24⁺ from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184⁻/CD44⁻/CD15^LOW/CD24⁺ and a population of glia that was CD184⁺/CD44⁺ were subsequently purified from cultures of differentiating NSC. Purified neurons were viable, expressed mature and subtype-specific neuronal markers, and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo.

Conclusions/Significance

These findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC, glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural cell populations.     

RGCC balances self‐renewal and neuronal differentiation of neural stem cells in the developing mammalian neocortex

During neocortical development, neural stem cells (NSCs) divide symmetrically to self‐renew at the early stage and then divide asymmetrically to generate post‐mitotic neurons. The molecular mechanisms regulating the balance between NSC self‐renewal and neurogenesis are not fully understood. Using mouse in utero electroporation (IUE) technique and in vitro human NSC differentiation models including cerebral organoids (hCOs), we show here that regulator of cell cycle (RGCC) modulates NSC self‐renewal and neuronal differentiation by affecting cell cycle regulation and spindle orientation. RGCC deficiency hampers normal cell cycle process and dysregulates the mitotic spindle, thus driving more cells to divide asymmetrically. These modulations diminish the NSC population and cause NSC pre‐differentiation that eventually leads to brain developmental malformation in hCOs. We further show that RGCC might regulate NSC spindle orientation by affecting the organization of centrosome and microtubules. Our results demonstrate that RGCC is essential to maintain the NSC pool during cortical development and suggest that RGCC defects could have etiological roles in human brain malformations.     

A voltage-sensitive dye-based assay for the identification of differentiated neurons derived from embryonic neural stem cell cultures

Background

Pluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC) and embryonic neural stem cell (NSC) cultures provide a valuable tool to study the processes of neural differentiation, which can be assessed using immunohistochemistry, gene expression, Ca²⁺-imaging or electrophysiology. However, indirect methods such as protein and gene analysis cannot provide direct evidence of neuronal functionality. In contrast, direct methods such as electrophysiological techniques are well suited to produce direct evidence of neural functionality but are limited to the study of a few cells on a culture plate.

Methodology/Principal Findings

In this study we describe a novel method for the detection of action potential-capable neurons differentiated from embryonic NSC cultures using fast voltage-sensitive dyes (VSD). We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators. In addition, VSD changes in fluorescence translated precisely to action potential kinetics as assessed by the injection of simulated slow and fast sodium currents using the dynamic clamp technique. We further demonstrate the use of a finite element model of the NSC culture cover slip for optimizing electrical stimulation parameters.

Conclusions/Significance

Our method allows for a repeatable fast and accurate stimulation of neurons derived from stem cell cultures to assess their differentiation state, which is capable of monitoring large amounts of cells without harming the overall culture.     

Iron Labeling and Pre-Clinical MRI Visualization of Therapeutic Human Neural Stem Cells in a Murine Glioma Model

Background

Treatment strategies for the highly invasive brain tumor, glioblastoma multiforme, require that cells which have invaded into the surrounding brain be specifically targeted. The inherent tumor-tropism of neural stem cells (NSCs) to primary and invasive tumor foci can be exploited to deliver therapeutics to invasive brain tumor cells in humans. Use of the strategy of converting prodrug to drug via therapeutic transgenes delivered by immortalized therapeutic NSC lines have shown efficacy in animal models. Thus therapeutic NSCs are being proposed for use in human brain tumor clinical trials. In the context of NSC-based therapies, MRI can be used both to non-invasively follow dynamic spatio-temporal patterns of the NSC tumor targeting allowing for the optimization of treatment strategies and to assess efficacy of the therapy. Iron-labeling of cells allows their presence to be visualized and tracked by MRI. Thus we aimed to iron-label therapeutic NSCs without affecting their cellular physiology using a method likely to gain United States Federal Drug Administration (FDA) approval.

Methodology

For human use, the characteristics of therapeutic Neural Stem Cells must be clearly defined with any pertubation to the cell including iron labeling requiring reanalysis of cellular physiology. Here, we studied the effect of iron-loading of the therapeutic NSCs, with ferumoxide-protamine sulfate complex (FE-Pro) on viability, proliferation, migratory properties and transgene expression, when compared to non-labeled cells. FE-Pro labeled NSCs were imaged by MRI at tumor sites, after intracranial administration into the hemisphere contralateral to the tumor, in an orthotopic human glioma xenograft mouse model.

Conclusion

FE-Pro labeled NSCs retain their proliferative status, tumor tropism, and maintain stem cell character, while allowing in vivo cellular MRI tracking at 7 Tesla, to monitor their real-time migration and distribution at brain tumor sites. Of significance, this work directly supports the use of FE-Pro-labeled NSCs for real-time tracking in the clinical trial under development: “A Pilot Feasibility Study of Oral 5-Fluorocytosine and Genetically modified Neural Stem Cells Expressing Escherichia coli Cytosine Deaminase for Treatment of Recurrent High-Grade Gliomas”.     

In vivo tracking of human neural stem cells with 19F magnetic resonance imaging

Background

Magnetic resonance imaging (MRI) is a promising tool for monitoring stem cell-based therapy. Conventionally, cells loaded with ironoxide nanoparticles appear hypointense on MR images. However, the contrast generated by ironoxide labeled cells is neither specific due to ambiguous background nor quantitative. A strategy to overcome these drawbacks is ¹⁹F MRI of cells labeled with perfluorocarbons. We show here for the first time that human neural stem cells (NSCs), a promising candidate for clinical translation of stem cell-based therapy of the brain, can be labeled with ¹⁹F as well as detected and quantified in vitro and after brain implantation.

Methodology/Principal Findings

Human NSCs were labeled with perfluoropolyether (PFPE). Labeling efficacy was assessed with ¹⁹F MR spectroscopy, influence of the label on cell phenotypes studied by immunocytochemistry. For in vitro MRI, NSCs were suspended in gelatin at varying densities. For in vivo experiments, labeled NSCs were implanted into the striatum of mice. A decrease of cell viability was observed directly after incubation with PFPE, which re-normalized after 7 days in culture of the replated cells. No label-related changes in the numbers of Ki67, nestin, GFAP, or βIII-tubulin+ cells were detected, both in vitro and on histological sections. We found that 1,000 NSCs were needed to accumulate in one image voxel to generate significant signal-to-noise ratio in vitro. A detection limit of ∼10,000 cells was found in vivo. The location and density of human cells (hunu+) on histological sections correlated well with observations in the ¹⁹F MR images.

Conclusion/Significance

Our results show that NSCs can be efficiently labeled with ¹⁹F with little effects on viability or proliferation and differentiation capacity. We show for the first time that ¹⁹F MRI can be utilized for tracking human NSCs in brain implantation studies, which ultimately aim for restoring loss of function after acute and neurodegenerative disorders.     

Anterograde Degeneration along the Visual Pathway after Optic Nerve Injury

Purpose

To investigate anterograde degenerative changes along the visual pathway in a rat model of optic nerve axotomy.

Methods

Optic nerve transection was performed in adult Sprague-Dawley rats. Animals were sacrificed at regular time intervals and tissues harvested. Immunoblotting followed by densitometric analysis was used to determine the phosphorylation profile of Akt in the dorsal lateral geniculate nucleus (dLGN) and the primary visual cortex (V1). The neuronal cell size and cell density were measured in the dLGN and the V1 using Nissl staining. The prevalence of apoptosis was characterized by terminal deoxynucleotidyl-transferase-mediated biotin-dUTP nick end labelling (TUNEL) histochemistry. Caspase-3 antibodies were also used to identify apoptotic cells. Neurons and astrocytes were detected using NeuN and glial fibrillary acidic protein (GFAP), respectively.

Results

An early and sustained loss of Akt phosphorylation was observed after optic nerve transection in both dLGN and V1. At week one, a decrease in the neuronal cell size (50.5±4.9 vs 60.3±5.0 µm², P = 0.042) and an increase of TUNEL positive cells (7.9±0.6 vs 1.4±0.5 ×10² cells/mm², P<0.001) were evident in the dLGN but not in V1. A significant decline in neuronal cell number (14.5±0.1 vs 17.4±1.3 ×10² cells/mm², P = 0.048), cell size (42.5±4.3 vs 62.1±4.7 µm², P = 0.001) and an increase in apoptotic cells (5.6±0.5 vs 2.0±0.4 ×10² cells/mm², P<0.001) appeared in V1 initially at one month post-transection. The changes in the visual pathway continued through two months. Both neuronal cells and GFAP-positive glial cells were affected in this anterograde degeneration along the visual pathway.

Conclusions

Anterograde degeneration along the visual pathway takes place in target relay (LGN) and visual cortex following the optic nerve injury. Apoptosis was observed in both neural and adjacent glial cells. Reduction of Akt phosphorylation preceded cellular and apoptotic changes.     

Ethosuximide Induces Hippocampal Neurogenesis and Reverses Cognitive Deficits in an Amyloid-β Toxin-induced Alzheimer Rat Model via the Phosphatidylinositol 3-Kinase (PI3K)/Akt/Wnt/β-Catenin Pathway

Neurogenesis involves generation of new neurons through finely tuned multistep processes, such as neural stem cell (NSC) proliferation, migration, differentiation, and integration into existing neuronal circuitry in the dentate gyrus of the hippocampus and subventricular zone. Adult hippocampal neurogenesis is involved in cognitive functions and altered in various neurodegenerative disorders, including Alzheimer disease (AD). Ethosuximide (ETH), an anticonvulsant drug is used for the treatment of epileptic seizures. However, the effects of ETH on adult hippocampal neurogenesis and the underlying cellular and molecular mechanism(s) are yet unexplored. Herein, we studied the effects of ETH on rat multipotent NSC proliferation and neuronal differentiation and adult hippocampal neurogenesis in an amyloid β (Aβ) toxin-induced rat model of AD-like phenotypes. ETH potently induced NSC proliferation and neuronal differentiation in the hippocampus-derived NSC in vitro. ETH enhanced NSC proliferation and neuronal differentiation and reduced Aβ toxin-mediated toxicity and neurodegeneration, leading to behavioral recovery in the rat AD model. ETH inhibited Aβ-mediated suppression of neurogenic and Akt/Wnt/β-catenin pathway gene expression in the hippocampus. ETH activated the PI3K·Akt and Wnt·β-catenin transduction pathways that are known to be involved in the regulation of neurogenesis. Inhibition of the PI3K·Akt and Wnt·β-catenin pathways effectively blocked the mitogenic and neurogenic effects of ETH. In silico molecular target prediction docking studies suggest that ETH interacts with Akt, Dkk-1, and GSK-3β. Our findings suggest that ETH stimulates NSC proliferation and differentiation in vitro and adult hippocampal neurogenesis via the PI3K·Akt and Wnt·β-catenin signaling.