Apoptosis in Postmortal Tissues of Goat Spinal Cords and Survival of Resident Neural Progenitors.

Growing demand for therapeutic tissue repair recurrently focusses scientists' attention on critical assessment of postmortal collection of live cells, especially stem cells. Our study aimed to assess the survival of neuronal progenitors in postmortal spinal cord and their differentiation potential. Postmortal samples of spinal cords were obtained from human-sized animals (goats) at 6, 12, 24, 36, and 54 h after slaughter. Samples were studied by immunohistology, differentiation assay, Western blot and flow cytometry for the presence and location of GD2-positive neural progenitors and their susceptibility to cell death. TUNEL staining of the goat spinal cord samples over 6-54 h postmortem revealed no difference in the number of positive cells per cross-section. Many TUNEL-positive cells were located in the gray commissure around the central canal of the spinal cord; no increase in TUNEL-positive cells was recorded in either posterior or anterior horns of the gray matter where many GD2-positive neural progenitors can be found. The active caspase 3 amount as measured by Western blot at the same intervals was moderately increasing over time. Neuronal cells were enriched by magnetic separation with antibodies against CD24; among them, the GD2-positive neural progenitor subpopulation did not overlap with apoptotic cells having high pan-caspase activity. Apoptotic cell death events are relatively rare in postmortal spinal cords and are not increased in areas of the neural progenitor cell's location, within measured postmortal intervals, or among the CD24/GD2-positive cells. Data from our study suggest postmortal spinal cords as a valuable source for harvesting highly viable allogenic neural progenitor cells.

Implication of thermal signaling in neuronal differentiation revealed by manipulation and measurement of intracellular temperature.

Neuronal differentiation-the development of neurons from neural stem cells-involves neurite outgrowth and is a key process during the development and regeneration of neural functions. In addition to various chemical signaling mechanisms, it has been suggested that thermal stimuli induce neuronal differentiation. However, the function of physiological subcellular thermogenesis during neuronal differentiation remains unknown. Here we create methods to manipulate and observe local intracellular temperature, and investigate the effects of noninvasive temperature changes on neuronal differentiation using neuron-like PC12 cells. Using quantitative heating with an infrared laser, we find an increase in local temperature (especially in the nucleus) facilitates neurite outgrowth. Intracellular thermometry reveals that neuronal differentiation is accompanied by intracellular thermogenesis associated with transcription and translation. Suppression of intracellular temperature increase during neuronal differentiation inhibits neurite outgrowth. Furthermore, spontaneous intracellular temperature elevation is involved in neurite outgrowth of primary mouse cortical neurons. These results offer a model for understanding neuronal differentiation induced by intracellular thermal signaling.

Size-Optimized Layered Double Hydroxide Nanoparticles Promote Neural Progenitor Cells Differentiation of Embryonic Stem Cells Through the Regulation of M6A Methylation.

Purpose: The committed differentiation fate regulation has been a difficult problem in the fields of stem cell research, evidence showed that nanomaterials could promote the differentiation of stem cells into specific cell types. Layered double hydroxide (LDH) nanoparticles possess the regulation function of stem cell fate, while the underlying mechanism needs to be investigated. In this study, the process of embryonic stem cells (ESCs) differentiate to neural progenitor cells (NPCs) by magnesium aluminum LDH (MgAl-LDH) was investigated. Methods: MgAl-LDH with diameters of 30, 50, and 100 nm were synthesized and characterized, and their effects on the cytotoxicity and differentiation of NPCs were detected in vitro. Dot blot and MeRIP-qPCR were performed to detect the level of m6A RNA methylation in nanoparticles-treated cells. Results: Our work displayed that LDH nanoparticles of three different sizes were biocompatible with NPCs, and the addition of MgAl-LDH could significantly promote the process of ESCs differentiate to NPCs. 100 nm LDH has a stronger effect on promoting NPCs differentiation compared to 30 nm and 50 nm LDH. In addition, dot blot results indicated that the enhanced NPCs differentiation by MgAl-LDH was closely related to m6A RNA methylation process, and the major modification enzyme in LDH controlled NPCs differentiation may be the m6A RNA methyltransferase METTL3. The upregulated METTL3 by LDH increased the m6A level of Sox1 mRNA, enhancing its stability. Conclusion: This work reveals that MgAl-LDH nanoparticles can regulate the differentiation of ESCs into NPCs by increasing m6A RNA methylation modification of Sox1.

Features of Intracellular Signal Transduction in Neural Stem Cells under the Influence of Alkaloid Songorine, an Agonist of Fibroblast Growth Factor Receptors.

We performed a comparative in vitro study of the involvement of NF-κB, PI3K, cAMP, ERK1/2, p38, JAKs, STAT3, JNK, and p53-dependent intracellular signaling in the functioning of neural stem cells (NSC) under the influence of basic fibroblast growth factor (FGF) and FGF receptor agonist, diterpene alkaloid songorine. The significant differences in FGFR-mediated intracellular signaling in NSC were revealed for these ligands. In both cases, stimulation of progenitor cell proliferation occurs with the participation of NF-κB, PI3K, ERK1/2, JAKs, and STAT3, while JNK and p53, on the contrary, inhibit cell cycle progression. However, under the influence of songorin, cAMP- and p38-mediated cascades are additionally involved in the transmission of the NSC division-activating signal. In addition, unlike FGF, the alkaloid stimulates progenitor cell differentiation by activating ERK1/2, p38, JNK, p53, and STAT3.

A novel adipose loss-of-function mutant in Drosophila.

To identify genes required for brain growth, we took an RNAi knockdown reverse genetic approach in Drosophila. One potential candidate isolated from this effort is the anti-lipogenic gene adipose (adp). Adp has an established role in the negative regulation of lipogenesis in the fat body of the fly and adipose tissue in mammals. While fat is key to proper development in general, adp has not been investigated during brain development. Here, we found that RNAi knockdown of adp in neuronal stem cells and neurons results in reduced brain lobe volume and sought to replicate this with a mutant fly. We generated a novel adp mutant that acts as a loss-of-function mutant based on buoyancy assay results. We found that despite a change in fat content in the body overall and a decrease in the number of larger (>5 µm) brain lipid droplets, there was no change in the brain lobe volume of mutant larvae. Overall, our work describes a novel adp mutant that can functionally replace the long-standing adp60 mutant and shows that the adp gene has no obvious involvement in brain growth.

A conserved molecular logic for neurogenesis to gliogenesis switch in the cerebral cortex.

During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascl1, Egfr, and Olig2. The increased Ascl1 expression and appearance of Egfr+ and Olig2+ cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regulatory programs, including Pax6 and Gli3, prevent precocious expression of Olig2, a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells.

The effects of exosomes originating from different cell sources on the differentiation of bone marrow mesenchymal stem cells into schwann cells.

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) can differentiate into Schwann cells (SCs) during peripheral nerve injury; in our previous research, we showed that SC-derived exosomes (SC-exos) played a direct induction role while fibroblast-derived exosomes (Fb-exos) had no obvious induction role. The induction role of neural stem cell (NSC)-derived exosomes (NSC-exos) has also been widely confirmed. However, no studies have compared the induction effects of these three types of cells at the same time. Therefore, by investigating the effect of these three cell-derived exosomes upon the induction of BMSCs to differentiate into SCs, this study explored the role of different exosomes in promoting the differentiation of stem cells into SCs cells, and conducted a comparison between the two groups by RNA sequencing to further narrow the range of target genes and related gene pathways in order to study their related mechanisms. MATERIALS AND METHODS: We extracted exosomes from SCs, fibroblasts (Fb) and neural stem cells (NSC) and then investigated the ability of these exosomes to induce differentiation into BMSCs under different culture conditions. The expression levels of key proteins and gene markers were detected in induced cells by fluorescence immunoassays, western blotting and polymerase chain reaction (PCR); then, we statistically compared the relative induction effects under different conditions. Finally, we analyzed the three types of exosomes by RNA-seq to predict target genes and related gene pathways. RESULTS: BMSCs were cultured by three media: conventional (no induction), pre-induction or pre-induction + original induction medium (ODM) with exosomes of the same cell origin under different culture conditions. When adding the three different types of exosomes separately, the overall induction of BMSCs to differentiate into SCs was significantly increased (P < 0.05). The induction ability was ranked as follows: pre-induction + ODM + exosome group > pre-induction + exosome group > non-induction + exosome group. Using exosomes from different cell sources under the same culture conditions, we observed the following trends under the three culture conditions: RSC96-exos group ≥ NSC-exos group > Fb-exos group. The overall ability to induce BMSCs into SCs was significantly greater in the RSC96-exos group and the NSC-exos group. Although there was no significant difference in induction efficiency when comparing these two groups, the overall induction ability of the RSC96-exos group was slightly higher than that of the NSC-exos group. By combining the differentiation induction results with the RNA-seq data, the three types of exosomes were divided into three comparative groups: RSC vs. NSC, RSC vs. Fb and NSC vs. Fb. We identified 203 differentially expressed mRNA target genes in these three groups. Two differentially expressed genes were upregulated simultaneously, namely riboflavin kinase (RFK, ENSRNOG00000022273) and ribosomal RNA processing 36 (Rrp36, ENSRNOG00000017836). We did not identify any co-upregulated target genes for the miRNAs, but did identify one target gene of the lncRNAs, namely ENSRNOG00000065005. Analysis identified 90 GO terms related to nerves and axons in the mRNAs; in addition, KEGG enrichment and GASA analysis identified 13 common differential expression pathways in the three groups. CONCLUSIONS: Our analysis found that pre-induction + ODM + RSC96/NSC-exos culture conditions were most conducive with regards to induction and differentiation. RSC96-exos and NSC-exos exhibited significantly greater differentiation efficiency of BMSCs into SCs. Although there was no statistical difference, the data indicated a trend for RSC96-exos to be advantageous We identified 203 differentially expressed mRNAs between the three groups and two differentially expressed target mRNAs were upregulated, namely riboflavin kinase (RFK, ENSRNOG00000022273) and ribosomal RNA processing 36 (Rrp36, ENSRNOG00000017836). 90 GO terms were related to nerves and axons. Finally, we identified 13 common differentially expressed pathways across our three types of exosomes. It is hoped that the efficiency of BMSCs induction differentiation into SCs can be improved, bringing hope to patients and more options for clinical treatment.

Cerebral organoids display dynamic clonal growth and tunable tissue replenishment.

During brain development, neural progenitors expand through symmetric divisions before giving rise to differentiating cell types via asymmetric divisions. Transition between those modes varies among individual neural stem cells, resulting in clones of different sizes. Imaging-based lineage tracing allows for lineage analysis at high cellular resolution but systematic approaches to analyse clonal behaviour of entire tissues are currently lacking. Here we implement whole-tissue lineage tracing by genomic DNA barcoding in 3D human cerebral organoids, to show that individual stem cell clones produce progeny on a vastly variable scale. By using stochastic modelling we find that variable lineage sizes arise because a subpopulation of lineages retains symmetrically dividing cells. We show that lineage sizes can adjust to tissue demands after growth perturbation via chemical ablation or genetic restriction of a subset of cells in chimeric organoids. Our data suggest that adaptive plasticity of stem cell populations ensures robustness of development in human brain organoids.

Dynamic light-responsive RhoA activity regulates mechanosensitive stem cell fate decision in 3D matrices.

The behavior of stem cells is regulated by mechanical cues in their niche that continuously vary due to extracellular matrix (ECM) remodeling, pulsated mechanical stress exerted by blood flow, and/or cell migration. However, it is still unclear how dynamics of mechanical cues influence stem cell lineage commitment, especially in a 3D microenvironment where mechanosensing differs from that in a 2D microenvironment. In the present study, we investigated how temporally varying mechanical signaling regulates expression of the early growth response 1 gene (Egr1), which we recently discovered to be a 3D matrix-specific mediator of mechanosensitive neural stem cell (NSC) lineage commitment. Specifically, we temporally controlled the activity of Ras homolog family member A (RhoA), which is known to have a central role in mechanotransduction, using our previously developed Arabidopsis thaliana cryptochrome-2-based optoactivation system. Interestingly, pulsed RhoA activation induced Egr1 upregulation in stiff 3D gels only, whereas static light stimulation induced an increase in Egr1 expression across a wide range of 3D gel stiffnesses. Actin assembly inhibition limited Egr1 upregulation upon RhoA activation, implying that RhoA signaling requires an actin-involved process to upregulate Egr1. Consistently, static-light RhoA activation rather than pulsed-light activation restricted neurogenesis in soft gels. Our findings indicate that the dynamics of RhoA activation influence Egr1-mediated stem cell fate within 3D matrices in a matrix stiffness-dependent manner.

Decellularized extracellular matrix enriched with GDNF enhances neurogenesis and remyelination for improved motor recovery after spinal cord injury.

Motor functional improvement represents a paramount treatment objective in the post-spinal cord injury (SCI) recovery process. However, neuronal cell death and axonal degeneration following SCI disrupt neural signaling, impeding the motor functional recovery. In this study, we developed a multifunctional decellularized spinal cord-derived extracellular matrix (dSECM), crosslinked with glial cell-derived neurotrophic factor (GDNF), to promote differentiation of stem cells into neural-like cells and facilitate axonogenesis and remyelination. After decellularization, the immunogenic cellular components were effectively removed in dSECM, while the crucial protein components were retained which supports stem cells proliferation and differentiation. Furthermore, sustained release of GDNF from the dSECM facilitated axonogenesis and remyelination by activating the PI3K/Akt and MEK/Erk pathways. Our findings demonstrate that the dSECM-GDNF platform promotes neurogenesis, axonogenesis, and remyelination to enhance neural signaling, thereby yielding promising therapeutic effects for motor functional improvement after SCI. STATEMENT OF SIGNIFICANCE: The dSECM promotes the proliferation and differentiation of MSCs or NSCs by retaining proteins associated with positive regulation of neurogenesis and neuronal differentiation, while eliminating proteins related to negative regulation of neurogenesis. After crosslinking, GDNF can be gradually released from the platform, thereby promoting neural differentiation, axonogenesis, and remyelination to enhance neural signaling through activation of the PI3K/Akt and MEK/Erk pathways. In vivo experiments demonstrated that dSECM-GDNF/MSC@GelMA hydrogel exhibited the ability to facilitate neuronal regeneration at 4 weeks post-surgery, while promoting axonogenesis and remyelination at 8 weeks post-surgery, ultimately leading to enhanced motor functional recovery. This study elucidates the ability of neural regeneration strategy to promote motor functional recovery and provides a promising approach for designing multifunctional tissue for SCI treatment.

Treatment of Syringomyelia Characterized by Focal Dilatation of the Central Canal Using Mesenchymal Stem Cells and Neural Stem Cells.

BACKGROUND: Syringomyelia is a progressive chronic disease that leads to nerve pain, sensory dissociation, and dyskinesia. Symptoms often do not improve after surgery. Stem cells have been widely explored for the treatment of nervous system diseases due to their immunoregulatory and neural replacement abilities. METHODS: In this study, we used a rat model of syringomyelia characterized by focal dilatation of the central canal to explore an effective transplantation scheme and evaluate the effect of mesenchymal stem cells and induced neural stem cells for the treatment of syringomyelia. RESULTS: The results showed that cell transplantation could not only promote syrinx shrinkage but also stimulate the proliferation of ependymal cells, and the effect of this result was related to the transplantation location. These reactions appeared only when the cells were transplanted into the cavity. Additionally, we discovered that cell transplantation transformed activated microglia into the M2 phenotype. IGF1-expressing M2 microglia may play a significant role in the repair of nerve pain. CONCLUSION: Cell transplantation can promote cavity shrinkage and regulate the local inflammatory environment. Moreover, the proliferation of ependymal cells may indicate the activation of endogenous stem cells, which is important for the regeneration and repair of spinal cord injury.

3D hydrogel microfibers promote the differentiation of encapsulated neural stem cells and facilitate neuron protection and axon regrowth after complete transactional spinal cord injury.

Spinal cord injury (SCI) can cause permanent impairment to motor or sensory functions. Pre-cultured neural stem cell (NSC) hydrogel scaffolds have emerged as a promising approach to treat SCI by promoting anti-inflammatory effects, axon regrowth, and motor function restoration. Here, in this study, we performed a coaxial extrusion process to fabricate a core-shell hydrogel microfiber with high NSC density in the core portion. Oxidized hyaluronic acid, carboxymethyl chitosan, and matrigel blend were used as a matrix for NSC growth and to facilitate the fabrication process. During thein vitrodifferentiation culture, it was found that NSC microfibers could differentiate into neurons and astrocytes with higher efficiency compared to NSC cultured in petri dishes. Furthermore, duringin vivotransplantation, NSC microfibers were coated with polylactic acid nanosheets by electrospinning for reinforcement. The coated NSC nanofibers exhibited higher anti-inflammatory effect and lesion cavity filling rate compared with the control group. Meanwhile, more neuron- and oligodendrocyte-like cells were visualized at the lesion epicenter. Finally, axon regrowth across the whole lesion site was observed, demonstrating that the microfiber could guide renascent axon regrowth. Experiment results indicate that the NSC microfiber is a promising bioactive treatment for complete SCI treatment with superior outcomes.

Early neural specification of stem cells is mediated by a set of SOX2-dependent neural-associated enhancers.

SOX2 is a transcription factor involved in the regulatory network maintaining the pluripotency of embryonic stem cells in culture as well as in early embryos. In addition, SOX2 plays a pivotal role in neural stem cell formation and neurogenesis. How SOX2 can serve both processes has remained elusive. Here, we identified a set of SOX2-dependent neural-associated enhancers required for neural lineage priming. They form a distinct subgroup (1,898) among 8,531 OCT4/SOX2/NANOG-bound enhancers characterized by enhanced SOX2 binding and chromatin accessibility. Activation of these enhancers is triggered by neural induction of wild-type cells or by default in Smad4-ablated cells resistant to mesoderm induction and is antagonized by mesodermal transcription factors via Sox2 repression. Our data provide mechanistic insight into the transition from the pluripotency state to the early neural fate and into the regulation of early neural versus mesodermal specification in embryonic stem cells and embryos.

Foxp1 suppresses cortical angiogenesis and attenuates HIF-1alpha signaling to promote neural progenitor cell maintenance.

Neural progenitor cells within the cerebral cortex undergo a characteristic switch between symmetric self-renewing cell divisions early in development and asymmetric neurogenic divisions later. Yet, the mechanisms controlling this transition remain unclear. Previous work has shown that early but not late neural progenitor cells (NPCs) endogenously express the autism-linked transcription factor Foxp1, and both loss and gain of Foxp1 function can alter NPC activity and fate choices. Here, we show that premature loss of Foxp1 upregulates transcriptional programs regulating angiogenesis, glycolysis, and cellular responses to hypoxia. These changes coincide with a premature destabilization of HIF-1α, an elevation in HIF-1α target genes, including Vegfa in NPCs, and precocious vascular network development. In vitro experiments demonstrate that stabilization of HIF-1α in Foxp1-deficient NPCs rescues the premature differentiation phenotype and restores NPC maintenance. Our data indicate that the endogenous decline in Foxp1 expression activates the HIF-1α transcriptional program leading to changes in the tissue environment adjacent to NPCs, which, in turn, might alter their self-renewal and neurogenic capacities.

YAP and TAZ differentially regulate postnatal cortical progenitor proliferation and astrocyte differentiation.

WW domain-containing transcription regulator 1 (WWTR1, referred to here as TAZ) and Yes-associated protein (YAP, also known as YAP1) are transcriptional co-activators traditionally studied together as a part of the Hippo pathway, and are best known for their roles in stem cell proliferation and differentiation. Despite their similarities, TAZ and YAP can exert divergent cellular effects by differentially interacting with other signaling pathways that regulate stem cell maintenance or differentiation. In this study, we show in mouse neural stem and progenitor cells (NPCs) that TAZ regulates astrocytic differentiation and maturation, and that TAZ mediates some, but not all, of the effects of bone morphogenetic protein (BMP) signaling on astrocytic development. By contrast, both TAZ and YAP mediate the effects on NPC fate of ß1-integrin (ITGB1) and integrin-linked kinase signaling, and these effects are dependent on extracellular matrix cues. These findings demonstrate that TAZ and YAP perform divergent functions in the regulation of astrocyte differentiation, where YAP regulates cell cycle states of astrocytic progenitors and TAZ regulates differentiation and maturation from astrocytic progenitors into astrocytes.

Neutral competition explains the clonal composition of neural organoids.

Neural organoids model the development of the human brain and are an indispensable tool for studying neurodevelopment. Whole-organoid lineage tracing has revealed the number of progenies arising from each initial stem cell to be highly diverse, with lineage sizes ranging from one to more than 20,000 cells. This high variability exceeds what can be explained by existing stochastic models of corticogenesis and indicates the existence of an additional source of stochasticity. To explain this variability, we introduce the SAN model which distinguishes Symmetrically diving, Asymmetrically dividing, and Non-proliferating cells. In the SAN model, the additional source of stochasticity is the survival time of a lineage's pool of symmetrically dividing cells. These survival times result from neutral competition within the sub-population of all symmetrically dividing cells. We demonstrate that our model explains the experimentally observed variability of lineage sizes and derive the quantitative relationship between survival time and lineage size. We also show that our model implies the existence of a regulatory mechanism which keeps the size of the symmetrically dividing cell population constant. Our results provide quantitative insight into the clonal composition of neural organoids and how it arises. This is relevant for many applications of neural organoids, and similar processes may occur in other developing tissues both in vitro and in vivo.

An improved method for generating human spinal cord neural stem cells.

Neural stem cells have exhibited efficacy in pre-clinical models of spinal cord injury (SCI) and are on a translational path to human testing. We recently reported that neural stem cells must be driven to a spinal cord fate to optimize host axonal regeneration into sites of implantation in the injured spinal cord, where they subsequently form neural relays across the lesion that support significant functional improvement. We also reported methods of deriving and culturing human spinal cord neural stem cells derived from embryonic stem cells that can be sustained over serial high passage numbers in vitro, providing a potentially optimized cell source for human clinical trials. We now report further optimization of methods for deriving and sustaining cultures of human spinal cord neural stem cell lines that result in improved karyotypic stability while retaining anatomical efficacy in vivo. This development improves prospects for safe human translation.

Numb positively regulates Hedgehog signaling at the ciliary pocket.

Hedgehog (Hh) signaling relies on the primary cilium, a cell surface organelle that serves as a signaling hub for the cell. Using proximity labeling and quantitative proteomics, we identify Numb as a ciliary protein that positively regulates Hh signaling. Numb localizes to the ciliary pocket and acts as an endocytic adaptor to incorporate Ptch1 into clathrin-coated vesicles, thereby promoting Ptch1 exit from the cilium, a key step in Hh signaling activation. Numb loss impedes Sonic hedgehog (Shh)-induced Ptch1 exit from the cilium, resulting in reduced Hh signaling. Numb loss in spinal neural progenitors reduces Shh-induced differentiation into cell fates reliant on high Hh activity. Genetic ablation of Numb in the developing cerebellum impairs the proliferation of granule cell precursors, a Hh-dependent process, resulting in reduced cerebellar size. This study highlights Numb as a regulator of ciliary Ptch1 levels during Hh signal activation and demonstrates the key role of ciliary pocket-mediated endocytosis in cell signaling.

Lineage-specific changes in mitochondrial properties during neural stem cell differentiation.

Neural stem cells (NSCs) reside in discrete regions of the adult mammalian brain where they can differentiate into neurons, astrocytes, and oligodendrocytes. Several studies suggest that mitochondria have a major role in regulating NSC fate. Here, we evaluated mitochondrial properties throughout NSC differentiation and in lineage-specific cells. For this, we used the neurosphere assay model to isolate, expand, and differentiate mouse subventricular zone postnatal NSCs. We found that the levels of proteins involved in mitochondrial fusion (Mitofusin [Mfn] 1 and Mfn 2) increased, whereas proteins involved in fission (dynamin-related protein 1 [DRP1]) decreased along differentiation. Importantly, changes in mitochondrial dynamics correlated with distinct patterns of mitochondrial morphology in each lineage. Particularly, we found that the number of branched and unbranched mitochondria increased during astroglial and neuronal differentiation, whereas the area occupied by mitochondrial structures significantly reduced with oligodendrocyte maturation. In addition, comparing the three lineages, neurons revealed to be the most energetically flexible, whereas astrocytes presented the highest ATP content. Our work identified putative mitochondrial targets to enhance lineage-directed differentiation of mouse subventricular zone-derived NSCs.

Reversal of Postnatal Brain Astrocytes and Ependymal Cells towards a Progenitor Phenotype in Culture.

Astrocytes and ependymal cells have been reported to be able to switch from a mature cell identity towards that of a neural stem/progenitor cell. Astrocytes are widely scattered in the brain where they exert multiple functions and are routinely targeted for in vitro and in vivo reprogramming. Ependymal cells serve more specialized functions, lining the ventricles and the central canal, and are multiciliated, epithelial-like cells that, in the spinal cord, act as bi-potent progenitors in response to injury. Here, we isolate or generate ependymal cells and post-mitotic astrocytes, respectively, from the lateral ventricles of the mouse brain and we investigate their capacity to reverse towards a progenitor-like identity in culture. Inhibition of the GSK3 and TGFß pathways facilitates the switch of mature astrocytes to Sox2-expressing, mitotic cells that generate oligodendrocytes. Although this medium allows for the expansion of quiescent NSCs, isolated from live rats by "milking of the brain", it does not fully reverse astrocytes towards the bona fide NSC identity; this is a failure correlated with a concomitant lack of neurogenic activity. Ependymal cells could be induced to enter mitosis either via exposure to neuraminidase-dependent stress or by culturing them in the presence of FGF2 and EGF. Overall, our data confirm that astrocytes and ependymal cells retain a high capacity to reverse to a progenitor identity and set up a simple and highly controlled platform for the elucidation of the molecular mechanisms that regulate this reversal.