Properties Of Neural Progenitor Cells Research Articles

Biophysical and biomechanical properties of neural progenitor cells as indicators of developmental neurotoxicity.

Conventional in vitro toxicity studies have focused on identifying IC50 and the underlying mechanisms, but how toxicants influence biophysical and biomechanical changes in human cells, especially during developmental stages, remain understudied. Here, using an atomic force microscope, we characterized changes in biophysical (cell area, actin organization) and biomechanical (Young's modulus, force of adhesion, tether force, membrane tension, tether radius) aspects of human fetal brain-derived neural progenitor cells (NPCs) induced by four classes of widely used toxic compounds, including rotenone, digoxin, N-arachidonoylethanolamide (AEA), and chlorpyrifos, under exposure up to 36h. The sub-cellular mechanisms (apoptosis, mitochondria membrane potential, DNA damage, glutathione levels) by which these toxicants induced biochemical changes in NPCs were assessed. Results suggest a significant compromise in cell viability with increasing toxicant concentration (p < 0.01), and biophysical and biomechanical characteristics with increasing exposure time (p < 0.01) as well as toxicant concentration (p < 0.01). Impairment of mitochondrial membrane potential appears to be the most sensitive mechanism of neurotoxicity for rotenone, AEA and chlorpyrifos exposure, but compromise in plasma membrane integrity for digoxin exposure. The surviving NPCs remarkably retained stemness (SOX2 expression) even at high toxicant concentrations. A negative linear correlation (R2 = 0.92) exists between the elastic modulus of surviving cells and the number of living cells in that environment. We propose that even subtle compromise in cell mechanics could serve as a crucial marker of developmental neurotoxicity (mechanotoxicology) and therefore should be included as part of toxicology assessment repertoire to characterize as well as predict developmental outcomes.

Comparison of methods for deriving neural progenitor cells from nonhuman primate embryonic stem cells

Background: Feeder-free monolayer culture and the suspension culture of embryoid bodies (EB) followed by adherent culture, rosette selection, and expansion are 2 methods for deriving neural progenitor cells (NPCs). Direct comparison of these 2 methods has not yet been reported. Objectives: To compare the influence of NPC derivation methods on the properties of NPCs derived from rhesus monkey (Macaca mulatta) embryonic stem cells (rhESCs). Methods: rhESCs were used to derive NPC lines using 2 different methods. EB were produced from a suspension culture of rhESC clumps and rhESCs were cultured in feeder-free monolayers on poly-L-ornithine/laminin coated plates. NPCs were derived by exposure to induction factors. Cell morphology, neural and nonneural lineage markers were evaluated. We measured the expression of nuclear receptor tailless (TLX), which acts as repressor of glial fibrillary acidic protein (GFAP) expression. Results: NPCs were successful derived using either method, with homogenous populations based on the expression of nestin (>97%) and Pax6 (>99%) as shown by flow cytometry. No significant difference in NPC specific markers or ability to differentiate into neurons in vitro was found between the methods. However, the expression of GFAP was >400-fold higher in cells produced by the feeder-free method. This distinction was consistent with the lower expression of TLX. Conclusions: NPCs derived by feeder-free and EB methods share similar morphology and properties. The elevated expression of GFAP and reduced expression of TLX in NPCs derived using the feeder-free method may explain their greater heterogeneity and tendency to differentiate toward cells of an astrocyte lineage. Keywords: Embryonic stem cells, neural progenitor cells, neural progenitor cell derivation, nonhuman primate,TLX

Maintenance of the self-renewal properties of neural progenitor cells cultured in three-dimensional collagen scaffolds by the REDD1-mTOR signal pathway

Three-dimensional (3-D) culture, compared with traditional two-dimensional (2-D) cell culture, can provide physical signals and 3-D matrix close to the in vivo microenvironments. Here, sponge-like collagen scaffolds were used to assess how 3-D culture would affect the differentiation and self-renewal of neural progenitor cells (NPCs). Cultured in differentiation medium without growth factors, cells in 3-D collagen scaffolds yielded much higher clone formation efficiency and expressed less neuron marker, TUJ1, compared with cells cultured on 2-D plates. mTOR inactivation was identified and showed to supported the self-renewal of NPCs in 3-D culture. At the same time, REDD1 was highly expressed in cells cultured in 3-D conditions, which blocks the activity of mTOR. Moreover, knocking-down REDD1 induced the differentiation of NPCs in 3-D collagen scaffolds. These results indicated that mTOR inactivation by REDD1 mediated the self-renewal regulation of NPCs in 3-D cultures. Thus, 3-D collagen scaffolds maintained self-renewal properties of NPCs, and the inhibitory regulator of mTOR (such as REDD1) played an important role in the regulation of self-renewal and differentiation of NPCs.

Bmp7 Regulates the Survival, Proliferation, and Neurogenic Properties of Neural Progenitor Cells during Corticogenesis in the Mouse

Bone morphogenetic proteins (BMPs) are considered important regulators of neural development. However, results mainly from a wide set of in vitro gain-of-function experiments are conflicting since these show that BMPs can act either as inhibitors or promoters of neurogenesis. Here, we report a specific and non-redundant role for BMP7 in cortical neurogenesis in vivo using knockout mice. Bmp7 is produced in regions adjacent to the developing cortex; the hem, meninges, and choroid plexus, and can be detected in the cerebrospinal fluid. Bmp7 deletion results in reduced cortical thickening, impaired neurogenesis, and loss of radial glia attachment to the meninges. Subsequent in vitro analyses of E14.5 cortical cells revealed that lack of Bmp7 affects neural progenitor cells, evidenced by their reduced proliferation, survival and self-renewal capacity. Addition of BMP7 was able to rescue these proliferation and survival defects. In addition, at the developmental stage E14.5 Bmp7 was also required to maintain Ngn2 expression in the subventricular zone. These data demonstrate a novel role for Bmp7 in the embryonic mouse cortex: Bmp7 nurtures radial glia cells and regulates fundamental properties of neural progenitor cells that subsequently affect Ngn2-dependent neurogenesis.

MicroRNA-9 Reveals Regional Diversity of Neural Progenitors along the Anterior-Posterior Axis

SummaryNeural progenitors self-renew and generate neurons throughout the central nervous system. Here, we uncover an unexpected regional specificity in the properties of neural progenitor cells, revealed by the function of a microRNA—miR-9. miR-9 is expressed in neural progenitors, and its knockdown results in an inhibition of neurogenesis along the anterior-posterior axis. However, the underlying mechanism differs—in the hindbrain, progenitors fail to exit the cell cycle, whereas in the forebrain they undergo apoptosis, counteracting the proliferative effect. Among several targets, we functionally identify hairy1 as a primary target of miR-9, regulated at the mRNA level. hairy1 mediates the effects of miR-9 on proliferation, through Fgf8 signaling in the forebrain and Wnt signaling in the hindbrain, but affects apoptosis only in the forebrain, via the p53 pathway. Our findings show a positional difference in the responsiveness of progenitors to miR-9 depletion, revealing an underlying divergence of their properties.

A comparison of epithelial and neural properties in progenitor cells derived from the adult human ciliary body and brain

Cells isolated from the ciliary body (CB) of the adult human eye possess properties of retinal stem/progenitor cells and can be propagated as spheres in culture. As these cells are isolated from a non-neural epithelium which has neuroepithelial origin, they may have both epithelial and neural lineages. Since it is the properties of neural progenitor cells that are sought after in a future scenario of autotransplantation, we wanted to directly compare human CB spheres with neurospheres derived from the human subventricular zone (SVZ), which is the best characterized neural stem cell niche in the CNS of adults. The CB epithelium was dissected from donor eyes ( n = 8). Biopsies from the ventricular wall were harvested during neurosurgery due to epilepsy ( n = 7). CB and SVZ tissue were also isolated from Brown Norwegian rats. Dissociated single cells were cultivated in a sphere-promoting medium and passaged every 10–30 days. Fixed spheres were studied by immunohistochemistry, quantitative RT-PCR and scanning/transmission electron microscopy. We found that both CB and SVZ spheres contained a mixed population of cells embedded in extracellular matrix. CB spheres, in contrast to SVZ neurospheres, contained pigmented cells with epithelial morphology that stained for cytokeratins (3/12 + 19), were connected through desmosomes and tight-junctions and produced PEDF. Markers of neural progenitors (nestin, Sox-2, GFAP) were significantly lower expressed in human CB compared to SVZ spheres, and nestin positive cells in the CB spheres also contained pigment. There was higher expression of EGF and TGF-β receptors in human CB spheres, and a comparative greater activation of the canonical Wnt pathway. These results indicate that adult human CB spheres contain progenitor cells with epithelial properties and limited expression of neural progenitor markers compared to CNS neurospheres. Further studies mapping the regulation between epithelial and neural properties in the adult human CB spheres are vital to fully utilize them as a clinical source of retinal progenitor cells in the future.

Neural progenitors from isolated postnatal rat myenteric ganglia: Expansion as neurospheres and differentiation in vitro

Identification of the stem cell niche is crucial for understanding the factors that regulate these cells. Rodent enteric neural crest-derived stem cells have previously been isolated by flow cytometry and culture of cell suspensions from the outer smooth muscle layers or the entire gut wall from postnatal and adult animals. Such cell suspensions contain a mixture of cell types, including smooth muscle, fibroblasts and cells associated with the vasculature and extrinsic innervation. Thus these preparations may be contaminated by stem cells associated with extrinsic sensory and autonomic nerves and by other types of stem cell that reside in the gut. Here we describe a different approach, similar to that recently used for infant human gut, to obtain enteric ganglion-derived cells, with properties of neural progenitor cells, using isolated myenteric ganglia from postnatal rat ileum. Myenteric ganglia were separated from the gut wall, dispersed and resulting cell dissociates were plated in non-adherent culture conditions with EGF and FGF-2. Under these conditions neurosphere-like bodies (NLB) developed. Cells in NLB incorporated BrdU and expressed the stem cell marker nestin but not the pan-neuronal marker PGP 9.5. Upon growth factor withdrawal some BrdU-immunopositive cells assumed the morphology of neurons and expressed PGP 9.5; others were flattened and expressed the glial cell marker GFAP. This work therefore provides evidence that neural crest-derived progenitors in the postnatal rat gut are located in the myenteric plexus, and shows that these cells can be expanded and differentiated in NLB in vitro.

IQGAP1 Regulates Adult Neural ProgenitorsIn Vivoand Vascular Endothelial Growth Factor-Triggered Neural Progenitor MigrationIn Vitro

In the germinative zone of the adult rodent brain, neural progenitors migrate into niches delimited by astrocyte processes and differentiate into neuronal precursors. In the present study, we report a modulating role for the scaffolding protein IQGAP1 in neural progenitor migration. We have identified IQGAP1 as a new marker of amplifying neural progenitor and neuronal precursor cells of the subventricular zone (SVZ) and the rostral migratory stream (RMS) in the adult mouse brain. To determine functions for IQGAP1 in neural progenitors, we compared the properties of neural progenitor cells from wild-type and Iqgap1-null mutant mice in vivo and in vitro. The in vivo studies reveal a delay in the transition of de novo neural progenitors into neuronal precursor cells in Iqgap1-null mice. In vitro, we demonstrated that IQGAP1 acts as a downstream effector in the vascular endothelial growth factor (VEGF)-dependent migratory response of neural progenitors that also impacts on their neuronal differentiation. The Rho-family GTPases cdc42/Rac1 and Lis1 are major partners of IQGAP1 in this migratory process. Finally, astrocytes of the neurogenic SVZ and RMS are shown to express VEGF. We propose that VEGF synthesized by astrocytes could be involved in the guidance of neural progenitors to neurogenic niches and that IQGAP1 is an effector of the VEGF-dependent migratory signal.

Biological properties of neural progenitor cells isolated from the hippocampus of adult cynomolgus monkeys

The existence of neurogenesis in the hippocampus of adult nonhuman primates has been confirmed in recent years, however, the biological properties of adult neural stem cells or neural progenitor cells (NPCs) from this region remain to be extensively explored. The present work was to investigate on the expansion of NSCs/NPCs from the hippocampus of adult cynomolgus monkeys and the examination of their characteristics in vitro. NPCs isolated from the hippocampus of adult cynomolgus monkeys were expanded in vitro in serum-free media containing growth factors, and were then allowed to differentiate by removing mitotic factors. The expansion capacity of NPCs and their differentiation potential were assayed by immunohistochemical and immunocytochemical analysis. During primary culture, NPCs underwent cell division, proliferation and aggregation to form neurospheres that were growing in suspension. Without mitotic stimulation, most neurospheres adhered to the culture dish and started to differentiate. Eventually, nearly 12% of the differentiated cells expressed neuron specific marker-beta III-tubulin (Tuj1) and 84% expressed astrocyte specific marker-fibrillary acidic protein (GFAP). In addition, the expression of a neural stem cell marker, nestin, was found both in NPCs and in the subgranular zone of adult monkey hippocampus, where NPCs were originally derived. NPCs from the hippocampus of adult cynomolgus monkeys can be expanded to some extent in vitro and are capable of differentiating into neurons and astrocytes. Further experiments to promote the in vitro proliferation capacity of NPCs will be required before adult NPCs can be used as a useful cell model for studying adult neurogenesis and cell replacement therapy using adult stem cells.

SOX2 Functions to Maintain Neural Progenitor Identity

Neural progenitors of the vertebrate CNS are defined by generic cellular characteristics, including their pseudoepithelial morphology and their ability to divide and differentiate. SOXB1 transcription factors, including the three closely related genes Sox1, Sox2, and Sox3, universally mark neural progenitor and stem cells throughout the vertebrate CNS. We show here that constitutive expression of SOX2 inhibits neuronal differentiation and results in the maintenance of progenitor characteristics. Conversely, inhibition of SOX2 signaling results in the delamination of neural progenitor cells from the ventricular zone and exit from cell cycle, which is associated with a loss of progenitor markers and the onset of early neuronal differentiation markers. The phenotype elicited by inhibition of SOX2 signaling can be rescued by coexpression of SOX1, providing evidence for redundant SOXB1 function in CNS progenitors. Taken together, these data indicate that SOXB1 signaling is both necessary and sufficient to maintain panneural properties of neural progenitor cells.

Numb and Numblike control cell number during vertebrate neurogenesis

A fundamental problem in nervous system development is how to control progenitor-cell divisions to generate the correct number of cells for the mature brain and spinal cord. A recent report by Petersen and colleagues has identified an essential role for Numb and a related protein, Numblike, in this process. This study demonstrates that Numb and Numblike function to maintain the self-renewal properties of neural progenitor cells in the vertebrate neural tube.

Differentiation of serum‐free mouse embryo cells into astrocytes is accompanied by induction of glutamine synthetase activity

Serum-free mouse embryo (SFME) cells derived in a defined serum-free medium have been cultured for more than 200 generations and display properties of neural progenitor cells. SFME cells express the neuroepithelial stem cell marker nestin in defined serum-free medium. Exposure of SFME cells to transforming growth factor beta (TGF-beta) or serum decreases nestin expression and induces the astrocyte marker glial fibrillary acidic protein, suggesting that SFME cells differentiate into astrocytes upon exposure to TGF-beta or serum. We examined the expression by SFME cells of the functional central nervous system (CNS) astrocyte marker glutamine synthetase (GS). GS activity is induced in SFME cells upon exposure to TFG-beta or serum. The induction of GS activity was dose- and time-dependent and was reversible. Retinoic acid, hydrocortisone, and dibutyryl cyclic AMP also induced GS expression. The induction of GS activity was accompanied by an increase in the level of GS mRNA and protein. This work provides further evidence that SFME cells represent neural progenitor cells which differentiate into functional astrocytes upon exposure to TGF-beta or serum.