Neural Stem Cell Markers Research Articles

Enumeration of Neural Stem Cells Using Clonal Assays.

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages — astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages — astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Hypoxia Enhances Differentiation of Hair Follicle-Associated-Pluripotent (HAP) Stem Cells to Cardiac-Muscle Cells.

We have previously demonstrated that the neural stem-cell marker nestin is expressed in hair-follicle stem cells located in the bulge area which are termed hair-follicle-associated pluripotent (HAP) stem cells. HAP stem cells from mouse and human could form spheres in culture, termed hair spheres, which are keratin 15-negative and nestin-positive and could differentiate to neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Subsequently, we demonstrated that nestin-expressing stem cells could effect nerve and spinal cord regeneration in mouse models. Recently, we demonstrated that HAP stem cells differentiated to beating cardiac muscle cells. We recently observed that isoproterenol directs HAP stem cells to differentiate to cardiac-muscle cells in large numbers in culture compared to HAP stem cells not supplemented with isoproterenol. The addition of activin A, bone morphogenetic protein 4, and basic fibroblast growth factor, along with isoproternal, induced the cardiac muscle cells to form tissue sheets of beating heart muscle cells. In the present study, we report that, under hypoxic conditions, HAP stem cells differentiated to troponin-positive cardiac-muscle cells at a higher rate that under normoxic conditions. Hypoxia did not influence the differentiation to other cell types. For future use of HAP stem cells for cardiac muscle regeneration, hypoxia should enhance the rate of differentiation thereby providing patients more opportunities to use their own HAP stem cells which are easily accessible, for this purpose. J. Cell. Biochem. 118: 554-558, 2017. © 2016 Wiley Periodicals, Inc.

From hair to heart: Nestin-expressing hair follicle associated pluripotent (HAP) stem cells differentiate to beating cardiac muscle cells

We have previously demonstrated that the neural stem-cell marker nestin is expressed in hair follicle stem cells located in the bulge area which are termed hair-follicle-associated pluripotent (HAP) stem cells. HAP stem cells from mouse and human could form spheres in culture, termed hair spheres, which are keratin 15-negative and CD34-positive and could differentiate to neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Subsequently, we demonstrated that nestin-expressing stem cells could effect nerve and spinal cord regeneration in mouse models. In the present study, we demonstrated that HAP stem cells differentiated to beating cardiac muscle cells. We separated the mouse vibrissa hair follicle into 3 parts (upper, middle, and lower), and suspended each part separately in DMEM containing 10% FBS. All three parts of hair follicle differentiated to beating cardiac muscle cells as well as neurons, glial cells, keratinocytes and smooth muscle cells. The differentiation potential to cardiac muscle is greatest in the upper part of the follicle. The beat rate of the cardiac muscle cells was stimulated by isoproterenol and inhibited by propanolol. HAP stem cells have potential for regenerative medicine for heart disease as well as nerve and spinal cord repair.

Hedgehog Controls Quiescence and Activation of Neural Stem Cells in the Adult Ventricular-Subventricular Zone.

SummaryIdentifying the mechanisms controlling quiescence and activation of neural stem cells (NSCs) is crucial for understanding brain repair. Here, we demonstrate that Hedgehog (Hh) signaling actively regulates different pools of quiescent and proliferative NSCs in the adult ventricular-subventricular zone (V-SVZ), one of the main brain neurogenic niches. Specific deletion of the Hh receptor Patched in NSCs during adulthood upregulated Hh signaling in quiescent NSCs, progressively leading to a large accumulation of these cells in the V-SVZ. The pool of non-neurogenic astrocytes was not modified, whereas the activated NSC pool increased after a short period, before progressively becoming exhausted. We also showed that Sonic Hedgehog regulates proliferation of activated NSCs in vivo and shortens both their G1 and S-G2/M phases in culture. These data demonstrate that Hh orchestrates the balance between quiescent and activated NSCs, with important implications for understanding adult neurogenesis under normal homeostatic conditions or during injury.

Elasticity Measurement of High-Metastatic Breast Cancer Cells That Highly Express Intermediate Filament Protein Nestin

Nestin is an intermediate filament protein that is used for a neural stem cell marker. Since its high expression was reported in several high-metastatic cancer cells, nestin is considered to be involved in metastasis of cancer cells1. However, relationship between nestin and metastatic mechanism of cancer cells is unknown. Cancer cells are generally softer than benign cells2. Because intermediate filaments contribute to cell mechanical property by binding with other cytoskeletal proteins, nestin expression could affect elastic modulus of cancer cell. Previously, we have obtained a highly metastatic murine breast cancer cell line, SC2 which expressed three-fold nestin of parent strain. In this study, we established nestin-knockout SC2 cell by CRISPR/Cas9-mediated genome editing to evaluate an effect of nestin disruption on the cell. Intermediate filaments are composed by three domains and rod domain in the N-terminus is necessary to form bundles. In order to disrupt nestin gene, sgRNA was designed to target the rod domain of mouse nestin. Plasmid expressing the sgRNA and Cas9 was transfected into the SC2 cells by lipofection. After transfection, the cells were cultured in RPMI 1640 medium including G418 (500 μg/mL) for 10 days. Obtained clones were confirmed by immunostaining and three clones were found to be disrupted nestin. Analysis of genome sequence revealed that frameshift mutation was occurred by deletion of nucleotides in nestin gene of the obtained clones. These results indicated that nestin knockout succeeded by CRISPR/Cas9-mediated genome editing. To examine in vivo metastatic activity of nestin knockout cells, the SC2 and knockout cells were intravenously injected into mice. Survival time of mice injected with nestin-knockout cell was significantly prolonged, suggesting that nestin knockout reduced metastatic ability of the SC2 cells. We measured elastic modulus of cell body by using of atomic force microscopy (AFM) and cylindrical-shaped AFM probe with 2.2 μm in diameter. Force curve obtained when the probe was contacted with cell surface was fitted by Herz model and young’s modulus was calculated. Since the young’s modulus of the nestin-knockout cell was significantly higher than that of the SC2, nestin knockout caused increase in mechanical property. In the metastasis process, cancer cells invade surrounding tissue. Thus, expression of nestin reduces the elastic modulus in the cell body and leads high metastatic activity. (1) Kleeberger, W. et al. Cancer research 67, 9199-9206, 2007. (2) Cross, E. S. et sl. Nature nanotechnology 2, 780-783, 2007.

In Vivo Generation of Neural Stem Cells Through Teratoma Formation

Pluripotent stem cells have the potential to differentiate into all cell types of the body in vitro through embryoid body formation or in vivo through teratoma formation. In this study, we attempted to generate in vivo neural stem cells (NSCs) differentiated through teratoma formation using Olig2-GFP transgenic embryonic stem cells (ESCs). After 4 to 6 weeks of injection with Olig2-GFP transgenic ESCs, Olig2-GFP(+) NSCs were identified in teratomas formed in immunodeficient mice. Interestingly, 4-week-old teratomas contained higher percentage of Olig2-GFP(+) cells (∼11%) than 6-week-old teratomas (∼3%). These in vivo-derived NSCs expressed common NSC markers (Nestin and Sox2) and differentiated into terminal neuronal and glial lineages. These results suggest that pure NSC populations exhibiting properties similar to those of brain-derived NSCs can be established through teratoma formation.

CD114: A New Member of the Neural Crest-Derived Cancer Stem Cell Marker Family.

The neural crest is a population of cells in the vertebrate embryo that gives rise to a wide range of tissues and cell types, including components of the peripheral nervous system and the craniofacial skeleton as well as melanocytes and the adrenal medulla. Aberrations in neural crest development can lead to numerous diseases, including cancers such as melanoma and neuroblastoma. Cancer stem cells (CSCs) have been identified in these neural crest-derived tumors, and these CSCs demonstrate resistance to treatment and are likely key contributors to disease relapse. Patients with neural crest-derived tumors often have poor outcomes due to frequent relapses, likely due to the continued presence of residual treatment-resistant CSCs, and therapies directed against these CSCs are likely to improve patient outcomes. CSCs share many of the same genetic and biologic features of primordial neural crest cells, and therefore a better understanding of neural crest development will likely lead to the development of effective therapies directed against these CSCs. Signaling through STAT3 has been shown to be required for neural crest development, and granulocyte colony stimulating factor (GCSF)-mediated activation of STAT3 has been shown to play a role in the pathogenesis of neural crest-derived tumors. Expression of the cell surface marker CD114 (the receptor for GCSF) has been identified as a potential marker for CSCs in neural crest-derived tumors, suggesting that CD114 expression and function may contribute to disease relapse and poor patient outcomes. Here we review the processes of neural crest development and tumorigenesis and we discuss the previously identified markers for CSC subpopulations identified in neural crest tumors and their role in neural crest tumor biology. We also discuss the potential for CD114 and downstream intracellular signaling pathways as potential targets for CSC-directed therapy. J. Cell. Biochem. 118: 221-231, 2017. © 2016 Wiley Periodicals, Inc.

Nestin regulates neural stem cell migration via controlling the cell contractility

Neural stem cells (NSCs) migration is essential for neurogenesis and neuroregeneration after brain injury. Nestin, a widely used marker of NSCs, is expressed abundantly in several cancers, where it may correlate with tumor migration and invasion. However, it is not yet known whether nestin participates in NSC migration. Here, we show that nestin down-regulation significantly inhibits the migration and contraction of murine neural stem cells, but does not obviously influence the proliferation, filamentous actin (F-actin) content, distribution or focal adhesion assembly of these cells. Mechanistically, nestin knockdown was found to affect the phosphorylation state of myosin regulatory light chain (MRLC) and regulate the activity of myosin light chain kinase (MLCK). Co-immunoprecipitation experiments showed that it interacts with MLCK and MRLC. Together, our results indicate that nestin may increase NSC motility via elevating MLCK activity through direct binding and provide new insight into the roles of nestin in NSC migration and repair.

Increased expression of suppressor of cytokine signaling 2 in the subventricular zone after transient focal cerebral ischemia in adult rats

Suppressor of cytokine signaling 2 (SOCS2) is a well-established negative regulator of growth hormone signaling that acts on adult hippocampal neurogenesis during ischemic insults. To explore whether SCOS2 is involved in poststroke neurogenesis, we studied the temporal expression of SOCS2 mRNA in the subventricular zone (SVZ) of rats after transient focal cerebral ischemia. We found that SOCS2 expression was upregulated in the SVZ of the infarcted hemisphere. The number of SOCS2-expressing cells was significantly increased in the ipsilateral SVZ compared with that on the contralateral side on days 7–10 after reperfusion, and SOCS2-expressing cells were highly proliferative, coinciding both spatially and temporally with stroke-induced neurogenesis. Almost all SOCS2-expressing cells in the SVZ were colabeled with the neural stem cell markers nestin and musashi1 and the neural/glial progenitor transcription factor Sox-2. In addition, SOCS2 was highly expressed in newly generated neurons that were immunoreactive for polysialic acid-neural cell adhesion molecule, indicating that SOCS2 expression may be persistent during neuronal differentiation. Thus, our data demonstrated that SOCS2 mRNA was highly expressed in proliferating neural stem/precursor cells and postmitotic migratory neuroblasts in the SVZ niche after focal cerebral ischemia, suggesting that SOCS2 may be actively involved in regulating adult neurogenesis induced by ischemic stroke.

Characterization of p75 neurotrophin receptor expression in human dental pulp stem cells

Human adult dental pulp stem cells (DPSC) are a heterogeneous stem cell population, which are able to differentiate down neural, chondrocyte, osteocyte and adipocyte lineages. We studied the expression pattern of p75 neurotrophin receptors (p75NTR), a marker of neural stem cells, within human DPSC populations from eight donors. p75NTR are expressed at low levels (<10%) in DPSC. Importantly, p75+ DPSC represent higher expression levels of SOX1 (neural precursor cell marker), SOX2 (cell pluripotency marker) and nestin (neural stem cell marker) in comparison to p75− DPSC. Our results suggest that p75+ hDPSC may denote a subpopulation with greater neurogenic potential.

Adult Human Peripheral Blood Mononuclear Cells Are Capable of Producing Neurocyte or Photoreceptor-Like Cells That Survive in Mouse Eyes After Preinduction With Neonatal Retina.

This study provides support for the use of peripheral blood mononuclear cells (PBMCs) as a potential source of pluripotent stem cells for treating retinal degeneration. First, this study demonstrated that PBMCs can differentiate into retinal neuron-like cells in vitro and in vivo. Second, some transplanted cells expressed markers for neural progenitors, mature neurons, or photoreceptors at 1, 3, and 6 months after subretinal injection. Finally, this study showed that PBMC transplantation can improve the function of a degenerated retina.

The expression of P-glycoprotein in leukemia cells is associated with the upregulated expression of nestin, a class 6 filament protein

Multidrug resistance (MDR) is a serious obstacle to the effective chemotherapeutic treatment of leukemia. Expression of plasma membrane P-glycoprotein (P-gp), a transporter involved in drug efflux, is the most frequently observed molecular causality of MDR. We observed the coexpression of P-gp and the filament protein nestin in the acute myeloid leukemia (AML) cell lines SKM-1 and MOLM-13 following the induction of P-gp expression using vincristine. Nestin is considered a marker of neural stem cells and neural progenitor cells. The aim of this study was to determine whether there is causal relationship between the expression of P-glycoprotein and the expression of nestin in both of these AML cell lines. The expression of P-gp was induced in SKM-1 cells by selective pressure using vincristine (VCR), mitoxantrone (MTX), azacytidine (AzaC) and lenalidomide (LEN). Whereas the selective pressure of VCR, MTX and AzaC also induced P-gp expression in MOLM-13 cells, LEN was found to be ineffective in this regard. In all cases in which P-gp expression was induced in SKM-1 and MOLM-13 cells, its expression was associated with the induction of nestin mRNA expression and the presence of a 200–220kDa nestin-immunoreactive protein band in western blots. Silencing P-gp expression using s10418 siRNA (known as the P-gp silencer) was associated with the downregulation of the nestin transcript level, demonstrated using RT-PCR. Nestin mRNA was also observed in two P-gp-positive variants of L1210 cells that were obtained either by selection with VCR or by transfection with a retrovirus encoding human P-gp. Detectable levels of nestin transcripts were not observed in P-gp-negative parental L1210 cells. Taken together, these results indicated that the induction of P-gp expression is causally associated with the expression of nestin in leukemia cells.

Abstract 909: How glioma stem cells maintain stemness outside their niche

Abstract Glioma stem cells (GSCs) have higher self-renewal capacity than neural stem cells (NSCs). However it is still not clear how GSCs maintain their self-renewal outside their niches and whether or not enhanced self-renewal alone without affecting cell proliferation can promote gliomagenesis. Here we report the generation of a novel conditional KO allele of QKI, a tumor suppressor in GBM with RNA binding motif. When QKI was deleted in NSCs isolated from subventricular zone of Nestin-CreERT2 QKIL/L mice, multiple NSC markers were dramatically increased, including Notch1, Sox2,beta-catenin, ID1 and BLBP. When QKI was deleted in adult Nestin-CreERT2 QKIL/L mice, the NSC population, which is characterized by long-term BrdU retention and GFAP+/Nestin+ double positive staining, was greatly increased, indicating that deletion of QKI enhances NSC self-renewal. Surprisingly, we found that deletion of QKI also decreases cell proliferation rate of NSCs/GSCs, suggesting that QKI deletion makes NSCs/GSCs more quiescent. When QKI−/− NSCs were cultured in differentiation medium, they were not able to differentiate like QKI+/+ NSCs, indicating that QKI is required for NSC lineage determination. To determine whether QKI KO promotes gliomagenesis, we generated a Nestin-CreERT2 QKIL/L PtenL/L P53L/L cohort, in which 92% of the mice developed glioblastoma starting at 2 months. However, the Nestin-CreERT2 PtenL/L P53L/L cohort did not develop any glioma up to a year; therefore QKI deletion greatly promotes gliomagenesis. Transcriptomic and proteomic profiling coupled with PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) analyses revealed that genes involved in receptor trafficking were greatly enriched by QKI deletion. Specifically, 34% of the genes up-regulated by QKI deletion are involved in receptor delivery, and 39% of the genes down-regulated by QKI deletion are subunits of receptor degradation machineries such as endosomes and lysosomes. High level of receptor delivery and low level of receptor degradation concomitantly enrich receptors on the membrane and enhance the activity of the receptors that are involved in maintaining stemness, including RTK, Notch1 and Frizzled. Inhibition of the activity of receptors such as EGFR can diminish the enhanced self-renewal and decreased differentiation caused by QKI deletion. Taken together, our data suggest that QKI deletion increases receptor delivery and decreases endocytosis-mediated degradation, consequently enhancing the receptor activity and self-renewal capacity. Therapeutically, because QKI deletion greatly reduces lysosome level and protein degradation capacity, QKI-low cells are under much higher ER-stress compared with QKI-high cells; hence high level of ER-stress may provide an Achilles’ heel for QKI-low glioblastoma, and pharmacologically inducing more ER-stress could render cytotoxicity to QKI-low glioma cells while keep normal cells intact. Citation Format: Jian Hu, Takashi Shingu, Liang Yuan. How glioma stem cells maintain stemness outside their niche. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 909.

Induction of Perivascular Neural Stem Cells and Possible Contribution to Neurogenesis Following Transient Brain Ischemia/Reperfusion Injury.

Recent therapeutic advances have increased the likelihood of recanalizing the obstructed brain arteries in patients with stroke. Therefore, it is important to understand the fate of neural cells under transient ischemia/reperfusion injury. Accumulating evidence shows that neurogenesis occurs in perivascular regions following brain injury, although the precise mechanism and origin of these newborn neurons under transient ischemia/reperfusion injury remain unclear. Using a mouse model of transient brain ischemia/reperfusion injury, we found that neural stem cells (NSCs) develop within injured areas. This induction of NSCs following ischemia/reperfusion injury was observed even in response to nonlethal ischemia, although massive numbers of NSCs were induced by lethal ischemia. Immunohistochemical and immunoelectron microscopic studies indicated that platelet-derived growth factor receptor beta-positive (PDGFRβ+) pericytes within injured areas following nonlethal ischemia began to express the NSC marker nestin as early as 3days after transient ischemia/reperfusion. Some PDGFRβ+ pericytes expressed the immature neuronal marker doublecortin at day 7. These findings indicate that brain pericytes are a potential source of the perivascular NSCs that generate neuronal cells under lethal and nonlethal ischemic conditions following transient ischemia/reperfusion. Thus, brain pericytes might be a target for neurogenesis mediation in patients with nonlethal and lethal ischemia following transient ischemia/reperfusion injury.

Functional roles of glycoconjugates in the maintenance of stemness and differentiation process of neural stem cells.

Neural stem cells (NSCs) possess a high proliferative potential and capacity for self-renewal with retention of multipotency to differentiate into brain-forming cells. NSCs have gained a considerable attention because of their potential application in treatment strategies on the basis of transplantation for neurodegenerative disorders and nerve injuries. Although several signaling pathways have been reportedly involved in the fate determination process of NSCs, the molecular mechanisms underlying the maintenance of neural cell stemness and differentiation process remain largely unknown. Glycoconjugates expressed in the NSC niche in the brain offer markers of NSCs; moreover, they serve as cell regulators, which are actively involved in the modulation of signal transduction. The glycans function on NCS surfaces by recruiting growth factor receptors to specific microdomains as components of glycolipids, thereby mediating the ligand-receptor interactions both indirectly and directly as components of proteoglycans and interacting with specific lectin-type receptors as components of ligand glycoproteins. In this review, we outline current knowledge of the possible functional mechanisms of glycoconjugates to determine cell fates, which are associated with their expression pattern and structural characteristic features.

Electrical Property Characterization of Neural Stem Cells in Differentiation.

Electrical property characterization of stem cells could be utilized as a potential label-free biophysical approach to evaluate the differentiation process. However, there has been a lack of technology or tools that can quantify the intrinsic cellular electrical markers (e.g., specific membrane capacitance (Cspecific membrane) and cytoplasm conductivity (σcytoplasm)) for a large amount of stem cells or differentiated cells. In this paper, a microfluidic platform enabling the high-throughput quantification of Cspecific membrane and σcytoplasm from hundreds of single neural stem cells undergoing differentiation was developed to explore the feasibility to characterize the neural stem cell differentiation process without biochemical staining. Experimental quantification using biochemical markers (e.g., Nestin, Tubulin and GFAP) of neural stem cells confirmed the initiation of the differentiation process featured with gradual loss in cellular stemness and increased cell markers for neurons and glial cells. The recorded electrical properties of neural stem cells undergoing differentiation showed distinctive and unique patterns: 1) in the suspension culture before inducing differentiation, a large distribution and difference in σcytoplasm among individual neural stem cells was noticed, which indicated heterogeneity that may result from the nature of suspension culture of neurospheres; and 2) during the differentiation in adhering monolayer culture, significant changes and a large difference in Cspecific membrane were located indicating different expressions of membrane proteins during the differentiation process, and a small distribution difference in σcytoplasm was less significant that indicated the relatively consistent properties of cytoplasm during the culture. In summary, significant differences in Cspecific membrane and σcytoplasm were observed during the neural stem cell differentiation process, which may potentially be used as label-free biophysical markers to monitor this process.

Down-regulation of nestin in mesenchymal stem cells derived from peripheral blood through blocking bone morphogenesis pathway.

Different signaling pathways are implicated in proliferation and differentiation of stem cells. Bone Morphogenesis Pathway (BMP) signaling was known to display an important function in osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs). In the present study, the authors investigated whether blocking BMP signaling was associated with down regulation of Nestin expression as neural stem cell marker in peripheral blood derived mesenchymal stem cells (PB-MSCs). At first, MSCs were isolated from peripheral blood by plastic adherent ability and flow cytometry analysis. After reaching the confluence, the cells were treated with medium containing Noggin as antagonist of BMP signaling upon 8days. Real time PCR analysis indicated that the expression of Nestin was diminished in PB-MSCs by attenuating BMP signaling. The obtained results suggested that BMP signaling might have a regulatory function on the Nestin expression in mesenchymal stem cells.

Icariin promotes cell proliferation and regulates gene expression in human neural stem cells invitro.

Icariin (ICA), which is an essential bioactive component extracted from the herb Epimedium, possesses neuroprotective properties. The aim of the present study was to investigate the regulatory roles of ICA in cell proliferation and gene expression in human neural stem cells (NSCs) invitro. Single cells were isolated from the corpus striatum of 16‑20‑week human fetuses obtained following spontaneous abortion. The cells were cultured in Dulbecco's modified Eagle's medium/F12 complete medium and were characterized by immunostaining and cell differentiation assay. NSCs were treated with ICA, and cell proliferation was assessed using the Cell Counting kit‑8 cell proliferation assay kit. In addition, neurosphere formation was comparatively studied between the ICA‑treated and control cells. cDNA microarray analysis was performed to examine the effects of ICA on gene expression. Altered expression of genes important for regulating NSC proliferation was further analyzed by quantitative polymerase chain reaction (qPCR). The results demonstrated that typical neurospheres appeared after 7‑10days of culturing of individual cells isolated from the corpus striatum. These cells expressed nestin, an important NSC marker, and in the presence of differentiation medium they expressed β‑III‑tubulin, a specific neuronal marker, and glial fibrillary acidic protein, an astrocyte marker. Treatment with ICA enhanced NSC proliferation and the formation of neurospheres. Microarray data and pathway analysis revealed that the genes regulated by ICA were involved in several signaling pathways, including the Wnt and basic fibroblast growth factor (bFGF) pathways, which are important for the regulation of NSC function. Upregulation of frizzled class receptor7 and dishevelled segment polarity protein3, which are key players in the Wnt pathway, and fibroblast growth factor receptor1, which is the receptor for bFGF, and downregulation of glycogen synthase kinase‑3β, which is a Wnt pathway inhibitor, was further validated by qPCR. In conclusion, ICA promoted proliferation and regulated gene expression in human NSCs, thus suggesting that ICA may exert its neuroprotective effects by regulating NSC activity.

Grafted Subventricular Zone Neural Stem Cells Display Robust Engraftment and Similar Differentiation Properties and Form New Neurogenic Niches in the Young and Aged Hippocampus.

: As clinical application of neural stem cell (NSC) grafting into the brain would also encompass aged people, critical evaluation of engraftment of NSC graft-derived cells in the aged hippocampus has significance. We examined the engraftment and differentiation of alkaline phosphatase-positive NSCs expanded from the postnatal subventricular zone (SVZ), 3 months after grafting into the intact young or aged rat hippocampus. Graft-derived cells engrafted robustly into both young and aged hippocampi. Although most graft-derived cells pervasively migrated into different hippocampal layers, the graft cores endured and contained graft-derived neurons expressing neuron-specific nuclear antigen (NeuN) and γ-amino butyric acid in both groups. A fraction of migrated graft-derived cells in the neurogenic subgranular zone-granule cell layer also expressed NeuN. Neuronal differentiation was, however, occasionally seen amid graft-derived cells that had migrated into non-neurogenic regions, where substantial fractions differentiated into S-100β+ astrocytes, NG2+ oligodendrocyte progenitors, or Olig2+ putative oligodendrocytes. In both age groups, graft cores located in non-neurogenic regions displayed many doublecortin-positive (DCX+) immature neurons at 3 months after grafting. Analyses of cells within graft cores using birth dating and putative NSC markers revealed that DCX+ neurons were newly born neurons derived from engrafted cells and that putative NSCs persisted within the graft cores. Thus, both young and aged hippocampi support robust engraftment and similar differentiation of SVZ-NSC graft-derived cells. Furthermore, some grafted NSCs retain the "stemness" feature and produce new neurons even at 3 months after grafting, implying that grafting of SVZ-NSCs into the young or aged hippocampus leads to establishment of new neurogenic niches in non-neurogenic regions. The results demonstrate that advanced age of the host at the time of grafting has no major adverse effects on engraftment, migration, and differentiation of grafted subventricular zone-neural stem cells (SVZ-NSCs) in the intact hippocampus, as both young and aged hippocampi promoted excellent engraftment, migration, and differentiation of SVZ-NSC graft-derived cells in the present study. Furthermore, SVZ-NSC grafts showed ability for establishing neurogenic niches in non-neurogenic regions, generating new neurons for extended periods after grafting. This phenomenon will be beneficial if these niches can continuously generate new neurons and glia in the grafted hippocampus, as newly generated neurons and glia are expected to improve, not only the microenvironment, but also the plasticity and function of the aged hippocampus. Overall, these results have significance because the potential application of NSC grafting for treatment of neurodegenerative disorders at early stages of disease progression and age-related impairments would mostly involve aged persons as recipients.