Behavior of hippocampal stem/progenitor cells following grafting into the injured aged hippocampus

Multipotent neural stem/progenitor cells (NSCs) from the embryonic hippocampus are potentially useful as donor cells to repopulate the degenerated regions of the aged hippocampus after stroke, epilepsy, or Alzheimer's disease. However, the efficacy of the NSC grafting strategy for repairing the injured aged hippocampus is unknown. To address this issue, we expanded FGF-2-responsive NSCs from the hippocampus of embryonic day 14 green fluorescent protein-expressing transgenic mice as neurospheres in vitro and grafted them into the hippocampus of 24-month-old F344 rats 4 days after CA3 region injury. Engraftment, migration, and neuronal/glial differentiation of cells derived from NSCs were analyzed 1 month after grafting. Differentiation of neurospheres in culture dishes or after placement on organotypic hippocampal slice cultures demonstrated that these cells had the ability to generate considerable numbers of neurons, astrocytes, and oligodendrocytes. Following grafting into the injured aged hippocampus, cells derived from neurospheres survived and dispersed, but exhibited no directed migration into degenerated or intact hippocampal cell layers. Phenotypic analyses of graft-derived cells revealed neuronal differentiation in 3%-5% of cells, astrocytic differentiation in 28% of cells, and oligodendrocytic differentiation in 6%-10% cells. The results demonstrate for the first time that NSCs derived from the fetal hippocampus survive and give rise to all three CNS phenotypes following transplantation into the injured aged hippocampus. However, grafted NSCs do not exhibit directed migration into lesioned areas or widespread neuronal differentiation, suggesting that direct grafting of primitive NSCs is not adequate for repair of the injured aged brain without priming the microenvironment.     

Hepatocyte growth factor promotes neuronal differentiation of neural stem cells derived from embryonic stem cells

We previously reported that hepatocyte growth factor (HGF) promoted proliferation of neurospheres and neuronal differentiation of neural stem cells (NSCs) derived from mouse embryonic brain. In this study, spheres from mouse embryonic stem (ES) cells were generated by floating culture following co-culture on PA6 stromal cells. In contrast to the behavior of the neurospheres derived from embryonic brain, addition of HGF to the growth medium of the floating cultures decreased the number of spheres derived from ES cells. When spheres were stained using a MAP-2 antibody, more MAP-2-positive cells were observed in spheres cultured with HGF. When HGF was added to the growth and/or differentiation medium, more MAP-2-positive cells were also obtained. These results suggest that HGF promotes neuronal differentiation of NSCs derived from ES cells.     

不同促细胞分裂因子对人神经干细胞定向分化的影响

目的观察不同促细胞分裂因子对人神经干细胞(NSCs)增殖及定向分化的影响。方法对人NSCs用无血清DMEM培养基行原代培养的同时,分别加入表皮生长因子(EGF)、碱性成纤维生长因子(bFGF)、神经生长因子(NGF)、维甲酸(RA)等因子,观察其对NSCs定向分化的作用。结果EGF培养的NSCs,克隆球形成慢且较松散,经血清诱导分化后,主要为星形胶质细胞,仅有少数神经元。而bFGF培养的NSCs则生长良好。在加血清诱导分化后,不同浓度bFGF培养的NSCs分化的细胞不同。bFGF与EGF共同培养的NSCs,其生长及神经球形成良好。在加血清诱导分化后,分化的神经细胞比例更接近脑内神经细胞的组分。NGF对神经球的形成无明显影响,但可促使其向神经元分化。RA使神经克隆球形成,可使NSCs直接分化为神经元细胞的比例明显增加。结论不同促细胞分裂因子对人NSCs的增殖及定向分化均有一定影响。     

Hepatocyte growth factor promotes proliferation and neuronal differentiation of neural stem cells from mouse embryos

Hepatocyte growth factor (HGF), originally cloned as a hepatocyte mitogen, has recently been reported to exhibit neurotrophic activity in addition to being expressed in different parts of the nervous system. At present, the effects of HGF on neural stem cells (NSCs) are not known. In this study, we first report the promoting effect of HGF on the proliferation of neurospheres and neuronal differentiation of NSCs. Medium containing only HGF was capable of inducing neurosphere formation. Addition of HGF to medium containing fibroblast growth factor 2 or epidermal growth factor increased both the size and number of newly formed neurospheres. More neurons were also obtained when HGF was added in differentiation medium. In contrast, neurosphere numbers were reduced after repeated subculture by mechanical dissociation, suggesting that HGF-formed neurospheres comprised predominantly progenitor cells committed to neuronal or glial lines. Together, these results suggest that HGF promotes proliferation of neurospheres and neuronal differentiation of NSCs derived from mouse embyos.     

Effects of lead exposure on proliferation and differentiation of neural stem cells derived from different regions of embryonic rat brain

Lead is a potent neurotoxin, causing brain damage and cognitive deficits in children even at low exposure levels. Although lead neurotoxicity can occur after prenatal or postnatal exposure, little is known of the effects of lead on embryonic neural stem cells (NSCs) or the extent to which NSCs originating in different brain regions may be differentially sensitive to the effects of lead exposure. The present study examined the effects of lead on proliferation and differentiation of neural stem cells (NSCs) originating from E15 rat cortex (CX), striatum (ST) or ventral mesencephalon (VM). Free-floating neurospheres were grown under standard conditions or in lead (0.01-100 microM)-containing conditioned media for 5 days and proliferation assessed by 3H-thymidine uptake. In other studies, control and lead-exposed neurospheres were collected, dissociated and re-plated in control or lead-containing differentiation media for 7 days. Cells were immunostained for visualization of mature neural and glial markers and counted. Lead exposure (0.01-10 microM) had no effect on neurosphere viability but caused a significant dose-dependent inhibition of proliferation in VM and ST but not CX neurospheres. The number of MAP2 positive neurons differentiated from lead-exposed neurospheres of VM and ST origin (but not CX) was significantly decreased from control as were the number of oligodendrocytes obtained, regardless of their region of origin. In contrast, lead exposure significantly increased the number of astrocytes obtained regardless of site of origin. These data suggest that even low levels of lead can differentially affect proliferation and differentiation of embryonic NSCs originating from different brain regions and supports the need for prevention of prenatal lead exposure.     

Regional specification of rodent and human neurospheres

Neural precursor cells were isolated from various regions of the developing rat and human brain and grown in culture as aggregates termed neurospheres. We asked whether cells within human and rodent neurospheres are identical, or whether they have species specific characteristics or differences based on their region of origin. Under our culture conditions, rodent neurospheres isolated from the cortex (ctxNS) and striatum (strNS) grew faster than those from the mesencephalon (mesNS), but stopped growing after only eight to ten population doublings. In contrast, human neurospheres under identical culture conditions, continued to grow for over 40 population doublings. Following migration and differentiation of both rodent and human cultures, ctxNS and strNS generated high numbers of small neurons whereas mesNS generated small numbers of large neurons with many long fibres. Only very rare neurons from mesNS expressed dopaminergic markers, and thus may require further signals to fully mature. While the rat neurospheres generated high numbers of oligodendrocytes, very few were found to develop from human neurospheres from any region after a few weeks of passaging. FACS analysis revealed a unique population of smaller cells within human strNS and ctxNS, which appeared to be neuronal progenitors. However, large cells within neurospheres were capable of generating these small neuronal progenitors following further proliferation. Together, our data show that rat and human neurospheres have unique characteristics with regard to growth and differentiation, and that the majority of precursor cells within neurospheres are regionally specified to generate set numbers of neurons. These findings have important implications for understanding the nature of proliferating neural precursors isolated from the developing CNS, and their potential for brain repair.     

The effect of bone marrow stromal cells on neuronal differentiation of mesencephalic neural stem cells in Sprague-Dawley rats

There are numerous parallels between the heamatolymphopoietic and nervous systems in terms of the mechanisms regulating their development. We proposed that neural stem cells (NSCs) may respond to the microenvironmental signals provided by bone marrow stromal cells (BMSCs) which regulate the differentiation and maturation of hematolymphopoietic stem cells. First, we isolated and proliferated BMSCs from the femur and tibia, and NSCs from the midbrain of Sprague-Dawley (SD) rats, and then investigated the effects of BMSCs on the differentiation of NSCs into neurons, astrocytes and oligodendrocytes by directly plating neurospheres on BMSC monolayers in serum-free conditions. The results confirmed that BMSCs induced NSCs to differentiate selectively into neurons. The percentage of neurons significantly increased in 7 days in vitro co-cultures of NSCs and BMSCs as compared to NSCs cultures alone. When the duration of the cultures was extended to 12 days in vitro, BMSCs enhanced the survival of neurons derived from these NSCs; our investigation then focused on the underlying mechanism for this effect of BMSCs. NSCs were cultured with BMSC conditioned-medium and co-cultured with membrane fragments of live BMSCs or paraformaldehyde fixed BMSCs, the inducing activity of BMSCs was solely detectable in BMSC conditioned-medium, indicating that soluble factors secreted by BMSCs were responsible for its effect on the neuronal differentiation of NSCs. Therefore, BMSCs may provide a powerful tool for therapeutic neurological applications.     

Induction of neuronal phenotypes from NG2+ glial progenitors by inhibiting epidermal growth factor receptor in mouse spinal cord injury

Besides neural stem cells, some glial cells, such as GFAP+ cells, radial glia, and oligodendrocyte progenitor cells can produce neuronal cells. Attractively, NG2+ glial progenitors exhibit lineage plasticity, and they rapidly proliferate and differentiate in response to central nervous system (CNS) injuries. These attributes of NG2+ glial progenitors make them a promising source of neurons. However, the potential of neuronal regeneration from NG2+ glial progenitors in CNS pathologies remains to be investigated. In this study, we showed that antagonizing epidermal growth factor receptor (EGFR) function with EGFR inhibitor caused a significant number of proliferative NG2+ glial progenitors to acquire neuronal phenotypes in contusive spinal cord injury (SCI), which presumably led to an accumulation of newly generated neurons and contributed to the improved neural behavioral performance of animals. In addition, the neuronal differentiation of glial progenitors induced by EGFR inhibitor was further confirmed with two different cell lines either in vitro or through ex vivo transplantation experiment. The inhibition of EGFR signaling pathway under the gliogenic conditions could induce these cells to acquire neuronal phenotypes. Furthermore, we find that the Ras‐ERK axis played a key role in neuronal differentiation of NG2+ glial progenitors upon EGFR inhibition. Taken together, our studies suggest that the EGFR inhibitor could promote neurogenesis post SCI, mainly from the NG2+ glial progenitors. These findings support the possibility of evoking endogenous neuronal replacement from NG2+ glial progenitors and suggest that EGFR inhibition may be beneficial to CNS trauma. © 2012 Wiley Periodicals, Inc.     

The adult spinal cord harbors a population of GFAP‐positive progenitors with limited self‐renewal potential

Adult neural stem cells (aNSCs) of the forebrain are GFAP‐expressing cells that are intercalated within ependymal cells of the subventricular zone (SVZ). Cells showing NSCs characteristics in vitro can also be isolated from the periaqueductal region in the adult spinal cord (SC), but contradicting results exist concerning their glial versus ependymal identity. We used an inducible transgenic mouse line (hGFAP‐CreERT2) to conditionally label GFAP‐expressing cells in the adult SVZ and SC periaqueduct, and directly and systematically compared their self‐renewal and multipotential properties in vitro. We demonstrate that a population of GFAP⁺ cells that share the morphology and the antigenic properties of SVZ‐NSCs mostly reside in the dorsal aspect of the central canal (CC) throughout the spinal cord. These cells are non‐proliferative in the intact spinal cord, but incorporate the S‐phase marker EdU following spinal cord injury. Multipotent, clonal YFP‐expressing neurospheres (i.e., deriving from recombined GFAP‐expressing cells) were successfully obtained from both the intact and injured spinal cord. These spheres however showed limited self‐renewal properties when compared with SVZ‐neurospheres, even after spinal cord injury. Altogether, these results demonstrate that significant differences exist in NSCs lineages between neurogenic and non‐neurogenic regions of the adult CNS. Thus, although we confirm that a population of multipotent GFAP⁺ cells co‐exists alongside with multipotent ependymal cells within the adult SC, we identify these cells as multipotent progenitors showing limited self‐renewal properties. GLIA 2013;61:2100–2113     

Nestin‐expressing cells in the gut give rise to enteric neurons and glial cells

Background Neuronal stem cells (NSCs) are promising for neurointestinal disease therapy. Although NSCs have been isolated from intestinal musclularis, their presence in mucosa has not been well described. Mucosa‐derived NSCs are accessible endoscopically and could be used autologously. Brain‐derived Nestin‐positive NSCs are important in endogenous repair and plasticity. The aim was to isolate and characterize mucosa‐derived NSCs, determine their relationship to Nestin‐expressing cells and to demonstrate their capacity to produce neuroglial networks in vitro and in vivo. Methods Neurospheres were generated from periventricular brain, colonic muscularis (Musc), and mucosa–submucosa (MSM) of mice expressing green fluorescent protein (GFP) controlled by the Nestin promoter (Nestin‐GFP). Neuronal stem cells were also grown as adherent colonies from intestinal mucosal organoids. Their differentiation potential was assessed using immunohistochemistry using glial and neuronal markers. Brain and gut‐derived neurospheres were transplanted into explants of chick embryonic aneural hindgut to determine their fate. Key Results Musc‐ and MSM‐derived neurospheres expressed Nestin and gave rise to cells of neuronal, glial, and mesenchymal lineage. Although Nestin expression in tissue was mostly limited to glia co‐labelled with glial fibrillary acid protein (GFAP), neurosphere‐derived neurons and glia both expressed Nestin in vitro, suggesting that Nestin+/GFAP+ glial cells may give rise to new neurons. Moreover, following transplantation into aneural colon, brain‐ and gut‐derived NSCs were able to differentiate into neurons. Conclusions & Inferences Nestin‐expressing intestinal NSCs cells give rise to neurospheres, differentiate into neuronal, glial, and mesenchymal lineages in vitro, generate neurons in vivo and can be isolated from mucosa. Further studies are needed for exploring their potential for treating neuropathies.     

Role of brominated diphenly ether-209 in the differentiation of neural stem cells in vitro

Brominated diphenly ether-209 (BDE-209, decaBDE) is among the most common flame retardants. In a previous study, it was confirmed that exposure to BDE-209 can decrease learning and memory in mice. However, it is still unknown whether BDE-209 has an effect on cultured neural stem cells (NSCs). To analyse the role of BDE-209 in the differentiation of neural stem cells, NSCs obtained from neonatal rats were cultured as neurospheres in DMEM/F12 medium that contained different concentrations of BDE-209 for 7 days. BDE-209 was found to inhibit neurite outgrowth and the differentiation of NSCs into neurons in a concentration-dependent manner. BDE-209 also enhanced the ratio of differentiation of NSCs into glial cells.     

Stromal cell-derived inducing activity does not promote dopaminergic differentiation, but enhances differentiation and proliferation of neural stem cell-derived astrocytes

Previous evidence has shown that stromal cell-derived inducing activity (SDIA), produced by the mouse PA6 stromal cell line, promotes dopaminergic differentiation of mouse, monkey and human embryonic stem cells in vitro. To examine whether PA6 stromal cells can enhance the yield of dopaminergic differentiation from neural progenitors, we generated neurospheres from embryonic day 11.5 (E11.5) (midbrain and forebrain) and E14.5 (ventral mesencephalon and cortex) rat embryos and allowed them to differentiate in co-culture with PA6 cells or poly-l-lysine/laminin-coated dishes. We observed that SDIA did not promote dopaminergic differentiation of E11.5 and E14.5 neurospheres but more prominently, enhanced astrocyte differentiation, cell survival and astrocyte proliferation. Our results suggest that PA6 cells do not have a general capacity to promote differentiation into dopaminergic neurons from all types of stem cells, but that they may specifically induce dopaminergic differentiation of highly uncommitted stem cells such as embryonic stem cells.     

Grafted lineage-restricted precursors differentiate exclusively into neurons in the adult spinal cord

Multipotent neural stem cells (NSCs) have the potential to differentiate into neuronal and glial cells and are therefore candidates for cell replacement after CNS injury. Their phenotypic fate in vivo is dependent on the engraftment site, suggesting that the environment exerts differential effects on neuronal and glial lineages. In particular, when grafted into the adult spinal cord, NSCs are restricted to the glial lineage, indicating that the host spinal cord environment is not permissive for neuronal differentiation. To identify the stage at which neuronal differentiation is inhibited we examined the survival, differentiation, and integration of neuronal restricted precursor (NRP) cells, derived from the embryonic spinal cord of transgenic alkaline phosphatase rats, after transplantation into the adult spinal cord. We found that grafted NRP cells differentiate into mature neurons, survive for at least 1 month, appear to integrate within the host spinal cord, and extend processes in both the gray and white matter. Conversely, grafted glial restricted precursor cells did not differentiate into neurons. We did not observe glial differentiation from the grafted NRP cells, indicating that they retained their neuronal restricted properties in vivo. We conclude that the adult nonneurogenic CNS environment does not support the transition of multipotential NSCs to the neuronal commitment stage, but does allow the survival, maturation, and integration of NRP cells.     

Maturing neurons are selectively sensitive to human immunodeficiency virus type 1 exposure in differentiating human neuroepithelial progenitor cell cultures

Human immunodeficiency virus type 1 (HIV-1) infection of the brain is associated with neuronal injury manifested by dendritic pruning, aberrant neurofilament metabolism, and decreased synaptic density. The central nervous system (CNS) responds to neuronal injury by differentiating new neurons and astrocytes from resident populations of multipotent neuroepithelial progenitor cells (NEP) located in regions such as the subventricular zone or hippocampus. In vitro studies have demonstrated that the HIV-1 virion or envelope glycoprotein gp120 can injure differentiated human neurons and astrocytes, suggesting that HIV-1 proteins could similarly injure NEP or NEP-derived glial and neuronal lineage-committed precursor cells. To answer this question, human fetal brain-derived "neurospheres" containing NEP and NEP-derived precursor cells were cultured in low serum differentiation medium containing lymphotropic HIV-1(SF2), macrophage-tropic HIV-1(SF128A), or recombinant gp120SF2 from HIV-1(SF2). These experiments indicate that exposure to HIV-1 does not affect the ability of the NEP to differentiate into cells expressing either astrocyte-specific or neuron-specific cytoskeletal antigens. However prolonged exposure to HIV-1 does selectively decrease expression of neuronal antigens (microtubule beta-III-tubulin and intermediate filament neurofilament-L) but not astrocyte antigens (intermediate filament glial fibrillary acidic protein). The effects of continuous exposure to HIV-1 or gp120 may result from injury to developing neurons and/or impairment of the neuronal developmental process itself. By depressing neuronal microtubule and neurofilament protein expression, HIV-1 and gp120 exposure compromise the potential for postmitotic neuronal dendrite and axon development.     

体外神经干细胞培养特性观察

目的 探讨神经干细胞(NSCs)体外分离培养和增殖的特性.方法 从新生24h内的SD大鼠脑组织分离NSCs,采用无血清悬浮培养法进行NSCs体外扩增培养.倒置相差显微镜观察细胞形态,通过绘制细胞生长曲线观察NSCs的自我更新和增殖能力,采用免疫细胞化学法检测NSCs标志物神经上皮干细胞蛋白(Nestin)的表达及分化后细胞神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)和2,3-环核甘酸磷酸二脂酶(CNP)的表达.结果 从新生SD大鼠脑组织分离的细胞在无血清的培养基中形成悬浮的神经球.神经球具有自我更新和表达Nestin的能力,分化后的细胞能表达神经元、星型胶质细胞及少突胶质细胞的特异性抗原.结论 从新生大鼠的脑组织中成功分离出NSCs,其具有在体外自我更新和增殖、多向分化潜能及表达Nestin的能力.     

Neurosphere generation from dental pulp of adult rat incisor

Dental pulp is a potential source of cells that can be used in cell replacement therapy for various nervous system disorders. Here we report that adult rat dental pulp cells have the ability to form neurospheres when cultured in serum-free culture medium on super-hydrophilic plates. The cells within small spheres continued to grow, and the dental pulp-derived cells generated large spheres. Sphere formation was dependent on exogenously supplied basic-fibroblast growth factor, but not on epidermal growth factor, and the formation and growth of dental pulp-derived spheres were negatively regulated by transforming growth factor-β. Plating cells that were dissociated from spheres on an adhesive substrate resulted in differentiation into Tuj1- and MAP2-positive neuronal cells. Analysis of the three-dimensional structure of dental pulp-derived spheres shows that they contained nestin-positive progenitors, Tuj1-positive neuronal cells and S100-positive glial cells. We found that spheres contained CD81 (TAPA1) and nestin double-positive cells, and identified a small population of CD81 and nestin double-positive cells in the odontoblast layer of the dental pulp. Flow cytometric analysis showed that CD81-positive cells were enriched in the spheres compared with the dental pulp tissue. Bromodeoxyuridine (BrdU) staining showed that nestin- and BrdU-positive cells were located only in the apical portion of the dental pulp, and the apical portion produced a large number of large-sized spheres. These data suggest that the CD81 and nestin double-positive cells localized in the odontoblast layer of the apical portion of the dental pulp may have the ability to grow and form neurospheres.