Trophism of neural progenitor cells to embryonic stem cells: neural induction and transplantation in a mouse ischemic stroke model

Embryonic stem cell (ESC)-derived products have emerged as a promising cell source for neuroregeneration. C17.2 neural precursor cells were noted to express genes of neurotrophins and neuroprotective factors and to be enable to enhance proliferation, neuritogenesis, and differentiation of SH-SY5Y and SK-N-AS neuroblasts, suggesting their neurotrophic potential. We used C17.2 cells as neurotrophic chaperones to induce ESCs, D3, and E14TG2a into neural lineage cells. Significantly greater numbers of Sox-2(+), Musashi-1(+), and nestin(+) neurospheres developed in noncontact cocultures than in cultures of ESCs without C17.2 support or with 50% conditioned medium after 8 days. Immunoreactivity of the neuronal, astrocytic and oligodendrocytic markers was evident in cultures further differentiated for 10 days. Expression of Pax-6, Otx-1, and Nurr-1 genes suggested neuroectodermal precursors in products encompassing neural stem cells, dopaminergic neurons, astrocytes, and oligodendrocytes. Alpha-fetoprotein, GATA-4, Brachyury, Nkx-2.5, and Myf-5 genes were not detected, indicating any mesodermal and endodermal cells. However, weak expression of Oct-4 was noted. Behavioral assessment of ischemic mice 2 weeks after transplantation revealed significant improvement in cognitive function compared with that in ischemic sham-operated mice. Tracking bromodeoxyuridine-labeled products demonstrated that mostly implanted cells were localized along the needle track of the injection in the brain parenchyma, whereas some migrated to the striatum, cortex, nerve fiber bundle of the corpus callosum, and hippocampus in the ipsilateral hemisphere. One episode (of 22) of teratoma development was noted. Data from this study suggest a paradigm of trophism of neural progenitor cells for induction of ESCs into neural lineage cells.     

胚胎大鼠纹状体神经干细胞的体外培养和分化

目的 :旨在探讨纹状体神经干细胞 (striatum neuralstemcells ,strNSCs)的培养及分化鉴定方法。方法 :选择性分离纹状体的神经干细胞 ,体外培养、扩增和诱导分化 ,并采用免疫荧光细胞化学检查鉴定。结果 :从胚鼠纹状体分离的细胞具有连续克隆能力 ,绝大多数细胞表达巢蛋白。诱导分化后的细胞表达成熟神经细胞和星形胶质细胞特异性的蛋白。结论 :用此方法分离的细胞具有自我更新、增殖和分化潜能 ,具备中枢神经系统干细胞的一般特征。     

Transplantation of rat neural stem cells reduces stereotypic behaviors in rats after intrastriatal microinfusion of Tourette syndrome sera

Tourette syndrome (TS) is a heterogenous neuropsychiatric disorder. In most cases, tics are self-limited or can be treated by behavioral or pharmacological therapy. However, for some individuals, tics can cause lifelong impairment and life-threatening symptoms, which are intractable to traditional treatment. Neural stem cell (NSC) is a potential tool to treat certain neurological diseases. In this study, we proposed to use neural stem cell transplantation as a novel therapy to treat TS and discussed its efficacy. Wistar rats were microinfused with TS sera into the striatum followed by the transplantation of NSCs or vehicle at the infusion site. The sera of the TS patients were identified to have enriched antineural antibodies. Prior to grafting, rat embryonic NSCs were co-cultured with 5-bromodeoxyuridine (Brdu) for 24 h. Stereotypic behaviors were counted at 1, 7, 14 and 21 days after transplantation of NSCs. Morphological analyses revealed that NSCs survived and differentiated into neurons and astrocytes in the striatum 3 weeks after grafting. To sum it up, rat embryonic neural stem cell grafts survived and differentiated in the striatum of TS rat may help relieve stereotypic behaviors of the host. Our results suggest that transplantation of NSCs intrastriatum may have therapeutic potential for TS.     

Human fetal cortical and striatal neural stem cells generate region-specific neurons in vitro and differentiate extensively to neurons after intrastriatal transplantation in neonatal rats

Human fetal brain is a potential source of neural stem cells (NSCs) for cell replacement therapy in neurodegenerative diseases. We explored whether NSCs isolated from cortex and striatum of human fetuses, aged 6-9 weeks post-conception, maintain their regional identity and differentiate into specific neuron types in culture and after intrastriatal transplantation in neonatal rats. We observed no differences between cortex- and striatum-derived NSCs expanded as neurospheres in proliferative capacity, growth rate, secondary sphere formation, and expression of neural markers. After 4 weeks of differentiation in vitro, cortical and striatal NSCs gave rise to similar numbers of GABAergic and VMAT2- and parvalbumin-containing neurons. However, whereas cortical NSCs produced higher number of glutamatergic and tyrosine hydroxylase- and calretinin-positive neurons, several-fold more neurons expressing the striatal projection neuron marker, DARPP-32, were observed in cultures of striatal NSCs. Human cortical and striatal NSCs survived and migrated equally well after transplantation. The two NSC types also generated similar numbers of mature NeuN-positive neurons, which were several-fold higher at 4 months as compared to at 1 month after grafting. At 4 months, the grafts contained cells with morphologic characteristics of neurons, astrocytes, and oligodendrocytes. Many of neurons were expressing parvalbumin. Our data show that NSCs derived from human fetal cortex and striatum exhibit region-specific differentiation in vitro, and survive, migrate, and form mature neurons to the same extent after intrastriatal transplantation in newborn rats.     

Flow cytometric analysis of neural stem cells in the developing and adult mouse brain

Despite recent progress in the neural stem cell biology, their cellular characteristics have not been described well. We investigated various characteristics of neural stem cells (NSCs) in vivo during CNS development, using FACS to identify the NSCs. We first examined stage-dependent changes in the physical parameters, using forward scatter (FSC) and side scatter (SSC) profiles, of NSCs from the developing striatum, where they appear to be active throughout the life of mammals. NSCs were divided into several fractions according to their FSC/SSC profile. With development, their number decreased in the FSC(high) fractions but increased in the FSC(low)/SSC(high) fraction, whereas NSCs were significantly concentrated in the fraction containing the largest cells (about 20 microm in diameter) at any stage, which were mostly the cells with the highest nestin-enhancer activity. Furthermore, we demonstrated that, at all stages examined, the "side population" (SP), defined as the Hoechst 33342 low/negative fraction, which is known to be a stem cell-enriched population in bone marrow, was also enriched for Notch1-positive immature neural cells (about 60%) from the developing striatum. However, these immature SP cells were not detected in the large-cell fraction, however, but were concentrated instead in the FSC(low/mid) fractions. FACS analysis showed that SP cells from adults were included to some extent in the CD24(low)/PNA(low) fraction, where NSCs were greatly concentrated. Collectively, the characteristics of NSCs were not uniform and changed developmentally.     

Overexpression of Wnt3a facilitates the proliferation and neural differentiation of neural stem cells in vitro and after transplantation into an injured rat retina

Neural stem cell‐based therapy is a promising option for repair after injury. However, poor stem cell proliferation and insufficient differentiation of the stem cells into neurons are still difficult problems. The present study investigated whether transplantation of neural stem cells (NSCs) genetically modified to express Wnt3a is a promising approach to overcome these difficulties. We explored the possibility that Wnt3a might contribute to the therapeutic effect of NSC transplantation in retinal repair. The relative promotion of proliferation and neural differentiation by modified NSCs was investigated in a rat model of optic nerve crush. A recombinant lentivirus (Lenti‐Wnt3a) was engineered to express Wnt3a. NSCs infected with control lentivirus (Lenti‐GFP) or Lenti‐Wnt3a were transplanted into the subretinal space immediately after the optic nerve crush. The proliferation and neural differentiation activity of the NSCs were assessed in vitro and in vivo. Overexpression of Wnt3a in NSCs induced activation of Wnt signaling, promoted proliferation, and directed the differentiation of the NSCs into neurons both in vitro and in vivo. Our study suggests that Wnt3a can potentiate the therapeutic benefits of NSC‐based therapy in the injured retina. © 2013 Wiley Periodicals, Inc.     

Neural stem cells may be uniquely suited for combined gene therapy and cell replacement: Evidence from engraftment of Neurotrophin-3-expressing stem cells in hypoxic-ischemic brain injury

Previously, we reported that, when clonal neural stem cells (NSCs) were transplanted into brains of postnatal mice subjected to unilateral hypoxic-ischemic (HI) injury (optimally 3-7 days following infarction), donor-derived cells homed preferentially (from even distant locations) to and integrated extensively within the large ischemic areas that spanned the hemisphere. A subpopulation of NSCs and host cells, particularly in the penumbra, "shifted" their differentiation towards neurons and oligodendrocytes, the cell types typically damaged following asphyxia and least likely to regenerate spontaneously and in sufficient quantity in the "post-developmental" CNS. That no neurons and few oligodendrocytes were generated from the NSCs in intact postnatal cortex suggested that novel signals are transiently elaborated following HI to which NSCs might respond. The proportion of "replacement" neurons was approximately 5%. Neurotrophin-3 (NT-3) is known to play a role in inducing neuronal differentiation during development and perhaps following injury. We demonstrated that NSCs express functional TrkC receptors. Furthermore, the donor cells continued to express a foreign reporter transgene robustly within the damaged brain. Therefore, it appeared feasible that neuronal differentiation of exogenous NSCs (as well as endogenous progenitors) might be enhanced if donor NSCs were engineered prior to transplantation to (over)express a bioactive gene such as NT-3. A subclone of NSCs transduced with a retrovirus encoding NT-3 (yielding >90% neurons in vitro) was implanted into unilaterally asphyxiated postnatal day 7 mouse brain (emulating one of the common causes of cerebral palsy). The subclone expressed NT-3 efficiently in vivo. The proportion of NSC-derived neurons increased to approximately 20% in the infarction cavity and >80% in the penumbra. The neurons variously differentiated further into cholinergic, GABAergic, or glutamatergic subtypes, appropriate to the cortex. Donor-derived glia were rare, and astroglial scarring was blunted. NT-3 likely functioned not only on donor cells in an autocrine/paracrine fashion but also on host cells to enhance neuronal differentiation of both. Taken together, these observations suggest (1) the feasibility of taking a fundamental biological response to injury and augmenting it for repair purposes and (2) the potential use of migratory NSCs in some degenerative conditions for simultaneous combined gene therapy and cell replacement during the same procedure in the same recipient using the same cell (a unique property of cells with stem-like attributes).     

菲立磁标记兔骨髓基质源神经干细胞自体移植入纹状体后的形态学观察

目的将体外标记的骨髓基质源神经干细胞经单细胞悬液微移植后观察其在兔纹状体的存活、迁移、分化和整合情况，为细胞移植治疗疾病奠定基础。方法分离兔骨髓基质细胞，利用神经干细胞培养基、白血病抑止因子和碱性成纤维母细胞生长因子进行细胞扩增并诱导成骨髓基质源神经干细胞，再经菲立磁和活细胞荧光染料PKH67标记后．采用微移植的方法，通过脑立体定位仪，用微玻璃针将干细胞分别植入兔脑纹状体内。存活1、4、8周后处死动物，组织切片，利用光镜和电镜观察标记细胞在脑内的形态学情况。结果菲立磁标记的兔骨髓基质源神经干细胞经微移植后可在兔脑内纹状体区域存活，移植的干细胞可向周围的脑实质内迁移和整合，迁移细胞沿特定的纹状体结构分布。少量菲立磁标记的干细胞可以分化成神经元。结论骨髓基质源神经干细胞移植后．可在脑实质内存活、迁移、分化和整合，这种细胞可能成为中枢神经系统自体移植的细胞来源。