Mesencephalic human neural progenitor cells transplanted into the neonatal hemiparkinsonian rat striatum differentiate into neurons and improve motor behaviour

Neural stem cell transplantation is a promising strategy for the treatment of neurodegenerative diseases. To evaluate the differentiation potential of human neural progenitor cells (hNPCs) as a prerequisite for clinical trials, we intracerebrally transplanted in vitro expanded fetal mesencephalic hNPCs into hemiparkinsonian rats. On postnatal day one (P1), 17 animals underwent a unilateral intraventricular 6-hydroxydopamine injection into the right lateral ventricle. At P3, animals (n = 10) received about 100,000 hNPCs (1 microL) in the right striatum. Five weeks after birth, animals underwent behaviour tests prior to fixation, followed by immunohistochemistry on brain slices for human nuclei, glial fibrillary acidic protein, S100beta, neuronal nuclei antigen, neuron-specific enolase and tyrosine hydroxylase. Compared with the apomorphine-induced rotations in the lesioned-only group (7.4 +/- 0.5 min(-1)), lesioned and successfully transplanted animals (0.3 +/- 0.1 min(-1)) showed a significant therapeutic improvement. Additionally, in the cylinder test, the lesioned-only animals preferred to use the ipsilateral forepaw. Conversely, the lesioned and transplanted animals showed no significant side bias similar to untreated control animals. Transplanted human nuclei-immunoreactive cells were found to survive and migrate up to 2000 microm into the host parenchyma, many containing the pan-neuronal markers neuronal nuclei antigen and neuron-specific enolase. In the striatum, tyrosine hydroxylase-immunoreactive somata were also found, indicating a dopaminergic differentiation capacity of transplanted hNPCs in vivo. However, the relative number of tyrosine hydroxylase-immunoreactive neurons in vivo seemed to be lower than in corresponding in vitro differentiation. To minimize donor tissue necessary for transplantation, further investigations will aim to enhance dopaminergic differentiation of transplanted cells in vivo.     

Migration and differentiation of adult rat subventricular zone progenitor cells transplanted into the adult rat striatum

Adult brain subventricular zone progenitor cells undergo neurogenesis in the olfactory bulb. We tested the hypothesis that cultured adult subventricular zone progenitor cells migrate and differentiate into neurons when transplanted into the adult striatum. Cells in the adult rat subventricular zone were isolated and cultured for 8 days in medium containing basic fibroblast growth factor. These cells proliferated as assayed by bromodeoxyuridine immunostaining, and the majority of them were neuron-specific class III beta-tubulin (TuJ1) immunoreactive at 8 days of culture. These cultured cells were labeled in vitro with bromodeoxyuridine or with lipophilic dye-coated particles and were transplanted into the adult rat striatum. Twenty-eight days after transplantation, the cells migrated 0.5-1.5 mm from the midline of the graft to the surrounding host striatum. Migration of grafted cells in the host striatum was also detected on magnetic resonance imaging in living rats. Morphological analysis revealed that many of these migrated cells exhibited multibranched processes from the cell soma resembling host medium-size striatal projection neurons. Only a few astrocyte-like cells were detected. Double immunostaining showed that many bromodeoxyuridine immunoreactive cells were microtubule-associated protein 2 or immunoreactive with a mouse monoclonal antibody against neuronal nuclear protein, whereas only a few bromodeoxyuridine immunoreactive cells had glial fibrillary acidic protein immunoreactivity. Morphology of bromodeoxyuridine and microtubule-associated protein 2 immunoreactive cells was similar to those of host microtubule-associated protein 2 immunoreactive cells. These results suggest that transplanted cultured adult subventricular zone progenitor cells can migrate and differentiate in response to guidance cues within the adult striatum.     

Desert Hedgehog诱导大鼠胚胎中脑神经前体细胞定向分化为多巴胺能神经元

目的 研究Desert Hedgehog(DHH)对胚胎中脑神经前体细胞(NPCs)向多巴胺能神经元分化的诱导作用。方法 大鼠DHH病毒上清感染COS7、NIH/3T3和9L细胞, 用免疫荧光、实时定量PCR和Western blot方法检测DHH表达。分离培养大鼠胚胎中脑神经前体细胞，免疫荧光鉴定神经前体细胞的增殖分化特性。条件细胞表达DHH成功后分别与上述NPCs共培养10d，免疫荧光检测酪氨酸羟化酶(TH)的表达。结果 DHH在COS7、NIH/3T3和9L细胞中成功表达；同时，分离培养的大鼠胚胎中脑神经前体细胞具有良好的增殖分化特性，但两者共培养10d后偶见TH阳性的细胞。结论 DHH编码的蛋白不能或不能单独诱导与其共培养的NPCs定向分化为多巴胺能神经元。     

Proliferation and differentiation of nerve cells transplanted from human supraoptic and paraventricular nuclei into adult rat brain

Department of Biology and Department of Obstetrics and Gynecology, N. I. Pirogov Second Moscow Medical Institute. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 113, No. 6, pp. 651–653, June, 1992.     

Proliferation and differentiation of fetal liver epithelial progenitor cells after transplantation into adult rat liver

To identify cells that have the ability to proliferate and differentiate into all epithelial components of the liver lobule, we isolated fetal liver epithelial cells (FLEC) from ED 14 Fischer (F) 344 rats and transplanted these cells in conjunction with two-thirds partial hepatectomy into the liver of normal and retrorsine (Rs) treated syngeneic dipeptidyl peptidase IV mutant (DPPIV(-)) F344 rats. Using dual label immunohistochemistry/in situ hybridization, three subpopulations of FLEC were identified: cells expressing both alpha-fetoprotein (AFP) and albumin, but not CK-19; cells expressing CK-19, but not AFP or albumin, and cells expressing AFP, albumin, and cytokeratins-19 (CK-19). Proliferation, differentiation, and expansion of transplanted FLEC differed significantly in the two models. In normal liver, 1 to 2 weeks after transplantation, mainly cells with a single phenotype, hepatocytic (expressing AFP and albumin) or bile ductular (expressing only CK-19), had proliferated. In Rs-treated rats, in which the proliferative capacity of endogenous hepatocytes is impaired, transplanted cells showed mainly a dual phenotype (expressing both AFP/albumin and CK-19). One month after transplantation, DPPIV(+) FLEC engrafted into the parenchyma exhibited an hepatocytic phenotype and generated new hepatic cord structures. FLEC, localized in the vicinity of bile ducts, exhibited a biliary epithelial phenotype and formed new bile duct structures or were incorporated into pre-existing bile ducts. In the absence of a proliferative stimulus, ED 14 FLEC did not proliferate or differentiate. Our results demonstrate that 14-day fetal liver contains lineage committed (unipotential) and uncommitted (bipotential) progenitor cells exerting different repopulating capacities, which are affected by the proliferative status of the recipient liver and the host site within the liver where the transplanted cells become engrafted. These findings have important implications in future studies directed toward liver repopulation and ex vivo gene therapy.     

Development of human brain neural/progenitor cells after transplantation into the brain of adult rats

The purpose of the present study was the investigation of human neural stem/progenitor cells (SPC) cultured in vitro, with special reference to their capacity for grafting, migration and differentiation after transplantation into adult rat brain. SPC were isolated from the brain of 9-week-old human embryos and were cultured in a selective medium for 3 weeks. For transplantation, cell suspension or whole neurospheres were used; they were studied 4 weeks following the transplantation in hippocampus, striatum and lateral ventricle of adult rat brain. For the analysis of transplanted SPC, various histological and immunohistochemical staining methods were applied (bisbenzidine, BrdU, antibodies against human nuclei, vimentin, beta-tubulin, neurofilaments, GFAP), that allowed an independent evaluation of their state and differentiation. Transplanted human brain SPC were shown to survive well for one month in all the areas of adult rat brain without immunosuppression. Cells from suspension transplants actively migrated and differentiated into neurons and glial cells. Meanwhile, cell migration from the transplanted whole neurospheres was limited or absent due to the formation of glial barrier.     

Microtransplantation of neural cells into adult rat brain

Epithelioid ("type 1") astrocytes and hippocampal neurons have been stereotaxically grafted into adult rat brain by a new "microtransplantation" method and the results of grafting assessed at various survival times by conventional histological methods using light and electron microscopes. The extent of bleeding that accompanied grafting was assessed by peroxidase staining of extravascular red cells. Grafts were small, compact and reproducible in terms of their volume and location. Implantation was achieved with very little trauma to host tissue. There was no apparent disruption of host tissue adjacent to the graft and very little damage to host blood vessels from earliest survival times. Donor astrocytes were in direct contact with cells of the host immediately after grafting. This situation was compared with conventional implantation of astrocytes in which much damage, necrosis and bleeding occurred and for which cells of graft and host were initially isolated from one another. Microtransplanted astrocytes were observed to migrate. Similar, comparatively non-traumatic microtransplantation of hippocampal neurons was also carried out. Long-term survival of grafted neurons was demonstrated.     

Significance of remyelination by neural stem/progenitor cells transplanted into the injured spinal cord

Previous reports of functional recovery from spinal cord injury (SCI) in rodents and monkeys after the delayed transplantation of neural stem/progenitor cells (NS/PCs) have raised hopes that stem cell therapy could be used to treat SCI in humans. More research is needed, however, to understand the mechanism of functional recovery. Oligodendrocytes derived from grafted NS/PCs remyelinate spared axons in the injured spinal cord. Here, we studied the extent of this remyelination's contribution to functional recovery following contusive SCI in mice. To isolate the effect of remyelination from other possible regenerative benefits of the grafted cells, NS/PCs obtained from myelin-deficient shiverer mutant mice (shi-NS/PCs) were used in this work alongside wild-type NS/PCs (wt-NS/PCs). shi-NS/PCs behaved like wt-NS/PCs in vitro and in vivo, with the exception of their myelinating potential. shi-NS/PC-derived oligodendrocytes did not express myelin basic protein in vitro and formed much thinner myelin sheaths in vivo compared with wt-NS/PC-derived oligodendrocytes. The transplantation of shi-NS/PCs promoted some locomotor and electrophysiological functional recovery but significantly less than that afforded by wt-NS/PCs. These findings establish the biological importance of remyelination by graft-derived cells for functional recovery after the transplantation of NS/PCs into the injured spinal cord.     

Infusion of epidermal growth factor and basic fibroblast growth factor into the striatum of parkinsonian rats leads to in vitro proliferation and differentiation of adult neural progenitor cells

This study investigated the proliferation and differentiation of adult neural progenitor cells (aNPCs) derived from the striatum and substantia nigra (SN) of parkinsonian rats. We found that aNPCs isolated from the two areas of parkinsonian rats readily formed nestin-enriched neurospheres in vitro and exhibited an ability to differentiate into either neurons or astrocytes. Injection of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) into the striatum of parkinsonian rats prior to the harvesting striatal aNPCs significantly increased the neurosphere formation rate and multiple differentiation capacity of these aNPCs when cultured in vitro. These data suggest that striatal and nigral adult NPCs in parkinsonian rats retain the abilities of proliferation and differentiation in vitro. In addition, exogenously applied growth factors could up-regulate the developmental potential of aNPCs. We conclude that our data supports the notion that endogenous cell replacement therapies may be useful for the future treatment of Parkinson's disease (PD).     

Survival of neural progenitor cells from the subventricular zone of the adult rat after transplantation into the host spinal cord of the same strain of adult rat

The subventricular zone (SVZ) of the lateral ventricle of the mammalian forebrain is the major site in which neural progenitor cells (NPC) persist in the adult brain. The NPC are located beneath ventricular ependymal cells and have the capacity to self-renew and continuously produce neurons and glial cells. We have shown previously that neurospheres can be obtained from the brain of deceased adult rats and that neurosphere cells survive after transplantation into the spinal cord. In the present study, we investigated whether fresh NPC from living adult rats can survive and be integrated into host tissues after transplantation into the adult rat spinal cord of the same strain. We used rats expressing transgenic green fluorescent protein (GFP) as a donor to identify the transplanted NPCs. The SVZ tissues were obtained from the striatal wall of the lateral ventricle of adult GFP-rats and were grafted into lesions of the spinal cord at the cervical level. Two to 3 weeks after grafting, NPC migrated through the host tissue 0.5-1 mm away from the implantation site, and were integrated into the white matter of the host spinal cord. Surviving NPC exhibited immunohistochemical phenotypes of astrocytes (glial fibrillary acidic protein), but not for neurons (alpha-tubulin III) or oligodendrocytes (Rip; Hybridoma Bank, Iowa City, IA, USA). Thus, NPC from the SVZ of adult rats can survive and differentiate into at least astrocytes, which can then be integrated into host tissue after transplantation into spinal cord lesions in the adult rat.     

The influence of piezoelectric scaffolds on neural differentiation of human neural stem/progenitor cells

Human neural stem/progenitor cells (hNSCs/NPCs) are a promising cell source for neural tissue engineering because of their ability to differentiate into various neural lineages. In this study, hNSC/NPC differentiation was evaluated on piezoelectric, fibrous scaffolds. These smart materials have an intrinsic material property where transient electric potential can be generated in the material upon minute mechanical deformation. hNSCs/NPCs cultured on the scaffolds and films differentiated into β-III tubulin-positive cells, a neuronal cell marker, with or without the presence of inductive factors. In contrast, hNSCs/NPCs cultured on laminin-coated plates were predominantly nestin positive, a NSC marker, in the control medium. Gene expression results suggest that the scaffolds may have promoted the formation of mature neural cells exhibiting neuron-like characteristics. hNSCs/NPCs differentiated mostly into β-III tubulin-positive cells and had the greatest average neurite length on micron-sized, annealed (more piezoelectric), aligned scaffolds, demonstrating their potential for neural tissue-engineering applications.     

Enhanced axonal growth from fetal human bcl-2 transgenic mouse dopamine neurons transplanted to the adult rat striatum

Embryonic neurons transplanted to the adult CNS extend axons only for a developmentally defined period. There are certain intercellular factors that control the axonal extension, one of which may be the expression of the bcl-2 protein. In this study, rats with complete striatal dopamine fiber denervation received embryonic day 14 mouse ventral mesencephalon cells overexpressing human bcl-2 or control wild-type ventral mesencephalon cells. All rats were treated with cyclosporine to prevent rejection and the surviving grafts were analyzed for cell survival and outgrowth of dopaminergic fibers. The results demonstrate that bcl-2 overexpression does not enhance neuronal graft survival. However, the bcl-2 overexpressing neurons had a higher number of dopaminergic fibers that grew longer distances.These results show that overexpression of bcl-2 can result in longer distance axonal growth of transplanted fetal dopaminergic neurons and that genetic modification of embryonic donor cells may enhance their ability to reinnervate a neuronal target territory.     

Soluble factors from neocortical astrocytes enhance neuronal differentiation of neural progenitor cells from adult rat hippocampus on micropatterned polymer substrates

Rat adult hippocampal progenitor cells (AHPCs) are self-renewing, multipotent neural progenitors that have the ability to differentiate into neurons and glia. Previously, we demonstrated that coculture of AHPCs with postnatal day 2, type 1 cortical astrocytes on laminin-coated micropatterned polymer substrates facilitates selective neuronal differentiation of the AHPCs (Recknor et al., Biomaterials 2006;27:4098-4108). Under this condition, multidimensional cell-cell and/or cell-extracellular matrix interactions, as well as possible soluble factors released from astrocytes provided spatial and temporal control selectively enhancing neuronal differentiation and neurite alignment on topographically different regions of the same substrate. To investigate the potential role of astrocyte-derived soluble factors as cues involved in neuronal differentiation, a noncontact coculture system was used. Under control conditions, approximately 14% of the AHPCs were immunoreactive (IR) for the neuronal marker, class III beta-tubulin (TUJ1-IR). When cocultured in physical contact with astrocytes, neuronal differentiation increased significantly to about 25%, consistent with our previous results. Moreover, under noncontact coculture conditions using Transwell insert cultures, neuronal differentiation was dramatically increased to approximately 64%. Furthermore, neurite outgrowth from neuronal cell bodies was considerably greater on the patterned substrate when compared with the nonpatterned planar substrate under noncontact coculture conditions. Taken together, our results demonstrate that astrocyte-derived soluble factors provide cues for specific neuronal differentiation of AHPCs cultured on micropatterned substrates. In addition, a suppressive influence on neuronal differentiation appears to be mediated by contact with cocultured astrocytes. These results provide important insights into mechanisms for controlling neural progenitor/stem cell differentiation and facilitate development of strategies for CNS repair.     

大鼠骨髓间充质干细胞向多巴胺样神经细胞分化

目的探索大鼠骨髓间充质干细胞向多巴胺能样细胞分化。方法全骨髓培养法分离大鼠骨髓间充质干细胞;体外培养至第3代,用碱性成纤维细胞生长因子,抗坏血酸和表皮生长因子诱导分化;免疫荧光法鉴定胞质中的多巴胺神经元相关蛋白的表达;RT-PCR鉴定多巴胺神经元相关基因的表达;ELISA法检测上清及胞质中的多巴胺。结果诱导后,免疫荧光法检测到诱导后的细胞表达多巴胺神经元相关蛋白:酪氨酸羟化酶、多巴胺转运蛋白和神经核蛋白;RT-PCR检测到诱导后的细胞表达多巴胺神经细胞相关基因TH、AADC;ELISA法检测到诱导后的上清及胞质中有多巴胺分泌。结论间充质干细胞具有向多巴胺能样细胞分化的能力。     

The Notch pathway mediates expansion of a progenitor pool and neuronal differentiation in adult neural progenitor cells after stroke

Molecular mechanisms by which stroke increases neurogenesis have not been fully investigated. Using neural progenitor cells isolated from the subventricular zone (SVZ) of the adult rat subjected to focal cerebral ischemia, we investigated the Notch pathway in regulating proliferation and differentiation of adult neural progenitor cells after stroke. During proliferation of neural progenitor cells, ischemic neural progenitor cells exhibited substantially increased levels of Notch, Notch intracellular domain (NICD), and hairy enhancer of split (Hes) 1, which was associated with a significant increase of proliferating cells. Blockage of the Notch pathway by short interfering ribonucleic acid (siRNA) against Notch or a γ secretase inhibitor significantly reduced Notch, NICD and Hes1 expression and cell proliferation induced by stroke. During differentiation of neural progenitor cells, Notch and Hes1 expression was downregulated in ischemic neural progenitor cells, which was coincident with a significant increase of neuronal population. Inhibition of the Notch pathway with a γ secretase inhibitor further substantially increased neurons, but did not alter astrocyte population in ischemic neural progenitor cells. These data suggest that the Notch signaling pathway mediates adult SVZ neural progenitor cell proliferation and differentiation after stroke.     

Engineering of adult human neural stem cells differentiation through surface micropatterning

Interaction between differentiating neural stem cells and the extracellular environment guides the establishment of cell polarity during nervous system development. Developing neurons read the physical properties of the local substrate in a contact-dependent manner and retrieve essential guidance cues. To restore damage brain area by tissue engineering, the biomaterial scaffold has to mimic this microenvironment to allow organized tissue regeneration. To establish the validity of using microgrooved surfaces in order to simultaneously provide to primary adult human neural stem cells a permissive growth environment and a guide for neurite outgrowth in a pre-established direction, we have studied the long-term culture of adult human neural stem cells from patient biopsies on microgrooved polymers. By exploiting polymer moulding techniques, we engineered non-cytotoxic deep microstructured surfaces of polydimethylsiloxane (PDMS) exhibiting microchannels of various widths. Our results demonstrate that precoated micropatterned PDMS surfaces can serve as effective neurite guidance surfaces for human neural stem cells. Immunocytochemistry analysis show that channel width can impact strongly development and differentiation. In particular we found an optimal microchannel width, that conciliates a high differentiation rate with a pronounced alignment of neurites along the edges of the microchannels. The impact of the microstructures on neurite orientation turned out to be strongly influenced by cell density, attesting that cell/surface interactions at the origin of the alignment effect, are in competition with cell/cell interactions tending to promote interconnected networks of cells. Considering all these effects, we have been able to design appropriate structures allowing to obtain neuron development and differentiation rate comparable to a plane unpatterned surface, with an efficient neurite guidance and a long-term cell viability.