Neural precursor cells differentiated from mouse embryonic stem cells relieve symptomatic motor behavior in a rat model of Parkinson's disease

Pluripotent embryonic stem (ES) cells are the most versatile cells, with the potential to differentiate into all types of cell lineages including neural precursor cells (NPCs), which can be expanded in large numbers for significant periods of time to provide a reliable cell source for transplantation in neurodegenerative disorders such as Parkinson's disease (PD). In the present study, we used the MESPU35 mouse ES cell line, which expresses enhanced green fluorescent protein that enables one to distinguish between transplanted cells and cells of host origin. Embryoid bodies (EBs) were formed and were induced to NPCs in N2 selection medium plus fibronectin. Praxiology and immunohistochemistry methods were used to observe the survival, differentiation, and therapeutic effect of NPCs after grafted into the striatum of PD rats. We found that mouse ESc were differentiated into nestin-positive NPCs 6 days after the EBs formed and cultured in the N2 selection medium. The number of survival NPCs was increased significantly by fibronectin. About 23.76+/-2.29% of remaining cells were tyrosine hydroxylase (TH)-positive 12 days after NPCs were cultured in N2 selective medium. The survival rates of NPCs were 2.10+/-0.41% and about 90.90+/-3.00% of the engrafted NPCs were TH-positive 6 weeks after transplantation into the striatum of PD rats. The rotation of PD rats was relieved 3 weeks after the NPCs transplantation and this effect was kept for at least 6 weeks. It suggests that most of the survival NPCs derived from ES cells differentiated into TH-positive neurons after grafted into the striatum of PD rats, which produces therapeutic effect on PD.     

Neural precursor cells differentiated from mouse embryonic stem cells relieve symptomatic motor behavior in a rat model of Parkinson’s disease

Pluripotent embryonic stem (ES) cells are the most versatile cells, with the potential to differentiate into all types of cell lineages including neural precursor cells (NPCs), which can be expanded in large numbers for significant periods of time to provide a reliable cell source for transplantation in neurodegenerative disorders such as Parkinson’s disease (PD). In the present study, we used the MESPU35 mouse ES cell line, which expresses enhanced green fluorescent protein that enables one to distinguish between transplanted cells and cells of host origin. Embryoid bodies (EBs) were formed and were induced to NPCs in N2 selection medium plus fibronectin. Praxiology and immunohistochemistry methods were used to observe the survival, differentiation, and therapeutic effect of NPCs after grafted into the striatum of PD rats. We found that mouse ESc were differentiated into nestin-positive NPCs 6 days after the EBs formed and cultured in the N2 selection medium. The number of survival NPCs was increased significantly by fibronectin. About 23.76 ± 2.29% of remaining cells were tyrosine hydroxylase (TH)-positive 12 days after NPCs were cultured in N2 selective medium. The survival rates of NPCs were 2.10 ± 0.41% and about 90.90 ± 3.00% of the engrafted NPCs were TH-positive 6 weeks after transplantation into the striatum of PD rats. The rotation of PD rats was relieved 3 weeks after the NPCs transplantation and this effect was kept for at least 6 weeks. It suggests that most of the survival NPCs derived from ES cells differentiated into TH-positive neurons after grafted into the striatum of PD rats, which produces therapeutic effect on PD.     

Integration of Grafted Neural Progenitor Cells in a Host Hippocampal Circuitry after Ischemic Injury

The induction of development of neurons and glial cells from neural progenitor cells (NPCs) is at present considered a promising strategy for recoverу after ischemic insult-evoked damage to the brain. To estimate whether grafted NPCs can develop morphological properties of the mature neurons and become functionally integrated within a host hippocampal circuitry, immunohistochemical approaches at the light and electron microscopy levels have been used. Ischemic insult in FVB-strain mice was evoked by 20-min-long occlusion of both carotid arteries. One day after occlusion, NPCs from GFP-transgenic fetuses were suboccipitally transplanted into the ischemic brain. We found that 44.7 ± 3.8% (mean ± s.e.m) of the grafted GFP-positive cells differentiated 3 months after transplantation into cells demonstrating morphological features of hippocampal pyramidal neurons. Moreover, grafted cells demonstrated manifestations of rather intense formation of the synapses between host and donor neural cells. Thus, our observations show that the NPC-based transplantation approach may be promising in the treatmen t of ischemic insult.     

Bax siRNA promotes survival of cultured and allografted granule cell precursors through blockade of caspase-3 cleavage

Transplantation of neuronal precursor cells (NPCs) into the central nervous system could represent a powerful therapeutical tool against neurodegenerative diseases. Unfortunately, numerous NPCs die shortly after transplantation, predominantly due to caspase-dependent apoptosis. Using a culture of cerebellar neuronal precursors, we have previously demonstrated protective effect of the neuropeptide PACAP, which suppresses ceramide-induced apoptosis by blockade of the mitochondrial apoptotic pathway. The main objective of this study was to determine whether Bax repression can promote survival of NPCs allotransplanted into a host animal. In vivo and ex vivo experiments revealed that C2-ceramide increases Bax expression, while PACAP reverses this effect. In vitro tests using cerebellar NPCs demonstrated that the Bax-specific small interfering RNA (siRNA) could reduce their death and caspase-3 cleavage within the first 24 h. BrdU-labelled NPCs were subjected to transfection procedure with or without siRNA introduction before using for in vivo transplantation. Twenty-four hours after, the allografted NPCs containing siRNA showed significantly reduced level of caspase-3 cleavage, and the volume of their implants was almost twofold higher than in the case of empty-transfected precursors. These data evidence an important role of Bax in life/death decision of grafted NPCs and suggest that RNA interference strategy may be applicable for maintaining NPCs survival within the critical first hours after their transplantation.     

Proliferation and differentiation of nerve cells of the human NN. supraopticus et paraventricularis transplanted to the brain of adult rats]

Human fetal hypothalamic neurosecretory cells (NSC) were studied after transplantation into the third ventricle of the adult rat brain. 3H-thymidine uptake data have shown that the time of origin and proliferation of NSC of grafted tissue and of supraoptic and paraventricular nuclei in normal embryogenesis was similar. Specific staining revealed that the start of neurosecretory function in grafted tissue corresponded to the date when NSC began functioning in normal ontogenesis.     

Trimethyltin Modulates Reelin Expression and Endogenous Neurogenesis in the Hippocampus of Developing Rats

Reelin is an extracellular matrix glycoprotein involved in the modulation of synaptic plasticity and essential for the proper radial migration of cortical neurons during development and for the integration and positioning of dentate granular cell progenitors; its expression is down-regulated as brain maturation is completed. Trimethyltin (TMT) is a potent neurotoxicant which causes selective neuronal death mainly localised in the CA1-CA3/hilus hippocampal regions. In the present study we analysed the expression of reelin and the modulation of endogenous neurogenesis in the postnatal rat hippocampus during TMT-induced neurodegeneration (TMT 6 mg/kg). Our results show that TMT administration induces changes in the physiological postnatal decrease of reelin expression in the hippocampus of developing rats. In particular, quantitative analysis of reelin-positive cells evidenced, in TMT-treated animals, a persistent reelin expression in the stratum lacunosum moleculare of Cornu Ammonis and in the molecular layer of Dentate Gyrus. In addition, a significant decrease in the number of bromodeoxyuridine (BrdU)-labeled newly-generated cells was also detectable in the subgranular zone of P21 TMT-treated rats compared with P21 control animals; no differences between P28 TMT-treated rats and age-matched control group were observed. In addition the neuronal commitment of BrdU-positive cells appeared reduced in P21 TMT-treated rats compared with P28 TMT-treated animals. Thus TMT treatment, administrated during development, induces an early reduction of endogenous neurogenesis and influences the hippocampal pattern of reelin expression in a temporally and regionally specific manner, altering the physiological decrease of this protein.     

神经祖细胞与纤维蛋白支撑物移植对老年痴呆模型鼠的记忆认知功能改善及其机理探究

目的探讨阿尔茨海默病（AD）模型鼠双侧海马区移植含多因子孵育的神经祖细胞（NPCs）后记忆认知功能改善情况及NPCs移植后迁移定位和分化能力。 方法取胎龄10?d的C57BL/6J孕鼠,分离得胎鼠NPCs,NPCs体外分化及鉴定,AD模型鼠分3组：NPCs+因子组、因子组及PBS组,对照组为同月龄C57BL/6J小鼠;Morris水迷宫及新物体识别实验检测AD模型鼠移植NPCs细胞前,及移植1至6个月后记忆行为变化情况;通过免疫荧光,免疫组化和Western-blot检测海马区移植的NPCs向神经元和胆碱能神经元分化及迁移能力。组间比较采用F检验。 结果Morris水迷宫实验中,NPCs+因子组找到平台前的逃避潜伏期时间（14.12±7.45）s要明显低于注射PBS的AD模型鼠组[（39.65±4.64）?s,F = 2.578,P = 0.0094],因子组时间（15.68±5.34）s同样低于PBS组[（39.65±4.64）s,F?= 1.324,P = 0.0016],24 h撤去平台后,NPCs+因子组逃避潜伏期时间（15.12±3.52）s仍低于PBS组[（37.17±2.18）?s,F = 2.598,P = 0.0003],因子组时间（16.62±3.23）s同样低于PBS组[（37.17±2.18）s,F = 2.186,P = 0.0004）];新物体识别实验中,各实验组对新物体探究时间占总探究时间百分比结果中,NPCs+因子组（68.46±2.4）%要高于PBS组[（54.47±4.79）%,F =3.983,P = 0.018],因子组（65.20±1.03）%同样高于PBS组[（54.47±4.79）%,F = 21.63,P = 0.042];实验结果表明,通过移植细胞与因子AD模型鼠的记忆认知功能在早期均得到改善,随着时间的增长,移植NPCs组的记忆改善情况持续时间更长久;Western blot结果显示AD模型鼠海马区胆碱能神经元与正常C57BL/6J鼠相比表达减少,移植NPCs后,AD模型鼠脑内胆碱能神经元增多;免疫荧光与免疫组化结果显示,移植的NPCs在AD模型鼠脑内移植区存活,并向胆碱能神经元分化。 结论AD模型鼠双侧海马区移植的含多因子孵育的NPCs,通过分化成功能性的胆碱能神经元来改善AD鼠的记忆认知功能。     

Elimination of the geomagnetic field stimulates the proliferation of mouse neural progenitor and stem cells

Living organisms are exposed to the geomagnetic field (GMF) throughout their lifespan. Elimination of the GMF, resulting in a hypogeomagnetic field (HMF), leads to central nervous system dysfunction and abnormal development in animals. However, the cellular mechanisms underlying these effects have not been identified so far. Here, we show that exposure to an HMF (<200 nT), produced by a magnetic field shielding chamber, promotes the proliferation of neural progenitor/stem cells (NPCs/NSCs) from C57BL/6 mice. Following seven-day HMF-exposure, the primary neurospheres (NSs) were significantly larger in size, and twice more NPCs/NSCs were harvested from neonatal NSs, when compared to the GMF controls. The self-renewal capacity and multipotency of the NSs were maintained, as HMF-exposed NSs were positive for NSC markers (Nestin and Sox2), and could differentiate into neurons and astrocyte/glial cells and be passaged continuously. In addition, adult mice exposed to the HMF for one month were observed to have a greater number of proliferative cells in the subventricular zone. These findings indicate that continuous HMF-exposure increases the proliferation of NPCs/NSCs,in vitro and in vivo. HMF-disturbed NPCs/ NSCs production probably Affects brain development and function, which provides a novel clue for elucidating the cellular mechanisms of the bio-HMF response.     

Fate and protective effect of marrow stromal cells after subretinal transplantation

Engraftment of marrow stromal cells (MSCs) has been proposed as a therapeutic approach for degenerative diseases. In this study we investigated the fate and dynamic progress of grafted MSCs in living retina with the aim of evaluating the use of transplanted MSCs to treat retinal degeneration. Approximately 1×10⁵ gfp -MSCs in 2 μl phosphate-buffered saline were injected into the subretinal space of adult Sprague-Dawley rats. Two weeks later, approximately 0.174%±0.082% of the transplanted cells had survived and diffused into the subretinal space. Nine weeks after transplantation the surviving gfp -MSCs accounted for 0.049%±0.023% of the number of cells injected and were mainly located at the injection site. The same number of MSCs were transplanted into the left eye subretinal space of 3-week-old hereditary retinal degenerative Royal College of Surgeons rats, and phosphate-buffered saline was injected into their right eyes as a control. Five weeks after transplantation, the amount of rudimentary photo-receptors was more significantly increased in grafted eyes than in control eyes. The results indicated that grafted MSCs could survive and rescue retinal degeneration.     

The frequency of neural stem cells in in vitro culture systems: insights from simple modeling

Neural stem cell (NSC) culture offers a renewable resource for cell replacement treatment of neurodegenerative diseases. In a NSC culture, the frequency of NSCs is estimated to be only a few percent, at most, while the majority of cells are neuroprogenitor cells (NPCs), the progeny of NSCs with a limited capability to proliferate. Here, the effects of several cell culture parameters on the cell composition of NSC cultures were illustrated over time using a simple model. The model shows thatvarious initial cell compositions in NSC cultures converge into a single steady state. The model also implies that the rate of increase in total cell number entirely depends on the self-renewal rate of NSCs, regardless of the proliferative capacity of NPCs. Furthermore, the model predicts that difference sin cell passage methods between neurosphere and monolayer cultures results in a change in the frequency of NSCs in culture.     

ApoTransferrin: dual role on adult subventricular zone-derived neurospheres

Neural stem and progenitor cells (NSC/NPCs) are multipotent self-renewing cells that are able to generate neurons, astrocytes and oligodendrocytes (OLs) within the adult central nervous system. We cultured NSC/NPCs from the rat subventricular zone as neurospheres (NS) and studied apoTransferrin (aTf) effects on oligodendroglial specification and maturation. Our findings suggest that aTf acts at different stages during progression from NSC to mature oligodendrocytes. On the one hand, an early event associated with the activation of NSC/NPCs proliferation and commitment toward the oligodendroglial fate, as indicated by increased BrdU incorporation, larger neurospheres production, and higher ability to generate OL precursors (OPCs) from undifferentiated cultures. On the other hand, aTf exposure during differentiating conditions favours OL maturation from OPCs by promoting OL morphological development. This evidence supports a key role of Tf on the generation of OL from NSC/NPCs and highlights its potential in demyelinating disorder treatment.     

神经干细胞脑内移植后的存活、迁移和分化

从胚胎或成体大鼠脑组织、人胚脑组织均能分离到神经干细胞 ,将它们进行体外原代培养扩增或永生化后植入脑内 ,均能观察到其在脑内的迁移和分化现象。其分化能力主要取决于移植部位的脑内微环境 ,但这种影响作用是相对的。同时 ,体外培养环境如培养时间和细胞融合程度、维甲酸类诱导分化剂处理、NGF转导处理再移植或与嗜铬细胞 (分泌NGF)共移植等 ,也能决定神经干细胞脑内移植后向神经元方向分化的能力。神经干细胞移植为中枢神经系统功能重建和神经再生带来新的希望。     

Treatment of Aganglionic Megacolon Mice via Neural Stem Cell Transplantation

To explore a potential methodology for treating aganglionic megacolon, neural stem cells (NSCs) expressing engineered endothelin receptor type B (EDNRB) and glial cell-derived neurotrophic factor (GDNF) genes were transplanted into the aganglionic megacolon mice. After transplantation, the regeneration of neurons in the colon tissue was observed, and expression levels of differentiation-related genes were determined. Primary culture of NSCs was obtained from the cortex of postnatal mouse brain and infected with recombinant adenovirus expressing EDNRB and GDNF genes. The mouse model of aganglionic megacolon was developed by treating the colon tissue with 0.5 % benzalkonium chloride (BAC) to selectively remove the myenteric nerve plexus that resembles the pathological changes in the human congenital megacolon. The NSCs stably expressing the EDNRB and GDNF genes were transplanted into the benzalkonium chloride-induced mouse aganglionic colon. Survival and differentiation of the implanted stem cells were assessed after transplantation. Results showed that the EDNRB and GDNF genes were able to be expressed in primary culture of NSCs by adenovirus infection. One week after implantation, grafted NSCs survived and differentiated into neurons. Compared to the controls, elevated expression of EDNRB and GDNF was determined in BAC-induced aganglionic megacolon mice with partially improved intestinal function. Those founding indicated that the genes transfected into NSCs were expressed in vivo after transplantation. Also, this study provided favorable support for the therapeutic potential of multiple gene-modified NSC transplantation to treat Hirschsprung’s disease, a congenital disorder of the colon in which ganglion cells are absent.     

Enhanced cell transplantation: preventing apoptosis increases cell survival and ventricular function

Cell transplantation prevents cardiac dysfunction after myocardial infarction. However, because most implanted cells are lost to ischemia and apoptosis, the benefits of cell transplantation on heart function could be improved by increasing cell survival. To examine this possibility, male Lewis rat aortic smooth muscle cells (SMCs; 4 x 10(6)) were pretreated with antiapoptotic Bcl-2 gene transfection or heat shock and then implanted into the infarcted myocardium of anesthetized, syngenic female rats (n = 23 per group). On the first day after transplantation, apoptotic SMCs were quantified by using transferase-mediated dUTP nick-end labeling staining. On days 7 and 28, grafted cell survival was quantified by using real-time PCR, and heart function was assessed with the use of echocardiography and the Langendorff apparatus. SMCs given antiapoptotic pretreatments exhibited improvements in each measure relative to controls. Apoptosis was reduced in Bcl-2-treated cells relative to all other groups (P < 0.05), whereas survival (P < 0.01) was increased. Heat shock also significantly decreased apoptosis and increased survival relative to control groups (P < 0.05 for group effect), although these effects were less pronounced than in the Bcl-2-treated group. Further, scar areas were reduced in both Bcl-2- and heat shock-treated groups relative to controls (P < 0.05), and fractional area change and cardiac function were greater (P < 0.05 for both measures). These results indicate that antiapoptosis pretreatments reduced grafted SMC loss after transplantation and enhanced grafted cell survival and ventricular function, which was directly related (r = 0.72; P = 0.002) to the number of surviving engrafted cells.     

Long-Term Potentiation Promotes Proliferation/Survival and Neuronal Differentiation of Neural Stem/Progenitor Cells

Neural stem cell (NSC) replacement therapy is considered a promising cell replacement therapy for various neurodegenerative diseases. However, the low rate of NSC survival and neurogenesis currently limits its clinical potential. Here, we examined if hippocampal long-term potentiation (LTP), one of the most well characterized forms of synaptic plasticity, promotes neurogenesis by facilitating proliferation/survival and neuronal differentiation of NSCs. We found that the induction of hippocampal LTP significantly facilitates proliferation/survival and neuronal differentiation of both endogenous neural progenitor cells (NPCs) and exogenously transplanted NSCs in the hippocampus in rats. These effects were eliminated by preventing LTP induction by pharmacological blockade of the N-methyl-D-aspartate glutamate receptor (NMDAR) via systemic application of the receptor antagonist, 3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP). Moreover, using a NPC-neuron co-culture system, we were able to demonstrate that the LTP-promoted NPC neurogenesis is at least in part mediated by a LTP-increased neuronal release of brain-derived neurotrophic factor (BDNF) and its consequent activation of tropomysosin receptor kinase B (TrkB) receptors on NSCs. Our results indicate that LTP promotes the neurogenesis of both endogenous and exogenously transplanted NSCs in the brain. The study suggests that pre-conditioning of the host brain receiving area with a LTP-inducing deep brain stimulation protocol prior to NSC transplantation may increase the likelihood of success of using NSC transplantation as an effective cell therapy for various neurodegenerative diseases.     

Dynamic changes in connexin expression following engraftment of neural stem cells to striatal tissue

Gap-junctional intercellular communication between grafted neural stem cells (NSCs) and host cells seem to be essential for many of the beneficial effects associated with NSC engraftment. Utilizing murine NSCs (mNSCs) grafted into an organotypic ex vivo model system for striatal tissue we examined the prerequisites for formation of gap-junctional couplings between graft and host cells at different time points following implantation. We utilized flow cytometry (to quantify the proportion of connexin (Cx) 26 and 43 expressing cells), immunohistochemistry (for localization of the gap-junctional proteins in graft and host cells), dye-transfer studies with and without pharmacological gap-junctional blockers (assaying the functionality of the formed gap-junctional couplings), and proliferation assays (to estimate the role of gap junctions for NSC well-being) to this end.Immunohistochemical staining and dye-transfer studies revealed that the NSCs already form functional gap junctions prior to engraftment, thereby creating a substrate for subsequent graft and host communication. The expression of Cx43 by grafted NSCs was decreased by neurotrophin-3 overexpression in NSCs and culturing of grafted tissue in serum-free Neurobasal B27 medium. Cx43 expression in NSC-derived cells also changed significantly following engraftment. In host cells the expression of Cx43 peaked following traumatic stimulation and then declined within two weeks, suggesting a window of opportunity for successful host cell rescue by NSC engraftment.Further investigation of the dynamic changes in gap junction expression in graft and host cells and the associated variations in intercellular communication between implanted and endogenous cells might help to understand and control the early positive and negative effects evident following neural stem cell transplantation and thereby optimize the outcome of future clinical NSC transplantation therapies.     

Effects of human cultural neuronal and mesenchymal stem cells on the rat learning and brain state after acute hypoxia

The aim of the study was to compare the effects of neurotransplantation of cultural neural stem cells (NSC) and mesenchymal stem cells (MSC) on the rat behaviour and brain state after acute hypoxia. It was shown that development of two-way avoidance defensive conditioning in a shuttle box improved in rats-recipients with NSC, but not MSC as compared to control. Both the transplants of NSC and transplants of MSC exert neuroprotective influence on the rat brain. NSC both in vitro (before transplantation) and in vivo (on day 27 after transplantation) gave rise to all neural cell types: stem/progenitor cells, precursors of neurons and glia, neurons and glial cells. MSC population in vitro and in vivo (on day 10 after transplantation) consisted of fibroblast-like cells which were eliminated by day 20 after transplantation and were surrounded by reactive glia. We suggest that effects of NSC may be connected with their good survival and potential to differentiate into neurons and with trophic influence on the brain of recipient, whereas MSC only have possible positive trophic effect at early stages after transplantation.     

Mesenchymal cells from human amniotic fluid survive and migrate after transplantation into adult rat brain

Amniotic fluid has been recently suggested as an alternative source of mesenchymal stem cells. However, the fate of amniotic fluid-derived mesenchymal stem cells (AF-MSCs) after in vivo transplantation has yet to be determined. In the present study we explored whether human AF-MSCs could survive and migrate following transplantation into the striatum of normal and ischemic rat. We found that the grafted cells could survive and migrate towards multiple brain regions in the normal animals, while they moved towards the injured region in the ischemic rat. Double-immunostaining analyses showed that the implanted human AF-MSCs express markers for immature neurons (Doublecortin) at 10 days, and for astrocytes (GFAP) at 10, 30 and 90 after transplantation. This study provides the first evidence that human amniotic fluid contains cells having the potential to survive and integrate into adult rat brain tissue and, therefore, to function as effective stem cells for therapeutic strategies.