Transfection of the glial cell line-derived neurotrophic factor gene promotes neuronal differentiation简

Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro- tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su- pernatant were significantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes- enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein743 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen- chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.     

底蜕膜间充质干细胞分化为多巴胺能样神经元

目的 探讨人胎盘底蜕膜间充质干细胞体外向多巴胺能样神经元分化的潜能,并优化诱导方案.方法 体外分离培养底蜕膜间充质干细胞,用表皮生长因子(EGF)+人碱性成纤维细胞生长因子(bFGF)+ B27添加剂和人音猬因子(SHH)+成纤维细胞生长因子8(FGF8)+forskolin+脑源性神经营养因子(BDNF)分两个阶段对其进行诱导;免疫细胞化学先后检测干细胞标记nestin和CD133、成熟神经元标记神经元特异性烯醇化酶(NSE)、神经胶质细胞标记胶质原纤维酸性蛋白(GFAP)、多巴胺能神经元标记酪氨酸羟化酶(TH)的表达;Western blot验证诱导后TH蛋白的表达;高效液相色谱-电化学检测诱导前后多巴胺的分泌.结果 经第一阶段诱导后,细胞形成漂浮生长的神经球,nestin和CD133均呈阳性表达;第二阶段诱导后,出现明显的神经元样形态,NSE、GFAP和TH均阳性表达,Western blot也显示TH蛋白的表达,多巴胺分泌量相比诱导前明显增加(P＜0.001).结论 底蜕膜间充质干细胞体外可分化为多巴胺能样神经元,可能成为帕金森病干细胞移植治疗新的种子细胞来源.     

In vitro differentiation of neural stem cells derived from human olfactory bulb into dopaminergic‐like neurons

This study describes a new accessible source of neuronal stem cells that can be used in Parkinson's disease cell transplant. The human olfactory bulb contains neural stem cells (NSCs) that are responsible for neurogenesis in the brain and the replacement of damaged cellular components throughout life. NSCs are capable of differentiating into neuronal and glial cells. We isolated NSCs from the olfactory bulb of brain‐death donors and differentiated them into dopaminergic neurons. The olfactory bulb tissues obtained were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F12, B27 supplemented with basic fibroblast growth factor, epidermal growth factor and leukemia inhibitory factor. The NSCs and proliferation markers were assessed. The multipotentiality of olfactory bulb NSCs was demonstrated by their capacity to differentiate into neurons, oligodendrocytes and astrocytes. To generate dopaminergic neurons, olfactory bulb NSCs were differentiated in neurobasal medium, supplemented with B27, and treated with sonic hedgehog, fibroblast growth factor 8 and glial cell‐derived neurotrophic factor from the 7th to the 21st day, followed by detection of dopaminergic neuronal markers including tyrosine hydroxylase and aromatic l ‐amino acid decarboxylase. The cells were expanded, established in continuous cell lines and differentiated into the two classical neuronal phenotypes. The percentage of co‐positive cells (microtubule‐associated protein 2 and tyrosine hydroxylase; aromatic l‐amino acid decarboxylase and tyrosine hydroxylase) in the treated cells was significantly higher than in the untreated cells. These results illustrate the existence of multipotent NSCs in the adult human olfactory bulb that are capable of differentiating toward putative dopaminergic neurons in the presence of trophic factors. Taken together, our data encourage further investigations of the possible use of olfactory bulb NSCs as a promising cell‐based therapeutic strategy for Parkinson's disease.     

Human umbilical cord mesenchymal stem cells promote peripheral nerve repair via paracrine mechanisms

Human umbilical cord-derived mesenchymal stem cells(h UCMSCs) represent a promising young-state stem cell source for cell-based therapy. h UCMSC transplantation into the transected sciatic nerve promotes axonal regeneration and functional recovery. To further clarify the paracrine effects of h UCMSCs on nerve regeneration, we performed human cytokine antibody array analysis, which revealed that h UCMSCs express 14 important neurotrophic factors. Enzyme-linked immunosorbent assay and immunohistochemistry showed that brain-derived neurotrophic factor, glial-derived neurotrophic factor, hepatocyte growth factor, neurotrophin-3, basic fibroblast growth factor, type I collagen, fibronectin and laminin were highly expressed. Treatment with h UCMSC-conditioned medium enhanced Schwann cell viability and proliferation, increased nerve growth factor and brain-derived neurotrophic factor expression in Schwann cells, and enhanced neurite growth from dorsal root ganglion explants. These findings suggest that paracrine action may be a key mechanism underlying the effects of h UCMSCs in peripheral nerve repair.     

音猬因子诱导恒河猴间充质干细胞向神经样细胞的分化**☆◆

背景：骨髓间充质干细胞诱导分化为神经细胞，是神经系统疾病细胞治疗的有效手段，但目前的时效尚不完善。 目的：应用神经发育音猬因子诱导恒河猴骨髓间充质干细胞向神经样细胞分化。 方法：应用经典的维甲酸方案与音猬因子方案两种方法诱导恒河猴骨髓间充质干细胞分化为神经样细胞，采用密度梯度离心法分离培养恒河猴骨髓间充质干细胞，倒置相差显微镜观察生长情况，MTT法测定细胞生长曲线，流式细胞仪鉴定细胞表型，免疫组织化学鉴定分化细胞标志，透射电镜和扫描电镜观察分化细胞超微结构。 结果与结论：体外分离培养的恒河猴骨髓间充质干细胞，经流式细胞仪表型鉴定，具有较高均一性。通过音猬因子诱导方案诱导处理7 d后，分化细胞多数表现为NSE、NF-M、Tau和Nestin染色阳性，经图像统计分析发现经音猬因子诱导方案神经干细胞标志物Nestin阳性率显著高于维甲酸诱导方案(P < 0.01)，另一方面经维甲酸诱导方案诱导的细胞表现GFAP阳性率高于音猬因子诱导方案，差异具有显著性意义(P < 0.05)。提示音猬因子诱导方案是一种诱导恒河猴骨髓间充质干细胞向神经样细胞分化的有效途径。     

细胞密度与骨髓间充质干细胞向神经元样细胞的分化

景：细胞种植密度是影响干细胞分化的因素之一,对于细胞种植密度在骨髓间充质干细胞向神经元样细胞分化过程中的作用尚缺乏深入研究。 目的：观察细胞种植密度对骨髓间充质干细胞诱导向神经元样细胞分化的影响。 方法：采用贴壁培养法分离大鼠骨髓间充质干细胞,传至第4代后将其按2×102,2×103,4×103,8×103,2×104,4×104/cm2种植于六孔板,每组均加入碱性成纤维细胞生长因子+表皮生长因子+维甲酸诱导向神经元样细胞分化,并通过免疫组织化学染色鉴定,计算每组细胞出现神经元样细胞的比例,比较各组的分化率。 结果与结论：各组骨髓间充质干细胞加入诱导剂后均出现神经元样细胞,Nestin、NSE、GFAP细胞化学染色呈阳性。不同种植密度组出现神经元样细胞比例不同,以8×103/cm2组神经元样细胞比例最高,且神经元样存活时间最长,达7 d。结果说明骨髓间充质干细胞向神经元样细胞分化与细胞接种密度有关,过高或过低细胞密度均不利分化。     

Influence of endogenous ciliary neurotrophic factor on neural differentiation of adult rat hippocampal progenitors

Ciliary neurotrophic factor is the only known neurotrophic factor that can promote differentiation of hippocampal neural progenitor cells to glial cells and neurons in adult rats. This process is similar to spontaneous differentiation. Therefore, ciliary neurotrophic factor may be involved in spontaneous differentiation of neural stem cells. To verify this hypothesis, the present study isolated neural progenitor cells from adult male rats and cultured them in vitro. Results showed that when neural progenitor cells were cultured in the absence of mitogen fibroblast growth factor-2 or epidermal growth factor, they underwent spontaneous differentiation into neurons and glial cells. Western blot and immunocytochemical staining showed that exogenous ciliary neurotrophic factor strongly induced adult hippocampal progenitor cells to differentiate into neurons and glial cells. Moreover, passage 4 adult hippocampal progenitor cells expressed high levels of endogenous ciliary neurotrophic factor, and a neutralizing antibody against ciliary neurotrophic factor prevented the spontaneous neuronal and glial differentiation of adult hippocampal progenitor cells. These results suggest that the spontaneous differentiation of adult hippocampal progenitor cells is mediated partially by endogenous ciliary neurotrophic factor.     

Effects of lateral ventricular transplantation of bone marrow-derived mesenchymal stem cells modified with brain-derived neurotrophic factor gene on cognition in a rat model of Alzheimer's disease

In the present study, transplantation of bone marrow-derived mesenchymal stem cells modified with brain-derived neurotrophic factor gene into the lateral ventricle of a rat model of Alzheimer’s disease, resulted in significant attenuation of nerve cell damage in the hippocampal CA1 region. Furthermore, brain-derived neurotrophic factor and tyrosine kinase B mRNA and protein levels were significantly increased, and learning and memory were significantly improved. Results indicate that transplantation of bone marrow-derived mesenchymal stem cells modified with brain-derived neurotrophic factor gene can significantly improve cognitive function in a rat model of Alzheimer’s disease, possibly by increasing the levels of brain-derived neurotrophic factor and tyrosine kinase B in the hippocampus.     

Peripheral glial cell differentiation from neurospheres derived from adipose mesenchymal stem cells

Mesenchymal stem cells derived from bone marrow and adipose tissue are being considered for use in neural repair because they can differentiate after appropriate induction in culture into neurons and glia. The question we asked was if neurospheres could be harvested from adipose-derived stem cells and if they then could differentiate in culture to peripheral glial-like cells. Here, we demonstrate that adipose-derived mesenchymal stem cells can form nestin-positive non-adherent neurosphere cellular aggregates when cultured with basic fibroblast growth factor and epidermal growth factor. Dissociation of these neurospheres and removal of mitogens results in expression of the characteristic Schwann cell markers S100 and p75 nerve growth factor receptor and GFAP. The simultaneous expression of these glia markers are characteristic features of Schwann cells and olfactory ensheathing cells which have unique properties regarding remyelination and enhancement of axonal regeneration. When co-cultured with dorsal root ganglion neurons, the peripheral glial-like cells derived from adipose mesenchymal stem cells aligned with neuritis and stimulated neuritic outgrowth. These results indicate that neurospheres can be generated from adipose-derived mesenchymal stem cells, and upon mitogen withdrawal can differentiate into peripheral glial cells with neurotrophic effects.     

丹参诱导大鼠骨髓间充质干细胞分化为神经元样细胞中的钙离子检测

目的 检测大鼠骨髓间充质干细胞经丹参注射液诱导分化的神经元样细胞内钙离子浓度,以期为骨髓间充质干细胞应用于神经系统疾病的治疗提供理论依据.方法 从成年大鼠骨髓中获取骨髓间充质干细胞,体外扩增培养,经碱性成纤维生长因子预诱导后施加10mL/L丹参注射液于骨髓间充质干细胞培养液中.运用免疫荧光检测神经元特异性核蛋白(NeuN)在诱导后细胞与经新生大鼠海马获取的体外培养海马神经元中的表达.激光共聚焦技术检测诱导后的细胞内钙离子浓度.并与原代培养海马神经元内的钙离子浓度进行比较.结果 大鼠骨髓间充质干细胞经碱性成纤维生长因子和丹参注射液处理后,可表达NeuN,并具有神经元样的表型.诱导分化的神经元样细胞内钙离子浓度为984.75±79.51,原代培养海马神经元内钙离子浓度为769.42±60.93,两者比较差异无统计学意义(P>0.05).结论 丹参注射液诱导骨髓间充质干细胞分化的神经元样细胞具有神经元的某些特征.     

Transplantation of autologous bone marrow-derived mesenchymal stem cells for traumatic brain injury

Results from the present study demonstrated that transplantation of autologous bone marrow-derived mesenchymal stem cells into the lesion site in rat brain significantly ameliorated brain tissue pathological changes and brain edema, attenuated glial cell proliferation, and increased brain-derived neurotrophic factor expression. In addition, the number of cells double-labeled for 5-bromodeoxyuridine/glial fibrillary acidic protein and cells expressing nestin increased. Finally, blood vessels were newly generated, and the rats exhibited improved motor and cognitive functions. These results suggested that transplantation of autologous bone marrow-derived mesenchymal stem cells promoted brain remodeling and improved neurological functions following traumatic brain injury.     

成人骨髓间充质干细胞的体外培养和定向神经分化

目的 探讨成人骨髓间充质干细胞(MSCs)的体外培养和定向神经诱导分化的条件.方法 从正常成人志愿者髂骨中分离获取MSCs,体外培养扩增纯化后传代于塑料培养皿中,以含有脑源性生长因子(BDNF)联合维A酸(RA)的培养液对传至3～5代的细胞进行体外诱导分化,采用免疫细胞化学法对诱导后的细胞鉴定.结果 诱导1h有部分细胞表达神经干细胞标志巢蛋白nestin,6h后大部分细胞具有典型神经元形态.表达神经元特异性烯醇化酶(NSE)阳性细胞占细胞总数的(46.45±2.54)%.结论 MSCs可通过体外培养并纯化,应用BDNF联合RA可以在体外诱导MSCs成为神经元样细胞.     

Combining acellular nerve allografis with brain- derived neurotrophic factor transfected bone marrow mesenchymal stem cells restores sciatic nerve injury better than either intervention alone

In this study, we chemically extracted acellular nerve allografts from bilateral sciatic nerves, and repaired 10-mm sciatic nerve defects in rats using these grafts and brain-derived neurotrophic factor transfected bone marrow mesenchymal stem cells. Experiments were performed in three groups： the acellular nerve allograft bridging group, acellular nerve allograft ＋ bone marrow mesenchymal stem cells group, and the acellular nerve allograft ＋ brain-derived neurotrophic factor transfected bone marrow mesenchyrnal stem cells group. Results showed that at 8 weeks after bridging, sciatic functional index, triceps wet weight recovery rate, myelin thickness, and number of myelinated nerve fibers were significantly changed in the three groups. Variations were the largest in the acellular nerve allograft ＋ brain-derived neurotrophic factor transfected bone marrow mesenchymal stem cells group compared with the other two groups. Experimental findings suggest that chemically extracted acellular nerve allograft combined nerve factor and mesenchymal stem cells can promote the restoration of sciatic nerve defects. The repair effect seen is better than the single application of acellular nerve allograft or acellular nerve allograft combined mesenchymal stem cell transplantation.     

Acetylcholine secretion by motor neuron-like cells from umbilical cord mesenchymal stem cells

Umbilical cord mesenchymal stem cells were isolated by a double enzyme digestion method.The third passage of umbilical cord mesenchymal stem cells was induced with heparin and/or basic fibroblast growth factor.Results confirmed that cell morphology did not change after induction with basic fibroblast growth factor alone.However,neuronal morphology was visible,and microtubule-associated protein-2 expression and acetylcholine levels increased following induction with heparin alone or heparin combined with basic fibroblast growth factor.Hb9 and choline acetyltransferase expression was high following inductive with heparin combined with basic fibroblast growth factor.Results indicate that the inductive effect of basic fibroblast growth factor alone was not obvious.Heparin combined with basic fibroblast growth factor noticeably promoted the differentiation of umbilical cord mesenchymal stem cells into motor neuron-like cells.Simultaneously,umbilical cord mesenchymal stem cells could secrete acetylcholine.     

Overexpression of microRNA-124 promotes the neuronal differentiation of bone marrow-derived mesenchymal stem cells

microRNAs(miRNAs) play an important regulatory role in the self-renewal and differentiation of stem cells. In this study, we examined the effects of miRNA-124(miR-124) overexpression in bone marrow-derived mesenchymal stem cells. In particular, we focused on the effect of overexpression on the differentiation of bone marrow-derived mesenchymal stem cells into neurons. First, we used GeneChip technology to analyze the expression of miRNAs in bone marrow-derived mesenchymal stem cells, neural stem cells and neurons. miR-124 expression was substantially reduced in bone marrow-derived mesenchymal stem cells compared with the other cell types. We constructed a lentiviral vector overexpressing miR-124 and transfected it into bone marrow-derived mesenchymal stem cells. Intracellular expression levels of the neuronal early markers β-III tubulin and microtubule-associated protein-2 were significantly increased, and apoptosis induced by oxygen and glucose deprivation was reduced in transfected cells. After miR-124-transfected bone marrow-derived mesenchymal stem cells were transplanted into the injured rat spinal cord, a large number of cells positive for the neuronal marker neurofilament-200 were observed in the transplanted region. The Basso-Beattie-Bresnahan locomotion scores showed that the motor function of the hind limb of rats with spinal cord injury was substantially improved. These results suggest that miR-124 plays an important role in the differentiation of bone marrow-derived mesenchymal stem cells into neurons. Our findings should facilitate the development of novel strategies for enhancing the therapeutic efficacy of bone marrow-derived mesenchymal stem cell transplantation for spinal cord injury.     

Neuronal-like cell differentiation of non-adherent bone marrow cell-derived mesenchymal stem cells*

Non-adherent bone marrow cell-derived mesenchymal stem cells from C57BL/6J mice were separated and cultured using the "pour-off" method.Non-adherent bone marrow cell-derived mesenchymal stem cells developed colony-forming unit-fibroblasts,and could be expanded by supplementation with epidermal growth factor.Immunocytochemistry showed that the non-adherent bone marrow cell-derived mesenchymal stem cells exposed to basic fibroblast growth factor/epidermal growth factor/nerve growth factor expressed the neuron specific markers,neurofilament-200 and NeuN,in vitro.Non-adherent bone marrow cell-derived mesenchymal stem cells from β-galactosidase transgenic mice were also transplanted into focal ischemic brain (right corpus striatum) of C57BL/6J mice.At 8 weeks,cells positive for LacZ and β-galactosidase staining were observed in the ischemic tissues,and cells co-labeled with both β-galactosidase and NeuN were seen by double immunohistochemical staining.These findings suggest that the non-adherent bone marrow cell-derived mesenchymal stem cells could differentiate into neuronal-like cells in vitro and in vivo.     

Umbilical cord-derived mesenchymal stem cells retain immunomodulatory and anti-oxidative activities after neural induction

The immunomodulatory and anti-oxidative activities of differentiated mesenchymal stem cells contribute to their therapeutic efficacy in cell-replacement therapy. Mesenchymal stem cells were isolated from human umbilical cord and induced to differentiate with basic fibroblast growth factor, nerve growth factor, epidermal growth factor, brain-derived neurotrophic factor and forskolin. The mesenchymal stem cells became rounded with long processes and expressed the neural markers, Tuj1, neurofilament 200, microtubule-associated protein-2 and neuron-specific enolase. Nestin expression was significantly reduced after neural induction. The expression of immunoregulatory and anti-oxidative genes was largely unchanged prior to and after neural induction in mesenchymal stem cells. There was no significant difference in the effects of control and induced mesenchymal stem cells on lymphocyte proliferation in co-culture experiments. However, the expression of human leukocyte antigen-G decreased significantly in induced neuron-like cells. These results suggest that growth factor-based methods enable the differentiation of mesenchymal stem cell toward immature neuronal-like cells, which retain their immunomodulatory and anti-oxidative activities.     

Repair of peripheral nerve defects with chemically extracted acellular nerve allografts loaded with neurotrophic factors-transfected bone marrow mesenchymal stem cells

Chemically extracted acellular nerve allografts loaded with brain-derived neurotrophic factor-transfected or ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells have been shown to repair sciatic nerve injury better than chemically extracted acellular nerve allografts alone, or chemically extracted acellular nerve allografts loaded with bone marrow mesenchymal stem cells. We hypothesized that these allografts compounded with both brain-derived neurotrophic factor- and ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells may demonstrate even better effects in the repair of peripheral nerve injury. We cultured bone marrow mesenchymal stem cells expressing brain-derived neurotrophic factor and/or ciliary neurotrophic factor and used them to treat sciatic nerve injury in rats. We observed an increase in sciatic functional index, triceps wet weight recovery rate, myelin thickness, number of myelinated nerve fibers, amplitude of motor-evoked potentials and nerve conduction velocity, and a shortened latency of motor-evoked potentials when allografts loaded with both neurotrophic factors were used, compared with allografts loaded with just one factor. Thus, the combination of both brain-derived neurotrophic factor and ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells can greatly improve nerve injury.