脑脊液中细胞源性囊泡对间充质干细胞向神经前体细胞分化的影响

目的探讨脑脊液微囊泡在间充质干细胞(MSCs)向神经干细胞分化中的作用。方法从人脑脊液中分离微囊泡,从小鼠骨髓中分离原代MSCs。将MSCs传代后在神经分化诱导培养基和脑脊液微囊泡作用下神经分化诱导。细胞形态学观察细胞突起数目和长度,western blot检测细胞神经特异性蛋白表达水平变化。结果单纯神经诱导液组单位面积内细胞突起长度为(2.2±0.4)mm,突起数量为(94±12)个;神经诱导液加脑脊液微囊泡诱导的细胞中细胞突起更长(5.7±1.2)mm,数目也更多(178.2±32)个,差异具有统计学意义(P0.01)。微囊泡组细胞中NSE、Nestin和MAP-2表达水平高于单纯神经诱导液组细胞,差异具有统计学意义(P0.01)。结论脑脊液微囊泡可有效促进MSCs向神经干细胞分化,有望为干细胞移植治疗神经功能缺损提供了一个新的思路。     

An effective inducer of dopaminergic neuron-like differentiation

Rat bone marrow-derived mesenchymal stem cells were cultured and passaged in vitro. After induction with basic fibroblast growth factor for 24 hours, passage 3 bone marrow-derived mesenchymal stem cells were additionally induced into dopaminergic neurons using three different combinations with basic fibroblast growth factor as follows: 20% Xiangdan injection; all-trans retinoic acid + glial-derived neurotrophic factor; or sonic hedgehog + fibroblast growth factor 8. Results suggest that the bone marrow-derived mesenchymal stem cells showed typical neuronal morphological characteristics after induction. In particular, after treatment with sonic hedgehog + fibroblast growth factor 8, the expressions of nestin, neuron-specific enolase, microtubule- associated protein 2, tyrosine hydroxylase and vesicular monoamine transporter-2 in cells were significantly increased. Moreover, the levels of catecholamines in the culture supernatant were significantly increased. These findings indicate that Xiangdan injection, all-trans retinoic acid + glial-derived neurotrophic factor, and sonic hedgehog + fibroblast growth factor 8 can all induce dopaminergic neuronal differentiation from bone marrow-derived mesenchymal stem cells. In particular, the efficiency of sonic hedgehog + fibroblast growth factor 8 was highest.     

Human umbilical cord blood-derived mesenchymal stem cells promote regeneration of crush-injured rat sciatic nerves

Several studies have demonstrated that human umbilical cord blood-derived mesenchymal stem cells can promote neural regeneration following brain injury. However, the therapeutic effects of human umbilical cord blood-derived mesenchymal stem cells in guiding peripheral nerve regeneration remain poorly understood. This study was designed to investigate the effects of human umbilical cord blood-derived mesenchymal stem cells on neural regeneration using a rat sciatic nerve crush injury model. Human umbilical cord blood-derived mesenchymal stem cells (1 × 10 6 ) or a PBS control were injected into the crush-injured segment of the sciatic nerve. Four weeks after cell injection, brain-derived neurotrophic factor and tyrosine kinase receptor B mRNA expression at the lesion site was increased in comparison to control. Furthermore, sciatic function index, Fluoro Gold-labeled neuron counts and axon density were also significantly increased when compared with control. Our results indicate that human umbilical cord blood-derived mesenchymal stem cells promote the functional recovery of crush-injured sciatic nerves.     

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.     

Brain-derived neurotrophic factor stimulates the neural differentiation of human umbilical cord blood-derived mesenchymal stem cells and survival of differentiated cells through MAPK/ERK and PI3K/Akt-dependent signaling pathways

Brain-derived neurotrophic factor (BDNF) plays an important role in the differentiation, development, and survival of neural stem cells. In this study, we analyzed its effects on the stimulation of human umbilical cord blood-derived mesenchymal stem cells in terms of their potential to differentiate into neuron-like cells, their survival characteristics, and the molecular mechanisms involved. The treatment of cells with neural induction medium (NIM) and BDNF generated more cells that were neuron-like and produced stronger expression of neural-lineage markers than cells treated with NIM and without BDNF. Raf-1 and ERK phosphorylation and p35 expression levels increased significantly in cells treated with both NIM and BDNF. This treatment also effectively blocked cell death following neural induction and increased Akt phosphorylation and Bcl2 expression compared with cells treated with NIM without BDNF. Inhibition of ERKs inhibited the BDNF-stimulated up-regulation of p35 and Bcl2. In addition, the inhibition of PI3K abrogated Akt phosphorylation and Bcl2 expression, but not p35 expression. Thus, MAPK/ERK-dependent p35 up-regulation and MAPK/ERK-dependent and PI3K/Akt-dependent Bcl2 up-regulation contribute to BDNF-stimulated neural differentiation and to the survival of differentiated cells.     

Transplantation of placenta-derived mesenchymal stem cell-induced neural stem cells to treat spinal cord injury

Because of their strong proliferative capacity and multi-potency, placenta-derived mesenchymal stem cells have gained interest as a cell source in the field of nerve damage repair. In the present study, human placenta-derived mesenchymal stem ceils were induced to differentiate into neural stem cells, which were then transplanted into the spinal cord after local spinal cord injury in rats. The motor functional recovery and pathological changes in the injured spinal cord were observed for 3 successive weeks. The results showed that human placenta-derived mesenchymal stem cells can differentiate into neuron-like cells and that induced neural stem cells contribute to the restoration of injured spinal cord without causing transplant rejection. Thus, these cells promote the recovery of motor and sensory functions in a rat model of spinal cord injury. Therefore, human placenta-derived mesenchymal stem cells may be useful as seed cells during the repair of spinal cord injury.     

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.     

人脐带间充质干细胞向神经细胞分化过程中ILK的表达变化及意义

目的 研究在人脐带间充质干细胞(hUCMSC)向神经干细胞样细胞(hucNSC)、神经细胞(Nc)诱导分化中整合素连接激酶(ILK)的表达变化及意义.方法 将体外培养、传至第3代的hUCMSC,用无血清培养基加入bFGF、EGF和B27预诱导分化为hucNSC,继之将其用BHA、弗司扣林等向神经细胞定向诱导分化,并用AF488分别标记hUCMSC、hucNSC、NC细胞骨架变化过程加以证实,免疫组化及RT-PCR检测ILK在hUCMSC、hucNSC、NC中的表达,并用实时定量PCR加以定量分析.结果 hUCMSC被诱导后首先聚集成细胞球样悬浮生长,表达nestin、CD133,ILK表达明显增强;再被诱导后表达NSE、TH,GFAP,并持续表达ILK.hUCMSC细胞骨架呈丝状规律排列,hucNSC时则成巢状,定向诱导后的神经细胞骨架转变成多突起的树枝状.实时定量PCR检测ILK在hUCMSC中微量表达,hucNSC中表达明显上调,是hUCMSC表达水平的12倍;在NC中ILK表达较hucNSC降低,但仍高于hUCMSC 8.6倍(P＜0.05).结论 人脐带间充质干细胞向神经干细胞样细胞、神经细胞诱导分化及细胞形态重塑中ILK可能发挥重要作用.     

Effects of cytokines and chemokines on migration of mesenchymal stem cells following spinal cord injury

We investigated the effects of cytokines and chemokines and their associated signaling pathways on mesenchymal stem cell migration after spinal cord injury, to determine their roles in the curative effects of mesenchymal stem cells. This study reviewed the effects of tumor necrosis factor-α, vascular endothelial growth factor, hepatocyte growth factor, platelet-derived growth factor, basic fibroblast growth factor, insulin like growth factor-1, stromal cell-derived factor and monocyte chemoattractant protein-1, 3 during mesenchymal stem cell migration to damaged sites, and analyzed the signal transduction pathways involved in their effects on mesenchymal stem cell migration. The results confirmed that phosphatidylinositol 3-kinase/serine/threonine protein kinases and nuclear factor-κB play crucial roles in the migration of mesenchymal stem cells induced by cytokines and chemokines.     

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.     

Differentiation of mesenchymal stem cells into neuronal cells on fetal bovine acellular dermal matrix as a tissue engineered nerve scaffold

The purpose of this study was to assess fetal bovine acellular dermal matrix as a scaffold for supporting the differentiation of bone marrow mesenchymal stem cells into neural cells following induction with neural differentiation medium.We performed long-term,continuous observation of cell morphology,growth,differentiation,and neuronal development using several microscopy techniques in conjunction with immunohistochemistry.We examined specific neuronal proteins and Nissl bodies involved in the differentiation process in order to determine the neuronal differentiation of bone marrow mesenchymal stem cells.The results show that bone marrow mesenchymal stem cells that differentiate on fetal bovine acellular dermal matrix display neuronal morphology with unipolar and bi/multipolar neurite elongations that express neuronal-specific proteins,including βIII tubulin.The bone marrow mesenchymal stem cells grown on fetal bovine acellular dermal matrix and induced for long periods of time with neural differentiation medium differentiated into a multilayered neural network-like structure with long nerve fibers that was composed of several parallel microfibers and neuronal cells,forming a complete neural circuit with dendrite-dendrite to axon-dendrite to dendrite-axon synapses.In addition,growth cones with filopodia were observed using scanning electron microscopy.Paraffin sectioning showed differentiated bone marrow mesenchymal stem cells with the typical features of neuronal phenotype,such as a large,round nucleus and a cytoplasm full of Nissl bodies.The data suggest that the biological scaffold fetal bovine acellular dermal matrix is capable of supporting human bone marrow mesenchymal stem cell differentiation into functional neurons and the subsequent formation of tissue engineered nerve.     

Directional induction of dopaminergic neurons from neural stem cells using substantia nigra homogenates and basic fibroblast growth factor

To date, complex components of available reagents have been used for directional induction of neural stem cells into dopaminergic neurons, resulting in a poor ability to repeat experiments. This study sought to investigate whether a homogenate of the substantia nigra of adult rats and/or basic fibroblast growth factor could directionally induce neural stem cells derived from the subventricular zone of embryonic rats to differentiate into dopaminergic neurons. Tyrosine hydroxylase-positive cells were observed exclusively after induction with the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor for 48 hours in vitro. However, in the groups treated with homogenate supernatant or basic fibroblast growth factor alone, tyrosine hydroxylase expression was not observed. Moreover, the content of dopamine in the culture medium of subventricular zone neurons was significantly increased at 48 hours after induction with the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor. Experimental findings indicate that the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor could directionally induce neural stem cells derived from the subventricular zone of embryonic rats to differentiate into dopaminergic neurons in the substantia nigra with the ability to secrete dopamine.     

Analysis of neural potential of human umbilical cord blood-derived multipotent mesenchymal stem cells in response to a range of neurogenic stimuli

We investigated the neurogenic potential of full-term human umbilical cord blood (hUCB)-derived multipotent mesenchymal stem cells (MSCs) in response to neural induction media or coculture with rat neural cells. Phenotypic and functional changes were assessed by immunocytochemistry, RT-PCR, and whole-cell patch-clamp recordings. Naive MSCs expressed both mesodermal and ectodermal markers prior to neural induction. Exposure to retinoic acid, basic fibroblast growth factor, or cyclic adenosine monophosphate (cAMP) did not stimulate neural morphology, whereas exposure to dibutyryl cAMP and 3-isobutyl-1-methylxanthine stimulated a neuron-like morphology but also appeared to be cytotoxic. All protocols stimulated increases in expression of the neural precursor marker nestin, but expression of mature neuronal or glial markers MAP2 and GFAP was not observed. Nestin expression increases were serum level dependent. Electrophysiological properties of MSCs were studied with whole-cell patch-clamp recordings. The MSCs possessed no ionic currents typical of neurons before or after neural induction protocols. Coculture of hUCB-derived MSCs and rat neural cells induced some MSCs to adopt an astrocyte-like morphology and express GFAP protein and mRNA. Our data suggest hUCB-derived MSCs do not transdifferentiate into mature functioning neurons in response to the above neurogenic protocols; however, coculture with rat neural cells led to a minority adopting an astrocyte-like phenotype.     

Brain and spinal cord affected by amyotrophic lateral sclerosis induce differential growth factors expression in rat mesenchymal and neural stem cells

C. Nicaise, D. Mitrecic and R. Pochet (2011) Neuropathology and Applied Neurobiology 37, 179–188
Brain and spinal cord affected by amyotrophic lateral sclerosis induce differential growth factors expression in rat mesenchymal and neural stem cells Stem cell research raises hopes for incurable neurodegenerative diseases. In amyotrophic lateral sclerosis (ALS), affecting the motoneurones of the central nervous system (CNS), stem cell‐based therapy aims to replace dying host motoneurones by transplantation of cells in disease‐affected regions. Moreover, transplanted stem cells can serve as a source of trophic factors providing neuroprotection, slowing down neuronal degeneration and disease progression. Aim: To determine the profile of seven trophic factors expressed by mesenchymal stem cells (MSC) and neural stem cells (NSC) upon stimulation with CNS protein extracts from SOD1‐linked ALS rat model. Methods: Culture of rat MSC, NSC and fibroblasts were incubated with brain and spinal cord extracts from SOD1(G93A) transgenic rats and mRNA expression of seven growth factors was measured by quantitative PCR. Results: MSC, NSC and fibroblasts exhibited different expression patterns. Nerve growth factor and brain‐derived neurotropic factor were significantly upregulated in both NSC and MSC cultures upon stimulation with SOD1(G93A) CNS extracts. Fibroblast growth factor 2, insulin‐like growth factor and glial‐derived neurotropic factor were upregulated in NSC, while the same factors were downregulated in MSC. Vascular endothelial growth factor A upregulation was restricted to MSC and fibroblasts. Surprisingly, SOD1(G93A) spinal cord, but not the brain extract, upregulated brain‐derived neurotropic factor in MSC and glial‐derived neurotropic factor in NSC. Conclusions: These results suggest that inherent characteristics of different stem cell populations define their healing potential and raise the concept of ALS environment in stem cell transplantation.     

Soluble mediators from human neural stem cells play a critical role in suppression of T‐cell activation and proliferation

Human neural stem cells (hNSCs) can control inflammation in the central nervous system, although the underlying mechanisms are not understood fully. We investigated the immunomodulatory effect of hNSCs on human T cells and the underlying mechanisms. Culture supernatant from an immortalized hNSC cell line, HB1.F3, which has a therapeutic effect on acute stroke and intracerebral hemorrhage, suppressed the proliferation of allogeneically or mitogenically stimulated human peripheral T cells, including the CD3⁺CD103⁺ subpopulation. CFSE labeling and flow cytometry showed that the suppression of proliferation was caused by cell cycle arrest and induction of apoptosis. The lack of significant change in caspase‐8 levels and the significant reduction in Bcl‐2 expression in the affected T cells suggest that the intrinsic pathway plays a major role in soluble‐factor‐mediated T‐cell apoptosis. The addition of culture supernatant from hNSCs to activated T cells reduced the expression of the activation markers CD69 and CD25 at 24 hr after activation, but at 48 hr only CD69 was down‐regulated. A cytometry bead assay showed that the secretion of interleukin (IL)‐2 decreased significantly, whereas that of IL‐4, IL‐10, tumor necrosis factor‐α, and interferon‐γ increased. These results show that hNSCs can negatively affect human peripheral T cells by suppressing their activation and proliferation through soluble mediators, suggesting that hNSCs have a bystander immunomodulatory effect on T cells. © 2009 Wiley‐Liss, Inc.     

Chitosan-collagen porous scaffold and bone marrow mesenchymal stem cell transplantation for ischemic stroke

In this study, we successfully constructed a composite of bone marrow mesenchymal stem cells and a chitosan-collagen scaffold in vitro, transplanted either the composite or bone marrow mesenchymal stem cells alone into the ischemic area in animal models, and compared their effects. At 14 days after co-transplantation of bone marrow mesenchymal stem cells and the hitosan-collagen scaffold, neurological function recovered noticeably. Vascular endothelial growth factor expression and nestin-labeled neural precursor cells were detected in the ischemic area, surrounding tissue, hippocampal dentate gyrus and subventricular zone. Simultaneously, a high level of expression of glial fibrillary acidic protein and a low level of expression of neuron-specific enolase were visible in Brd U-labeled bone marrow mesenchymal stem cells. These findings suggest that transplantation of a composite of bone marrow mesenchymal stem cells and a chitosan-collagen scaffold has a neuroprotective effect following ischemic stroke.     

Thrombospondin 1 promotes synaptic formation in bone marrow-derived neuron-like cells

In this study, a combination of growth factors was used to induce bone marrow mesenchymal stem cells differentiation into neuron-like cells, in a broader attempt to observe the role of thrombospondin 1 in synapse formation. Results showed that there was no significant difference in the differentiation rate of neuron-like cells between bone marrow mesenchymal stem cells with thrombospondin induction and those without. However, the cell shape was more complex and the neurites were dendritic, with unipolar, bipolar or multipolar morphologies, after induction with thrombospondin 1. The induced cells were similar in morphology to normal neurites. Immunohistochemical staining showed that the number of positive cells for postsynaptic density protein 95 and synaptophysin 1 protein was significantly increased after induction with thrombospondin 1. These findings indicate that thrombospondin 1 promotes synapse formation in neuron-like cells that are differentiated from bone marrow mesenchymal stem cells.