Directed differentiation and transplantation of human embryonic stem cell-derived motoneurons

Motoneurons represent a specialized class of neurons essential for the control of body movement. Motoneuron loss is the cause of a wide range of neurological disorders including amyotrophic lateral sclerosis and spinal muscular atrophy. Embryonic stem cells are a promising cell source for the study and potential treatment of motoneuron diseases. Here, we present a novel in vitro protocol of the directed differentiation of human embryonic stem cells (hESCs) into engraftable motoneurons. Neural induction of hESCs was induced on MS5 stromal feeders, resulting in the formation of neural rosettes. In response to sonic hedgehog and retinoic acid, neural rosettes were efficiently directed into spinal motoneurons with appropriate in vitro morphological, physiological, and biochemical properties. Global gene expression analysis was used as an unbiased measure to confirm motoneuron identity and type. Transplantation of motoneuron progeny into the developing chick embryo resulted in robust engraftment, maintenance of motoneuron phenotype, and long-distance axonal projections into peripheral host tissues. Transplantation into the adult rat spinal cord yielded neural grafts comprising a large number of human motoneurons with outgrowth of choline acetyltransferase positive fibers. These data provide evidence for in vivo survival of hESC-derived motoneurons, a key requirement in the development of hESC-based cell therapy in motoneuron disease. Disclosure of potential conflicts of interest is found at the end of this article.     

维甲酸、音猬因子对大鼠脊髓神经干细胞向运动神经元样细胞分化的影响

目的:分离大鼠胚胎的脊髓神经干细胞进行体外培养,探讨维甲酸(RA)、音猬因子(Shh)诱导其向运动神经元样细胞分化. 方法:应用无血清培养技术分离培养脊髓神经干细胞,通过5-溴-2-脱氧尿苷标记、免疫荧光显色检测细胞增殖、分化情况;培养液分组添加Shh、 RA或Shh+RA,观察神经干细胞向运动神经元样细胞的分化情况. 结果:大鼠胚胎脊髓可成功分离神经干细胞,分化后可表达神经元、星形胶质细胞的特异性抗原;诱导分化后结果显示Shh组未检测到胆碱乙酰转移酶阳性细胞,RA组为20.63%, Shh+RA组为66.84%,其差异具统计学意义.结论:从大鼠胚胎脊髓可成功分离神经干细胞,Shh、 RA可不同程度地诱导脊髓神经干细胞分化为运动神经元样细胞.     

Involvement of brain-derived neurotrophic factor and sonic hedgehog in the spinal cord plasticity after neurotoxic partial removal of lumbar motoneurons

Adult mammals could spontaneously achieve a partial sensory-motor recovery after spinal cord injury, by mechanisms including synaptic plasticity. We previously showed that this recovery is associated to the expression of synapsin-I, and that sonic hedgehog and Notch-1 could be also involved in plasticity. The role of brain-derived neurotrophic factor and glutamate receptors in regulating synaptic efficacy has been explored in the last decade but, although these mechanisms are now well-defined in the brain, the molecular mechanisms underlying the so called "spinal learning" are still less clear. Here, we measured the expression levels of choline acetyltransferase, synapsin-I, sonic hedgehog, Notch-1, glutamate receptor subunits (GluR1, GluR2, GluR4, NMDAR1) and brain-derived neurotrophic factor, in a motoneuron-depleted mouse spinal lesion model obtained by intramuscular injection of cholera toxin-B saporin. The lesion caused the down-regulation of the majority of analysed proteins. Moreover, we found that in lesioned but not in control spinal tissue, synapsin-I expression is associated to that of both brain-derived neurotrophic factor and sonic hedgehog, whereas GluR2 expression is linked to that of Shh. These results suggest that brain-derived neurotrophic factor and sonic hedgehog could collaborate in modulating synaptic plasticity after the removal of motoneurons, by a mechanism involving both pre- and post-synaptic processes. Interestingly, the involvement of sonic hedgehog showed here is novel, and offers new routes to address spinal cord plasticity and repair.     

Stage-dependent Olig2 expression in motor neurons and oligodendrocytes differentiated from embryonic stem cells

Although embryonic stem (ES) cells are capable of forming any cell type in the body, the mechanisms that control cell type-specific differentiation are largely unknown. In the present study, we examined the process of differentiation to motor neurons and oligodendrocytes from mouse (Olig2GFP) ES cells. Mouse ES cells undergo a sequential process of differentiation over a 3-week period to generate motor neurons and oligodendrocytes. At day 7 of differentiation, Olig2-expressing cells are biased to a neuronal lineage. However, further differentiation (day 32) resulted in the majority of Olig2-expressing cells exhibiting an oligodendrocyte phenotype as well as a reduced ability to make motor neurons. Exposure of human ES cells to Sonic hedgehog (Shh) likewise resulted in enhanced motor neuron differentiation. Our results establish the requirements for directing ES cells to become motor neurons and oligodendrocytes and show that ES cell-derived Olig2 + cells can give rise to both motor neurons and oligodendrocytes, depending on the time at which differentiation is initiated.     

The effects of soluble growth factors on embryonic stem cell differentiation inside of fibrin scaffolds

The goal of this research was to determine the effects of different growth factors on the survival and differentiation of murine embryonic stem cell-derived neural progenitor cells (ESNPCs) seeded inside of fibrin scaffolds. Embryoid bodies were cultured for 8 days in suspension, retinoic acid was applied for the final 4 days to induce ESNPC formation, and then the EBs were seeded inside of three-dimensional fibrin scaffolds. Scaffolds were cultured in the presence of media containing different doses of the following growth factors: neurotrophin-3 (NT-3), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF)-AA, ciliary neurotrophic factor, and sonic hedgehog (Shh). The cell phenotypes were characterized using fluorescence-activated cell sorting and immunohistochemistry after 14 days of culture. Cell viability was also assessed at this time point. Shh (10 ng/ml) and NT-3 (25 ng/ml) produced the largest fractions of neurons and oligodendrocytes, whereas PDGF (2 and 10 ng/ml) and bFGF (10 ng/ml) produced an increase in cell viability after 14 days of culture. Combinations of growth factors were tested based on the results of the individual growth factor studies to determine their effect on cell differentiation. The incorporation of ESNPCs and growth factors into fibrin scaffolds may serve as potential treatment for spinal cord injury.     

鸡胚发育过程中Shh超表达对脊髓神经纤维投射的抑制作用

目的探讨鸡胚发育过程中,sonic hedgehog(Shh)在脊髓中超表达后对神经纤维投射的影响。方法在鸡胚龄2.5~3d(E2.5~E3)时,利用鸡胚活体原位电转基因技术将Shh表达质粒和携带有报告基因绿色荧光蛋白(GFP)的质粒共转入鸡胚脊髓,E6时取材切片,至少取3个材料,免疫荧光技术检验Shh的超表达,利用Open Book技术研究Shh在鸡胚脊髓中超表达后对神经纤维投射的影响。结果在脊髓组织切片水平上,免疫荧光结果表明,Shh在鸡胚脊髓中成功超表达,无论在组织切片上还是通过Open Book技术进行观察,与对照组相比,超表达后转染侧神经纤维都无法正常通过底板投射到对侧,表明Shh在鸡胚脊髓神经纤维投射方面具有重要作用。结论在鸡胚发育过程中,脊髓中Shh超表达后对神经纤维投射具有抑制作用。     

Induction of mice adult bone marrow mesenchymal stem cells into functional motor neuron-like cells

The differentiation of mesenchymal stem cells (MSC) into acetylcholine secreted motor neuron-like cells, followed by elongation of the cell axon, is a promising treatment for spinal cord injury and motor neuron cell dysfunction in mammals. Differentiation is induced through a pre-induction step using Beta- mercaptoethanol (BME) followed by four days of induction with retinoic acid and sonic hedgehog. This process results in a very efficient differentiation of BM-MSCs into motor neuron-like cells. Immunocytochemistry showed that these treated cells had specific motor neural markers: microtubule associated protein-2 and acetylcholine transferase. The ability of these cells to function as motor neuron cells was assessed by measuring acetylcholine levels in a culture media during differentiation. High-performance liquid chromatography (HPLC) showed that the differentiated cells were functional. Motor neuron axon elongation was then induced by adding different concentrations of a nerve growth factor (NGF) to the differentiation media. Using a collagen matrix to mimic the natural condition of neural cells in a three-dimensional model showed that the MSCs were successfully differentiated into motor neuron-like cells. This process can efficiently differentiate MSCs into functional motor neurons that can be used for autologous nervous system therapy and especially for treating spinal cord injuries.     

Ectopic expression of Hoxd10 in thoracic spinal segments induces motoneurons with a lumbosacral molecular profile and axon projections to the limb.

Hox genes encode anterior-posterior identity during central nervous system development. Few studies have examined Hox gene function at lumbosacral (LS) levels of the spinal cord, where there is extensive information on normal development. Hoxd10 is expressed at high levels in the embryonic LS cord but not the thoracic cord. To test the hypothesis that restricted expression of Hoxd10 contributes to the attainment of an LS identity, and specifically an LS motoneuron identity, Hoxd10 was ectopically expressed in thoracic segments in chick embryos by means of in ovo electroporation. Regional motoneuron identity was assessed after the normal period of motoneuron differentiation. Subsets of motoneurons in transfected thoracic segments developed a molecular profile normally shown by LS motoneurons, including Lim 1 and RALDH2 expression. In addition, motoneurons in posterior thoracic segments showed novel axon projections to two muscles in the anterodorsal limb, the sartorius and anterior iliotibialis muscles. At thoracic levels, we also found a decrease in motoneuron numbers and a reduction in gonad size. These last findings suggest that early and high levels of Hox expression impeded motoneuron development and neural-mesodermal interactions. Despite these adverse effects, our data indicate that Hoxd10 expression is sufficient to induce LS motoneuron identity and axon trajectories characteristic of motoneurons in the LS region.     

An approach to experimental synaptic pathology using green fluorescent protein-transgenic mice and gene knockout mice to show mitochondrial permeability transition pore-driven excitotoxicity in interneurons and motoneurons

Researchers used transgenic mice expressing enhanced-green fluorescent protein (eGFP) driven by either the glycine transporter-2 gene promoter to specifically visualize glycinergic interneurons or the homeobox-9 (Hb9) gene promoter to visualize motoneurons for assessing their vulnerabilities to excitotoxins in vivo. Stereotaxic excitotoxic lesions were made in adult male and female mouse lumbar spinal cord with the specific N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid (QA) and the non-NMDA ion channel glutamate receptor agonist kainic acid (KA). QA and KA induced large-scale degeneration of glycinergic interneurons in spinal cord. Glycinergic interneurons were more sensitive than motoneurons to NMDA receptor-mediated and non-NMDA glutamate receptor-mediated excitotoxicity. Outcome after spinal cord excitotoxicity was gender-dependent, with males showing greater sensitivity than females. Excitotoxic degeneration of spinal interneurons resembled apoptosis, while motoneuron degeneration appeared non-apoptotic. Perikaryal mitochondrial accumulation was antecedent to both NMDA and non-NMDA receptor-mediated excitotoxic stimulation of interneurons and motoneurons. Genetic ablation of cyclophilin D, a regulator of the mitochondrial permeability transition pore (mPTP), protected both interneurons and motoneurons from excitotoxicity. The results demonstrate in adult mouse spinal cord that glycinergic interneurons are more sensitive than motoneurons to excitotoxicity that stimulates mitochondrial accumulation, and that the mPTP has pro-death functions mediating apoptotic and non-apoptotic neuronal degeneration in vivo.     

Human motor neuron differentiation from human embryonic stem cells

The therapeutic potential of embryonic stem (ES) cells is promising, but in many cases limited by our inability to promote their differentiation to specific cell types, such as motor neurons. Here we provide the first report of the successful differentiation of human ES cells to cells of a motor neuron phenotype. A renewable source of neuroepithelial cells was generated from human ES cells. Extracellular signals were then employed to induce motor neuron differentiation and related gene expression by these cells. OLIG2 and HLXB9 gene expression increased upon the addition of basic fibroblast growth factor, retinoic acid, and sonic hedgehog, as a motor neuron phenotype expressing Islet1 and choline acetyltransferase (ChAT) developed. This study demonstrates that neuroepithelial cells derived from human ES cells are renewable progenitors capable of generating motor neurons at levels that may be therapeutically useful. Sonic hedgehog, basic fibroblast growth factor, and retinoic acid differentially influence human motor neuron differentiation by mechanisms that remain to be defined.     

CEPU-1, an immunoglobulin superfamily molecule, has cell adhesion activity and shows dynamic expression patterns in chick embryonic spinal cord

In an attempt to isolate novel molecules involved in motoneuron differentiation and target muscle innervation during embryogenesis, we performed mRNA differential display analysis by comparing cDNAs of motoneurons purified by immunopanning from different portions along the rostro-caudal axis of chick embryonic spinal cord, and cloned an immunoglobulin superfamily protein named C30. By sequence comparison, C30 was shown to be an alternatively spliced isoform of CEPU-1, which was formerly reported as a member of the immunoglobulin superfamily specifically expressed in cerebellar Purkinje cells (Spaltmann and Brummendorf, 1996, J. Neurosci. 16, 1770-1779). We analyzed the expression pattern of CEPU-1 both at the mRNA and protein levels in the spinal cord of the chick embryo. Until stage 23, CEPU-1 was expressed faintly in the ventral part of the neural tube but gradually it became localized to a specific group of cells. In the motor column, CEPU-1 was expressed transiently in many columnar layers. A C30-transfected cell line showed Ca(2+)-independent cell-cell binding activity. These results suggest a role for CEPU-1 in specific axon guidance and/or fasciculation of motoneurons during development.     

异种脊髓前角匀浆对豚鼠前角运动神经元的影响

目的：观察豚鼠脊髓前角运动神经元在异种脊髓前角匀浆免疫后的变化。方法：猪脊髓前角匀浆免疫豚鼠后,豚鼠脊髓前角以苏木精-伊红、甲苯胺蓝及IgG免疫组化染色,同时电镜下观察前角运动神经元的超微变化。结果：豚鼠脊髓前角运动神经元存在变性和丢失,以神经元固缩、胶质细胞围绕破坏的神经元形成卫星现象及小墓穴为主,脊髓前角运动神经元胞质内IgG沉积呈颗粒状分布。运动神经元胞质内内质网扩张、线粒体肿胀。结论：猪脊髓前角匀浆作为抗原可引起豚鼠下运动神经元损伤,说明猪与豚鼠运动神经元存在共同抗原,自身免疫机制可能参与了运动神经元变性过程。     

鸡胚发育过程中音猬因子超表达对Pax家族基因表达的影响

目的 探讨鸡胚发育过程中,音猬因子（Shh）基因在脊髓中超表达后对配对框（Pax）基因家族Pax3、Pax6以及Pax7蛋白表达的影响。 方法 在鸡胚龄2.5～3.0d（E2.5～E3）时,实验组利用鸡胚脊髓活体电转基因技术将携带Shh基因的质粒和携带有绿色荧光蛋白（GFP）报告基因的质粒共转入鸡胚脊髓,对照组则只转入报告基因质粒。E4时取材,每组至少取3例,冷冻切片后采用原位杂交技术检测Shh基因的mRNA表达水平,利用荧光免疫技术检测Shh在鸡胚脊髓中超表达后对Pax3,Pax6以及Pax7蛋白表达水平的影响,并将对照组及实验组中3种基因在脊髓两侧的表达量进行统计学分析。 结果 原位杂交结果表明,E4时Shh的mRNA主要表达在脊髓底板和脊索,实验组结果显示,利用鸡胚脊髓活体电转技术成功建立了Shh超表达模型。对照组中荧光免疫结果表明,Pax3主要表达于脊髓翼板、生肌节、背根以及背根神经节;Pax6主要表达于跨越翼板和基板的脊髓中间部位;Pax7则主要均匀表达于翼板、顶板和生肌节。Shh超表达后,脊髓转染侧与未转染侧相比Pax3、Pax6以及Pax7的表达量差异极显著（P<0.01）,这3种基因在脊髓中的表达均被抑制。 结论 在鸡胚发育过程中,Pax家族Pax3、Pax6以及Pax7 3种基因在鸡胚脊髓中具有不同的表达模式,Shh超表达后对其均具有明显的抑制作用,显示这3种Pax基因受Shh信号通路调控。     

Human neural stem cell-derived cholinergic neurons innervate muscle in motoneuron deficient adult rats

Motoneuron damage occurs in spinal cord injury and amyotrophic lateral sclerosis. Current advances offer hope that human embryonic stem cells [Science 282 (1998) 1145] or neural stem cells (NSC) [Exp Neurol 161 (2000) 67; Exp Neurol 158 (1999) 265; J Neurosci Methods 85 (1998) 141; Proc Natl Acad Sci USA 97 (2000) 14720; Exp Neurol 156 (1999) 156 ] may be donors to replace lost motoneurons. Previously, we developed a priming procedure that produced cholinergic cells that resemble motoneurons from human NSCs grafted into adult rat spinal cord [Nat Neurosci 5 (2002a) 1271]. However, effective replacement therapy will ultimately rely on successful connection of new motoneurons with their muscle targets. In this study, we examined the potential of human fetal NSC transplantation to replace lost motoneurons in an animal model of chronic motoneuron deficiency (newborn sciatic axotomy) [J Comp Neurol 224 (1984) 252; J Neurobiol 23 (1992) 1231]. We found, for the first time, that human neural stem cell-derived motoneurons send axons that pass through ventral root and sciatic nerve to form neuromuscular junctions with their peripheral muscle targets. Furthermore, this new cholinergic innervation correlates with partial improvement of motor function.     

Investigating differentiation mechanisms of the constituent cells of sex cord-stromal tumours of the ovary

SOX-9, an essential factor for male sexual development, can be induced by prostaglandin D2 in a Sry-independent mechanism. Recent data suggest that the hedgehog pathway is involved in the differentiation of normal Sertoli and Leydig cells. The purpose of our study was to investigate the mechanisms involved in the differentiation of ovarian sex cord-stromal tumour (SCST) cells. Two Sertoli-Leydig cell tumours and two granulosa cell tumours with a minor Sertoli element were studied using immunohistochemistry on paraffin-embedded tissue sections. Sertoli cells expressed anti-Mullerian hormone (AMH), SOX-9, prostaglandin D synthase (Pgds) and bcl-2 (in four of four cases); sonic hedgehog (Shh) and p53 (in three of four cases) and androgen receptors (AR; in one of four cases). Ki-67 index ranged from 10% to 50%. Leydig cells expressed Shh and AR (two of two cases), while they showed no expression of p53, bcl-2 and 0% Ki-67 index. Granulosa cells expressed AMH, Pgds, Shh, estrogen receptors, progesterone receptors, AR and bcl-2 (in two of two cases) and p53 (in one of two cases). Ki-67 index was 10% and 40%, respectively. Further investigation is required to clarify the role of the molecules outlined above in the histogenesis of ovarian SCST, as Pgds-mediated SOX-9 upregulation could provide a reasonable explanation for the presence of testicular differentiation in ovarian SCST.     

Implanted spike wave electric stimulation promotes survival of the bone marrow mesenchymal stem cells and functional recovery in the spinal cord injured rats

Transplantation of bone marrow-derived mesenchymal stromal cells (BMSCs) into the injured spinal cord may provide therapeutic benefit, but its application is limited by their poor survival and low differentiation rate into neurons. Electrical stimulation (ES) has been reported to promote survival and differentiation of the BMSCs. Therefore we investigated whether implanted spike wave ES could improve survival of BMSCs after transplantation and result in functional improvement in animals with spinal cord injury. Our results showed that the number and ratio of survived BMSCs near the lesion site were significantly increased in the BMSCs+ES-treated group as compared to BMSCs transplantation or ES treatment alone group. Furthermore, results from BBB scales, SSEP and DTI demonstrated a significant improved functional recovery in the BMSCs+ES group. This indicated that implanted spike wave ES could promote the bioactivity of BMSCs and their survival. This represents a new therapeutic potential of the combination of BMSCs transplantation with implanted spike wave ES to treat spinal cord injury.     

Axonal growth of the spinal cord interneurons expressing a homophilic adhesion molecule SC1 ectopically

SC1/DM-GRASP/BEN, a cell adhesion molecule of the immunoglobulin super family, promotes the extension of neurites from neurons that express SC1 in culture, presumably by direct homophilic interactions. SC1 is specifically and transiently expressed on motoneurons during the period of their axonal growth, suggesting that it plays an important role in this growth. To explore this possibility, we expressed SC1 ectopically on the spinal cord interneurons of quail and chick embryos by in ovo electroporation at E3, when the motoneuron axonal growth starts. The axonal growth of the interneurons expressing chick SC1 was analyzed by immunohistochemistry with chick-specific anti-SC1 monoclonal antibody in quail, and by retrograde labeling with a dye in chick and quail embryos at E5. The majority of the axons of SC1-positive interneurons passed through the motor column and extended normally along the spinal cord basement membrane, but a few appeared to grow out from the cord. However, a dye back-labeling of the spinal nerves revealed that none of the interneurons were both SC1 and dye positive. These results suggest that the expression of SC1/DM-GRASP/BEN alone is insufficient for regulating the first step of the selective axonal pathfinding of motoneurons.