Adherent self-renewable human embryonic stem cell-derived neural stem cell line: functional engraftment in experimental stroke model

Background

Human embryonic stem cells (hESCs) offer a virtually unlimited source of neural cells for structural repair in neurological disorders, such as stroke. Neural cells can be derived from hESCs either by direct enrichment, or by isolating specific growth factor-responsive and expandable populations of human neural stem cells (hNSCs). Studies have indicated that the direct enrichment method generates a heterogeneous population of cells that may contain residual undifferentiated stem cells that could lead to tumor formation in vivo.

Methods/Principal Findings

We isolated an expandable and homogenous population of hNSCs (named SD56) from hESCs using a defined media supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory growth factor (LIF). These hNSCs grew as an adherent monolayer culture. They were fully neuralized and uniformly expressed molecular features of NSCs, including nestin, vimentin and radial glial markers. These hNSCs did not express the pluripotency markers Oct4 or Nanog, nor did they express markers for the mesoderm or endoderm lineages. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. The SD56 hNSCs differentiated into neurons, astrocytes and oligodendrocytes throughout multiple passages in vitro, as well as after transplantation. Together, these criteria confirm the definitive NSC identity of the SD56 cell line. Importantly, they exhibited no chromosome abnormalities and did not form tumors after implantation into rat ischemic brains and into naïve nude rat brains and flanks. Furthermore, hNSCs isolated under these conditions migrated toward the ischemia-injured adult brain parenchyma and improved the independent use of the stroke-impaired forelimb two months post-transplantation.

Conclusions/Significance

The SD56 human neural stem cells derived under the reported conditions are stable, do not form tumors in vivo and enable functional recovery after stroke. These properties indicate that this hNSC line may offer a renewable, homogenous source of neural cells that will be valuable for basic and translational research.     

Rat mesenchymal stem cells increase tyrosine hydroxylase expression and dopamine content in ventral mesencephalic cells in vitro

Mesenchymal stem cells (MSCs) are pluripotent adult stem cells. It has been shown that MSCs secrete neurotrophic factors involving nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Also, these neurotrophic factors can upregulate tyrosine hydroxylase (TH) gene expression in PC12 cells and neural stem cells. Here, we investigated the effect of co-culturing rat E13.5 ventral mesencephalic cells (VMCs) with MSCs from rat bone marrow on TH expression and dopamine (DA) content. The study consisted of 3 groups: MSC, VMC and a combined MSC+VMC group. All groups were cultured in serum-free neuro-basal medium for 3 days. Thereafter, each group was analyzed by RT-PCR, western blotting, and HPLC. The co-culture group showed a higher expression at TH and DA than the VMC group. However, TH and DA were not present in the MSC group. These observations suggest that MSCs could be an alternative source for treating neurodegenerative diseases such as Parkinson's disease (PD).     

体外诱导人骨髓间充质干细胞向多巴胺神经元分化的研究

通过体外诱导人骨髓间充质干细胞(bone marrow mesenchymal stemcells,BMSCs)向多巴胺(dopamine,DA)神经元分化,探讨人BMSCs来源的DA神经元的功能特征及其分化机制,为临床上细胞移植替代治疗诸如帕金森氏病(parkinson’sdisease,PD)等神经精神性疾病提供一种理想的细胞来源。通过密度梯度离心获取人骨髓中的单个核细胞,贴壁培养纯化BMSCs。50μmol/L脑源性神经营养因子(brain derivedneurotrophy factor,BDNF),10μmol/Lforskolin(FSK)和10μmol/LDA联合对BMSCs进行诱导。电子显微镜观察诱导2周后细胞是否具有神经元的超微结构特点;免疫细胞化学染色和RT-PCR检测DA神经元分化过程中的标志物酪氨酸羟化酶(tyrosine hydroxylase,TH)的表达以及转录因子Nurr1、Ptx3和Lmx1b的表达;高效液相色谱(highperformance liquid chromatogram,HPLC)检测诱导2周后的细胞多巴胺的释放水平。结果表明,诱导2周后,电镜下细胞胞浆中有大量密集的呈扁平囊状的粗面内质网及其间的一些游离核糖体以及神经微丝的形成。RT-PCR结果显示NSE(neuron specificenolase)、Nurr1、Ptx3、Lmx1b和TH的mRNA均有表达;免疫细胞化学染色结果表明诱导2周后TH阳性细胞(24·80±3·36)%的表达较诱导3d后(3·77±1·77)%明显提高(P<0·01);HPLC检测到诱导2周后的细胞DA释放水平[(1·22±0·36)μg/mL(n=6)]高于未经诱导的细胞[(0·75±0·22)μg/mL(n=6)(t=-2·79,P=0·038)]。由此得出,BDNF、FSK和DA可以在体外诱导人BMSCs向DA神经元分化,并具有DA神经元的功能特征,是临床用于治疗神经精神性疾病的理想细胞来源。     

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson’s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.     

CD marker expression profiles of human embryonic stem cells and their neural derivatives,determined using flow-cytometric analysis,reveal a novel CD marker for exclusion of pluripotent stem cells

Human embryonic stem cells (hESCs) are pluripotent cells that can differentiate into neural cell lineages. These neural populations are usually heterogeneous and can contain undifferentiated pluripotent cells that are capable of producing teratomas in cell grafts. The characterization of surface protein profiles of hESCs and their neural derivatives is important to determine the specific markers that can be used to exclude undifferentiated cells from neural populations. In this study, we analyzed the cluster of differentiation (CD) marker expression profiles of seven undifferentiated hESC lines using flow-cytometric analysis and compared their profiles to those of neural derivatives. Stem cell and progenitor marker CD133 and epithelial adhesion molecule marker CD326 were more highly expressed in undifferentiated hESCs, whereas neural marker CD56 (NCAM) and neural precursor marker (chemokine receptor) CD184 were more highly expressed in hESC-derived neural cells. CD326 expression levels were consistently higher in all nondifferentiated hESC lines than in neural cell derivatives. In addition, CD326-positive hESCs produced teratomas in SCID mouse testes, whereas CD362-negative neural populations did not. Thus, CD326 may be useful as a novel marker of undifferentiated hESCs to exclude undifferentiated hESCs from differentiated neural cell populations prior to transplantation.     

17β‐Oestradiol promotes differentiation of human embryonic stem cells into dopamine neurons via cross‐talk between insulin‐like growth factors‐1 and oestrogen receptor β

Human embryonic stem cells (hESCs) can self‐renew and differentiate into all cell lineages. E2 is known to exhibit positive effects on embryo development. Although the importance of E2 in many physiological processes has been reported, to date few researchers have investigated the effects of E2 on hESCs differentiation. We studied the effects of E2 on dopamine (DA) neuron induction of hESCs and its related signalling pathways using the three‐stage protocol. In our study, 0.1 μM E2 were applied to hESCs‐derived human embryoid bodies (hEBs) and effects of E2 on neural cells differentiation were investigated. Protein and mRNA level assay indicated that E2 up‐regulated the expression of insulin‐like growth factors (IGF)‐1, ectoderm, neural precursor cells (NPC) and DA neuron markers, respectively. The population of hESC‐derived NPCs and DA neurons was increased to 92% and 93% to that of DMSO group, respectively. Furthermore, yield of DA neuron‐secreted tyrosine hydroxylase (TH) and dopamine was also increased. E2‐caused promotion was relieved in single inhibitor (ICI or JB1) group partly, and E2 effects were repressed more stronger in inhibitors combination (ICI plus JB1) group than in single inhibitor group at hEBs, hNPCs and hDA neurons stages. Owing to oestrogen receptors regulate multiple brain functions, when single or two inhibitors were used to treat neural differentiation stage, we found that oestrogen receptor (ER)β but not ERα is strongly repressed at the hNPCs and hDA neurons stage. These findings, for the first time, demonstrate the molecular cascade and related cell biology events involved in E2‐improved hNPC and hDA neuron differentiation through cross‐talk between IGF‐1 and ERβ in vitro.     

Soluble amyloid precursor protein induces rapid neural differentiation of human embryonic stem cells

Human embryonic stem cells (hESCs) offer tremendous potential for not only treating neurological disorders but also for their ability to serve as vital reagents to model and investigate human disease. To further our understanding of a key protein involved in Alzheimer disease pathogenesis, we stably overexpressed amyloid precursor protein (APP) in hESCs. Remarkably, we found that APP overexpression in hESCs caused a rapid and robust differentiation of pluripotent stem cells toward a neural fate. Despite maintenance in standard hESC media, up to 80% of cells expressed the neural stem cell marker nestin, and 65% exhibited the more mature neural marker β-3 tubulin within just 5 days of passaging. To elucidate the mechanism underlying the effects of APP on neural differentiation, we examined the proteolysis of APP and performed both gain of function and loss of function experiments. Taken together, our results demonstrate that the N-terminal secreted soluble forms of APP (in particular sAPPβ) robustly drive neural differentiation of hESCs. Our findings not only reveal a novel and intriguing role for APP in neural lineage commitment but also identify a straightforward and rapid approach to generate large numbers of neurons from human embryonic stem cells. These novel APP-hESC lines represent a valuable tool to investigate the potential role of APP in development and neurodegeneration and allow for insights into physiological functions of this protein.     

Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model

Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson''s disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.     

Cytokine-induced conversion of mesencephalic-derived progenitor cells into dopamine neurons

We have previously shown that a combination of the cytokines interleukin (IL)-1, IL-11, leukemia inhibitory factor (LIF), and glial cell line-derived neurotrophic factor (GDNF) can convert rat fetal (E14.5) mesencephalic progenitor cells into tyrosine hydroxylase (TH)-immunoreactive (ir) neurons in vitro. The experiments described here characterize the mesencephalic progenitor cells and their cytokine-induced conversion into dopamine (DA) neurons. For all experiments, we used bromodeoxyuridine (BrdU)-ir cultures of (E14.5) mesencephalic progenitor cells that had been expanded at least 21 days. We first demonstrated that IL-1 induced DA neuron conversion in mesencephalic progenitors, but not in striatal progenitors (P < 0.001). Thus, these cells should be classified as lineage-restricted progenitors, and not omnipotent stem cells. To further characterize cell populations in these cultures, we used monoclonal antibodies against Hu (an early marker for neurons), growth-associated protein (GAP)-43 (a marker for neuronal process extension), TH (a marker for DA neurons), and glial fibrillary acidic protein (GFAP, a marker for astrocytes). We assessed (E14.5) mesencephalic progenitor cell cultures (plated at 125,000 cells/cm2) incubated in the cytokine mixture (described above) or in complete media (CM, negative control). Following 7 days incubation, GFAP-positive cells formed a nearly confluent carpet in both types of cultures. However, numbers of Hu-ir and GAP-43-ir cells in the cytokine-incubated cultures far exceeded those in CM-incubated controls (P = 0.0003, P = 0.0001, respectively), while numbers of TH-ir cells were 58-fold greater in the cytokine-incubated cultures versus CM-incubated controls. The TH phenotype persisted for 7 days following withdrawal of the differentiation media. Numerous double-labeled cells that were BrdU-ir and also TH-ir, or Hu-ir and also TH-ir, were observed in the cytokine-incubated cultures. These data suggest that cytokines "drive" the conversion of progenitor cells into DA neurons.     

Co-Transplantation of GDNF-Overexpressing Neural Stem Cells and Fetal Dopaminergic Neurons Mitigates Motor Symptoms in a Rat Model of Parkinson’s Disease

Striatal transplantation of dopaminergic (DA) neurons or neural stem cells (NSCs) has been reported to improve the symptoms of Parkinson’s disease (PD), but the low rate of cell survival, differentiation, and integration in the host brain limits the therapeutic efficacy. We investigated the therapeutic effects of intracranial co-transplantation of mesencephalic NSCs stably overexpressing human glial-derived neurotrophic factor (GDNF-mNSCs) together with fetal DA neurons in the 6-OHDA rat model of PD. Striatal injection of mNSCs labeled by the contrast enhancer superparamagnetic iron oxide (SPIO) resulted in a hypointense signal in the striatum on T2-weighted magnetic resonance images that lasted for at least 8 weeks post-injection, confirming the long-term survival of injected stem cells in vivo. Co-transplantation of GDNF-mNSCs with fetal DA neurons significantly reduced apomorphine-induced rotation, a behavioral endophenotype of PD, compared to sham-treated controls, rats injected with mNSCs expressing empty vector (control mNSCs) plus fetal DA neurons, or rats injected separately with either control mNSCs, GDNF-mNSCs, or fetal DA neurons. In addition, survival and differentiation of mNSCs into DA neurons was significantly greater following co-transplantation of GDNF-mNSCs plus fetal DA neurons compared to the other treatment groups as indicated by the greater number of cell expressing both the mNSCs lineage tracer enhanced green fluorescent protein (eGFP) and the DA neuron marker tyrosine hydroxylase. The success of cell-based therapies for PD may be greatly improved by co-transplantation of fetal DA neurons with mNSCs genetically modified to overexpress trophic factors such as GDNF that support differentiation into DA cells and their survival in vivo.     

Generation of dopamine neurons from human embryonic stem cells in vitro

The aim of the study was to generate dopaminergic (DA) neurons from human embryonic stem cells (ESC) in vitro. It was shown that human ESCs are able to differentiated into DA neurons without co-culture with stromal cells. Terminal differentiation into DA neurons was reached by successive application of noggin and bFGF growth factors on collagen and matrigel substrates during 3-4 weeks. Differentiation efficiency was evaluated by the number of colonies with cells expressing tyrosine hydroxylase (TH), a DA neuron marker, and by the number of TH-positive cells in cell suspension using flow cytometry. No cells with pluripotent markers were detected in DA-differentiated cultures. It makes possible to propose that the protocol of human ESC differentiation might be applied to generate DA neurons for their transplantation into the animals modeling neurodegenerative (Parkinson) disease without the risk of tumor growth.     

Efficient derivation of human neuronal progenitors and neurons from pluripotent human embryonic stem cells with small molecule induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging(1-3). Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells(1-3). Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration(1,4-7). However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity(7-10). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic(11-13). To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules(14) (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders(1, 14). And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons(1, 14, 15). However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.     

Dopaminergic‐like cells from epigenetically reprogrammed mesenchymal stem cells

A number of recent studies have examined the ability of stem cells derived from different sources to differentiate into dopamine‐producing cells and ameliorate behavioural deficits in Parkinsonian models. Recently, using the approach of cell reprogramming by small cell‐permeable biological active compounds that involved in the regulation of chromatin structure and function, and interfere with specific cell signalling pathways that promote neural differentiation we have been able to generate neural‐like cells from human bone marrow (BM)‐derived MSCs (hMSCs). Neurally induced hMSCs (NI‐hMSCs) exhibited several neural properties and exerted beneficial therapeutic effect on tissue preservation and locomotor recovery in spinal cord injured rats. In this study, we aimed to determine whether hMSCs neuralized by this approach can generate dopaminergic (DA) neurons. Immunocytochemisty studies showed that approximately 50–60% of NI‐hMSCs expressed early and late dopaminergic marker such as Nurr‐1 and TH that was confirmed by Western blot. ELISA studies showed that NI‐hMSCs also secreted neurotrophins and dopamine. Hypoxia preconditioning prior to neural induction increased hMSCs proliferation, viability, expression TH and the secretion level of dopamine induced by ATP. Taken together, these studies demonstrated that hMSCs neurally modified by this original approach can be differentiated towards DA‐like neurons.     

Generation of dopamine neurons from human embryonic stem cells in vitro

The aim of the study was to generate dopaminergic (DA) neurons from human embryonic stem cells (ESCs) in vitro. It was shown that human ESCs can be differentiated into DA neurons without co-culture with stromal cells. Terminal differentiation into DA neurons was reached by the successive application of noggin and bFGF growth factors and collagen and matrigel substrates for 3–4 weeks. The efficiency of differentiation was evaluated by the number of colonies with cells that express tyrosine hydroxylase (TH), a DA neuron marker, and by the number of TH-positive cells in cell suspension estimated by flow cytometry. No cells with pluripotent markers were detected in DA-differentiated cultures. The lack of pluripotent cells in population at the final stage of differentiation is encouraging and shows that this protocol of human ESC differentiation may be applied to generate DA neurons for their transplantation into the animals modeling neurodegenative (Parkinson) disease without the risk of tumor growth.     

Dopaminergic differentiation using pluripotent stem cells

Parkinson's disease (PD) is the second most common neurodegenerative disorder. The motor symptoms of PD are caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta of mesencephalon. The causes for death of DA neurons are not well understood, but the strongest risk factor is increasing age. There is no cure currently available for PD, and treatment is limited to management of PD symptoms in patients. Primary DA neurons are virtually unobtainable from living patients and animal studies have proven inadequate for studying the mechanism of PD development. Pluripotent stem cells (PSC) are primary self‐renewing cells capable of differentiating into all cell types of an organism, including DA neurons. PSCs represent an abundant source of cells that can be genetically modified or isolated from patients with complex diseases, enabling the production of large quantities of DA neurons for disease modeling, drug screening, and gene function studies. Furthermore, since PD arises as a result of deterioration of DA neurons in a specific brain region, it has been suggested that a relatively small number of cells could restore normal function. PSCs could provide a source of DA neurons for cell replacement therapy. In this Prospects article, we focus on the development and in vitro derivation of DA neurons from PSCs, as well as current applications of the technological advances, with the emphasis on future directions and efforts in the field. J. Cell. Biochem. 113: 3610–3619, 2012. © 2012 Wiley Periodicals, Inc.