Comparative study on the therapeutic potential of neurally differentiated stem cells in a mouse model of multiple sclerosis

Background

Transplantation of neural stem cells (NSCs) is a promising novel approach to the treatment of neuroinflammatory diseases such as multiple sclerosis (MS). NSCs can be derived from primary central nervous system (CNS) tissue or obtained by neural differentiation of embryonic stem (ES) cells, the latter having the advantage of readily providing an unlimited number of cells for therapeutic purposes. Using a mouse model of MS, we evaluated the therapeutic potential of NSCs derived from ES cells by two different neural differentiation protocols that utilized adherent culture conditions and compared their effect to primary NSCs derived from the subventricular zone (SVZ).

Methodology/Principal Findings

The proliferation and secretion of pro-inflammatory cytokines by antigen-stimulated splenocytes was reduced in the presence of SVZ-NSCs, while ES cell-derived NSCs exerted differential immunosuppressive effects. Surprisingly, intravenously injected NSCs displayed no significant therapeutic impact on clinical and pathological disease outcomes in mice with experimental autoimmune encephalomyelitis (EAE) induced by recombinant myelin oligodendrocyte glycoprotein, independent of the cell source. Studies tracking the biodistribution of transplanted ES cell-derived NSCs revealed that these cells were unable to traffic to the CNS or peripheral lymphoid tissues, consistent with the lack of cell surface homing molecules. Attenuation of peripheral immune responses could only be achieved through multiple high doses of NSCs administered intraperitoneally, which led to some neuroprotective effects within the CNS.

Conclusion/Significance

Systemic transplantation of these NSCs does not have a major influence on the clinical course of rMOG-induced EAE. Improving the efficiency at which NSCs home to inflammatory sites may enhance their therapeutic potential in this model of CNS autoimmunity.     

Evaluation of the differentiation status of neural stem cells based on cell morphology and the expression of Notch and Sox2

Neural stem cells (NSCs) isolated from a variety of sources are being developed as cellular therapies aimed at treating neurodegenerative diseases. During NSC culture and expansion it is important the cells do not differentiate prematurely because this may have an unfavorable effect on product quality and yield. In our study, we evaluated the use of Notch and Sox2 as markers for undifferentiated human and mouse NSCs. The expression of Notch2 and Sox2 during extensive-passage, low-oxygen culture and differentiation conditions were analyzed to confirm that the presence of these signature proteins directly correlates with the ability of NSCs to form new neurospheres and differentiate into multiple cell types. Using expression of Notch1, Notch2 and Sox2 as a reference, we then used flow cytometry to identify a specific morphological profile for undifferentiated murine and human NSCs. Our studies show that Notch and Sox2 expression, along with flow cytometry analysis, can be used to monitor the differentiation status of NSCs grown in culture for use in cellular therapies.     

胚胎大鼠脑和脊髓神经干细胞的分离和培养

研究采用显微解剖、无血清细胞培养和免疫荧光细胞化学染色等实验技术 ,成功地建立了胚胎大鼠脑和脊髓神经干细胞 (NSCs)的分离和培养方法。结果显示 ,( 1)在含成纤维细胞生长因子 2 (FGF 2 )和表皮生长因子(EGF)的无血清培养液中 ,两种来源的NSCs经体外培养 8- 10代后 ,其细胞数呈指数级增加 ,其中脑来源的NSCs数由原代培养时的 1× 10 6 增加至 1× 10 12 ,脊髓来源的NSCs数从 1× 10 6 增加至 1× 10 11。增殖的细胞表达神经上皮干细胞蛋白 (nestin) ;( 2 )在含 1%胎牛血清 (FBS)的培养条件下 ,它们都能被诱导分化为神经元、少突胶质细胞和星型胶质细胞。但其分化比例可随细胞传代次数的增加而改变 ,其中 ,大脑来源的NSCs分化为神经元的比例从第二代 (P2 )的 11 95± 2 5 %下降至第五代 (P5)的 1 97± 1 16% (P <0 0 1) ,而少突胶质细胞的分化比例则基本保持不变 ,这一分化格局同样可在脊髓来源的NSCs中发现。结果表明 ,我们所分离和培养的细胞在体外经多次传代后仍具有很强的增殖能力和多向分化潜能 ,它们都表达nestin ,属于中枢神经系统的干细胞     

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.     

Culture of neural stem cells in calcium alginate beads

Neural stem cells (NSCs) with the capacity of extensive self-renewal and multilineage differentiation have attracted more and more attention in research as NSCs will play an important role in the nerve disease treatment and nerve injury repair. The shortage of NSCs, both their sources and their numbers, however, is the biggest challenge for their clinic application, and hence, in vitro culture and expansion of NSCs is vitally important to realize their potentials. In this work, mouse-derived NSCs were cultured in three-dimensional calcium alginate beads (Ca-Alg-Bs). Gelling conditions, cell density, and cell harvest were determined by the exploration of formation and dissociation parameters for Ca-Alg-Bs. Additionally, the recovered and the subsequent induced cells were identified by immunofluorescence staining of Nestin, beta-tubulin, and GFAP. The results show that the 2-mm diameter Ca-Alg-Bs, prepared with 1.5% sodium alginate solution and 3.5% CaCl2 solution and with gelling for 10 min, is suitable for the NSCs culture. The seeding density of 0.8 x 10(5) cells x mL-1 for the encapsulation of NSCs resulted in the most expansion, and the NSCs almost doubled during the experiment. The average cell recovery rate is over 88.5%, with the Ca-Alg-Bs dissolving in 55 mM sodium citrate solution for 10 min. The recovered cells cultured in the Ca-Alg-Bs still expressed Nestin and had the capacity of multilineage differentiation into neurons and glial cells and, thus, remained to be NSCs. These results demonstrate that NSC expansion within Ca-Alg-Bs is feasible and provides further possibilities for NSC expansion in bioreactors of the scale of clinical relevance.     

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.     

Cellular characteristics of head and neck cancer stem cells in type IV collagen-coated adherent cultures

Although head and neck squamous carcinoma cancer stem cells (HNSC-CSCs) can be enriched in serum-free suspension cultures, it is difficult to stably expand HNSC-CSC lines in suspension due to spontaneous apoptosis and differentiation. Here, we investigated whether HNSC-CSCs can be expanded without loss of stem cell properties by adherent culture methods. Cell culture plates were coated with type IV collagen, laminin, or fibronectin. We examined cancer stem cell traits of adherent HNSC-CSCs grown on these plates using immunocytochemistry for stem cell marker expression and analyses of chemo-resistance and xenograft tumorigenicity. We also assessed the growth rate, apoptosis rate, and gene transduction efficiency of adherent and suspended HNSC-CSCs. HNSC-CSCs grew much faster on type IV collagen-coated plates than in suspension. Adherent HNSC-CSCs expressed putative stem cell markers (OCT4 and CD44) and were chemo-resistant to various cytotoxic drugs (cisplatin, fluorouracil, paclitaxel, and docetaxel). Adherent HNSC-CSCs at the limiting dilution (1000 cells) produced tumors in nude mice. Adherent HNSC-CSCs also showed less spontaneous apoptotic cell death and were more competent to lentiviral transduction than suspended HNSC-CSCs. In conclusion, compared to suspension cultures, adherence on type IV collagen-coated culture plates provides better experimental conditions for HNSC-CSC expansion, which should facilitate various refined cellular studies.     

Survival of Neural Stem Cells Undergoing DNA Damage-Induced Astrocytic Differentiation in Self-Renewal-Promoting Conditions In Vitro

We recently reported that neural stem cells (NSCs) become senescent and commit to astrocytic differentiation upon X-ray irradiation. Surprisingly, under self-renewing culture conditions, some of these senescent cells undergo p53-independent apoptosis, which can be suppressed by caspase inhibition and BCL2 overexpression. Inhibition of apoptosis proved beneficial for astroglial differentiation efficiency; hence the toxicity of DNA damage on NSCs was specifically tested in context of the culture conditions. In this regard, self-renewal-promoting culture conditions proved incompatible with terminal astrocyte differentiation and impacted negatively on the viability of NSCs following DNA damage-induced cell cycle exit. On the contrary, a switch to differentiation-supporting conditions ablated apoptosis and conveyed tolerance to DNA damage. Thus, stem cell death has likely not originated from DNA break toxicity, while the potentially confounding effect of stem cell niche should always be taken in consideration in stem cell irradiation experiments.     

Scalable GMP compliant suspension culture system for human ES cells

Suspension bioreactors are an attractive alternative to static culture of human embryonic stem cells (hESCs) for the generation of clinically relevant cell numbers in a controlled system. In this study, we have developed a scalable suspension culture system using serum-free defined media with spinner flasks for hESC expansion as cell aggregates. With optimized cell seeding density and splitting interval, we demonstrate prolonged passaging and expansion of several hESC lines with overall expansion, yield, viability and maintenance of pluripotency equivalent to adherent culture. Human ESCs maintained in suspension as aggregates can be passaged at least 20 times to achieve over 1×10(13) fold calculated expansion with high undifferentiation rate and normal karyotype. Furthermore, the aggregates are able to differentiate to cardiomyocytes in a directed fashion. Finally, we show that the cells can be cryopreserved in serum-free medium and thawed into adherent or suspension cultures to continue passaging and expansion. We have successfully used this method under cGMP or cGMP-equivalent conditions to generate cell banks of several hESC lines. Taken together, our suspension culture system provides a powerful approach for scale-up expansion of hESCs under defined and serum-free conditions for clinical and research applications.     

Generation of human cortical neurons from a new immortal fetal neural stem cell line

Isolation and expansion of neural stem cells (NSCs) of human origin are crucial for successful development of cell therapy approaches in neurodegenerative diseases. Different epigenetic and genetic immortalization strategies have been established for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new, clonal NSC (hc-NSC) line, derived from human fetal cortical tissue, based on v-myc immortalization. Using immunocytochemistry, we show that these cells retain the characteristics of NSCs after more than 50 passages. Under proliferation conditions, when supplemented with epidermal and basic fibroblast growth factors, the hc-NSCs expressed neural stem/progenitor cell markers like nestin, vimentin and Sox2. When growth factors were withdrawn, proliferation and expression of v-myc and telomerase were dramatically reduced, and the hc-NSCs differentiated into glia and neurons (mostly glutamatergic and GABAergic, as well as tyrosine hydroxylase-positive, presumably dopaminergic neurons). RT-PCR analysis showed that the hc-NSCs retained expression of Pax6, Emx2 and Neurogenin2, which are genes associated with regionalization and cell commitment in cortical precursors during brain development. Our data indicate that this hc-NSC line could be useful for exploring the potential of human NSCs to replace dead or damaged cortical cells in animal models of acute and chronic neurodegenerative diseases. Taking advantage of its clonality and homogeneity, this cell line will also be a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in human NSC biology.     

Characteristics of neural stem cells expanded in lowered oxygen and the potential role of hypoxia-inducible factor-1Alpha

It has recently been reported that hypoxia promotes the survival and proliferation of neural stem cells (NSCs). In the present study, we examine the differentiation ability of neural precursors expanded under lowered oxygen conditions, and the potential role of hypoxia-inducible factor (HIF)-1alphain vitro, which is the key molecule in response to lowered oxygen. The NSCs were cultured in a 3% O(2) environment for 3 days, and differentiated with 1% fetal bovine serum (FBS) for another 5-7 days, and the cell lineage was evaluated by immunohistochemistry, flow cytometry and HPLC. Compared with the normal condition, the NSCs cultured in hypoxia (3% O(2)) displayed an increase in the percentage of neurons. Especially the percentage of TH-positive neurons differentiated from NSCs in lowered oxygen increased significantly; the dopamine (DA) content in the medium was higher than under normal conditions. These data indicate that lowered oxygen favors dopaminergic differentiation. We then examined the expression of HIF-1alpha during differentiation of NSCs. The levels of HIF-1alpha mRNA expression under 3% oxygen did not change as compared with those under normal conditions. However, HIF-1alpha protein expression was higher from 3 to 72 h during hypoxia than under normal conditions. Overexpression of HIF-1alpha significantly increased the number of TH-positive cells and the DA content in culture medium under normal conditions. These results suggest that HIF-1alpha is involved in the regulation of dopaminergic differentiation of NSCs in lowered oxygen. This study may also offer a new approach to yield DA neurons using a physical factor.     

The Effects of Co-Culture of Embryonic Stem Cells with Neural Stem Cells on Differentiation

Researching the technology for in vitro differentiation of embryonic stem cells (ESCs) into neural lineages is very important in developmental biology, regenerative medicine, and cell therapy. Thus, studies on in vitro differentiation of ESCs into neural lineages by co-culture are expected to improve our understanding of this process. A co-culture system has long been used to study interactions between cell populations, improve culture efficiency, and establish synthetic interactions between populations. In this study, we investigated the effect of a co-culture of ESCs with neural stem cells (NSCs) in two-dimensional (2D) or three-dimensional (3D) culture conditions. Furthermore, we examined the effect of an NSC-derived conditioned medium (CM) on ESC differentiation. OG2-ESCs lost the specific morphology of colonies and Oct4-GFP when co-cultured with NSC. Additionally, real-time PCR analysis showed that ESCs co-cultured with NSCs expressed higher levels of ectoderm markers Pax6 and Sox1 under both co-culture conditions. However, the differentiation efficiency of CM was lower than that of the non-conditioned medium. Collectively, our results show that co-culture with NSCs promotes the differentiation of ESCs into the ectoderm.     

低氧促进神经干细胞向多巴胺能神经元分化

神经干细胞（neural stem cells,NSCs）作为具有多向分化潜能的神经前体细胞,被广泛应用于细胞移植等研究,而低氧不但调节干细胞的体外增殖,在干细胞分化中也具有重要的作用。本文着重探讨了低氧对NSCs分化的调节作用。采用Wistar孕大鼠（E13．5d）,分离胚胎中脑NSCs,加入无血清DMEM／F12培养液（含20ng／mL EGF、20ng／mL bFGF、1％ N2和B27）,3～5d后传代,细胞培养至第三代进行诱导分化,分别在低氧（3％O2）和常氧（20％O2）条件下诱导分化3d,然后在常氧条件下分化成熟5～7d（DMEM／F12含1％FBS、N2和B27）后进行检测。Nestin、NeuN以及TH免疫组织化学鉴定NSCs;流式细胞术分析测定NSCs向TH阳性神经元方向的分化;高效液相色谱测定细胞培养上清液中多巴胺（dopamine,DA）含量。结果显示,分离培养的NSCs均为nestin阳性细胞;低氧可明显促进NSCs向神经元方向的分化;TH阳性神经元比例在常氧和低氧组分别为（10．25±1．03）％和（19．88±1．44）％。NSCs诱导分化7d后,低氧组细胞培养上清液中DA浓度明显增加,约为常氧组的2倍（P〈0．05,n=8）。上述结果表明,3％低氧可促进NSCs向神经元方向,特别是向DA能神经元方向分化。这为NSCs应用于临床治疗帕金森病提供了基础。     

Scalable culture and cryopreservation of human embryonic stem cells on microcarriers

As a result of their pluripotency and potential for unlimited self‐renewal, human embryonic stem cells (hESCs) hold tremendous promise in regenerative medicine. An essential prerequisite for the widespread application of hESCs is the establishment of effective and efficient protocols for large‐scale cell culture, storage, and distribution. At laboratory scales hESCs are cultured adherent to tissue culture plates; these culture techniques are labor‐intensive and do not scale to high cell numbers. In an effort to facilitate larger scale hESC cultivation, we investigated the feasibility of culturing hESCs adherent to microcarriers. We modified the surface of Cytodex 3 microcarriers with either Matrigel or mouse embryonic fibroblasts (MEFs). hESC colonies were effectively expanded in a pluripotent, undifferentiated state on both Matrigel‐coated microcarriers and microcarriers seeded with a MEF monolayer. While the hESC expansion rate on MEF‐microcarriers was less than that on MEF‐plates, the doubling time of hESCs on Matrigel‐microcarriers was indistinguishable from that of hESCs expanded on Matrigel‐coated tissue culture plates. Standard hESC cryopreservation methodologies are plagued by poor viability and high differentiation rates upon thawing. Here, we demonstrate that cryopreservation of hESCs adherent to microcarriers in cryovials provides a higher recovery of undifferentiated cells than cryopreservation of cells in suspension. Together, these results suggest that microcarrier‐based stabilization and culture may facilitate hESC expansion and storage for research and therapeutic applications. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Properties of a fetal multipotent neural stem cell (NEP cell)

Multipotent neural stem cells (NSCs) present in the developing neural tube (E10.5, neuroepithelial cells; NEP) were examined for the expression of candidate stem cell markers, and the expression of these markers was compared with later appearing precursor cells (E14.5) that can be distinguished by the expression of embryonic neural cell adhesion molecule (E-NCAM) and A2B5. NEP cells possess gap junctions, express connexins, and appear to lack long cilia. Most candidate markers, including Nestin, Presenilin, Notch, and Numb, were expressed by both NEP cells as well as other cell populations. Fibroblast growth factor receptor 4 (FGFR4), Frizzled 9 (Fz9), and SRY box-containing gene 2 (Sox2) as assessed by immunocytochemistry and in situ hybridization are markers that appear to distinguish NSCs from other precursor cells. Neither Hoechst 33342 nor rhodamine-123 staining, telomerase (Tert) expression, telomerase activity, or breakpoint cluster region protein 1 (Bcrp1) transporter expression could be used to distinguish NEP stem cells from other dividing cells. NEP cells, however, lacked expression of several lineage markers that are expressed by later appearing cells. These included absence of expression of CD44, E-NCAM, A2B5, epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor-alpha (PDGFR alpha), suggesting that negative selection using cell surface epitopes could be used to isolate stem cell populations from mixed cultures of cells. Using mixed cultures of cells isolated from E14.5 stage embryos, we show that NEP cells can be enriched by depleting differentiating cells that express E-NCAM or A2B5 immunoreactivity. Overall, our results show that a spectrum of markers used in combination can reliably distinguish multipotent NSCs from other precursor cells as well as differentiated cells present in the CNS.     

Efficient cultivation of neural stem cells with controlled delivery of FGF-2

Neural stem cells (NSCs) raised the hope for cell-based therapies in human neurodevelopmental and neurodegenerative diseases. Current research strategies aim to isolate, enrich, and propagate homogeneous populations of neural stem cells. Unfortunately, several concerns with NSC cultures currently may limit their therapeutic promise. Exhaustion of growth factors and/or their uncontrolled release with burst and fall in their concentration may greatly affect the in vitro behavior of NSCs. In this context, we investigate whether a device containing heparan sulfate (HS), which is a co-factor in growth factor-mediated cell proliferation and differentiation, could potentiate and prolong the delivery of fibroblast growth factor-2 (FGF-2) and thus improve in vitro NSC cultivation. We demonstrated that NSCs cultivated in media with a controlled release of FGF-2 from a polyelectrolyte polymer showed a higher proliferation rate, and reduced apoptosis and senescence. In these experimental conditions NSCs preserve their stemness properties for a longer period of time compared with controls. Also of interest is that cell fate properties are conserved as well. The controlled release of FGF-2 reduced the level of oxidative stress and this is associated with a lower level of damaged DNA. This result may explain the reduced level of senescence and apoptosis in NSCs cultivated in the presence of hydrogel-releasing FGF-2.     

Neural stem cells: involvement in adult neurogenesis and CNS repair

Recent advances in stem cell research, including the selective expansion of neural stem cells (NSCs) in vitro, the induction of particular neural cells from embryonic stem cells in vitro, the identification of NSCs or NSC-like cells in the adult brain and the detection of neurogenesis in the adult brain (adult neurogenesis), have laid the groundwork for the development of novel therapies aimed at inducing regeneration in the damaged central nervous system (CNS). There are two major strategies for inducing regeneration in the damaged CNS: (i) activation of the endogenous regenerative capacity and (ii) cell transplantation therapy. In this review, we summarize the recent findings from our group and others on NSCs, with respect to their role in insult-induced neurogenesis (activation of adult NSCs, proliferation of transit-amplifying cells, migration of neuroblasts and survival and maturation of the newborn neurons), and implications for therapeutic interventions, together with tactics for using cell transplantation therapy to treat the damaged CNS.     

Isolation and characterization of neural stem cells from human fetal striatum

This paper described that neural stem cells (hsNSCs) were isolated and expanded rapidly from human fetal striatum in adherent culture. The population was serum- and growth factor-dependent and expressed neural stem cell markers. They were capable of multi-differentiation into neurons, astrocytes, and oligodendrocytes. When plated in the dopaminergic neuron inducing medium, human striatum neural stem cells could differentiate into tyrosine hydroxylase positive neurons. hsNSCs were morphologically homogeneous and possessed high proliferation ability. The population doubled every 44.28h and until now it has divided for more than 82 generations in vitro. Normal human diploid karyotype was unchanged throughout the in vitro culture period. Together, this study has exploited a method for continuous and rapid expansion of human neural stem cells as pure population, which maintained the capacity to generate almost fifty percent neurons. The availability of such cells may hold great interest for basic and applied neuroscience.