Extrusion-based bioprinting (EBB) is a rapidly developing technique that has made substantial progress in the fabrication of constructs for cartilage tissue engineering (CTE) over the past decade. With this technique, cell-laden hydrogels or bio-inks have been extruded onto printing stages, layer-by-layer, to form three-dimensional (3D) constructs with varying sizes, shapes, and resolutions. This paper reviews the cell sources and hydrogels that can be used for bio-ink formulations in CTE application. Additionally, this paper discusses the important properties of bio-inks to be applied in the EBB technique, including biocompatibility, printability, as well as mechanical properties. The printability of a bio-ink is associated with the formation of first layer, ink rheological properties, and crosslinking mechanisms. Further, this paper discusses two bioprinting approaches to build up cartilage constructs, i.e., self-supporting hydrogel bioprinting and hybrid bioprinting, along with their applications in fabricating chondral, osteochondral, and zonally organized cartilage regenerative constructs. Lastly, current limitations and future opportunities of EBB in printing cartilage regenerative constructs are reviewed. Bioprinting of personalized complex tissue grafts is promising for overcoming the current challenges of cartilage tissue engineering (CTE). Cartilage is a highly hydrated and specialized tissue to provide a low-friction, wear-resistant, and load-bearing surface in diarthrodial joints for efficient joint movement . UnfortuAlthough some reviews on bioprinting of tissues and organs are available, investigation into the extrusion-based bioprinting (EBB) of cartilage constructs from bio-inks has not been well-documented. This article presents a brief review of the application of EBB for fabricating cartilage constructs from bio-inks, covering its working principles, applicable cell sources and materials, printability, printed cartilage constructs, as well as future perspectives of bioprinting cartilage.Rapid prototyping (RP), also known as solid freeform fabrication, refer to a series of techniques that manufacture objects through sequential delivery of energy and/or material in a layer-by-layer manner per computer aided design (CAD) data. The external shape and internal architecture of the scaffold can be defined by either 3D computer models or clinical imaging data  ,18. OnceAmong various RP techniques, EBB stands out for its unique advantages. It allows for production of 3D tissue constructs from bio-inks by a layer-by-layer deposition process in a designed way . EBB alsHydrogel precursors and living cells are two important components of bio-ink formulations. Cell sources and hydrogel types employed for encapsulating chondrogenic cells are reviewed below.The choice of cells is a central problem to any modality of TE. For cartilage bioprinting, several factors need to be taken into consideration when choosing suitable cell sources: (i) cells must be robust enough to survive any shear stress and pressure during the printing process; (ii) cells must proliferate well; (iii) cells must possess biosynthesis levels  comparable with native chondrocytes so they can maintain their biological functions . So far,Consistent with the distinct zonal structure of native articular cartilage , chondro® F127, PEG, and PVA). Hydrogels that are biocompatible for encapsulating stem cells or chondrogenic cells for CTE are summarized and reviewed  between generated constructs and CAD data ,126.The printing and formation of the first layer of bio-inks play an important role for fabricating the whole construct. A relatively large contact angle between dispensed bio-inks and the substrate help to maintain the vertical dimension of printed bio-inks and avoid the flattening of the printed hydrogel precursor solution. The interaction between printed bio-inks and substrate is crucial, since suitable interaction helps to anchor the whole bioprinted construct on the printing surface and avoids possible deformation and undesired movement during the layer-by-layer bio-inks deposition process. Unfortunately, most receiving surfaces such as glass or plastic have poor contact angles with bio-inks and it is difficult to establish any interaction between receiving surface and dispensed bio-inks. These issues could be addressed by either printing hydrogels in a hydrophobic high-density fluid, such as perfluorotributylamine , or coatViscosity describes the internal resistance of a fluid to flow upon application of stress. The viscosity of a polymer solution is determined by its concentration, molecular weight, and temperature. Higher polymer concentration and molecular weight are associated with higher viscosity. Typically, sufficient viscosity of bio-inks leads to good printability, since it can help the bio-inks to overcome the surface-tension-driven droplet formation and be drawn to form continuous strands. Sufficient viscosity will also help the dispensed strands to maintain the cylinder shape and keep adjacent strands from merging together, which also explains why thermoplastic polymers are usually printed with higher accuracy and resolution than hydrogels. However, cells thrive best in an aqueous environment, in which their matrix deposition is not limited by the dense polymer network . Bio-inkThe viscosity of a bio-ink solution is mainly determined by the polymer concentration and molecular weight. Given that bio-inks with high concentrations may not be favorable for cell proliferation/migration and ECM formation , it is rShear thinning is another desirable feature for bio-inks that will help to improve the printability, and it refers to the fact that viscosity decreases as shear rate increases . PolymerThe printability is also influenced by how easily and efficiently materials can be crosslinked. EBB usually requires printing a cell-laden polymer solution followed by initiating gelation immediately after extrusion. The cell-laden polymer solution must be either prepared quite viscous or crosslinked rapidly after dispensing onto the printing surface to achieve good printability and shape fidelity. However, high viscosity is not ideal for its application in TE and impedes cells survival and proliferation ,133. TheEngineered cartilage should maintain sufficient mechanical properties after bioprinting to provide embedded cells with a stable environment for attachment, proliferation, and differentiation. Particularly for cartilage bioprinting in CTE, mechanical properties are crucial because the functions of cartilage mainly rely on their mechanical performance. Mechanical properties of hydrogel are intrinsically weak compared to cartilage . StrategResearch has supplemented hydrogel with mineral particles  to create composite hydrogels, by combining organic and inorganic phases to obtain desirable properties including the improvement of mechanical properties and enhancement of biological properties ,136. In A novel approach reinforced hydrogel constructs by incorporating printed polycaprolactone (PCL) scaffolds. Hydrogel precursors were poured and perfused into the printed porous PCL scaffold and crosslinked. In this way, the stiffness of the resulting constructs could be tailored to that of native cartilage by reinforcement with high-porosity PCL scaffolds . FabricaA higher mechanical strength can also be achieved by blending multiple polymers and varying the molar ratio of bio-ink components. From instance, nanocellulose and alginate composite bio-ink was synthesized and printed to fabricate chondrocyte-laden constructs. Increasing the alginate fraction in bio-ink formula would lead to an increase in compressive modulus of printed constructs .Making use of the crosslinking mechanism is also an efficient way to enhance the mechanical properties of the printed constructs. For example, a three-step method was used to crosslink alginate hydrogel for improved elastic stiffness; furthermore, the three steps are the primary calcium ionic cross-linking to increase the initial viscosity of alginate, secondary calcium ionic crosslinking to solidify the printed structure, and tertiary barium ionic crosslinking to strengthen elastic stiffness .Another effective way to enhance the mechanical properties is the use of hybrid bioprinting to co-deposit hydrogels and thermoplastic polymers alternately. Cell-laden hydrogels are supported by printed thermoplastic polymers; thus, these hybrid constructs possess mechanical characteristics that are mainly provided by the printed thermoplastic polymer frame, which is significantly higher than the hydrogel-only constructs . MeanwhiIf a scaffold is designed to initially promote engineered tissue formation in vitro prior to implantation in vivo, then they are not required to exactly match the mechanical properties of natural cartilage at the initial stage. Thereby, many hydrogel-based cartilage bioprinting research still focus on formulating bio-inks to favor the synthesis of cartilaginous ECM instead of their initial mechanical strength with the hope that the ECM generated by the cells in vitro provides sufficient mechanical properties upon implantation in vivo.Current cartilage constructs are mainly printed based on two approaches: (i) direct printing of cartilage constructs from bio-inks  and (ii) alternating printing of bio-inks and thermoplastic-polymer network . The advantages of self-supporting hydrogel bioprinting rests on their mild and physiological crosslinking conditions and its relatively simple process as compared to hybrid bioprinting. However, the self-supporting bioprinting requires a high level of printability of bio-inks and the printed hydrogel constructs typically have week mechanical properties . In contSelf-supporting hydrogel bioprinting approaches form cartilage constructs for CTE application by printing stem cell- or chondrocyte-laden natural and synthetic hydrogels . ChondroHyaluronic acid (HA), as an essential component of cartilage ECM, can mediate cellular signaling, wound repair, and ECM organization due to its structural and biological properties . More reAlthough gelatin gel has been shown to support chondrocyte viability and differentiation, its low viscosity and de-crosslinking at 37 °C make it hard to print . TherefoAcrylation is also commonly used with synthetic hydrogels to facilitate cartilage bioprinting. An example is printing poly (ethylene glycol) dimethacrylate (PEGDMA) together with human chondrocytes to repair defects with osteochondral plugs through a layer-by-layer manner. The printed construct showed a higher mechanical property of 395.73 kPa than most printed natural hydrogels. This study demonstrated that hydrogel bioprinting is a feasible approach of producing cartilage constructs with anatomic characteristics to accurate targeted locations. The embedded human chondrocyte viability was 89% and showed an elevated glycosaminoglycan (GAG) content. Additionally, printed cartilage constructs firmly attached to the surrounding tissue and showed even greater proteoglycan deposition at the interface of implant and native cartilage .Improving the integrity between the engineered cartilage and subchondral bone remains a challenge. In this regard, a self-supporting hydrogel construct was printed onto the printed bone paste  to mimic the cartilage and subchondral bone respectively . HeterogTo sum up, self-supporting hydrogel bioprinting of cartilage constructs can be processed under cytocompatible conditions and printed constructs are generally shown to support cartilage ECM biosynthesis. Current research emphasis is focused on formulating bio-inks to achieve high printability and improving the mechanical performance of printed constructs. The relatively weak mechanical properties of printed hydrogel-based cartilage constructs limit its application to regenerating focal cartilage defects, where most exerted force is born by its surrounding tissue. To overcome these issues, hybrid cartilage bioprinting by alternating printing of bio-inks and thermoplastic polymers fibers (hybrid bioprinting) has been brought forward.A hybrid construct combining advantages of hydrogel and thermoplastics has been brought forward, offering potential for application in CTE . ScaffolBy applying this state-of-the-art printing technology, human nasoseptal chondrocyte-laden alginate hydrogel with a supportive PCL structure was printed . The stuHybrid bioprinting also showed good suitability to fabricate osteochondral constructs, enabling researchers to use different bio-inks in cartilage portion and bone portion. A mechanically stable 3D dual cell-laden construct consisting of osteoblasts and chondrocytes for osteochondral tissue engineering using a multi-head extrusion-based printing system was successfully printed. Two different alginate solutions with encapsulated osteoblasts or chondrocytes were deposited into the previously printed PCL framework . A more These studies show the promise of hybrid bioprinting as an advanced fabrication technique for CTE. However, mechanical stimuli exerted on hybrid construct would probably be mainly withstood by the polymeric scaffolds instead of chondrocyte-laden hydrogel because of stress shielding . This miZonal cartilage constructs that reflect the native structural depth-dependent characteristics of articular cartilage could have advantages over homogeneous constructs. A zonal cartilage construct can be achieved by the following strategies: 1) using zonal chondrocyte subpopulations from different zones of cartilage; (2) using a single cell source combined with the correct biochemical and/or biomechanical cues; (3) using different biomaterials and smart scaffold designs. Zonal chondrocyte subpopulations from different zones of cartilage tissue can be harvested  using zo,167, butEBB is a convenient and promising technique that can print porous tissue-engineered constructs with structural and biological properties from a wide range of bio-inks. It still has several limitations, including limited biomaterials for bio-ink formulation, cell death during printing, low resolution as well as insufficient mechanical properties. Bio-inks formulation is restricted by limited printable biomaterials, which makes up only a small portion of biomaterials applied in TE. To alleviate this problem, development of new biomaterials for bio-ink formulation is needed. When formulating and processing new bio-inks, the properties discussed in Other printing parameters, including printing pressure, nozzle geometry and diameter, and bio-ink concentration, have also been shown to influence cell viability within bio-inks ,130. ManTo fabricate functional cartilage construct, suitable cell sources, biological cues, and construct organization are still needed to be determined for successful cartilage regeneration. Most present studies only focus on evaluating cell viability in different bioprinted hydrogels, while functionality of the engineered cartilage is not very well characterized. As such, we also urge that, for bioprinted engineered cartilage constructs, research should also emphasize the overall chondrogenesis within the constructs, either qualitatively  or quantitatively . Moreover, given that the mechanical performance of cartilage engineered from hydrogel is usually inferior to native cartilage, research on mechanical properties is also required for future cartilage bioprinting studies. Notably, current mechanical characterization of engineered cartilage constructs is mainly performed based on a single mechanical test . Howeverin situ” bioprinting has been performed to directly print alginate hydrogels into a defect on an explanted articular surface from a calf [Theoretically, the shape of scaffolds fabricated by bioprinting techniques can match personalized defects in vivo. Notably, current in vivo research is usually based on man-made regular defects, which can be made fitting with a bioprinted scaffold with exact shape and dimension. It could be difficult for the in vitro printed material to match perfectly with the defect that needs to be regenerated. Printed construct could deform during in vitro culture and defects may expand while waiting for implantation. Although defined defects can be created in clinic, this is not desirable since it further increases the area that needs to be regenerated. Therefore, the concept of “m a calf . This stIssues facing CTE is the inability to translate technologies into the clinic and lack of clinic standards of materials for human tissue biopriting . To moveEBB is an advanced fabrication technique to produce customized cell-laden hydrogel-based constructs for CTE so as to mimic chondral, osteochondral, and zonal organization of articular cartilage. Despite the advantages and opportunities provided by hydrogel-based EBB for cartilage bioprinting, there are still multiple challenges that need to be addressed. Bio-inks for EBB need to be synthesized and optimized in terms of their biocompatibility, formulation, processing, printability, and optimal cell sources. Self-supporting hydrogel bioprinting and hybrid bioprinting are two common approaches to fabricate cartilage constructs. The former technique provides a cell-friendly printing environment but limited mechanical strength, while the latter brings elevated mechanical properties but the stress shielding may disable external mechanical stimuli. Tackling the challenges revolving around bio-inks and mechanical performance of resulting cartilage constructs will foster biologically active and living bioprinted implants for future clinical applications.


Neural stem cells (NSCs) have a unique role in neural regeneration. Cell therapy based on NSC transplantation is a promising tool for the treatment of nervous system diseases. However, there are still many issues and controversies associated with the derivation and therapeutic application of these cells. In this review, we summarize the different sources of NSCs and their derivation methods, including direct isolation from primary tissues, differentiation from pluripotent stem cells and transdifferentiation from somatic cells. We also review the current progress in NSC implantation for the treatment of various neural defects and injuries in animal models and clinical trials. Finally, we discuss potential optimization strategies for NSC derivation and propose urgent challenges to the clinical translation of NSC-based therapies in the near future. NSCs are a promising treatment modality for diseases associated with the nervous system as they secrete soluble factors and differentiate into neurons, astrocytes and oligodendrocytes.NSCs can be derived from three different sources using recent technical advances: direct isolation from primary tissues, differentiation from pluripotent stem cells and transdifferentiation from somatic cells.Cell therapies based on NSC transplantation for the treatment of various neural defects and injuries in animal models and clinical trials have been widely investigated.Which NSC derivation strategy is most efficient and safe for clinical translation?How can NSC transplantation methods be translated from preclinical studies into clinical trials?What are the optimization strategies and urgent challenges for the clinical translation of NSC-based therapies in the near future?Nervous system diseases are refractory diseases that can cause loss of sensation, loss of motor function and memory failure, as well as directly threaten the life of a patient. Currently, the pathogenic factors involved in these diseases and their pathogenesis are unclear. Traditional drug treatments are used to delay disease progression and cannot restore function or regenerate tissues. Recent studies have indicated that the transplantation of neural stem cells (NSCs) is a promising treatment modality for diseases associated with the nervous system, for the regeneration of neural cells and for the restoration of the microenvironment at the injury site .The cell source is the first issue that must be addressed to enable the application of NSCs in clinical treatments because the cell dose required for adequate transplantation is very high. NSCs can be derived from three different sources using recent technical advances, including direct extraction from primary tissues, differentiation from pluripotent stem cells and transdifferentiation from somatic cells .in vitro.1 Two years later, they found that the subependymal region in the mouse brain is the source of NSCs in vivo.2 Based on previous results, Weiss further reported that EGF and basic fibroblast growth factor (bFGF) cooperatively induce the proliferation, self-renewal and expansion of NSCs isolated from adult mouse thoracic spinal cord.3 NSCs can grow in single-cell suspensions obtained by enzymatic digestion and form spherical clusters called neurospheres, which are non-adherent and can be re-plated in selective culture medium to obtain neural cells.5 Neurospheres can also be sub-cultured to expand the pool of NSCs for experimental or therapeutic purposes. Periventricular regions and olfactory bulb in adult mammalian brains are rich sources of NSCs.8 Beyond these methods of isolating NSCs through diverse culturing strategies, NSCs can also be directly isolated by cell sorting based on the expression of NSC surface markers.9 Belenguer and Guo have developed an optimized protocol for the isolation, culture and expansion of NSCs from mammalian animals.11 Although, a canonical protocol for obtaining human tissue-derived NSCs has not yet been established, the technical methods are generally similar to the ones applied in animals, and the tissues must be obtained in accordance with ethical guidelines.The establishment of cell isolation and cell culture techniques has led to the development of favorable experimental methods and the identification of a rich cell source for NSC research. In 1992, Reynolds and Weiss isolated NSCs from the striatum of the adult mouse brain and reported the first use of epidermal growth factor (EGF) to induce NSC proliferation Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can generate desired cells through differentiation, which are attractive alternatives to primary cell isolation. Generally, protocols for neural differentiation from pluripotent stem cells can be categorized into two major routes: embryoid body (EB) formation and monolayer culture.12 Zhang et al.13 first described the differentiation, enrichment and transplantation of neural precursor cells from human ESC-derived EBs in vitro. Later, Kozhich et al.14 developed a novel protocol suitable for standardized generation and differentiation of neural precursor cells from human pluripotent stem cells, making iPSC-derived NSCs an appealing source for cell-based therapies.15 Pluripotent stem cells can also directly differentiate into NSCs in monolayer culture via the neural rosettes stage, which is a striking feature here.18 A serum-free and nutrient-poor medium is utilized to initiate differentiation, and depending on the cell line, additional growth factors or inhibitors may be required to promote neural differentiation.19 Banda and Grabel20 have been focusing on using monolayer culture to directly differentiate human ESCs into neural progenitors, which includes four typical stages. Wen and Jin21 have developed a straightforward and useful strategy for the generation of NSCs from both human ESC and iPSC lines. Comparisons of NSC marker expression and morphology have indicated that there are no significant differences between the NSCs derived via EB formation and monolayer culture methods.In the protocol for EB formation, ESC and iPSC colonies are detached and grow in suspension to form EBs. Subsequently, EBs are plated on adhesive substrates with defined serum-free medium that promote the generation of neural tube-like rosettes and the selection of neural progenitor cells (NPCs).22 During this process, one type of mature somatic cell transforms into another type of mature somatic cell without undergoing an intermediate pluripotent state.23 This process is induced mainly by the exogenous expression of lineage-specific transcription factors (TFs) and by chemical compounds.The term transdifferentiation, also known as lineage reprogramming, was originally coined by Selman and Kafatos in 1974.25 which were called induced NSCs (iNSCs). This finding provides a new strategy for the generation of NSCs through direct cell transdifferentiation following virus-mediated exogenous gene expression. Aside from the pluripotency factors, NSC-specific TFs can also induce the generation of neural stem-like cells with self-renewal and tripotent differentiation potential.27 Furthermore, it is found that a single TF, Sox2 or ZFP521, can be used to generate iNSCs from mouse and human fibroblasts.29 Two Chinese teams, led by Zhang and Ding, have separately combined defined TFs with chemical cocktails that enable the generation of expandable iNSCs from both primate and human fibroblasts,31 thus suggesting that small-molecule chemicals can increase the efficiency of iNSC generation. In addition to fibroblasts, many other somatic cell types are considered to be ideal starting cells for NSC generation depending on the clinical situation, including Sertoli cells,32 adult liver cells and B lymphocytes,33 urine epithelial-like cells,34 astrocytes,35 and cord blood sample.36Ding and others first demonstrated that transient expression of pluripotency factors combined with the appropriate neural signaling inputs can successfully induce mouse fibroblasts to form expandable NSCs,In recent years, researchers have explored chemical reprogramming as a new method to manipulate cell fate. Compared with the conventional practice of importing exogenous viral genes to induce cell transdifferentiation, the use of small-molecule chemicals to elicit cell transdifferentiation has many obvious advantages in terms of safety and controllability.β, respectively, to generate chemically induced NPCs (ciNPCs) from mouse embryonic fibroblasts under hypoxic conditions.37 Further assays confirmed that mouse tail-tip fibroblasts and human urinary cells can also be induced into ciNPCs via treatment with the same chemical cocktail. This work demonstrates that direct lineage-specific conversion to NPCs can be achieved without introducing exogenous genes and that physiological hypoxia is essential for the initial transition process. In the absence of hypoxic conditions, Zhang et al.38 developed a cocktail of eight small-molecule components, namely CHIR99021, LDN193189 (an inhibitor of the BMP type I receptor ALK2/3), A83-01 (an inhibitor of the TGF-β type I receptor ALK4/5/7), Hh-Ag1.5 (a potent smoothened agonist), RA, SMER28 (an autophagy modulator), RG108 (a DNA methyltransferase inhibitor) and Parnate (a histone demethylase inhibitor), which can efficiently and specifically transdifferentiate mouse fibroblasts into NSC-like cells. These cells resemble primary NSCs in terms of their long-term self-renewal and tripotent differentiation abilities. Takayama et al.39 also developed a small-molecule cocktail composed of VPA, Forskolin (an adenylyl cyclase activator), Parnate, CHIR99021, Repsox, Dorsomorphin  and SB431542  to induce neural crest-like precursors from mouse embryonic fibroblasts. Zheng et al.40 showed that a combination of A83-01, Purmorphamine (a smoothened receptor agonist), VPA and Thiazovivin (a selective Rho-associated protein kinase inhibitor) can directly lead to the generation of ciNSCs. Despite these achievements, the mechanisms underlying chemically induced transdifferentiation remain largely unknown. Global gene expression profiles determined through microarray analysis have revealed that the small-molecule-based culture method strongly affects cell identity and specifically induces neural differentiation and development-related genes. Interestingly, small molecules targeting HDACs, GSK-3 and TGF-β are included in most of the abovementioned reports and may constitute the core chemicals. It can be speculated that HDAC inhibitors cause chromatin decondensation and induce cells into a plastic state, TGF-β inhibitors may regulate cell transition between the mesenchymal and epithelial states and promote cell fate conversion, and GSK-3 inhibitors probably activate Wnt signaling, which helps maintain stem cell properties.41We were the first to report using only a chemical cocktail containing valproic acid (VPA), CHIR99021 and Repsox, which inhibits histone deacetylases (HDACs), glycogen synthase kinase (GSK)-3 and transforming growth factor (TGF)-et al.42 successfully established a three-step induction protocol that generates highly purified neural stem-like cells from human adipose-derived mesenchymal stem cells (MSCs) by activating SOX1 with conditional medium, EGF and bFGF. In addition, Song and Sanchez-Ramos43 proposed a detailed protocol with which to generate neural-like progenitors from bone marrow-derived MSCs and umbilical cord blood-derived MSCs. Later, Ge et al.44 found that cerebrospinal fluid containing growth factors may be a better microenvironment for a more rapid transition of MSCs to a NSC fate. Recently, Gao et al.45 reported a method for neural precursor cell generation from mouse fibroblasts using physical stress and a few growth factors, including EGF, bFGF, leukemia inhibitory factor and heparin, which synergize to regulate the signaling pathways upstream and downstream of Sox2. During this direct induction process, cells first pass through a transient partially reprogrammed state, and then, cell transdifferentiation is achieved via a safe, non-integrated and efficient method.Regardless of the method used for NSC derivation, growth factors are utilized, which indicate the significance of these factors. Without the introduction of any exogenous genes and chemicals, Feng in vivo, a three-dimensional in vitro culture system should mimic the complex physical environment and enhance NSC self-renewal and multipotency compared with traditional two-dimensional culture conditions. Su et al.46 reported that mouse fibroblasts can be converted into three-dimensional spheres on non-adherent substrates and later exhibit the characteristics of neural progenitor-like cells in terms of cell morphology, specific marker expression and self-renewal ability. Immunocytochemical experiments have indicated that the expression of Sox2 is significantly upregulated in three-dimensional cultured mouse fibroblasts. This study has introduced a new paradigm for safer and more convenient cell transdifferentiation using physical tools. This study suggests that other three-dimensional scaffolds might also be used during iNSC generation. For example, graphene foam, a three-dimensional porous scaffold that is biocompatible and conducive to NSC proliferation, has shown great potential for NSC research, neural tissue engineering and neural prostheses.47Given that stem cells reside in specific niches Neurodegenerative diseases are caused by neural or glial cell defects in the brain or spinal cord, which lead to memory deterioration, cognitive disorders, dementia or body movement disorders and mainly include amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), Alzheimer's disease (AD) and Huntington’s disease (HD).48 Human NSCs secrete glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor, which induced the regeneration of motor neurons in a transgenic rat model of ALS.49 Besides, human iPSCs-derived NSCs effectively improve the function of neuromuscular and motor units and significantly increase the lifespan of ALS mice after intrathecal or intravenous injection.50 Beyond the animal models, NSC treatment for ALS has been at the clinical trial stage for years. The clinical results indicate the safety of this therapeutic approach via spinal cord injection.53ALS is characterized by degeneration and loss of motor neurons in the cerebral cortex, brain stem and spinal cord, thus resulting in muscle wasting, weakness and, eventually, death within 5 years.54 In toxin-induced animal models of PD, transplanted human NSCs stimulate the dedifferentiation of rat astrocytes and the secretion of exogenous growth factors, thus inhibiting the activation of microglial cells and slowing PD progression by modulating the lesion microenvironment.56 As Lmx1a contributes to NSC differentiation into dopamine neurons, the transplantation of Lmx1a-overexpressing iNSCs markedly enhances the efficiency of dopamine neuron production and elicits therapeutic effects in a PD mouse model.57 Although NSC transplantation in PD animal models has shown a certain degree of benefit,61 additional studies are required to elucidate its clinical efficacy and safety. To date, one report has verified that human parthenogenetic stem cell-derived NSCs (hpNSCs) can successfully engraft, survive long-term and increase dopamine levels in the brains of rodent and nonhuman primate models of PD. In addition, hpNSCs have negligible tumourigenic potential and are safe for clinical application, thus supporting the approval of an hpNSC-based phase I/IIa study for the treatment of PD.63PD is a disease characterized by the loss of dopamine neurons in the substantia nigra pars compacta and their terminals in the striatum.64 It has also been reported that hippocampal neuronal mitochondria levels are decreased in AD patients. Transplantation of exogenous NSCs into transgenic AD mice leads to a significant increase in the number of mitochondria and the expression of mitochondria-related proteins, as well as improvements in mouse cognitive function.65 The therapeutic effect of NSC transplantation can be further improved by combining with cerebrolysin controlling amyloid precursor protein metabolism,66 self-assembled peptides providing a protective niche67 and nerve growth factor nanoparticles.68 Human NSCs have also been intensively investigated as an AD treatment in transgenic animal models.70 Although many positive results have been reported based on those models, there were still negative consequences emerged. Marsh et al.71 used an immune-deficient AD model to examine the long-term effects of the transplantation of human NSC products and found that five months after transplantation, human NSCs had engrafted and migrated throughout the hippocampus; however, changes in brain-derived neurotrophic factor expression and increases in synaptic density were not observed. The disappointing result of this assay might be due to the failure of the human NSCs to terminally differentiate, which reinforces the notion that candidate cells need to be thoroughly evaluated for safety and efficacy before every transplantation. Considering these inconsistent data, to date, there have been no clinical study of NSC transplantation in AD patient.AD is characterized by increased levels of both soluble and insoluble amyloid beta peptides.72 Animal HD models can be established by injecting quinolinic acid into rodent and primate striatum to simulate excitotoxicity. Human fetal NSCs can differentiate into neurons and astrocytes following transplantation into the rat striatum, partially eliciting behavioral and anatomical recovery in HD rats.73 Furthermore, intracerebral transplantation of NSCs combined with trehalose has been found to not only alleviate polyglutamine aggregation formation and decrease striatal volume but also extend lifespan in transgenic mouse model of HD.74 To further promote the beneficial effects of transplanting NSCs in animal models of HD, the timing of transplantation and cell preparation, NSC activity and co-transplantation of NSCs with helper cells, such as MSCs that secrete brain-derived neurotrophic factor, need to be fully considered based on the pathological conditions.76HD is an autosomal-dominant inherited disease that induces caudate nucleus atrophy and is characterized by involuntary choreic movements, cognitive impairment and emotional disturbance.77 There are no effective conventional treatments for SCI, but transplantation of NSCs in a mouse model of SCI leads to significant improvements in motor function recovery, thus indicating that NSCs can survive in vivo, differentiate, and alter the microenvironment of early chronic injury sites.78 In a primate SCI model, transplanted NSCs have been found to differentiate into cells expressing neuronal markers, thereby improving hind limb performance.79 Considering that treatment of SCI with NSCs is regulated by a variety of cytokines and proteins, drugs that modulate these factors will be a helpful adjunctive therapy, such as etanercept having anti-inflammatory and anti-apoptotic effects,80 free radical scavenger edaravone,81 and erythropoietin.82 With the rapid development of tissue engineering techniques, biomaterials have gradually been applied to SCI treatment and have provided new prospects for NSC transplantation; for example, biodegradable scaffolds with aligned columns83 and gelatine sponge scaffold.84 In contrast to these positive results, Anderson et al.85 found that human CNS-derived stem cells failed to show preclinical efficacy in a pathway study of cervical SCI. In that study, no evidence of donor cell differentiation into the neuronal lineage was observed. This failure might be attributed to the insufficient characterization of the clinical cell line supplied by the sponsor using potency assays. However, in a phase I/IIa clinical trial on the transplantation of fetal cerebral NSCs into 19 traumatic cervical SCI patients, 17 patients regained sensorimotor function after 1 year, and 2 patients showed complete motor but incomplete sensory recovery. There was no evidence of cord damage; syrinx or tumor formation; neurological deterioration; and exacerbating neuropathic pain or spasticity.86Spinal cord injury (SCI) is a severe physical injury and often gives rise to severe loss of motor function and secondary damage.88 Transplanted mouse iPSC-derived or human fetus-derived NSC lines have been reported to provide neurotrophic factors and increase angiogenesis and neurogenesis in both ischemic and hemorrhagic stroke animal models.90 Besides, Li et al.91 demonstrated that compared with transplantation of NPCs alone, co-transplantation with vascular progenitor cells, which might support NPC survival, leads to more effective improvements in neurovascular recovery and attenuation of the infarct volume. Furthermore, the application of three-dimensional electrospun fibers as cell carriers has shown promising results and has been found to extend the survival rate of administered human NSCs by blocking microglial infiltration in an animal model of stroke induced by middle cerebral artery occlusion.92 Despite positive results from animal assays, caution should be exercised before clinical trials, because other findings have suggested that endogenous neurogenesis is decreased after NPC treatment via microglial activation.93 However, in pioneering work, the United Kingdom has already initiated phase I/II clinical trials on the treatment of ischemic stroke with CTX0E03, an immortalized human NSC line.94 Thirteen men were recruited for the phase I trial in which a single dose of up to 20 million cells was implanted via stereotactic ipsilateral putamen injection, and whereas neurological function was improved, no adverse events were observed. Based on the results of the phase I trial, the phase II trial was initiated and is still underway.Stroke is an acute cerebrovascular disease that includes ischemic and hemorrhagic stroke.95 TBI is extremely likely to cause cognitive and memory deficits as well as motor impairments. Previous study concluded that NSCs may stabilize the cortical microenvironment after TBI.96 Transplantation of mouse brain-derived NSCs into brain injury mice effectively prevents astroglial activation and microglial/macrophage accumulation while increasing oligodendrocytes and repairing and maintaining normal neuron function.97 A sodium hyaluronate collagen scaffold loaded with bFGF promotes the survival and differentiation of transplanted rat NSCs and promotes functional synapse formation to repair traumatic brain injuries in rats.98 In a clinical study, Zhu’s group labeled autologous cultured NSCs with superparamagnetic iron oxide nanoparticles and then stereotactically implanted them around the regions of brain trauma in TBI patients. Magnetic resonance imaging tracking images showed the accumulation and proliferation of cells around the lesion and even their migration from the primary sites of injection to the border of the damaged tissue.99The principal mechanisms of traumatic brain injury (TBI) are classified as focal brain damage and diffuse brain damage, which correspond to contact injury types and acceleration/deceleration injury types.γ-aminobutyric acid and astrocytes that secrete anticonvulsant factors, which slow the cognitive and emotional dysfunction caused by TLE.101Epilepsy is an abnormal discharge of cerebral neurons resulting from an imbalance between excitation and inhibition in the CNS; this imbalance leads to transient cerebral dysfunction, which is mainly related to changes in ion channel neurotransmitters and glial cells. Nearly 30% of patients with temporal lobe epilepsy (TLE) are resistant to antiepileptic drugs. Current reports demonstrate that NSC transplantation can inhibit spontaneous seizures. When transplanted into the epileptic sites of the hippocampus, exogenous NSCs produce a specific type of neuron that synthesizes the inhibitory neurotransmitter 102 At present, CP treatment includes many measures, but none of these treatments can cure CP patients. In recent years, the safety and efficacy of NSC/NPC therapy for CP has been evaluated. Based on current studies, transplantation of NSCs transduced with vascular endothelial growth factor can partially slow brain damage and has neuroprotective effects on neonatal rats with hypoxia-mediated CP, thus suggesting a new potential strategy for CP treatment.104 In clinic, motor development and cognition have been found to be significantly accelerated after aborted fetal tissue-derived NPCs were injected into the lateral ventricle of children with CP.105 Besides, CP patients undergo bone marrow MSC-derived neural stem-like cell transplantation were followed up for long term. Improvements in motor function have been observed following this procedure but not improvements in language quotient, demonstrating that neural stem-like cells derived from autologous MSCs may be a better cell type for CP therapy.106Cerebral palsy (CP) is a group of permanent movement disorders that appear in early childhood, which is due to the formation of non-progressive lesions in the developing central nervous system.108 Recent research has shown that mild hypothermia therapy for neonatal encephalopathy via NSC transplantation can attenuate disease progression and prevent long-term damage associated with HIE.109 The ginsenoside Rg1 has been reported to be effective in promoting the recovery of brain function after injury, and thus, Li et al.110 performed ginsenoside-induced NSC transplantation into HIE rats and reported improved behavioral capacity compared with the control group that received only saline, thus presenting a promising new treatment for brain injury. In addition, human embryonic NSC treatment significantly improves learning, memory and cognitive deficits in HIE rat.111 Based on these preclinical studies, much effort is required to translate the NSC transplantation techniques for evaluation in clinical trials involving HIE patients.Hypoxic-ischemic encephalopathy (HIE) is a condition that occurs when the entire brain is incompletely deprived of adequate oxygen supply.112 Although treatment options for AMD are limited, transplantation of human NPCs into a rodent model of retinal degeneration not only halted the progression of vision loss but also preserved photoreceptors and visual function long term.114 A phase I/II clinical study is investigating the safety and preliminary efficacy of unilateral subretinal transplantation of human NSCs in subjects with geographic atrophy secondary to AMD. In addition, intensive research and development have resulted in a series of NSC products and genetically modified NSCs entering the preclinical study phase and clinical testing phase for the treatment of malignant and non-malignant diseases, such as recurrent high-grade gliomas, metastatic encephaloma and lower limb ischemia (Age-related macular degeneration (AMD) is characterized by a progressive loss of photoreceptors, and it is the leading cause of vision loss.ischemia .117 In addition, NSCs secrete neurotrophic factors that improve the lesion microenvironment, thereby generating appropriate conditions for pathological repair. The above findings confirm the significant value of NSCs in basic research and in therapeutic applications. The therapeutic effect of NSC transplantation can be further improved with adjuvant pharmaceutical agents, genetic modification, three-dimensional grafts or helper cells, which may improve cell survival and differentiation into specific types of cells.NSCs show great plasticity in the treatment of nervous system diseases and can be induced to differentiate into mature neural cells of various types, including neurons, astrocytes and oligodendrocytes. Under defined conditions, NSCs have a broad differentiation repertoire and can even give rise to non-ectodermal cells, including hematopoietic cells and skeletal myogenic cells.118 into NSCs avoids the previous shortcomings and provides a very attractive strategy for the mass production of NSCs for clinical application.Initially, NSCs could be obtained only from embryonic brains, and therefore, the limited source, technical issues in isolation and low purity blocked the progression of NSC-based cell therapy. In addition, there were ethical and moral concerns. Currently, emerging technologies continuously improve the methods that can be used to obtain NSCs, especially the identification of ESCs and the development of iPSCs, thus enabling the acquisition of a large number of NSCs and promoting basic research on NSCs. However, the differentiation of pluripotent stem cells into NSCs with high purity usually requires a long time and is accompanied by safety issues, thus complicating the translation of this procedure into a clinical therapy. Somatic cell transdifferentiation, especially methods without a viral strategy or integration of exogenous genes such as chemical-, growth factor-, 3D culture- or microRNA-induced cell transdifferentiation,120 In addition, MSCs release paracrine signals that enhance neuronal cell proliferation and the differentiation of human NSCs in vitro in co-culture systems, except for the direct transition of MSCs to neural stem-like cells.123 Moreover, MSCs are a type of immune cell, which can be applied to decrease immune rejection, prolong the survival time of grafts and treat immune dysregulation. Importantly, MSCs alone have been used in many clinical trials for the treatment of neurodevelopmental disorders.125 Co-transplantation of bone marrow MSCs and adult NSCs in a transgenic rat model of HD has been found to confer long-term behavioral benefits and to improve survival of the transplanted NSCs.126 Thus, it should be anticipated that MSC-derived NSC-based therapy with or without MSCs will be a major direction for the treatment of nerve diseases in the future (MSCs have great value in regenerative medicine because autologous MSCs are easily harvested and can be effectively induced into a variety of specialized cells, including neural cells. MSC-derived neural stem-like cells have been found to exhibit significant neuroprotective effects.e future .in vivo if transplanted at high density; thus, the density of transplanted NSCs must be precisely controlled to avoid secondary damages to the injected tissues. Therapeutic routines for NSC administration, including local injection via intracranial or intraspinal routes and systemic injection via intravenous or intrathecal routes, are highly dependent on the lesion site. Second, stem cells that are used as seeds must be verified as safe both in vitro and in vivo. To accomplish this, we suggest carrying out deep sequencing and checking tumor formation potency in mice for every lot of manufactured NSC products. Third, the poor survival rate and the modest treatment effects of NSCs in vivo are major problems that still remain to be solved. Furthermore, the underlying mechanisms of stem cell therapy are still unclear, and hence, additional studies are required. Further basic research in related areas may help resolve the above-mentioned issues. Finally, advanced imaging techniques are required to monitor the physiological state of transplanted NSCs in vivo to exclude tumourigenicity and other pitfalls.Although NSC treatment has exhibited some success in a variety of animal disease models, many problems remain to be addressed before transition to clinical applications because of the substantial physiological differences between humans and animals. First, clinical treatments must abide by standardized protocols. Therefore, detailed and efficient standards must be established for therapeutic routines, including stem cell types, the time of transplantation and cell dosage. Purity of NSCs is the priority and needs to be addressed and approved, as contamination of other cells may cause unexpected side effects. The optimal transplantation time for NSCs should be evaluated for each type of acute or chronic disease. Along with others, we have found that even primary tissue-derived NSCs or NPCs can form a clot Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


We recently developed novel cell transplantation method “cell transfer technology” utilizing photolithography. Using this method, we can transfer ex vivo expanded cells onto scaffold material in desired patterns, like printing of pictures and letters on a paper. We have investigated the possibility of this novel method for cell-based therapy using several disease models. We first transferred endothelial cells in capillary-like patterns on amnion. The transplantation of the endothelial cell-transferred amnion enhanced the reperfusion in mouse ischemic limb model. The fusion of transplanted capillary with host vessel networks was also observed. The osteoblast- and periodontal ligament stem cell-transferred amnion were next transplanted in bone and periodontal defects models. After healing period, both transplantations improved the regeneration of bone and periodontal tissues, respectively. This method was further applicable to transfer of multiple cell types and the transplantation of osteoblasts and periodontal ligament stem cell-transferred amnion resulted in the improved bone regeneration compared with single cell type transplantation. These data suggested the therapeutic potential of the technology in cell-based therapies for reperfusion of ischemic limb and regeneration of bone and periodontal tissues. Cell transfer technology is applicable to wide range of regenerative medicine in the future. Recent progress in tissue engineering made it possible to treat various diseases using ex vivo expanded cells . The posPhotolithography is a word with a prefix “photo” meaning light to “lithography,” which is originated from lithograph. Literally, among various lithographic methods, photolithography uses the pattern made by light for document copy. Due to its precision, reproducibility, and mass productivity, photolithography is widely used in the precision machinery industry and printing. Photolithography consists mainly of two steps, namely the depiction of desired pattern on the substrate and “transfer” of the pattern to the product surface.We have developed “cell transfer technology” that enables transfer of cultured cells onto the surface of transplantation scaffold. Figure For surface coating of the transfer substrate, we first coated glass the plate with fluoroalkyl silane (FAS). However, because of the lower stability of patterning on the substrate, FAS was replaced with PEG/TEG. We observed longer stability of patterns made with PEG/TEG on the substrate compared with those made with FAS. The time needed for cell transfer was also found to be shorter for PEG/TEG substrate than that with FAS.Using cell transfer technology, ex vivo expanded cells were transferred to scaffold materials including hydrogels. Among these, we have used amnion, a part of amniotic membrane, as a scaffold material for cell transfer because of its elasticity, flexibility, and high success rate of cell transfer. Amniotic membrane is the biological membrane forming amniotic sac, which keeps and protects amniotic fluid and embryo inside . AmniotiAlmost 100% of cells on transfer substrate were successfully transferred onto amnion after several hours of incubation. Amnion is great scaffold material for cell transfer, as to facilitate high transfer efficiency. Moreover, the most prominent characteristics of amnion upon cell transfer is the stability of transferred cells on the membrane . The traOne of the notable features of photolithography is precise transfer of substances according to the fine pattering drawn on substrate. Taking advantage of this unique characteristic of photolithography, we are able to transfer cells in any desired patterns onto scaffold. We made patterns resembling to capillaries on glass substrate and tried to make capillaries by tissue engineering approach. We seeded bovine carotid artery endothelial cells (BCAECs) on transfer substrate with capillary-like pattering and transfer them onto amnion . BCAECs,5 cells/cm2 were transferred onto an amnion using the cell transfer technology. Furthermore, we transferred two different cell types in overlapping two layers using cell transfer technology and named it “double cell transfer” [On the other hand, we can also fabricate cell transfer substrate with whole cell adhesive characteristics. With this substrate, cells are transferred onto amnion in layer structure. In case of cell transplantation requires cell numbers, not positioning, this sheet-like cell transplantation material is useful. We succeeded in transfer of cells and fabricating cell sheet-like materials using various cell types such as fibroblasts, mesenchymal stem cells (MSC), osteoblasts, and endothelial cells –13. Apprransfer” . For thiVarious cell patterning and cell sheet formation methods have been reported. For cell patterning, two modalities have been mainly studied. One is to utilize the specificity of cell adhesion to the extracellular matrix for cell placement, and this can form sharp edge of patterns , 15. HowIschemic vascular diseases, including myocardial infarction, cerebral infarction, and peripheral arterial embolism, develop by the absence of blood flow due to blockage of the artery and veins , 21. Art4 cells were transplanted per defect. After 5 weeks, complete closure of bone defect was observed in osteoblast-transplanted defect while control defect did not show the healing. Compared with other test groups including injection of osteoblast, only amnion transplantation, and no treatment, significant bone regeneration was found in osteoblast-transferred amnion defects. Transplanted osteoblasts were found around newly formed bone 5 weeks post operation, and it is suggested that those osteoblasts are directly involved in the regeneration of bone. The firm adhesion of osteoblasts to amnion may contribute to the longer cell retention in bone defects and the prominent bone regeneration observed in osteoblast-amnion transplantation compared with that in cell injection. These results suggested the therapeutic potential of osteoblast-transferred amnion for the treatment of bone defect. We also observed the improvement in bone healing by transplanting double cell-transferred amnion, made using osteoblasts and MSC from periodontal ligament  [For bone defects caused by tumor resection and fracture, various treatment strategies have been investigated including local application of growth factor with bone-forming activity such as bone morphogenetic proteins and transplantation of autologous or allogeanic bone graft and artificial bone substitute materials such as hydroxyapatite and beta-tricalcium phosphate , 27. Rec, PDLSC) . We tranPeriodontal disease is characterized by the chronic inflammation and destruction of tooth supporting tissues including bone, periodontal ligament, and cementum mainly due to infection of gram-negative bacteria . ConventWe developed novel cell transplantation method “cell transfer technology” using photolithography technique. By transplanting cell transferred-amnion made by the method, we have demonstrated the therapeutic potential of the material in cell-based treatment including reperfusion of ischemic limb and regeneration of bone and periodontal tissues. Forming a fine patterning by cultured cell is the most unique characteristics of cell transfer technique. In this regard, capillary patterning grafting is taking full advantage of this technology. However, the cell transplantation, which requires cell patterning, might be rather limited. Therefore, cell sheet-like amnion, which we used for bone and periodontal defects, maybe widely applicable for tissue regeneration. Depending on the target tissue to be regenerated, we can select patterned or non-patterned cell transfer substrate. We also found that the transferred cells are firmly and stably adhered on amnion and withstand against the deformation and movement of the membrane by surgical manipulations. Additionally, the flexibility of amnion enables us to transplant cultured cell in direct contact with defects or tissue surface, which is vital importance in certain regenerative cases. Taking these advantages and unique features, cell transfer technology is applicable to wide range of regenerative medicine in the future.


Disseminated oligodendroglial-like leptomeningeal tumor is a recently acknowledged entity whose radiological characteristics have rarely been discussed before. Typical of the childhood period, it should be differentiated clinically and radiographically from granulomatous or infectious conditions such as tuberculous meningitis, which is more common in this age group. The key to the diagnosis, even at an early stage, might be the presence of tiny T2 hyperintense lesions on the surface of the brain or spine. When suspected, a meningeal biopsy should be performed to confirm the diagnostic. Disseminated oligodendroglial-like leptomeningeal tumors (DOLT), also called diffuse leptomeningeal oligodendrogliomatosis, are rare neoplasms with extensive dissemination in leptomeninges of brain and spine, with or without very small intraparenchymal lesions, most commonly depicted in the spinal cord . This raA four-year-old boy presented in October 2012 with a history of asthenia, vomiting, gait disturbance, and drowsiness lasting over two weeks. Neurologic examination revealed stiff neck, hyperreflexia in lower limbs, and moderate dysmetria. Blood samples showed no inflammatory syndrome. Brain and spine MRI demonstrated communicating hydrocephalus and diffuse leptomeningeal thickening and enhancement along the brain and spine, including few cranial nerves and nerve roots of the cauda equina Figure . LeptomeRepeated CSF analysis revealed slightly elevated proteins (82 mg/dl); bacteriology analysis was negative, and no neoplastic cells were found. PCR was positive for Mycobacterium tuberculosis. Intradermal test was negative.The placement of a venticuloperitoneal shunt and anti-tuberculosis treatment  associated with glucocorticoids resulted in a transient clinical improvement that lasted for two months. The lung lesion disappeared a month after the beginning of treatment. As from December 2012, the patient regularly suffered from early morning emesis, and in June 2013, he was admitted for neurological deterioration, including dysmetria, ataxia, and cranial nerve paresis associated with severe weight loss.He continued to clinically deteriorate despite a change in the anti-tuberculosis treatment and presented in May 2014 with repeated generalized seizures. MRI performed at that moment disclosed a raise in both the number and size of the small T2 hyperintense pseudocystic deposits, some of which were enhanced, along the parenchymal surface of the cerebellum, brainstem, anterior and medial temporal lobes, basal frontal lobes, thalami, and spinal cord Figure . The masSurgery with decompressive craniectomy of the posterior fossa, along with biopsy of meninges, was performed. Histopathological examination showed a leptomeningeal infiltration by oligodendrocyte-like cells with rounded nuclei and perinuclear halo, showing positive immunostaining for OLIG2 and GFAP Figure . Growth Chemotherapy (carboplatine and vincristine) was initiated, thereby helping to improve the patient’s clinical conditions. Follow-up MRI performed after 14 months of chemotherapy revealed no regression of the pseudocystic lesions along the surface of the brain and spine. Recent medical reports from October 2016 describe diffuse sensory-motor sequelae due to the diffuse leptomeningeal lesions as well as cerebellar disorders. Neurocognitive sequelae implied enrollment in a centre for children with special needs. Fits were also frequent due to an epileptogenic focus in the frontal lobe.A disorder similar to that of our patient was first reported in 1942, and it was designated as “oligodendrogliomatosis of the cerebrospinal pathway” . DiffuseA recent paper defined diffuse leptomeningeal oligodendroglioma-like neoplasm as a desmoplasia inciting tumor, principally or exclusively involving leptomeninges and made up of low-grade-oligodendroglioma-like cells that are extensively disseminated in the subarachnoid space . It typiThe neuroimaging profile is characteristic in advanced disease where innumerable, small pseudocystic implants are observed along the surface of the brain, mainly in the posterior fossa and spine, as reported in our case . The chaIn our case, the clinico-radiological settings associated to an apical pulmonary condensation and a PCR positive for mycobacterium tuberculosis initially led to anti-tuberculous treatment, even though other infectious tests were negative. The clinical improvement yet observed was probably related to the implantation of a ventricular shunt.To be able to suggest the diagnostic of DOLT early enough, the presence of pseudocystic T2 or FLAIR hyperintense lesions along the subpial surface of the spinal cord or the brain should be investigated. In our patient, those superficial pseudocystic lesions were seen in the initial MRI examination at the level of the cerebellum and brainstem.From an anatomopathologic point of view, these T2 or FLAIR hyperintense pseudocystic lesions along the central nervous system surface actually correspond to tumor cell infiltration with focal rarefaction of surrounding neural tissue, the tumor implants being embedded in a myxoid stroma 6. Micros5Widespread infiltration of basal cranial nerves by oligodendroglioma-like cells may be an additional feature that explains the enhancement along cranial nerves demonstrated in our patient . The preClinical course is generally marked by periods of stability or slow progression, although evolution towards anaplasia and aggressiveness has been described in few cases . Early tDiagnostic can only be confirmed through biopsy, since cerebrospinal fluid frequently merely shows increased protein levels while more rarely revealing lymphocytosis . CytologTo summarise, disseminated oligodendroglial–like leptomeningeal tumors are rare, low-grade, glial or glioneuronal tumors which predominate in the pediatric population. They are characterised by the presence of multiple, nodular cystic-like lesions scattered over the brain and spinal cord surface. Neuroradiologists and pediatric radiologists should be aware of the distinctive neuroimaging features of this entity and be able to recognise it even at an early stage. Indeed, this entity should not to be confused with an infectious or inflammatory condition, as early diagnosis is linked to better prognosis.


Cognitive decline poses a great concern to elderly people and their families. In addition to pharmacological therapies, several varieties of nonpharmacological intervention have been developed. Most training trials proved that a well-organized task is clinically effective in cognition improvement.We will first review clinical trials of cognitive training for healthy elders, MCI and AD patients, respectively. Besides, potential neuroprotective and compensatory mechanisms in animal models of AD are discussed. Despite controversy, cognitive training has promising effect on cognitive ability. In animal model of AD, environmental enrichment showed beneficial effect for cognitive ability, as well as neuronal plasticity. Neurotrophin, neurotransmitter and neuromodulator signaling pathway were also involved in the process. Well-designed cognitive activity could benefit cognitive function, and thus life quality of patients and their families.The positive effects of cognitive activity is closely related with neural plasticity, neurotrophin, neurotransmitter and neuromodulator signaling pathway changes. Cognitive decline is age-specific or related with dementia. Alzheimer disease (AD) was the most common types of dementia. In 2006, the global prevalence of AD was 26.6 million . It is eAging and aging-related diseases pose a major threat to individuals’ life quality, and cause high economic burden on families and the whole society. Prevalence of MCI in population-based epidemiological studies ranges from 3 to 20% in adults older than 60 or 65 years old –7. Some Unfortunately, there is no cure or robust pharmacologic treatment for AD. So far, the primary focus is slowing down the decline of neurological and associated behavioral functions, by providing medications, training and caregiver support.Due to the increasing prevalence of MCI and AD, pharmacological and non-pharmacological treatments have been greatly concerned about their effects. Donepezil, rivastigmine, galantamine, and memantine are the drugs presently approved by the Food and Drug Administration (FDA) for treatment of AD. Meta-analysis of cholinesterase inhibitors (ChEIs) showed their effects by a small improvement in activities of daily living . AntigluAs well as medications, non-pharmacological interventions also aim to delay the loss of cognitive abilities, to help people stay independent in everyday life as long as possible, and to increase their well-being and quality of life. Because of its readiness and few side effects, not only AD or MCI patients, healthy elder adults can also participate at home or in the community. There are various approaches, including mental exercise, diet control and physical exercises , 15. HowThere have been a large number of clinical trials and reviews about the effect of cognitive therapy for aging populations –18. CompIn this paper, we first review clinical trials of cognitive training for people with different extent of cognitive ability. The characteristics and special outcomes of the clinical trials of cognitive intervention were discussed, as well as shortcomes of clinical studies. Then, much attention was devoted to neural mechanisms of these training, exploring valuable information from animal studies.We take two steps in the literature review. In step one, we searched relevant meta-analysis for a quick look at the effect of cognitive intervention. In step two, we selected consolidated clinical trials for detailed analysis and discussion.Step One: Several systemic reviews and meta-analysis have summarized cognitive training trials. Firstly, we selected the work which fully complies with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) or Delphi list. We searched PubMed- and PsycINFO-based literature for relevant meta-analysis using the following Boolean strategy: “cognitive stimulation” OR “cognitive rehabilitation” OR “cognitive activity” OR “cognitive intervention” OR “training” OR “memory training” OR “mnemonic training”. After screened by title and abstract, meta analysis studies were included if they enrolled latest clinical trials that evaluated the effect of the cognitive intervention by assessing cognitive changes before and after intervention Table .Table 1MStep Two: In order to further take a close look at the entire literature and details, we manually searched consolidated evidence from the references of selected articles and earlier review . We alsoThe search strategy in PubMed  was shown as an example: (“Dementia”[Mesh] OR “Alzheimer Disease”[Mesh] OR “mild cognitive impairment”[Mesh] OR aging OR elder) AND  AND We selected studies by three steps based on eligibility criteria of PRISMA checklist : the title and abstract screening, full-text assessment for rationale and eligibility of methodology, and final evaluation of results. Two reviewers  independently checked the following factors before inclusion, and discrepancy was discussed to arrive at agreement: subjects, study design, blinding, withdrawals and dropouts, intervention method and duration. Studies were excluded if they only enrolled participants with other dementias or no clearly intervention description. Eligible studies were classified by cognition level of subjects, in order to find out training effect on people of different cognitive ability.Normal aging causes natural decline in multiple cognitive domains, and thus poses threat to maintaining independence and quality of life. Researchers tended to develop methods to slow down the speed of cognitive decline, based on the theory that brain retains some plasticity. Since 1980s, a number of studies had tried cognitive interventions for healthy older adults, in order to improve cognitive performance and quality of life –25. ThesAs memory loss is the major and early complaint from older adults, memory training was dominantly adopted in a number of studies as early as 1980s –29. WorkMore studies paid attention to comprehensive training covering more cognitive domains. Jobe et al. conducted a famous large-scale, randomized, controlled, single-masked trial (ACTIVE study) in 2001, which was designed to determine whether cognitive interventions could affect cognition-based daily functioning and basic trained abilities served as mediators . The ACTDonostia Longitudinal Study aimed at all cognitive functions. The authors compared structured training program with unstructured one and control in a 2-year time span. Each participant in experimental groups received 180 ninety-minute sessions (every session per week). Structured intervention covered the cognitive functions of attention and orientation, memory, language, visuoconstructive ability, executive functions, visuomanual coordination and praxia. Only the group that received structured intervention got higher scores in nearly all cognitive tests . SimilarIn addition to these training programs for particular one or more cognitive domains, social activities or everyday activity were also evaluated as a kind of intervention . Some maStudies varied in the interventions, study design, duration of sessions and sample size. All programs mainly aimed at explicit memory because individuals with MCI suffered from memory deficiency. Similarly to intervention for healthy old adults, attention, speed of processing, language, visual-spatial abilities and executive functions were also adopted –51, whilIn the early 2000s, Rapp et al. tried multi-component memory interventions. Patients received education about symptoms of memory loss, memory skills training, and memory-related beliefs. Treated group did better in memory assessment at the end of treatment and at a 6-month follow-up .In a prospective study, Rozzini et al. used training covered different cognitive functions for patients with MCI. After 1-year follow-up, subjects treated with training and cholinesterase inhibitors (ChEIs) medication showed significant improvements in memory, abstract reasoning and in behavioral disturbances. The combined treatment group showed its advantage over ChEIs group . WenischIn addition to memory, behavior rehabilitation was once believed to be a potential cognitive helper. Nevertheless, it is a bit controversial of its effect in MCI patients. Greenaway et al. tested a calendar/notebook system, including three sections: appointments; daily “to do” items and important events that happened that day. Patients compliant with the system had a medium effect size for improvement in functional ability. Subjects further reported improved independence, self-confidence, and mood . By contAnother critical factor in cognitive training is the use of strategy. Strategy acts as a compensator in functioning. Belleville et al. focused on episodic memory strategies by comparing pre-and post-intervention difference. Progress was remarkable in delayed list recall and face-name association tasks of strategy-intervention group, while no improvement was observed in MCI individuals without receiving the intervention . HampsteAfter being taught with memory strategies based on meta-memory, old adults had training-related gains in a recall task, as well as transfer benefits in short-term memory, long-term memory, working memory and motivation . StrategLondos et al. also aimed at developing compensatory memory strategies that can improve their cognition, as well as occupational performance and quality of life. The study compared cognitive function, occupational performance, and self-perceived quality of life before and after intervention. Significant improvements were seen in cognitive processing speed, occupational performance after participation in the program 2 days per week for 8 weeks . Troyer Since publication in the early 1980s by Zarit et al., much work has been done for cognitive stimulation in populations suffering from dementia, especially AD. The early detection and diagnosis of AD raised the importance of effective psychological intervention in the early stage. Types of stimulation programs covered all cognitive domains. Similarly to interventions for normal older adults and MCI patients, memory is the main target, as well as memory strategies and external memory aids. However, due to poor cooperation of AD patients, more delicate techniques were adopted.In contrary to its general application in MCI patients, memory intervention was questioned when it is applied in AD patients. Quayhagen et al. compared cognitive stimulation with other non-pharmacological interventions . Cognitive stimulation group demonstrated more improvement over time only in cognitive outcomes .Zarit et al. tried visual imagery for overcoming memory loss in senile dementia patients in community. Though recall performance (imagery techniques were taught) was improved for subjects in the intervention group, the author claimed its little practical value for caregivers . ImaginaIn a combined intervention consisting of face-name associations, spaced retrieval, and cognitive stimulation, 37 patients with probable AD were enrolled for 5 weeks. AD patients who received stimulation showed significant improvement in trained tasks, while no benefit was observed in the additional neuropsychological measures of dementia severity, verbal memory, visual memory, word generation, motor speed, or caregiver-assessed patient quality of life .Errorless learning was an instructional design introduced in the 1930s in order to create the most effective learning environment. Errors are once regarded as a function of learning and vice-versa. However, in the errorful learning, people with amnesia much more easily remember their own mistakes than they remember the correction . Errorless learning is an alternative way to get someone to learn something without the opportunity to make a mistake.In addition to traditional memory or cognitive intervention, errorless learning provides a useful additional strategy. Patients should be tailored to interventions, based on errorless learning principles and specific cognitive problems. In one study, five of the six participants showed significant improvement on the target measures, and maintained this improvement up to 6 months later .Spaced retrieval requires users to rehearse learned information at a certain time. Each new rehearsal is done with a longer or equal interval between itself and the previous rehearsal. At the end of every trial period there is a test phase . LandaueSchacter et al. applied the technique to people suffering from amnesia and other memory disorders. Participants were asked to remember some fact with increasing intervals. If the subject is able to recall the information correctly the time is doubled to further help them keep the information fresh in their mind to recall in the future. The findings showed that using spaced retrieval help name face association of young students, as well as individuals with memory disorders . The tecSpaced-retrieval method was combined with traditional memory stimulation tasks. The term “prospective memory” refers to the timely execution of a previously formed intention. In 1991, it was used for training four AD patients to remember and to implement an intention for future action. All participants were able to shift to new task requirement, and all learned three successive coupon colors successfully . A pilotNeuronal mechanisms underlying the effects of these interventions were investigated by functional magnetic resonance imaging (fMRI) and electroencephalography (EEG). When training and tasks in the fMRI were the same (name-face association) for 6 MCI patients, increased activation was observed in the medial frontal cortex, parietal and occipital lobes neighboring the temporal-parietal junction, left frontal operculum and some areas of the left temporal cortex. It also revealed increased generally connectivity after training, particularly involving the medial temporal gyrus and foci in the occipital and precuneus cortices . In a coCortex also involves in memory trainings, especially when training and tests in fMRI involved different cognitive processes. A combination of specialized areas (activated areas during pre-training and new areas during post-training) was activated in the frontal, temporal and occipital areas. Right inferior parietal lobe was the only activated area that correlated with performance . When taIn gist reasoning training, higher fractional anisotrophy was found after 1-year training using diffusion tensor imaging (DTI) MRI . DTI alsConnectivity between different brain areas may change during and after some specific cognitive manipulations. A 6-week training program for healthy older adults on Brain Fitness (an adaptive auditory perception computer game) showed improvement in the activities of daily living (a transfer effect from sensory processing to everyday problem solving). It also selectively increased the integrity of occipito-temporal white matter in the ventral attention network, and decreased connectivity between superior parietal cortex and inferior temporal lobe . This inNeurons interaction could also be evaluated by EEG performance, taking its advantage of high temporal resolution. EEG coherence indicates synchronization between different cortical areas , 82. AftEvent-related potentials (ERP)  were useDespite the methodological limitations in both fMRI and EEG studies, such as small sample sizes and lack of control groups, these evidence suggested that elder individuals exhibit high plasticity, which can be used as a clue to understand the effects of cognitive interventions.Trials above provided exciting results for clinical practice. However, there are several problems confusing and unsolved. We raise the following reasons that make us turn to laboratory for more help.At first, the selection and publication bias of clinical trials could not be ignored. AD has its owe pathological characteristics, and preclinical AD happened without any symptoms. Most of the clinical trials only lasted for a relatively short period and only evaluated participants by neuropsychological assessment. It is not sure that they enrolled participants of the correct diagnosis. Besides, some “normal aging” or MCI participants at baseline might progress to AD during follow-up, but few trial corrected the bias when enrollment. In this way, classic animal models of AD may help to find more consolidated evidence for cognitive intervention.We once reviewed and concluded several principles for an effective training program , based oAnother problem is the duration of effect since intervention ends. The benefits of name-face association training were observed after 1 week , or afteFinally, though functional MRI provided some evidence for brain activity changes, it has own methodological limitations. Blood oxygenation level dependent contrast (BOLD) in resting or task-related fMRI were mainly used to reflect brain activity. Various statistical methods were adopt to analyze BOLD data. Regional homogeneity, amplitude of low-frequency fluctuation (ALFF), fractional ALFF (fALFF) were once used as representation of regional activities in AD , 93, whiStudies above have shown that the progression of dementia could be slowed down in the patients who have cognitively stimulating activities. These discoveries point to the conclusion that cognitive interventions intellectually may not only improve memory performance but also prevent future cognitive decline. Some difficulties met in clinical trials above asked for further exploration in neurobiological way.We now manipulate mental exercise as stimulating brain which acts a positive role on AD and other forms of dementias via neuroprotective and compensatory mechanism. Existing animal models that are most relevant to our understanding of non-pharmacological therapy include those which utilize environmental or cognitive stimulation as experimental paradigms to alter levels of cognitive activity. In order to attempt to investigate the mechanistic underpinnings of mental exercise in cognitive function, we should first understand early experimental studies of brain stimulation- environmental enrichment (EE) on animal models. EE has defined broadly as the use of housing conditions that offer enhanced sensory, motor, and cognitive stimulation of brain in comparison with standard caging . In the From these early conclusions regarding the effects of EE, increasingly refined studies have progressed to showing effects at the cell and molecular levels. Adult rats were placed into enriched housing conditions for 1 year and showed significantly higher levels of nerve growth factor . AnotherNumerous cognitive studies about how EE affected the brain had been also proposed. In the intact animals, EE could dramatically improve cognitive abilities –115. In Many studies had shown that placing animals in complex environments for extended period improved their cognitive performance and brain activity in normal mice and rats. Whether did EE show beneficial effects on behavior and cognition in an animal model of AD? There were several mouse models of neurodegeneration like AD studying the modulating effects of environmental factors: transgenic mice overexpressing amyloid precursor protein (APP) and/or presenilin (PS)-1, AD11 mice expressing anti-nerve growth factor (NGF) antibodies and double transgenic TgCRND8 mice overexpressing the Swedish and Indiana mutations in the human APP. In these researches, the mouse models were given extensive enrichment such as cognitive stimulation or complex housing condition. Cognitive impairment and neuronal alterations elicited by neurodegenerative pathologies were evaluated to determine if they had been ameliorated or rescued by EE. If a long-lasting exposure to EE, the mouse model should display a delayed onset or progression of cognitive impairment.Before the onset of amyloid formation, APP/PS1 transgenic mice exposed to the long term of EE from 2 to 8 months of age would show mitigated learning and memory deficits. For example, long term EE led to improvement in cognitive function but without decreasing brain beta-amyloid deposition in the aged APPsw mice . JankowsAD11 mice developed age-dependent neurodegeneration including hallmarks of human AD and exhibited progressive memory impairment , 122. ExIn the TgCRND8 mice, EE had increased exploratory behavior and decreased anxiety-related behavior but could not clearly ameliorate deficits in learning and memory performance . More reAdult neurogenesis is shown to continue in two parts of brains: the subventricular zone (SVZ) lining the lateral ventricles and subgranular zone (SGZ). In fully adult mammals, new neurons born in SVZ migrate anteriorly into the olfactory bulb (OB), where they mature into local interneurons –133. AduHippocampal neurogenesis could be influenced by several environmental factors and stimuli , 141. WhPrevious studies in transgenic models of AD had generated mounting evidence supporting alterations in neurogenesis. Short-term exposure to EE led to a striking increase in new neurons and a substantial improvement in behavior performance . StudiesAlthough there are lots of studies reporting that EE could increase neurogenesis in DG of the adult hippocampus, neurogenesis in other parts of the brain- the subventricular zone(SVZ) or olfactory bulb (OB) system may be affected by other forms of enhanced stimulation significantly . OlfactoNeurons, neuroglia  and ependymal cells make up the complex structure of the adult central nervous system (CNS). Adult neurogenesis bridges between neuronal and glial neurobiology in an intriguing way. When the enrichment environment altered the neurogenesis, the differentiation and development of neuron and glial were always changed at the same time. Increased neuronal differentiation in DG was observed in the EE treated rats and the density of NeuN positive cells was enhanced without new neurons . As manyWe have now outlined various important structural and cellular changes that have been observed to occur in the animal brain exposure to EE. Evidence in support of such behavioral and cellular effects on molecular mechanisms has been gathered using a range of approaches. Examples of such molecular classes including specific neurotrophins, neurotransmitters and neuromodulator receptors, and synaptic signaling pathways have been validated via gene/protein studies.One early study demonstrated the attenuated expression of AP-2 in the CA2 and CA3 subfield of hippocampus after exposure to EE for 30 days . In adulA more detailed research analyzed gene expression changes in the cortex of mice and found a large number of genes changed in response to enrichment . AnotherFurthermore, there are still numbers of evidence that non-coding RNA species such as microRNAs (MiR) could also be modulated by EE. For example, MiR-183 expression could be upregulated by EE and reduce anxiety-like behavior in mice . MiR-124Previous studies have demonstrated that oxidative stress is an important factor contributing to the onset and progression of AD and the brain is sensitive to oxidative imbalance. Oxidative stress results from increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS)  and possModulation of neurotrophin expression and changes in neurotransmitters and neuromodulators are related to EE according to extensive findings. The primary effect of increased cognitive activity must be via enhanced synaptic and neuronal activity in the relevant neural circuitry. So, it is not difficult to image that molecular effects induced by EE have been shown to involve changes in neurotransmitters. Numbers of studies have shown that EE in animals increased the expression levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) .In rats with cognitive impairment, the levels of BDNF decreased in the hippocampus and EE exposure could up-regulate the decreased protein levels of BDNF . More stThe experience of APP/PS1 mice in EE would upregulate critical signaling that plays a major role in learning and memory, such as BDNF, IGF-1, N-methyl-D-aspartic acid receptor (NMDAR) and CREB transcripts . NGF is As one of non-pharmacological therapies, cognitive activity could benefit cognitive function, and thus life quality of patients and their families. These reviewed clinical studies above highlighted the positive effects of cognitive activity on cognitive ability, well-beings, behavior, and mood in older adults or patients with cognitive decline. We also reviewed the molecular and cellular mechanisms underlying this non-pharmacological therapy.Based on these clinical trials and meta analysis, we recommend some key features of training that often associated with positive outcomes. Firstly, multi-domain tasks contribute more to cognition , 192. AsHowever, the heterogeneity of cognitive intervention poses great difficulty for a safe conclusion. Combining data from trials of different sample size can result in overestimating the precision of smaller studies. Because of limitation in clinical trials mentioned in Section Evidence from epidemiology and animal model studies suggests that the onset of neurodegenerative diseases could be modulated by environmental factors . HoweverWe find EE has positive influence on normal aging and transgenic AD mouse, which could induce brain structure to produce a variety of changes. The change on anatomy includes the increases of brain weight, thickening of cerebral cortex and enlargement of hippocampus volume, etc. On cellular levels, EE could increase the proliferation of neural progenitor cells, production of newly generated neurons, the dendritic branches and tree density and the number of neuronal synapsis. These changes are most obvious in hippocampus and cerebral cortex. EE also could change the morphology of glia cells (including astrocyte and oligodendroglia cell) and promote the glial cell proliferation in the brain and cerebellum. EE could also induce various neural active substance changes, BDNF, VEGF, NGF and so on. These growth factors play important role on neurogenesis and neural network, which helps neuron development, differentiation and survival. In these ways, EE could improve the learning ability and cognition.Several questions remain unclear. Classic water maze was usually applied in mice around 8 months old. However, most EE studies used transgenic AD mice of relative young age, about 2–3 month-old. Thus, the effect of EE in elder mice was still unclarified. Maybe older mice should be used on EE in the following days. Besides, few study concerns how EE influence neuronal death and related mechanism for its non-ignorable role in neural circuit. A more difficult challenge is linking animal model data with clinical studies. Finally, the questions we raised in Section