Photo‐crosslinkable hydrogel‐based 3D microfluidic culture device

We developed the photo‐crosslinkable hydrogel‐based 3D microfluidic device to culture neural stem cells (NSCs) and tumors. The photo‐crosslinkable gelatin methacrylate (GelMA) polymer was used as a physical barrier in the microfluidic device and collagen type I gel was employed to culture NSCs in a 3D manner. We demonstrated that the pore size was inversely proportional to concentrations of GelMA hydrogels, showing the pore sizes of 5 and 25 w/v% GelMA hydrogels were 34 and 4 μm, respectively. It also revealed that the morphology of pores in 5 w/v% GelMA hydrogels was elliptical shape, whereas we observed circular‐shaped pores in 25 w/v% GelMA hydrogels. To culture NSCs and tumors in the 3D microfluidic device, we investigated the molecular diffusion properties across GelMA hydrogels, indicating that 25 w/v% GelMA hydrogels inhibited the molecular diffusion for 6 days in the 3D microfluidic device. In contrast, the chemicals were diffused in 5 w/v% GelMA hydrogels. Finally, we cultured NSCs and tumors in the hydrogel‐based 3D microfluidic device, showing that 53–75% NSCs differentiated into neurons, while tumors were cultured in the collagen gels. Therefore, this photo‐crosslinkable hydrogel‐based 3D microfluidic culture device could be a potentially powerful tool for regenerative tissue engineering applications.     

Transplantation of Wnt5a-modified NSCs promotes tissue repair and locomotor functional recovery after spinal cord injury

Traditional therapeutic strategies for spinal cord injury (SCI) are insufficient to repair locomotor function because of the failure of axonal reconnection and neuronal regeneration in the injured central nervous system (CNS). Neural stem cell (NSC) transplantation has been considered a potential strategy and is generally feasible for repairing the neural circuit after SCI; however, the most formidable problem is that the neuronal differentiation rate of NSCs is quite limited. Therefore, it is essential to induce the neuronal differentiation of NSCs and improve the differentiation rate of NSCs in spinal cord repair. Our results demonstrate that both Wnt5a and miRNA200b-3p could promote NSC differentiation into neurons and that Wnt5a upregulated miRNA200b-3p expression through MAPK/JNK signaling to promote NSC differentiation into neurons. Wnt5a could reduce RhoA expression by upregulating miRNA200b-3p expression to inhibit activation of the RhoA/Rock signaling pathway, which has been reported to suppress neuronal differentiation. Overexpression of RhoA abolished the neurogenic capacity of Wnt5a and miRNA200b-3p. In vivo, miRNA200b-3p was critical for Wnt5a-induced NSC differentiation into neurons to promote motor functional and histological recovery after SCI by suppressing RhoA/Rock signaling. These findings provide more insight into SCI and help with the identification of novel treatment strategies.Subject terms: Neural stem cells, Neuroscience     

Directed neural stem cell differentiation on polyaniline-coated high strength hydrogels

Stem-cell-based neural regeneration has received significant attention, as it has potential to restore functionality to diseased or damaged neural tissues that have a limited ability to self-repair or regenerate. Culturing neural stem cells (NSCs) on hydrogel substrates has been shown to facilitate differentiation to neural progenitors, but this has only been achieved on very soft hydrogels, greatly increasing the difficulty of manufacture and limiting their wide applications. Here, we realized the differentiation of NSCs to neural and glial progenitors on high-strength hydrogels. Hydrogen-bonding-strengthened conductive hydrogels (PVV-PANI) were synthesized through one-pot copolymerization of 2-vinyl-4,6-diamino-1,3,5-triazine, 1-vinylimidazole and polyethylene glycol diacrylate, followed by post-coating with polyaniline (PANI). Diaminotriazine-diaminotriazine hydrogen bonding dramatically increases their mechanical strength, while copolymerization with VI pronouncedly promotes the adsorption of PANI particles, endowing the hydrogels with electrical conductivity. These hydrogels exhibit tensile strengths up to 1.16 MPa, a 559% breaking strain, a 9.9 MPa compressive strength and up to 16.7 mS/cm conductivity. Importantly, PVV-PANI hydrogels support the attachment, proliferation, and differentiation of NSCs, and allow the efficient induction of neural and glial differentiation via electrical stimulation. This work demonstrates high-strength conductive hydrogels can serve as an electroactive soft-wet platform for modulating the specific differentiation of NSCs, a significant step towards cell-based therapies for neurological diseases.     

Amyloid-β oligomers regulate the properties of human neural stem cells through GSK-3β signaling

Alzheimer''s disease (AD) is the most common cause of age-related dementia. The neuropathological hallmarks of AD include extracellular deposition of amyloid-β peptides and neurofibrillary tangles that lead to intracellular hyperphosphorylated tau in the brain. Soluble amyloid-β oligomers are the primary pathogenic factor leading to cognitive impairment in AD. Neural stem cells (NSCs) are able to self-renew and give rise to multiple neural cell lineages in both developing and adult central nervous systems. To explore the relationship between AD-related pathology and the behaviors of NSCs that enable neuroregeneration, a number of studies have used animal and in vitro models to investigate the role of amyloid-β on NSCs derived from various brain regions at different developmental stages. However, the Aβ effects on NSCs remain poorly understood because of conflicting results. To investigate the effects of amyloid-β oligomers on human NSCs, we established amyloid precursor protein Swedish mutant-expressing cells and identified cell-derived amyloid-β oligomers in the culture media. Human NSCs were isolated from an aborted fetal telencephalon at 13 weeks of gestation and expanded in culture as neurospheres. Human NSCs exposure to cell-derived amyloid-β oligomers decreased dividing potential resulting from senescence through telomere attrition, impaired neurogenesis and promoted gliogenesis, and attenuated mobility. These amyloid-β oligomers modulated the proliferation, differentiation and migration patterns of human NSCs via a glycogen synthase kinase-3β-mediated signaling pathway. These findings contribute to the development of human NSC-based therapy for AD by elucidating the effects of Aβ oligomers on human NSCs.     

Human neural stem cell growth and differentiation in a gradient-generating microfluidic device

This paper describes a gradient-generating microfluidic platform for optimizing proliferation and differentiation of neural stem cells (NSCs) in culture. Microfluidic technology has great potential to improve stem cell (SC) cultures, whose promise in cell-based therapies is limited by the inability to precisely control their behavior in culture. Compared to traditional culture tools, microfluidic platforms should provide much greater control over cell microenvironment and rapid optimization of media composition using relatively small numbers of cells. Our platform exposes cells to a concentration gradient of growth factors under continuous flow, thus minimizing autocrine and paracrine signaling. Human NSCs (hNSCs) from the developing cerebral cortex were cultured for more than 1 week in the microfluidic device while constantly exposed to a continuous gradient of a growth factor (GF) mixture containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF). Proliferation and differentiation of NSCs into astrocytes were monitored by time-lapse microscopy and immunocytochemistry. The NSCs remained healthy throughout the entire culture period, and importantly, proliferated and differentiated in a graded and proportional fashion that varied directly with GF concentration. These concentration-dependent cellular responses were quantitatively similar to those measured in control chambers built into the device and in parallel cultures using traditional 6-well plates. This gradient-generating microfluidic platform should be useful for a wide range of basic and applied studies on cultured cells, including SCs.     

Patterning of neural stem cells on poly(lactic-co-glycolic acid) film modified by hydrophobin

Patterning of neural stem cells (NSCs) is of great importance for its potential applications in the therapy of nerve injuries. Due to the critical requirements and the great difficulty in NSCs cultivation, developing new methods for NSCs patterning is very challenging and has progressed slowly in recent years. In this study, we reported a new method for patterning NSCs on a hydrophobin II (HFBI) modified poly(lactic-co-glycolic acid) (PLGA) film by using microcontact printing (μCP) technique. HFBI modification converted the PLGA surface from hydrophobic to hydrophilic, which should facilitate the absorption of serum on it. Serum was transferred onto the modified PLGA film by microcontact printing (μCP) to promote NSCs adhesion on the PLGA surface. Since the serum-coated PLGA surface promoted NSCs adhesion and the serum-free PLGA surface inhibited NSCs adhesion, micro-patterns of NSCs were obtained by directly culturing NSCs on the PLGA surface patterned with serum. This method allows the precise control of NSCs adhesion on the PLGA film without using the conventional cell-repellent species, which is anticipated to make great contribution in the fields of therapy of nerve injuries.     

Microfluidics embedded with microelectrodes for electrostimulation of neural stem cells proliferation

The regeneration of the injured nerve and recovery of its function have brought attention in the medical field. Electrical stimulation(ES) can enhance the cellular biological behavior and has been widely studied in the treatment of neurological diseases. Microfluidic technology can provide a cell culture platform with the well-controlled environment. Here a novel microfluidic/microelectrode composite microdevice was developed by embedding the microelectrodes to the microfluidic platform, in whic...     

Uniform‐sized neurosphere‐mediated motoneuron differentiation in microwell arrays

We developed the photocrosslinkable hydrogel microwell arrays for uniform‐sized neurosphere‐mediated motoneuron differentiation. Neural stem cells (NSCs) were obtained from embryonic cerebral cortex and spinal cord. To generate uniform‐sized neurospheres in a homogeneous manner, the dissociated cells were cultured in the hydrogel microwell arrays for 3 days. Uniform‐sized neurospheres harvested from microwell arrays were replated into laminin‐coated substrate. In parallel, uniform‐sized neurospheres cultured in microwell arrays were encapsulated by photocrosslinkable gelatin methacrylate hydrogels in a three‐dimensional manner. We demonstrated the effect of hydrogel microwell sizes (e.g., 50, 100, 150 μm in diameter) on motoneuron differentiation, showing that the largest uniform‐sized neurospheres derived from embryonic spinal cord efficiently differentiated into motoneurons. Therefore, this hydrogel microwell array could be a powerful array to regulate the uniform‐sized neurosphere‐mediated motoneuron differentiation.     

Characterization and classification of rat neural stem cells and differentiated cells by comparative metabolic and lipidomic profiling

It is necessary to characterize and classify neural stem cells (NSCs) and differentiated cells (DCs) for potential use of NSC to treat neurodegenerative diseases. We therefore performed an analysis of NSCs and DCs using gas chromatography mass spectrometry (GC-MS) and direct infusion mass spectrometry (DI-MS) with elaborate multivariate statistical analysis for the characterization and classification of rat NSCs and DCs. GC-MS and DI-MS detected a total of 92 metabolites and lipids in NSCs and DCs, and the levels of 72 of them differed significantly between NSCs and DCs. The optimal model for partial least squares (PLS) discriminant analysis was constructed by applying 3 and 2 PLS components with a unit-variance scaling method for classifying NSCs and DCs based on the data obtained in the GC-MS and DI-MS analyses, respectively. The obtained results from PCA and PLS-DA suggest that creatinine, lactic acid, lysine, glutamine, glycine, pyroglutamic acid, PG 18:1/20:2, PS 18:0/20:2, PI 18:0/20:3, PC 16:0/20:4, PI 16:0/20:4, and PI 18:1/20:4 were the main contributors that provided distinct characteristics of NSCs and DCs. The results of this study suggest objective and complementary criteria for the characterization and classification of NSCs and DCs for potential clinical applications.

Graphical abstract

     

A two-photon ratiometric fluorescent probe for real-time imaging and quantification of NO in neural stem cells during activation regulation

Developing a novel tool capable of real-time monitoring and accurate quantification of NO is critical to understanding its role in physiological and pathological processes. Herein, a two-photon ratiometric fluorescent probe (NOP) was developed for real-time imaging and quantification of NO based on fluorescence resonance energy transfer-photoinduced electron transfer (FRET-PET). In this developed probe, coumarin (CM) and naphthalimide with o-phenylenediamine (NPM) were rationally designed as a fluorescent donor and acceptor, respectively, to enable a ratiometric fluorescence response to NO. The developed NO probe demonstrated good detection linearity with the concentration of NO in the range of 0.100–200 μM, with a detection limit of 19.5 ± 1.00 nM. Considering the advantages of high selectivity, good accuracy and rapid dynamic response (<15 s), the developed NO probe was successfully applied for real-time imaging and accurate quantification of NO in neural stem cells (NSCs) and different regions of mouse brain tissue with a penetration depth of 350 μm. Using this powerful tool, it was found that NO regulated the activation and differentiation of quiescent NSCs (qNSCs). In addition, NO-induced differentiation of qNSCs into neurons was found to be dose-dependent: 50.0 μM NO caused about 50.0% of qNSCs to differentiate into neurons. Moreover, different regions of the mouse brain were observed to be closely related to the concentration of NO, and the concentration of NO in the DG region was found to be lower than that in the S1BF, CA1, LD and CPu of the Alzheimer''s disease (AD) mouse brain. The symptoms of AD mice were significantly improved through the treatment with NO-activated NSCs in the DG region.

Developing a novel tool capable of real-time monitoring and accurate quantification of NO is critical to understanding its role in physiological and pathological processes.     

Enhancing computer-assisted structure elucidation with DFT analysis of J-couplings

Computer-assisted structure elucidation (CASE) is the class of expert systems that derives molecular structures primarily from one-dimensional and two-dimensional nuclear magnetic resonance data. Contemporary CASE systems, including Advanced Chemistry Development/Structure Elucidator (ACD/SE), consider cross-peaks in heteronuclear multiple bond coherence (HMBC) and correlation spectroscopy (COSY) spectra as two- or three-bond correlations by default. However, four and more bond correlations (nonstandard correlations [NSCs]) could be present in these spectra too. The indiscriminate addition of NSCs to the CASE computations is prohibitively expensive. To address this problem, the ACD/SE program performs a logical analysis of observed correlations and determines the minimum number of NSCs. Guided by this information, a more efficient fuzzy structure generation (FSG) algorithm is subsequently applied. Until now, the FSG algorithm was utilized without any verification of the reliability of found NSCs. Here, we report a verification method for NSCs based on the relationship between NSCs and J-couplings computed with high accuracy density functional theory (DFT) methods. We used the example of strychnine to show that 41 (32%) of 8-Hz HMBC cross-peaks were NSCs and were consistent with ^4–6J_CH couplings greater than 0.3 Hz. This cutoff value was largely confirmed by the analysis of NSCs in 11 real-world natural products elucidated by ACD/SE. Additionally, utilizing the example of the CASE study of cleospinol A, we showed that the DFT-computed J-couplings of NSCs can distinctively differentiate the correct structure among six proposed isomers. The proposed approach of NSC verification should further improve the robustness of CASE analysis and can help reveal potential problems with reported experimental data.     

A novel silk/PES hybrid nanofibrous scaffold promotes the in vitro proliferation and differentiation of adipose‐derived mesenchymal stem cells into insulin producing cells

Using stem cells to replace the lost beta cells is a hopeful strategy in the treatment of diabetic patients. Furthermore, during stem cell culture and therapy, it is a need to use a substrate to act as a supportive matrix to mimic 3D in vivo microenvironment. Therefore, in this study, human adipose‐derived stem cells were used to differentiate into insulin‐producing cells (IPCs) on a silk/polyethersulfone (PES) scaffold. After exposing to the differentiation media, 2D and 3D (silk/PES) cultured cells were gradually aggregated and formed spherical shaped clusters. The viability of cells was comparable in both 3D and 2D culture. As the results of gene expression assay in both RNA and protein level showed, the differentiation efficiency was higher in 3D culture. Furthermore, ELISA revealed that the release of C‐peptide and insulin was higher in 3D than 2D culture. It seems that silk/PES nanofibrous hybrid scaffold could provide an appropriate matrix to mimic in vivo microenvironment and therefore increases the IPC differentiation potency of stem cells.     

Study on induction of differentiation of a human megakaryoblastic leukemia cell line (HIMeg) by 1,25-dihydroxyvitamin D3

It is reported that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), a physiological factor, has an inductive effect on the differentiation of a novel human megakaryoblastic leukemia cell line (HIMeg) in vitro. At the concentrations ranging from 10(-9) to 10(-6) mol/L, 1,25(OH)2D3 showed inhibition of proliferation on HIMeg cells which was demonstrated by count of survival cells and cloning efficiency. Meanwhile, using light/electron microscopy, stain of cytochemistry (including immunoenzymatic technique) and flow cytometry, we found that HIMeg cells could be further induced into more mature cells in megakaryocytic lineage confirmed by a series of evidence, including the changes of cell morphology/structure and cytochemistry, increased expression of differentiation antigens on the cell surface, and polyploidization. So, it is possible for 1,25(OH)2D3 to promote the differentiation of the cells in megakaryocytic lineage in vivo and to be used to treat acute megakaryoblastic leukemia and other diseases with malignant megakaryocytosis.     

Catalytic asymmetric syntheses and biological activities of singly dehydroxylated 19-nor-1alpha,25-dihydroxyvitamin D(3) A-ring analogs in cancer cell differentiation and apoptosis

BACKGROUND: 1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) has been shown to modulate not only proliferation and differentiation, but also apoptosis in malignant cells, indicating that it could be useful for treating cancer. Little information is available concerning the structural motifs of the 1alpha, 25(OH)(2)D(3) molecule responsible for modulation of differentiation and apoptosis, however. We set out to synthesize singly dehydroxylated A-ring analogs of 19-nor-1alpha,25(OH)(2)D(3) in a catalytic asymmetric fashion, and to investigate their biological activities in leukemia HL-60 cells. RESULTS: A series of singly dehydroxylated 19-nor-1alpha,25-dihydroxyvitamin D(3) A-ring analogs were synthesized using a combinatiorial sequence of regioselective propiolate-ene reaction and catalytic asymmetric carbonyl-ene cyclization. Surprisingly, the analogs could be clearly divided into two categories; one group, bearing 1alpha-hydroxy or 3beta-hydroxy groups in the A-ring, were potent differentiators and the second group, bearing 1beta-hydroxy or 3alpha-hydroxy groups, were potent stimulators of apoptosis. CONCLUSIONS: We have clearly identified the structural motifs of 19-nor-1alpha,25(OH)(2)D(3) analogs responsible for differentiation and apoptosis in HL-60 cells. These findings will provide useful information not only for development of therapeutic agents for treatment of leukemia and other cancers, but also for structure-function studies of 1alpha,25(OH)(2)D(3).     

Migration of human neural stem cells toward an intracranial glioma

Many in vivo and in vitro studies have demonstrated the targeted migration of neural stem cells (NSC) to infiltrating brain tumors, including malignant glioma, highlighting a potential therapeutic approach. However, there is not enough information to apply this approach to clinical therapy. The most important things in stem cell therapy for brain tumors involve selecting the appropriate neural progenitor type and optimizing the efficiency of the cell engraftment. By histological analysis using two different live-dyes, human NSCs were shown to migrate away from the transplanted site in the direction of the expanding C6 glioma and to intermix with the tumor bed, especially with the tumor core. This intermixing occurred within 7 days when NSCs were implanted into glioma model. The time course of migratory HB1.F5 with the greatest mobility of three NSC lines was as follows. As early as 3 days after transplantation, several NSCs were found leaving the implant site, primarily approaching microsatellites and frontier cells located near the site of NSC implantation. Through 7 days post-transplantation, massive numbers of NSCs continued to be attracted to and interspersed with C6 glioma, and were finally distributed extensively throughout the whole tumor bed, including the core and penumbra of the tumor mass. However, NSCs appeared to penetrate into the tumor mass very well, whereas normal fibroblast cells could not migrate. These findings strengthen the potential for human NSCs as attractive vehicles to improve therapeutic gene delivery to cancer or glioma if they are optimized to selectively kill neoplastic cells.     

Induction of differentiation of human leukemia cells by various combinations of cytokines and low-molecular-weight inducers

To explore agents for differentiation therapy of leukemias, various combinations of cytokines and low-molecular-weight inducers were examined for differentiation-inducing activity toward three kinds of human leukemia-derived cell lines. The strongest differentiation inducing activity on promyelocytic HL60 cells and histiocytic U937 cells was obtained by combining recombinant tumor necrosis factor (rTNF), interferon-gamma (IFN-gamma), retinoic acid (RA), and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3). For myeloblastic ML1 cells, the combination of rTNF, IFN-gamma, and RA had the strongest differentiation-inducing activity.     

Mass Transfer Analysis of Growth and Substance Metabolism of NSCs Cultured in Collagen-Based Scaffold In Vitro

The aim of this study is to analyze the growth and substance metabolism of neural stem cells (NSCs) cultured in biological collagen-based scaffolds. Mass transfer and metabolism model of glucose, lactic acid, and dissolved oxygen (DO) were established and solved on MATLAB platform to obtain the concentration distributions of DO, glucose, and lactic acid in culture system, respectively. Calculation results showed that the DO influenced their normal growth and metabolism of NSCs mostly in the in vitro culture within collagen-based scaffolds. This study also confirmed that 2-mm thickness of collagen scaffold was capable of in vitro cultivation and growth of NSCs with an inoculating density of 1?×?10⁶ cells/mL.     

Sample preparation and current applications of liquid chromatography for the determination of non-structural carbohydrates in plants

This article summarizes the current methods of determination of non-structural carbohydrates (NSCs) in plant samples based on liquid chromatography (LC). NSCs comprise several types of carbohydrates: sugar alcohols (e.g., sorbitol), monosaccharides (e.g., glucose and fructose), disaccharides (e.g., sucrose), oligosaccharides (e.g., raffinose) and polysaccharides [e.g., starch and polyfructans (e.g., inulin)]. NSCs are important in plant metabolism and have to be strictly distinguished from all sorts of structural carbohydrates (e.g., polysaccharide cellulose) that make up the backbone of the plants. Consequently, preservation of structural carbohydrates is a crucial step during sample preparation for NSC determination and is therefore addressed.Sugar alcohols, monosaccharides, disaccharides and those oligosaccharides that are easily soluble in polar solvents can be analyzed directly by high-performance LC. They are also referred to as free carbohydrates (FCs).However, polysaccharides are generally submitted to hydrolyzation into monomers prior to their quantitative analysis. This can be done either chemically, using acids, or enzymatically - both methods are discussed. For identification and quantification of the NSCs after LC separation, the following detectors are used: pulsed amperometry, refractive index, evaporate light scattering and finally, mass spectrometry.