Yi-Chen Li

A neural stem/precursor cell monolayer for neural tissue engineering.

The purpose of this study was to prepare a monolayer of neural stem/precursor cells (NSPCs) for neural tissue engineering applications. Two components present in serum, fibronectin and epidermal growth factor (EGF) were added into DMEM/F12 medium (termed medium B) to examine the effect of the migration-, proliferation- and differentiation-promoting potential on the cultured NSPCs, isolated from embryonic rat cerebral cortex. Compared with the serum effect, medium B also permitted neurosphere attachment onto the substrate surface and cell migration out of neurospheres extensively, but enhanced more extensive cell division and slowed down NSPC differentiation to generate a confluent NSPC monolayer. It was found the medium B-treated NSPCs possessed the capability to form typical neurospheres or to undergo differentiation into neuron-related cell types on various biomaterial surfaces. Therefore, we proposed a two-stage process for wound healing or nerve conduit preparation. Extensive NSPC division and MAP2-positive neuron differentiation were manipulated in NSPCs cultured in the medium B followed by the neuronal differentiation-favorable medium. These results should be useful for controlling the proliferation and differentiation of NSPCs on various biomaterials and conduits in neuroscience research.

Combination of media, biomaterials and extracellular matrix proteins to enhance the differentiation of neural stem/precursor cells into neurons

The purpose of this study was to induce the differentiation of neural stem/precursor cells (NSPC) more towards neurons than glial cells by the combination of media, biomaterials and extracellular matrix (ECM) proteins. Considering the role of serum, 10% fetal bovine serum or its fractions were added to DMEM/F12 medium to examine the effect of the differentiation-promoting potential on cultured NSPC isolated from embryonic rat cerebral cortex. The NSPC were cultured for 7 days, after which differentiation was assayed using immunocytochemistry for lineage specific markers. It was demonstrated that molecules promoting neuron differentiation were present in serum with molecular weight <100 kDa, which could dominate the differentiation of NSPC principally into neurons in the presence of basic fibroblast growth factor. In contrast, NSPC were induced to differentiate predominantly into glial cell phenotypes in the presence of whole serum components. Based on medium containing serum fraction, semi-quantification showed that the MAP2-positive percentage of the immunoreactive ratio within migrated cells could be promoted over 85% by combining poly(ethylene-co-vinyl alcohol) biomaterial and fibronectin matrix protein. These results are very encouraging, since an environment favorable for neuronal differentiation should be useful in the development of strategies for controlling the behavior of NSPC in neuroscience research.

Differentiation of Neural Stem/Progenitor Cells Using Low-Intensity Ultrasound

Herein, we report the evaluation of apoptosis, cell differentiation, neurite outgrowth and differentiation of neural stem/progenitor cells (NSPCs) in response to low-intensity ultrasound (LIUS) exposure. NSPCs were cultured under different conditions, with and without LIUS exposure, to evaluate the single and complex effects of LIUS. A lactic dehydrogenase assay revealed that the cell viability of NSPCs was maintained with LIUS exposure at an intensity range from 100 to 500 mW/cm(2). Additionally, in comparison with no LIUS exposure, the cell survival rate was improved with the combination of medium supplemented with nerve growth factor and LIUS exposure. Our results indicate that LIUS exposure promoted NSPC attachment and differentiation on a glass substrate. Neurite outgrowth assays revealed the generation of longer, thicker neurites after LIUS exposure. Furthermore, LIUS stimulation substantially increased the percentage of differentiating neural cells in NSPCs treated with nerve growth factor in comparison with the unstimulated group. The high percentage of differentiated neural cells indicated that LIUS induced neuronal networks denser than those observed in the unstimulated groups. Furthermore, the release of nitric oxide, an important small-molecule neurotransmitter, was significantly upregulated after LIUS exposure. It is therefore reasonable to suggest that LIUS promotes the differentiation of NSPCs into neural cells, induces neurite outgrowth and regulates nitric oxide production; thus, LIUS may be a potential candidate for NSPC induction and neural cell therapy.

Gallium nitride induces neuronal differentiation markers in neural stem/precursor cells derived from rat cerebral cortex

In the present study, gallium nitride (GaN) was used as a substrate to culture neural stem/precursor cells (NSPCs), isolated from embryonic rat cerebral cortex, to examine the effect of GaN on the behavior of NSPCs in the presence of basic fibroblast growth factor (bFGF) in serum-free medium. Morphological studies showed that neurospheres maintained their initial shape and formed many long and thick processes with the fasciculate feature on GaN. Immunocytochemical characterization showed that GaN could induce the differentiation of NSPCs into neurons and astrocytes. Compared to poly-d-lysine (PDL), the most common substrate used for culturing neurons, there was considerable expression of synapsin I for differentiated neurons on GaN, suggesting GaN could induce the differentiation of NSPCs towards the mature differentiated neurons. Western blot analysis showed that the suppression of glycogen synthase kinase-3beta (GSK-3beta) activity was one of the effects of GaN-promoted NSPC differentiation into neurons. Finally, compared to PDL, GaN could significantly improve cell survival to reduce cell death after long-term culture. These results suggest that GaN potentially has a combination of electric characteristics suitable for developing neuron and/or NSPC chip systems.

Covalent bonding of GYIGSR to EVAL membrane surface to improve migration and adhesion of cultured neural stem/precursor cells.

In the present study, we modified poly (ethylene-co-vinyl alcohol) (EVAL) membranes with the covalent bonding of the laminin-derived peptides, GYIGSR by using carbodiimidazole (CDI) to activate the hydroxyl groups on the membrane surface. The resulting GYIGSR-immobilized EVAL (EVAL-GYIGSR) membrane was analyzed in terms of the effect of immobilized peptide sequence on the behaviors of neural stem/precursor cells (NSPCs), isolated from embryonic rat cerebral cortex, in the serum-free medium. Compared to the unmodified EVAL, GYIGSR immobilized on the EVAL membrane was shown to significantly increase NSPCs migrating out of neurospheres (p<0.05). In addition, NSPCs on the EVAL-GYIGSR membrane were able to differentiate into neural lineage cells and differentiated neurons expressed functional synaptic activity. Basically, there was no significant difference between GYIGSR-immobilized and laminin-coated substrates for their ability to enhance migration and differentiation of NSPCs, suggesting that the immobilization of GYIGSR on the EVAL membrane was successful and the laminin-derived peptide YIGSR and laminin had similar effect on NSPC behaviors. However, it is non-permanent modification for coating laminin on the substrates to support cell survival after a long-term culture. In this study, differentiated neurons could still adhere to the EVAL-GYIGSR surface with functional synaptic activity after incubation for 20 days. Therefore, the bioactive EVAL-GYIGSR provided an alternative approach to improve migration and survival of NSPCs for neural tissue engineering applications.

Pearl extract enhances the migratory ability of fibroblasts in a wound healing model

Context: For 2000 years, traditional Chinese medicine has been used as a remedy for general health improvement, including the fight against aging. Pearl powder has recently been used as a health food that has antioxidant, antiaging, antiradioactive, and tonic activities for cells; it is also applied to cure aphthous ulcer, gastric ulcer, and duodenal ulcer on clinical therapy. In addition, the mother of pearl, nacre, could enhance the cell adhesion and tissue regeneration of skin fibroblasts. Objective: Fibroblast is regarded as indispensable in the processes of wound healing. Therefore, the effect of pearl extract (PL) on fibroblasts is investigated in this study. Materials and methods: PL is produced by a room temperature super extraction system (Taiwan patent no. I271 220). DMEM medium containing PL (300 μg/mL) was used to examine the effect of migration-promoting potential on human fibroblast cell line or human primary fibroblast cells in a wound healing model in vitro. Results: Medium containing PL (300 μg/mL) demonstrated that the migratory cell numbers of fibroblasts were three times more than that without PL, and mRNA expression of collagen type III was higher than in collagen type I in fibroblasts. It revealed a migration-promoting potential of human fibroblasts in a wound healing model in vitro. Discussion and conclusion: The present study found that the migration-promoting effect in PL, which could be a supplement in cell culture. These data suggest PL could be useful for enhancing the wound healing of fibroblasts.

Induced pluripotent stem cells, form in vitro tissue engineering to in vivo allogeneic transplantation

Stem cell therapy brings a new hope and provides an alternative therapeutic strategy for treatment of human diseases in clinical therapy. Due to the stemness characteristics, stem cells are considered as a potential tool to understand and model many critical diseases such as Alzheimer’s (1), cardiovascular (2), cancer diseases (3) etc.

3,6-Bis(1-methyl-4-vinylpyridinium)-carbazole diiodide as a marker for tracking living neural stem/precursor cells

Until now, there has been no appropriate marker to specifically and effectively label living neural stem/precursor cells (NSPCs) to investigate NSPC migration and differentiation. Therefore, the purpose of this study was to develop a specific method for tracking NSPCs by a cell marker, 3,6-bis(1-methyl-4-vinylpyridinium)-carbazole diiodide (BMVC). It was found that the bright fluorescence spots could be rapidly observed in the nuclei of NSPCs, isolated from the embryonic rat cerebral cortex, after incubation with BMVC for just 5 min and could be maintained for exceeding 7 days without obvious decay. Even NSPCs were induced to undergo differentiation; their daughter cells also expressed bright fluorescence spots. In contrast, foreskin fibroblasts, adipose-derived stem cells (ADSCs), bone marrow mesenchymal stem cells (BMMSCs), neurons, and glial cells did not express the bright fluorescence spot in the presence of BMVC. In addition, BMVC-labeled NSPCs could be tracked in wound healing and decellularized rat brain models with minimal cell toxicity. Therefore, BMVC possessed a specific function to distinguish between NSPCs and other stem and neuron-related cells. These results are very encouraging, since a cell marker favorable for long-term labeling NSPCs should be useful in the development of strategies for tracking the development of NSPCs in neuroscience research.

INDUCTION OF NEURONAL DIFFERENTIATION OF EMBRYONIC RAT CORTICAL NEUROSPHERES BY NERVE GROWTH FACTOR AND FETAL BOVINE SERUM ON THE NONADHERENT AND ADHERENT SUBSTRATES

Regulating neural stem cells will greatly aid in developing regenerative medicine for the repair purpose of central nervous system (CNS). In this study, we explored the effect of nerve growth factor (NGF) on the differentiation of neural stem cells from embryonic rat cerebral cortex on nonadherent and adherent substrates at neurosphere level. The results showed that NGF could not promote adhesion and differentiation of neural stem cells when neurospheres were cultured on nonadherent tissue culture polystyrene (TCPS) substrates under serum-free condition. However, when 10% fetal bovine serum (FBS) was added to the culture system, FBS could induce the attachment of neurospheres onto an originally nonadherent substrate and almost all of the neurosphere-forming cells would migrate away from the spheres with the protoplasmic astrocyte morphology. Furthermore, when NGF was added into the FBS-containing medium, a significant number of differentiated neurons developed and distributed on the astrocyte layer. For comparison, adherent poly-D-lysine (PDL) substrates were also used. It was found that NGF could enhance differentiation of neural stem cells into neurons on PDL under serum-free condition but neurons and astrocytes could not migrate far away from the spheres. Similar to the TCPS results were also observed on PDL that differentiated neurons migrated out from the spheres and developed on the astrocyte layer when the medium contained NGF and FBS. Since the development of neurons needs glia cells to form a cellular substratum and to support neuronal migration to appropriate location, these results provide evidences that FBS-induced glia cells, serving as an architectural support layer, are essential for the growth and migration of NGF-induced neurons.