Dan Ge

Culture of neural stem cells in calcium alginate beads

Neural stem cells (NSCs) with the capacity of extensive self‐renewal and multilineage differentiation have attracted more and more attention in research as NSCs will play an important role in the nerve disease treatment and nerve injury repair. The shortage of NSCs, both their sources and their numbers, however, is the biggest challenge for their clinic application, and hence, in vitro culture and expansion of NSCs is vitally important to realize their potentials. In this work, mouse‐derived NSCs were cultured in three‐dimensional calcium alginate beads (Ca‐Alg‐Bs). Gelling conditions, cell density, and cell harvest were determined by the exploration of formation and dissociation parameters for Ca‐Alg‐Bs. Additionally, the recovered and the subsequent induced cells were identified by immunofluorescence staining of Nestin, β‐tubulin, and GFAP. The results show that the 2‐mm diameter Ca‐Alg‐Bs, prepared with 1.5% sodium alginate solution and 3.5% CaCl2 solution and with gelling for 10 min, is suitable for the NSCs culture. The seeding density of 0.8 × 105 cells·mL−1 for the encapsulation of NSCs resulted in the most expansion, and the NSCs almost doubled during the experiment. The average cell recovery rate is over 88.5%, with the Ca‐Alg‐Bs dissolving in 55 mM sodium citrate solution for 10 min. The recovered cells cultured in the Ca‐Alg‐Bs still expressed Nestin and had the capacity of multilineage differentiation into neurons and glial cells and, thus, remained to be NSCs. These results demonstrate that NSC expansion within Ca‐Alg‐Bs is feasible and provides further possibilities for NSC expansion in bioreactors of the scale of clinical relevance.

Protocatechuic acid promotes cell proliferation and reduces basal apoptosis in cultured neural stem cells

Protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia oxyphylla, showed anti-oxidant neuroprotective property in our previous study. However, it is still unknown whether PCA have effects on the cultured neural stem cells (NSCs). In this study, we investigated the roles of PCA in the survival and apoptosis of rat NSCs under normal conditions. NSCs obtained from 13.5-day-old rat embryos were propagated as neurospheres and cultured under normal conditions with or without PCA for 4 and 7 days. The cell viability was determined by the cell counting kit-8 (CCK-8) test, while cell proliferation was assayed by bromodeoxyuridine (BrdU) labeling. PCA increased the cellular viability of NSCs and stimulated cell proliferation in a dose- and time-dependent manner. Apoptotic cells were detected after 4 days by observing the nuclear morphological changes and flow cytometric analysis. Compared with the control on both culture days, treatment with PCA effectively reduced the levels of apoptosis of NSCs. At the same time, the reactive oxygen species (ROS) level in NSCs was depressed. In addition, PCA also significantly decreased the activity of elevated caspase-3, indicating that PCA may inhibit apoptosis of NSCs via suppression of the caspase cascade. These results suggest that PCA may be a potential growth inducer and apoptosis inhibitor for NSCs.

Induced pluripotent stem cells generated from human adipose-derived stem cells using a non-viral polycistronic plasmid in feeder-free conditions.

Induced pluripotent stem cells (iPSCs) can be generated from somatic cells by ectopic expression of defined transcription factors (TFs). However, the optimal cell type and the easy reprogramming approaches that minimize genetic aberrations of parent cells must be considered before generating the iPSCs. This paper reports a method to generate iPSCs from adult human adipose-derived stem cells (hADSCs) without the use of a feeder layer, by ectopic expression of the defined transcription factors OCT4, SOX2, KLF4 and C-MYC using a polycistronic plasmid. The results, based on the expression of pluripotent marker, demonstrated that the iPSCs have the characteristics similar to those of embryonic stem cells (ESCs). The iPSCs differentiated into three embryonic germ layers both in vitro by embryoid body generation and in vivo by teratoma formation after being injected into immunodeficient mice. More importantly, the plasmid DNA does not integrate into the genome of human iPSCs as revealed by Southern blotting experiments. Karyotypic analysis also demonstrated that the reprogramming of hADSCs by the defined factors did not induce chromosomal abnormalities. Therefore, this technology provides a platform for studying the biology of iPSCs without viral vectors, and can hopefully overcome immune rejection and ethical concerns, which are the two important barriers of ESC applications.

Slow-freezing cryopreservation of neural stem cell spheres with different diameters

Neural stem cells (NSCs) are of great value for clinical application and scientific research. The development of efficient cryopreservation protocols could significantly facilitate the storage and transportation for clinic applications. The objective of the present study is to improve the survival rate and viability of NSCs. Neural stem cells with three states of single-cell suspension, NSC spheres with diameters of 30-50 microm and 80-100 microm, were cryopreserved by slow-freezing method with the cryoprotective agent (CPA) of dimethyl sulfoxide (Me(2)SO), respectively. Then the post-thawing NSCs were tested for the survival rate and the differentiation ability. As a result, NSC spheres with diameter of 80-100 microm and Me(2)SO concentration of 8% achieve the survival rate of 82.9%, and the NSCs still sustain the multi-differentiation potentiality. These results indicated that both the subtle interaction among NSCs and sphere diameters may affect the survival rate together.

Protocatechuic acid promotes the neuronal differentiation and facilitates survival of phenotypes differentiated from cultured neural stem and progenitor cells

Protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia (A.) oxyphylla, plays crucial roles in the proliferation and neuroprotection of cultured neural stem and progenitor cells (NS/PCs) in our previous study. However, whether PCA modulates the differentiation of NS/PCs has remained to be elucidated. In this study, we show that PCA can promote the neuronal differentiation combined with fetal bovine serum (FBS) in vitro, although it cannot initiate the differentiation of NS/PCs by itself. Moreover, PCA is able to induce neuronal maturation and efficiently promote neurite outgrowth. On the other hand, PCA facilitates survival of phenotypes differentiated from cultured NS/PCs, which was associated with an increased percentage of the cellular viability and a decreased percentage of cells undergoing apoptosis under differentiation conditions. In addition, PCA-induced survival is also mediated with the activating of endogenous antioxidant enzymes. These results suggest that PCA may serve as a useful reference for future studies in designing stem cell strategies to promote brain recovery and repair in neurodegenerative diseases.

Cryoprotectants for the vitrification of corneal endothelial cells

The objective of this work was to select and test systematically possible cryoprotective agents (CPAs) and to obtain a suitable formula for vitrification of corneal endothelial cells (CECs). Fresh bovine CECs were isolated and tested with an optimized vitrification protocol with multi-step CPA loading and removal. Three types of CPAs components, i.e. the penetrating CPAs, sugars and macromolecular compounds, were experimentally evaluated using the viability assayed by trypan blue. Dimethyl sulfoxide, ethylene glycol (EG), 1,2-propanediol, 2,3-butanediol, acetamide and ethylene glycol monomethyl ether were chosen as the penetrating CPA components. Sugars including xylose, fructose, mannose, glucose, maltose, sucrose and trehalose were tested. Ficoll (MW 7kDa), dextran (MW 7kDa), chondroitin sulfate (CS, MW 18-30kDa), bovine serum albumin (MW 68kDa) and polyethylene glycol (MW 6kDa, 10kDa and 20kDa) were chosen as the macromolecular compounds. CECs were also preserved by slow freezing as a control. The results showed that EG was the most suitable penetrating CPA component and glucose the most suitable sugar, and CS the most suitable macromolecule. The optimized concentrations for each component in the vitrification solution were 52% (w/w) EG, 8% (w/w) glucose and 3% (w/w) CS. The CEC survival rate of 89.4+/-2.1% (mean+/-SD) was obtained using this formula and established vitrification protocol which was comparable to that by slow freezing.

Study of neuroprotective function of Ginkgo biloba extract (EGb761) derived-flavonoid monomers using a three-dimensional stem cell-derived neural model

An in vitro three‐dimensional (3D) cell culture system that can mimic organ and tissue structure and function in vivo will be of great benefit for drug discovery and toxicity testing. In this study, the neuroprotective properties of the three most prevalent flavonoid monomers extracted from EGb 761 (isorharmnetin, kaempferol, and quercetin) were investigated using the developed 3D stem cell‐derived neural co‐culture model. Rat neural stem cells were differentiated into co‐culture of both neurons and astrocytes at an equal ratio in the developed 3D model and standard two‐dimensional (2D) model using a two‐step differentiation protocol for 14 days. The level of neuroprotective effect offered by each flavonoid was found to be aligned with its effect as an antioxidant and its ability to inhibit Caspase‐3 activity in a dose‐dependent manner. Cell exposure to quercetin (100 µM) following oxidative insult provided the highest levels of neuroprotection in both 2D and 3D models, comparable with exposure to 100 µM of Vitamin E, whilst exposure to isorhamnetin and kaempferol provided a reduced level of neuroprotection in both 2D and 3D models. At lower dosages (10 µM flavonoid concentration), the 3D model was more representative of results previously reported in vivo. The co‐cultures of stem cell derived neurons and astrocytes in 3D hydrogel scaffolds as an in vitro neural model closely replicates in vivo results for routine neural drug toxicity and efficacy testing.

Thermo-responsive poly(N-isopropylacrylamide)-grafted hollow fiber membranes for osteoblasts culture and non-invasive harvest

Hollow fiber membrane (HFM) culture system is one of the most important bioreactors for the large-scale culture and expansion of therapeutic cells. However, enzymatic and mechanical treatments are traditionally applied to harvest the expanded cells from HFMs, which inevitably causes harm to the cells. In this study, thermo-responsive cellulose acetate HFMs for cell culture and non-invasive harvest were prepared for the first time via free radical polymerization in the presence of cerium (IV). ATR-FTIR and elemental analysis results indicated that the poly(N-isopropylacrylamide) (PNIPAAm) was covalently grafted on HFMs successfully. Dynamic contact angle measurements at different temperatures revealed that the magnitude of volume phase transition was decreased with increasing grafted amount of PNIPAAm. And the amount of serum protein adsorbed on HFMs surface also displayed the same pattern. Meanwhile osteoblasts adhered and spread well on the surface of PNIPAAm-grafted HFMs at 37 °C. And Calcein-AM/PI staining, AB assay, ALP activity and OCN protein expression level all showed that PNIPAAm-grafted HFMs had good cell compatibility. After incubation at 20 °C for 120 min, the adhering cells on PNIPAAm-grafted HFMs turned to be round and detached after being gently pipetted. These results suggest that thermo-responsive HFMs are attractive cell culture substrates which enable cell culture, expansion and the recovery without proteolytic enzyme treatment for the application in tissue engineering and regenerative medicine.

Chitosan/gelatin porous scaffolds assembled with conductive poly(3,4-ethylenedioxythiophene) nanoparticles for neural tissue engineering

Electroactive biomaterials are widely explored as scaffolds for nerve tissue regeneration. Poly(3,4-ethylenedioxythiophene) (PEDOT) is a conductive polymer that has been chosen to construct tissue engineered scaffolds because of its excellent conductivity and non-cytotoxicity. In the present study, an electrically conductive scaffold was prepared by assembling PEDOT on a chitosan/gelatin (Cs/Gel) porous scaffold surface via in situ interfacial polymerization. The hydrophilic Cs/Gel hydrogel was used as a template, and PEDOT nanoparticles were uniformly assembled on the scaffold surface. The static polymerization of the 3,4-ethylenedioxythiophene (EDOT) monomer at the interface between the aqueous phase and the organic phase was accompanied by the formation of the PEDOT-assembled Cs/Gel scaffolds. PEDOT/Cs/Gel scaffolds were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy. The results confirmed the deposition of PEDOT nanoparticles with the mean diameter of 50 nm on the Cs/Gel scaffold channel surface. Compared to the Cs/Gel scaffold, the incorporation of PEDOT on the scaffold increased the electrical conductivity, hydrophilicity, mechanical properties and thermal stability, whereas decreased the water absorption and biodegradation. For biocompatibility, PEDOT/Cs/Gel scaffolds, especially the 2PEDOT/Cs/Gel scaffold group, significantly promoted neuron-like rat pheochromocytoma (PC12) cell adhesion and proliferation. The results of both the gene expression and protein level assessments suggested that the PEDOT-assembled Cs/Gel scaffold enhanced the PC12 cellular neurite growth with higher protein and gene expression levels. This is the first report on the construction of a conductive PEDOT/Cs/Gel porous scaffold via an in situ interfacial polymerization method, and the results demonstrate that it may be a promising conductive scaffold for neural tissue engineering.

Pyrroloquinoline quinone against glutamate-induced neurotoxicity in cultured neural stem and progenitor cells

Pyrroloquinoline quinone (PQQ), as a well‐known redox enzyme cofactor, has been proven to play important roles in the regulation of cellular growth and development in mammals. Numerous physiological and medicinal functions of PQQ have so far been reported although its effect on neural stem and progenitor cells (NS/PCs) and the potential mechanism were even rarely investigated. In this study, the neuroprotective effects of PQQ were observed by pretreatment of NS/PCs with PQQ before glutamate injury, and the possible mechanisms were examined. PQQ stimulated cell proliferation and markedly attenuated glutamate‐induced cell damage in a dose‐dependent manner. By observing the nuclear morphological changes and flow cytometric analysis, PQQ pretreatment showed its significant effect on protecting NS/PCs against glutamate‐induced apoptosis/necrosis. PQQ neuroprotection was associated with the decrease of intracellular reactive oxygen species (ROS) production, the increase of glutathione (GSH) levels, and the decrease of caspase‐3 activity. In addition, pretreatment with PQQ also significantly enhanced the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in the NS/PCs exposed to glutamate. These results suggest that PQQ can protect NS/PCs against glutamate toxicity associated with ROS‐mediated mitochondrial pathway, indicating a useful chemical for the clinical application of NS/PCs.