Differentiation of embryonic stem cells into neural cells on 3D poly (D, L-lactic acid) scaffolds versus 2D cultures (original) (raw)
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Tissue Engineering Part C: Methods, 2009
In the present study, we examined the possible utility of a three-dimensional culture system using a thermoreversible gelation polymer to isolate and expand neural stem cells (NSCs). The polymer is a synthetic biologically inert polymer and gelates at temperatures higher than the gel-sol transition point (*208C). When fetal mouse brain cells were inoculated into the gel, spherical colonies were formed (*1% in primary culture and *9% in passage cultures). The spheroid-forming cells were positive for expression of the NSC markers nestin and Musashi. Under conditions facilitating spontaneous neural differentiation, the spheroid-forming cells expressed genes characteristic to astrocytes, oligodendrocytes, and neurons. The cells could be successively propagated at least to 80 poly-D-lysines over a period of 20 weeks in the gel culture with a growth rate higher than that observed in suspension culture. The spheroids formed by fetal mouse brain cells in the gel were shown to be of clonal origin. These results indicate that the spheroid culture system is a convenient and powerful tool for isolation and clonal expansion of NSCs in vitro.
Growth of human stem cell-derived neurons on solid three-dimensional polymers
Journal of Biochemical and Biophysical Methods, 2005
Understanding neural differentiation and the development of complex neurite networks in threedimensional matrices is critical for neural tissue engineering in vitro. In this study we describe for the first time the growth of human stem cell-derived neurons on solid polystyrene matrices coated with bioactive molecules. Highly porous foams were prepared from poly(styrene/divinylbenzene) using a high internal phase emulsion (HIPE) as a template to create the porous structure. The resulting polyHIPE matrices were readily coated with aqueous-based solutions including poly-d-lysine and laminin. Human neurons adhered well to poly-d-lysine coated surfaces and extended neural processes, however, neurite outgrowth was particularly enhanced when polymers also received a coating of laminin. These data clearly demonstrate the potential use of solid polystyrene scaffolds to create three-dimensional environments for cell growth and differentiation. We propose that these robust and stable matrices can be conveniently and routinely used in the tissue culture laboratory to study the behaviour of cells grown in three-dimensions. D
Scaffolds for 3D in vitro culture of neural lineage cells
Acta biomaterialia, 2017
Understanding how neurodegenerative disorders develop is not only a key challenge for researchers but also for the wider society, given the rapidly aging populations in developed countries. Advances in this field require new tools with which to recreate neural tissue in vitro and produce realistic disease models. This in turn requires robust and reliable systems for performing 3D in vitro culture of neural lineage cells. This review provides a state of the art update on three-dimensional culture systems for in vitro development of neural tissue, employing a wide range of scaffold types including hydrogels, solid porous polymers, fibrous materials and decellularised tissues as well as microfluidic devices and lab-on-a-chip systems. To provide some context with in vivo development of the central nervous system (CNS), we also provide a brief overview of the neural stem cell niche, neural development and neural differentiation in vitro. We conclude with a discussion of future directions...
Biomaterials, 2013
The onset of neurodegenerative disorders is characterized by the progressive dysfunction and loss of subpopulations of specialized cells within specific regions of the central nervous system (CNS). Since CNS has a limited ability for self-repair and regeneration under such conditions, clinical transplantation of stem cells has been explored as an alternative. Although embryonic stem cells (ESCs) offer a promising therapeutic platform to treat a variety of neurodegenerative disorders, the niche microenvironment, which could regulate their differentiation into specialized lineages on demand, needs to be optimized for successful clinical transplantation. Here, we evaluated the synergistic role of matrix microenvironment (type, architecture, composition, stiffness) and signaling molecules (type, dosage) on murine ESC differentiation into specific neural and glial lineages. ESCs were cultured as embryoid bodies on either 2D substrates or within 3D scaffolds, in the presence or absence of retinoic acid (RA) and sonic hedgehog (Shh). Results showed that ESCs maintained their stemness even after 4 days in the absence of exogenous signaling molecules, as evidenced by Oct-4 staining. RA at 1 mM dosage was deemed optimal for neural differentiation and neurite outgrowth on collagen-1 coated substrates. Significant neural differentiation with robust neurite outgrowth and branching was evident only on collagen-1 coated 2D substrates and within 3D matrigel scaffolds, in the presence of 1 mM RA. Blocking a6 or b1 integrin subunits on differentiating cells inhibited matrigel-induced effects on neural differentiation and neurite outgrowth. Hydrogel concentration strongly regulated formation of neural and astrocyte lineages in 1 mM RA additive cultures. When RA and Shh were provided, either alone or together, 3D collagen-1 scaffolds enhanced significant motor neuron formation, while 3D matrigel stimulated dopaminergic neuron differentiation. These results suggest a synergistic role of microenvironmental cues for ESC differentiation and maturation, with potential applications in cell transplantation therapy.
Stem Cell Research, 2013
Human embryonic stem cells (hESCs) are emerging as an attractive alternative source for cell replacement therapy since the cells can be expanded in culture indefinitely and differentiated into any cell types in the body. In order to optimize cell-to-cell interaction, cell proliferation and differentiation into specific lineages as well as tissue organization, it is important to provide a microenvironment for the hESCs which mimics the stem cell niche. One approach is to provide a three-dimensional (3D) environment such as encapsulation. We present an approach to culture and differentiate hESCs into midbrain dopamine (mdDA) neurons in a 3D microenvironment using alginate microcapsules for the first time. A detailed gene and protein expression analysis during neuronal differentiation showed an increased gene and protein expression of various specific DA neuronal markers, particularly tyrosine hydroxylase (TH) by N 100 folds after 2 weeks and at least 50% higher expression after 4 weeks respectively, compared to cells differentiated under conventional two-dimensional (2D) platform. The encapsulated TH + cells co-expressed mdDA neuronal markers, forkhead box protein A-2 (FOXA2) and pituitary homeobox-3 (PITX3) after 4 weeks and secreted approximately 60 pg/ml/10 6 cells higher DA level when induced. We propose that the 3D platform facilitated an early onset of DA neuronal generation compared to that with conventional 2D system which also secretes more DA under potassium-induction. It is a very useful model to study the proliferation and directed differentiation of hESCs to various lineages, particularly to mdDA neurons. This 3D system also allows the separation of feeder cells from hESCs during the process of differentiation and also has potential for immune-isolation during transplantation studies.
Enhanced neurite outgrowth by human neurons grown on solid three-dimensional scaffolds
Biochemical and Biophysical Research Communications, 2004
Growing and differentiating human stem cells in vitro can provide access to study the molecular mechanisms that control cellular development in a manner pertinent to human embryogenesis. To fully understand such processes, however, it is important to recreate culture conditions that most closely relate to those in living tissues. As step in this direction, we have developed a robust threedimensional cell culture system using inert highly porous solid matrices manufactured from polystyrene that can be routinely used to study the differentiation of human pluripotent stem cell-derived neurons in vitro. Neurite outgrowth was significantly enhanced when neurons were grown in a three-dimensional environment compared to traditional flat surfaces and resulted in the formation of extensive neural networks. These data suggest that the topography within the culture environment can significantly alter cell development and will therefore be an important feature when investigating the potential of human stem cells.
Differentiation of Mouse Stem Cells into Neural Cells on PLGA Microspheres Scaffold
The cellular therapy and nerve tissue engineering will probably become a major therapeutic strategy for promoting axonal growth through injured area in central nervous system and peripheral nervous system in the coming years. The stem cell carrier scaffolds in nerve tissue engineering resulted in strong survival of cells and suitable differentiation into neural cells, so this pathway should be created a favorable environment for axon regeneration. Poly lactic-co-glycolic acid (PLGA) has been widely used for manufacturing three dimentional scaffolds for tissue engineering. The pluripotent nature and proliferative capacity of embryonic carcinoma cells such as P19 also makes them an attractive cell source for tissue engineering. This study was initiated to evaluate potential of biodegradable PLGA microspheres for P19-derived neurons for neural tissue engineering and axon regeneration. The PLGA microspheres were prepared by using solvent evaporation, water in oil in water, technique. Th...
Scientific Reports, 2020
Synthetic biodegradable polymers including poly(lactic acid) (PLA) are attractive cell culture substrates because their surfaces can be micropatterned to support cell adhesion. The cell adhesion properties of a scaffold mainly depend on its surface chemical and structural features; however, it remains unclear how these characteristics affect the growth and differentiation of cultured cells or their gene expression. In this study, we fabricated two differently structured PLA nanosheets: flat and microgrooved. We assessed the growth and differentiation of mouse primary cultured cortical neurons on these two types of nanosheets after pre-coating with poly-D-lysine and vitronectin. Interestingly, prominent neurite bundles were formed along the grooves on the microgrooved nanosheets, whereas thin and randomly extended neurites were only observed on the flat nanosheets. Comparative RNA sequencing analyses revealed that the expression of genes related to postsynaptic density, dendritic shafts, and asymmetric synapses was significantly and consistently up-regulated in cells cultured on the microgrooved nanosheets when compared with those cultured on the flat nanosheets. These results indicate that microgrooved PLA nanosheets can provide a powerful means of establishing a culture system for the efficient and reproducible differentiation of neurons, which will facilitate future investigations of the molecular mechanisms underlying the pathogenesis of neurological disorders. Dissociated primary neuronal cultures are widely used not only for basic neuroscience research but also for drug discovery for neurological disorders 1-3. In such culture systems, scaffolds are one of the key factors providing the cells with structural support for attachment and subsequent growth and differentiation. Thus far, numerous synthetic polymers including polystyrene, poly(lactic acid) (PLA), poly(glycolic acid), and poly(lactic-co-glycolic acid) 4-6 have been developed to serve as scaffolds. Among these, PLA, a biodegradable and resorbable polyester, has recently come into the limelight for its utility in medical applications such as tissue regeneration 7. Polymeric ultrathin film consisting of PLA, hereinafter called "PLA nanosheet," is a thin, soft, and flexible material, with properties that allow it to adhere anywhere without any adhesive materials 8. Many studies have demonstrated that nanosheets can be used to dress wounds to avoid suture, prevent infection, promote bone regeneration, etc. for biomedical applications 8-13. Nanosheets are also suitable for use as a sheet substrate in cell
Biomaterials, 2011
The adult central nervous system (CNS) contains adult neural stem/progenitor cells (NSPCs) that possess the ability to differentiate into the primary cell types found in the CNS and to regenerate lost or damaged tissue. The ability to specifically and spatially control differentiation is vital to enable cell-based CNS regenerative strategies. Here we describe the development of a protein-biomaterial system that allows rapid, stable and homogenous linking of a growth factor to a photocrosslinkable material. A bioactive recombinant fusion protein incorporating pro-neural rat interferon-g (rIFN-g) and the AviTag for biotinylation was successfully expressed in Escherichia coli and purified. The photocrosslinkable biopolymer, methacrylamide chitosan (MAC), was thiolated, allowing conjugation of maleimideestrepatavidin via Michael-type addition. We demonstrated that biotinerIFN-g binds specifically to MAC-streptavidin in stoichiometric yields at 100 and 200 ng/mL in photocrosslinked hydrogels. For cell studies, NSPCs were photo-encapsulated in 100 ng/mL biotinerIFN-g immobilized MAC based scaffolds and compared to similar NSPC-seeded scaffolds combining 100 ng/mL soluble biotinerIFN-g vs. no growth factor. Cells were cultured for 8 days after which differentiation was assayed using immunohistochemistry for lineage specific markers. Quantification showed that immobilized biotin-rIFN-g promoted neuronal differentiation (72.8 AE 16.0%) similar to soluble biotinerIFN-g (71.8 AE 13.2%). The percentage of nestin-positive (stem/progenitor) cells as well as RIP-positive (oligodendrocyte) cells were significantly higher in scaffolds with soluble vs. immobilized biotinerIFN-g suggesting that 3-D immobilization results in a more committed lineage specification.