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ABSTRACT: Human-induced pluripotent stem cell-derived neural progenitors (hiPSC-NPs) have the ability to self-renew and differentiate into glial and neuronal lineages, which makes them an invaluable source in cell replacement therapy for neurological diseases. Therefore, their enhanced proliferation and neuronal differentiation are pivotal features that can be used in repairing neurological injuries. One of the main regulators of neural development is Wnt signaling, which results in the inhibition of glycogen synthase kinase 3 (GSK-3). Here, we assess the impact of GSK-3 inhibition by the small molecule CHIR99021 on the expansion and differentiation of hiPSC-NPs in an adherent condition and a defined medium. Cell proliferation analyses have revealed that inhibition of GSK-3 in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) increased the proliferation of hiPSC-NPs across 10 passages. The inhibition of β-catenin signaling by XAV and NOTCH signaling by DAPT reversed CHIR impact on hiPSC-NPs proliferation. The target genes of β-catenin, C-MYC and CYCLIN D1 as well as NOTCH target genes, HES1 and HES5 were upregulated. The treatment of NPs by CHIR in the absence of bFGF and EGF resulted in an increase of neuronal differentiation rather than proliferation by stabilization of β-catenin regardless of the NOTCH pathway. Thus, GSK-3 inhibition has been shown to promote proliferation of the NPs by activating β-catenin and NOTCH-related cell cycle genes in the presence of bFGF and EGF. Additionally, during GSK-3 inhibition, an absence of these growth factors allows for the switch to neuronal differentiation with a bias toward a dopaminergic fate. This may provide desired cells that can be used in therapeutic applications and offer insights into the etiology of some neurological disorders.
Stem cells and development 05/2012; · 4.15 Impact Factor
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ABSTRACT: In this study, a highly porous poly (D, L-lactic acid) (PDLLA) scaffold was designed and fabricated using dioxane and thermal-induced phase separation (TIPS) methods (liquid-liquid and solid-liquid). Additionally, we characterized the ability of mouse embryonic stem cells (ESCs) to differentiate into neural cells in PDLLA scaffold with uniform porosity, interconnectivity, and high porosity, and then compared them with cells seeded under conventional two-dimensional (2D) culture conditions. Histochemistry staining showed the migration of differentiated cells through the scaffold. Immunofluorescence analysis of the differentiated cells by counting positive cells revealed that the PDLLA scaffold resulted in a significantly greater number of neural markers, microtubule associated protein-2, ß-tubulin III, neurofilament protein, and glial fibrillary acidic protein (the astrocyte marker) when compared to those in 2D culture condition. Moreover, the expression of Nestin, Mash1, Pax6, and HB9 increased significantly in 3D scaffolds when compared with 2D cultures as detected by semi-quantitative RT-PCR. Scanning electron microscopy of differentiated neurons on scaffolds also demonstrated favorable results for neurite outgrowth. The results of this study demonstrated a promising effect of 3D scaffold culture for neural cell differentiation from ESCs in prospective tissue engineering applications.
The International journal of artificial organs 10/2011; 34(10):1012-23. · 1.86 Impact Factor
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ABSTRACT: Quantum dots (QDs), as new and promising fluorescent probes, hold great potential in long term non-invasive bio-imaging, however there are many uncovered issues regarding their competency. In the present study, different QDs (525, 585 and 800 nm) were used to label CD133, CD34, CD14 and mesenchymal stem cells (MSCs) using positively charged peptides. Results demonstrated highly efficient internalization with the possible involvement of macropinocytosis. As indicated by LDH release and the TUNEL assay, no measurable effects on cell viability were detected at a concentration of 10 nM. QDs did not have any deleterious effects on normal cell functionality where both labeled CD133(+) cells and MSCs remarkably differentiated along multiple lineages with the use of the colony forming assay and adipo/osteo induction, respectively. Our results regarding QD maintenance revealed that these nano-particles are not properly stable and various excretion times have been observed depending on particle size and cell type. In vitro co-culture system and transplantation of labeled cells to an animal model showed that QDs leaked out from labeled cells and the released nano-particles were able to re-enter adjacent cells over time. These data suggest that before any utilization of QDs in bio-imaging and related applications, an efficient intra-cellular delivery technique should be considered to preserve QDs for a prolonged time as well as eliminating their leakage.
Biomaterials 08/2011; 32(22):5195-205. · 7.40 Impact Factor
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ABSTRACT: In this study, the impact of randomly oriented electrospun polyamide nanofibrous architecture on neurogenic differentiation of human embryonic stem cells (hESCs) compared with the lack of nanofibrous features in vitro in a neural-inducing condition was examined. Flow cytometry analysis of hESC-derived neural ectoderm (NE) showed nanofibrous surfaces capable of supporting NE by expression of higher percentages of related markers NESTIN, SOX1, and PAX6 in addition to significantly greater total cell proliferation as shown by Ki67 in the neurogenic condition. After replating hESC-derived NE, the differentiated cells expressed higher neuronal markers (TUJ1 and MAP2) and motor neuron markers (HB9, ISL1, and ChAT) at both the protein and mRNA levels on nanofibers. The presence of developed spread neurites and plausible neurite connections were shown by scanning electron microscopy. Additionally, Na(+) and Ca(2+) currents in differentiated neurons on nanofibers were significantly greater than both control and generated action potentials. Moreover, less duration of inward currents, greater negative resting membrane potential, and enhanced expression and functionality of ionic channel genes were observed in neuronal cells on nanofibers. These results indicated that a nanofibrillar surface along with neurogenic growth factors provided a better environment for hESC neurogenic differentiation and function, which holds great promise in prospective tissue engineering applications.
Tissue Engineering Part A 07/2011; 17(23-24):3021-31. · 4.64 Impact Factor
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ABSTRACT: Stem cell technology combined with nano-scaffold surfaces provides a new tool for better induction involved in cell lineage differentiations and therefore for central nervous system repair. This study was undertaken to investigate appropriate neural cell-substrate interactions. Neural progenitors (NPs) were established from human embryonic stem cells (hESCs), as a first step, using an adherent system and a defined medium supplemented with a combination of factors. Next, the behavior of hESC-derived NPs (hESC-NPs) was evaluated on a synthetic, randomly oriented, three-dimensional nanofibrillar matrix composed of electrospun polyamide nanofibers (Ultra-Web™) using a variety of experimental approaches. We have demonstrated that homogenous, expandable, and self-renewable NPs can be easily generated from hESCs; they can express related markers Nestin, Sox1, and Pax6; and they can undergo multipotency differentiation to neurons and glials. Functionally, NPs cultured on nanofibers demonstrated an increase in the rate of migration, proliferation, morphology, and neurite length when compared with NPs cultured on two-dimensional culture surfaces. The results suggest that topographical features of the extracellular matrix of the cell environment have paved the way for a better understanding of human neuronal development, thus allowing for future clinical applications.
The International journal of artificial organs 07/2011; 34(7):559-70. · 1.86 Impact Factor
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ABSTRACT: Among the numerous attempts to integrate tissue engineering concepts into strategies to repair nearly all parts of the body, neuronal repair stands out. This is partially due to the complexity of the nervous anatomical system, its functioning and the inefficiency of conventional repair approaches, which are based on single components of either biomaterials or cells alone. Electrical stimulation has been shown to enhance the nerve regeneration process and this consequently makes the use of electrically conductive polymers very attractive for the construction of scaffolds for nerve tissue engineering. In this review, by taking into consideration the electrical properties of nerve cells and the effect of electrical stimulation on nerve cells, we discuss the most commonly utilized conductive polymers, polypyrrole (PPy) and polyaniline (PANI), along with their design and modifications, thus making them suitable scaffolds for nerve tissue engineering. Other electrospun, composite, conductive scaffolds, such as PANI/gelatin and PPy/poly(ε-caprolactone), with or without electrical stimulation, are also discussed. Different procedures of electrical stimulation which have been used in tissue engineering, with examples on their specific applications in tissue engineering, are also discussed.
Journal of Tissue Engineering and Regenerative Medicine 04/2011; 5(4):e17-35. · 3.28 Impact Factor
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ABSTRACT: Human induced pluripotent stem cells (hiPSCs) have led to an important revolution in stem cell research and regenerative medicine. To create patient-specific neural progenitors (NPs), we have established a homogenous, expandable, and self-renewable population of multipotent NPs from hiPSCs, using an adherent system and defined medium supplemented with a combination of factors. The established hiPSC-NPs highly expressed Nestin and Sox1. These NPs were continuously propagated for ~1 year without losing their potential to generate astrocytes, oligodendrocytes, and functional neurons and maintained a stable chromosome number. Voltage clamp analysis revealed outward potassium currents in hiPSC-NPs. The self-renewal characteristic of the NPs was demonstrated by a symmetrical mode of Nestin-positive cell division. Additionally, these hiPSC-NPs can be easily frozen and thawed in the presence of Rho-associated kinase (ROCK) inhibitor without losing their proliferation, karyotype stability, and developmental potential. The characteristics of our generated hiPSC-NPs provide the opportunity to use patient-specific or ready-to-use hiPSC-NPs in future biomedical applications.
Stem cells and development 03/2011; 20(3):503-14. · 4.15 Impact Factor
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ABSTRACT: Global gene expression analysis of human embryonic stem cells (hESCs) that differentiate into neural cells would help to further define the molecular mechanisms involved in neurogenesis in humans. We performed a comprehensive transcripteome analysis of hESC differentiation at three different stages: early neural differentiation, neural ectoderm, and differentiated neurons. We identified and validated time-dependent gene expression patterns and showed that the gene expression patterns reflect early ESC differentiation. Sets of genes are induced in primary ectodermal lineages and then in differentiated neurons, constituting consecutive waves of known and novel genes. Pathway analysis revealed dynamic expression patterns of members of several signaling pathways, including NOTCH, mTOR and Toll like receptors (TLR), during neural differentiation. An interaction network analysis revealed that the TGFβ family of genes, including LEFTY1, ID1 and ID2, are possible key players in the proliferation and maintenance of neural ectoderm. Collectively, these results enhance our understanding of the molecular dynamics underlying neural commitment and differentiation.
PLoS ONE 01/2011; 6(7):e22856. · 4.09 Impact Factor
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PLoS ONE 01/2011; 6(8). · 4.09 Impact Factor
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ABSTRACT: This study aims to differentiate human induced pluripotent stem cells (hiPSCs) into oligodendrocyte precursors and assess their recovery potential in a demyelinated optic chiasm model in rats.
We generated a cell population of oligodendrocyte progenitors from hiPSCs by using embryoid body formation in a defined medium supplemented with a combination of factors, positive selection and mechanical enrichment. Real-time polymerase chain reaction and immunofluorescence analyses showed that stage-specific markers, Olig2, Sox10, NG2, PDGFRα, O4, A2B5, GalC, and MBP were expressed following the differentiation procedure, and enrichment of the oligodendrocyte lineage. These results are comparable with the expression of stage-specific markers in human embryonic stem cell-derived oligodendrocyte lineage cells. Transplantation of hiPSC-derived oligodendrocyte progenitors into the lysolecithin-induced demyelinated optic chiasm of the rat model resulted in recovery from symptoms, and integration and differentiation into oligodendrocytes were detected by immunohistofluorescence staining against PLP and MBP, and measurements of the visual evoked potentials.
These results showed that oligodendrocyte progenitors generated efficiently from hiPSCs can be used in future biomedical studies once safety issues have been overcome.
PLoS ONE 01/2011; 6(11):e27925. · 4.09 Impact Factor
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ABSTRACT: Major advances in various disciplines of basic sciences including embryology, molecular and cell biology, genetics, and nanotechnology, as well as stem cell biology have opened new horizons for regenerative therapy. The unique characteristics of stem cells prompt a sound understanding for their use in modern regenerative therapies. This review article discusses stem cells, developmental stages of the eye field, eye field transcriptional factors, and endogenous and exogenous sources of stem cells. Recent studies and challenges in the application of stem cells for retinal pigment epithelial degeneration models will be summarized followed by obstacles facing regenerative therapy.
Journal of ophthalmic & vision research. 10/2010; 5(4):250-64.
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ABSTRACT: Several studies have reported functional improvement after transplantation of in vivo-derived neural progenitor cells (NPC) into injured spinal cord. However, the potential of human embryonic stem cell-derived NPC (hESC-NPC) as a tool for cell replacement of spinal cord injury (SCI) should be considered.
We report on the generation of NPC as neural-like tubes in adherent and feeder-free hESC using a defined media supplemented with growth factors, and their transplantation in collagen scaffolds in adult rats subjected to midline lateral hemisection SCI.
hESC-NPC were highly expressed molecular features of NPC such as Nestin, Sox1 and Pax6. Furthermore, these cells exhibited the multipotential characteristic of differentiating into neurons and glials in vitro. Implantation of xenografted hESC-NPC into the spinal cord with collagen scaffold improved the recovery of hindlimb locomotor function and sensory responses in an adult rat model of SCI. Analysis of transplanted cells showed migration toward the spinal cord and both neural and glial differentiation in vivo.
These findings show that transplantation of hESC-NPC in collagen scaffolds into an injured spinal cord may provide a new approach to SCI.
Cytotherapy 07/2009; 11(5):618-30. · 3.63 Impact Factor
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ABSTRACT: Understanding how to direct human embryonic stem cells (hESCs) toward a specific lineage pathway and generate appropriate cell types robustly is very important, not only for the study of developmental biology but also for potentially using these cells to treat human diseases. In this study, hESCs were differentiated to the neural lineage in defined adherent culture by retinoic acid and basic fibroblast growth factor. Our protocol seems to recapitulate the early steps of nervous system development in vivo in that undifferentiated hESCs organized into rosettes and then neural tube-like structures are formed. Differentiating cells expressed neuroectodermal and mature neuron markers during neural plate and tube formation and maturation, as shown by reverse transcriptase-PCR. More than 90% of differentiated cells expressed additional neuron-specific antigens (i.e., tubulin-III, MAP-2, synaptophysin and neurofilament protein). Ultrastructural analysis of differentiating neural tube-like structures in three dimensional collagen scaffolds showed an ependymal-like layer and neural structure with typical synapses. These results provide a simple and relatively defined system for differentiation of hESCs to neural lineages, particularly neurons with typical cellular, molecular and ultrastuctureal markers. The culture of neural precursor cells in a collagen scaffold may provide a new approach for the repair of spinal cord injury.
The International Journal of Developmental Biology 02/2007; 51(5):371-8. · 2.82 Impact Factor
