搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

The potential for directed differentiation of human-induced pluripotent stem (iPS) cells to functional postmitotic neuronal phenotypes is unknown. Following methods shown to be effective at generating motor neurons from human embryonic stem cells (hESCs),we found that once specified to a neural lineage,human iPS cells could be differentiated to form motor neurons with a similar efficiency as hESCs. Human iPS-derived cells appeared to follow a normal developmental progression associated with motor neuron formation and possessed prototypical electrophysiological properties. This is the first demonstration that human iPS-derived cells are able to generate electrically active motor neurons. These findings demonstrate the feasibility of using iPS-derived motor neuron progenitors and motor neurons in regenerative medicine applications and in vitro modeling of motor neuron diseases. View Publication

There is growing interest in developing drugs that specifically target glioblastoma tumor-initiating cells (TICs). Current cell culture methods,however,cannot cost-effectively produce the large numbers of glioblastoma TICs required for drug discovery and development. In this paper we report a new method that encapsulates patient-derived primary glioblastoma TICs and grows them in 3 dimension thermoreversible hydrogels. Our method allows long-term culture (˜50 days,10 passages tested,accumulative ˜>10(10)-fold expansion) with both high growth rate (˜20-fold expansion/7 days) and high volumetric yield (˜2.0%A-%10(7)%cells/ml) without the loss of stemness. The scalable method can be used to produce sufficient,affordable glioblastoma TICs for drug discovery. View Publication

Low cell recovery rate of human embryonic stem cells (hESCs) resulting from cryopreservation damages leads to the difficulty in their successful commercialization of clinical applications. Hence in this study,sensitivity of human embryonic stem cells (hESCs) to different cooling rates,ice seeding and cryoprotective agent (CPA) types was compared and cell viability and recovery after cryopreservation under different cooling conditions were assessed. Both extracellular and intracellular ice formation were observed. Reactive oxidative species (ROS) accumulation of hESCs was determined. Cryopreservation of hESCs at 1 °C/min with the ice seeding and at the theoretically predicted optimal cooling rate (TPOCR) led to lower level of intracellular ROS,and prevented irregular and big ice clump formation compared with cryopreservation at 1 °C/min. This strategy further resulted in a significant increase in the hESC recovery when glycerol and 1,2-propanediol were used as the CPAs,but no increase for Me2SO. hESCs after cryopreservation under all the tested conditions still maintained their pluripotency. Our results provide guidance for improving the hESC cryopreservation recovery through the combination of CPA type,cooling rate and ice seeding. View Publication

Glioblastoma multiforme (GBM) is an aggressive,Grade IV astrocytoma with a poor survival rate,primarily due to the GBM tumor cells migrating away from the primary tumor site along the nanotopography of white matter tracts and blood vessels. It is unclear whether this nanotopography influences the biomechanical properties (i.e. cytoskeletal stiffness) of GBM tumor cells. Although GBM tumor cells have an innate propensity to migrate,we believe this capability is enhanced due to the influence of nanotopography on the tumor cells' biomechanical properties. In this study,we used an aligned nanofiber film that mimics the nanotopography in the tumor microenvironment to investigate the mechanical properties of GBM tumor cells in vitro. The data demonstrate that the cytoskeletal stiffness,cell traction stress,and focal adhesion area were significantly lower in the GBM tumor cells compared to healthy astrocytes. Moreover,the cytoskeletal stiffness was significantly reduced when cultured on aligned nanofiber films compared to smooth and randomly aligned nanofiber films. Gene expression analysis showed that tumor cells cultured on the aligned nanotopography upregulated key migratory genes and downregulated key proliferative genes. Therefore,our data suggest that the migratory potential is elevated when GBM tumor cells are migrating along aligned nanotopographical substrates. View Publication

Little is known about monocyte differentiation in the lung mucosal environment and about how the epithelium shapes monocyte function. We studied the role of the soluble component of bronchial epithelial cells (BECs) obtained under basal culture conditions in innate and adaptive monocyte responses. Monocytes cultured in bronchial epithelial cell-conditioned media (BEC-CM) specifically upregulate CD141,CD123,and DC-SIGN surface levels andFLT3expression,as well as the release of IL-1β,IL-6,and IL-10. BEC-conditioned monocytes stimulate naive T cells to produce IL-17 through IL-1β mechanism and also trigger IL-10 production by memory T cells. Furthermore,monocytes cultured in an inflammatory environment induced by the cytokines IL-6,IL-8,IL-1β,IL-15,TNF-α,and GM-CSF also upregulate CD123 and DC-SIGN expression. However,only inflammatory cytokines in the epithelial environment boost the expression of CD141. Interestingly,we identified a CD141/CD123/DC-SIGN triple positive population in the bronchoalveolar lavage fluid (BALF) from patients with different inflammatory conditions,demonstrating that this monocyte population existsin vivo. The frequency of this monocyte population was significantly increased in patients with sarcoidosis,suggesting a role in inflammatory mechanisms. Overall,these data highlight the specific role that the epithelium plays in shaping monocyte responses. Therefore,the unraveling of these mechanisms contributes to the understanding of the function that the epithelium may playin vivo. View Publication

Bio-engineered organs for transplantation may ultimately provide a personalized solution for end-stage organ failure,without the risk of rejection. Building upon the process of whole organ perfusion decellularization,we aimed to develop novel,translational methods for the recellularization and regeneration of transplantable lung constructs. We first isolated a proliferative KRT5+TP63+ basal epithelial stem cell population from human lung tissue and demonstrated expansion capacity in conventional 2D culture. We then repopulated acellular rat scaffolds in ex vivo whole organ culture and observed continued cell proliferation,in combination with primary pulmonary endothelial cells. To show clinical scalability,and to test the regenerative capacity of the basal cell population in a human context,we then recellularized and cultured isolated human lung scaffolds under biomimetic conditions. Analysis of the regenerated tissue constructs confirmed cell viability and sustained metabolic activity over 7 days of culture. Tissue analysis revealed extensive recellularization with organized tissue architecture and morphology,and preserved basal epithelial cell phenotype. The recellularized lung constructs displayed dynamic compliance and rudimentary gas exchange capacity. Our results underline the regenerative potential of patient-derived human airway stem cells in lung tissue engineering. We anticipate these advances to have clinically relevant implications for whole lung bioengineering and ex vivo organ repair. View Publication

Adverse effect of alcohol on neural function has been well documented. Especially,the teratogenic effect of alcohol on neurodevelopment during embryogenesis has been demonstrated in various models,which could be a pathologic basis for fetal alcohol spectrum disorders (FASDs). While the developmental defects from alcohol abuse during gestation have been described,the specific mechanisms by which alcohol mediates these injuries have yet to be determined. Recent studies have shown that alcohol has significant effect on molecular and cellular regulatory mechanisms in embryonic stem cell (ESC) differentiation including genes involved in neural development. To test our hypothesis that alcohol induces molecular alterations during neural differentiation we have derived neural precursor cells from pluripotent human ESCs in the presence or absence of ethanol treatment. Genome-wide transcriptomic profiling identified molecular alterations induced by ethanol exposure during neural differentiation of hESCs into neural rosettes and neural precursor cell populations. The Database for Annotation,Visualization and Integrated Discovery (DAVID) functional analysis on significantly altered genes showed potential ethanol's effect on JAK-STAT signaling pathway,neuroactive ligand-receptor interaction,Toll-like receptor (TLR) signaling pathway,cytokine-cytokine receptor interaction and regulation of autophagy. We have further quantitatively verified ethanol-induced alterations of selected candidate genes. Among verified genes we further examined the expression of P2RX3,which is associated with nociception,a peripheral pain response. We found ethanol significantly reduced the level of P2RX3 in undifferentiated hESCs,but induced the level of P2RX3 mRNA and protein in hESC-derived NPCs. Our result suggests ethanol-induced dysregulation of P2RX3 along with alterations in molecules involved in neural activity such as neuroactive ligand-receptor interaction may be a molecular event associated with alcohol-related peripheral neuropathy of an enhanced nociceptive response. View Publication

Patients with dyskeratosis congenita (DC),a disorder of telomere maintenance,suffer degeneration of multiple tissues. Patient-specific induced pluripotent stem (iPS) cells represent invaluable in vitro models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity,which is accompanied by induction of the telomerase reverse transcriptase gene (TERT). We investigated whether defects in telomerase function would limit derivation and maintenance of iPS cells from patients with DC. Here we show that reprogrammed DC cells overcome a critical limitation in telomerase RNA component (TERC) levels to restore telomere maintenance and self-renewal. We discovered that TERC upregulation is a feature of the pluripotent state,that several telomerase components are targeted by pluripotency-associated transcription factors,and that in autosomal dominant DC,transcriptional silencing accompanies a 3' deletion at the TERC locus. Our results demonstrate that reprogramming restores telomere elongation in DC cells despite genetic lesions affecting telomerase,and show that strategies to increase TERC expression may be therapeutically beneficial in DC patients. View Publication

The use of induced pluripotent stem cells (iPSCs) is an exciting frontier in the study and treatment of human diseases through the generation of specific cell types. Here we show the derivation of iPSCs from human nonmobilized peripheral blood (PB) and bone marrow (BM) mononuclear cells (MNCs) by retroviral transduction of OCT3/4,SOX2,KLF4,and c-MYC. The PB- and BM-derived iPSCs were quite similar to human embryonic stem cells with regard to morphology,expression of surface antigens and pluripotency-associated transcription factors,global gene expression profiles,and differentiation potential in vitro and in vivo. Infected PB and BM MNCs gave rise to iPSCs in the presence of several cytokines,although transduction efficiencies were not high. We found that 5 × 10(5) PB MNCs,which corresponds to less than 1 mL of PB,was enough for the generation of several iPSC colonies. Generation of iPSCs from MNCs of nonmobilized PB,with its relative efficiency and ease of harvesting,could enable the therapeutic use of patient-specific pluripotent stem cells. View Publication

Human neural progenitor cells provide a source for cell replacement therapy to treat neurodegenerative diseases. Therefore,there is great interest in mechanisms and tools to direct the fate of multipotent progenitor cells during their differentiation to increase the yield of a desired cell type. We tested small molecule inhibitors of glycogen synthase kinase-3 (GSK-3) for their functionality and their influence on neurogenesis using the human neural progenitor cell line ReNcell VM. Here we report the enhancement of neurogenesis of human neural progenitor cells by treatment with GSK-3 inhibitors. We tested different small molecule inhibitors of GSK-3 i.e. LiCl,sodium-valproate,kenpaullone,indirubin-3-monoxime and SB-216763 for their ability to inhibit GSK-3 in human neural progenitor cells. The highest in situ GSK-3 inhibitory effect of the drugs was found for kenpaullone and SB-216763. Accordingly,kenpaullone and SB-216763 were the only drugs tested in this study to stimulate the Wnt/β-catenin pathway that is antagonized by GSK-3. Analysis of human neural progenitor differentiation revealed an augmentation of neurogenesis by SB-216763 and kenpaullone,without changing cell cycle exit or cell survival. Small molecule inhibitors of GSK-3 enhance neurogenesis of human neural progenitor cells and may be used to direct the differentiation of neural stem and progenitor cells in therapeutic applications. View Publication

Imatinib mesylate has shown remarkable efficacy in the treatment of patients in the chronic phase of chronic myeloid leukemia. However,despite an overall significant hematological and cytogenetic response,imatinib therapy may favor the emergence of drug-resistant clones,ultimately leading to relapse. Some imatinib resistance mechanisms had not been fully elucidated yet. In this study we used sensitive and resistant sublines from a Bcr-Abl positive cell line to investigate the putative involvement of telomerase in the promotion of imatinib resistance. We showed that sensitivity to imatinib can be partly restored in imatinib-resistant cells by targeting telomerase expression,either by the introduction of a dominant-negative form of the catalytic protein subunit of the telomerase (hTERT) or by the treatment with all-trans-retinoic acid,a clinically used drug. Furthermore,we showed that hTERT overexpression favors the development of imatinib resistance through both its antiapoptotic and telomere maintenance functions. Therefore,combining antitelomerase strategies to imatinib treatment at the beginning of the treatment should be promoted to reduce the risk of imatinib resistance development and increase the probability of eradicating the disease. View Publication

Therapeutic and industrial applications of pluripotent stem cells and their derivatives require large cell quantities generated in defined conditions. To this end,we have translated single cell-inoculated suspension cultures of human pluripotent stem cells (hPSCs; including human induced pluripotent stem cells [hiPS] and human embryonic stem cells [hESC]) to stirred tank bioreactors. These systems that are widely used in biopharmaceutical industry allow straightforward scale up and detailed online monitoring of key process parameters. To ensure minimum medium consumption,but in parallel functional integration of all probes mandatory for process monitoring,that is,for pO₂ and pH,experiments were performed in 100 mL culture volume in a mini reactor platform" consisting of four independently controlled vessels. By establishing defined parameters for tightly controlled cell inoculation and aggregate formation up to 2×10�?� hiPSCs/100 mL were generated in a single process run in 7 days. Expression of pluripotency markers and ability of cells to differentiate into derivates of all three germ layers in vitro was maintained� View Publication