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

As multiple sclerosis research progresses,it is pertinent to continue to develop suitable paradigms to allow for ever more sophisticated investigations. Animal models of multiple sclerosis,despite their continuing contributions to the field,may not be the most prudent for every experiment. Indeed,such may be either insufficient to reflect the functional impact of human genetic variations or unsuitable for drug screenings. Thus,we have established a cell- and patient-specific paradigm to provide an in vitro model within which to perform future genetic investigations. Renal proximal tubule epithelial cells were isolated from multiple sclerosis patients' urine and transfected with pluripotency-inducing episomal factors. Subsequent induced pluripotent stem cells were formed into embryoid bodies selective for ectodermal lineage,resulting in neural tube-like rosettes and eventually neural progenitor cells. Differentiation of these precursors into primary neurons was achieved through a regimen of neurotrophic and other factors. These patient-specific primary neurons displayed typical morphology and functionality,also staining positive for mature neuronal markers. The development of such a non-invasive procedure devoid of permanent genetic manipulation during the course of differentiation,in the context of multiple sclerosis,provides an avenue for studies with a greater cell- and human-specific focus,specifically in the context of genetic contributions to neurodegeneration and drug discovery. View Publication

Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here,we report that short-term expression of two components,NANOG and KLF2,is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling,are phenotypically stable,and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors,TFCP2L1 or KLF4,has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells. View Publication

This paper proposes a bio-driven algorithm that detects cell regions automatically in the human embryonic stem cell (hESC) images obtained using a phase contrast microscope. The algorithm uses both statistical intensity distributions of foreground/hESCs and background/substrate as well as cell property for cell region detection. The intensity distributions of foreground/hESCs and background/substrate are modeled as a mixture of two Gaussians. The cell property is translated into local spatial information. The algorithm is optimized by parameters of the modeled distributions and cell regions evolve with the local cell property. The paper validates the method with various videos acquired using different microscope objectives. In comparison with the state-of-the-art methods,the proposed method is able to detect the entire cell region instead of fragmented cell regions. It also yields high marks on measures such as Jacard similarity,Dice coefficient,sensitivity and specificity. Automated detection by the proposed method has the potential to enable fast quantifiable analysis of hESCs using large data sets which are needed to understand dynamic cell behaviors. View Publication

Generation of induced dopaminergic (iDA) neurons may provide a significant step forward towards cell replacement therapy for Parkinson's disease (PD). To study and compare transcriptional programs of induced cells versus primary DA neurons is a preliminary step towards characterizing human iDA neurons. We have optimized a protocol to efficiently generate iDA neurons from human pluripotent stem cells (hPSCs). We then sequenced the transcriptomes of iDA neurons derived from 6 different hPSC lines and compared them to that of primary midbrain (mDA) neurons. We identified a small subset of genes with altered expression in derived iDA neurons from patients with Parkinson's Disease (PD). We also observed that iDA neurons differ significantly from primary mDA neurons in global gene expression,especially in genes related to neuron maturation level. Results suggest iDA neurons from patient iPSCs could be useful for basic and translational studies,including in vitro modeling of PD. However,further refinement of methods of induction and maturation of neurons may better recapitulate full development of mDA neurons from hPSCs. View Publication

Nature Methods 8,293 (2011). doi:10.1038/nmeth0411-293 View Publication

DNA cytosine methylation is a central epigenetic modification that has essential roles in cellular processes including genome regulation,development and disease. Here we present the first genome-wide,single-base-resolution maps of methylated cytosines in a mammalian genome,from both human embryonic stem cells and fetal fibroblasts,along with comparative analysis of messenger RNA and small RNA components of the transcriptome,several histone modifications,and sites of DNA-protein interaction for several key regulatory factors. Widespread differences were identified in the composition and patterning of cytosine methylation between the two genomes. Nearly one-quarter of all methylation identified in embryonic stem cells was in a non-CG context,suggesting that embryonic stem cells may use different methylation mechanisms to affect gene regulation. Methylation in non-CG contexts showed enrichment in gene bodies and depletion in protein binding sites and enhancers. Non-CG methylation disappeared upon induced differentiation of the embryonic stem cells,and was restored in induced pluripotent stem cells. We identified hundreds of differentially methylated regions proximal to genes involved in pluripotency and differentiation,and widespread reduced methylation levels in fibroblasts associated with lower transcriptional activity. These reference epigenomes provide a foundation for future studies exploring this key epigenetic modification in human disease and development. View Publication

Differentiated neurons can be rapidly acquired,within days,by inducing stem cells to express neurogenic transcription factors. We developed a protocol to maintain long-term cultures of human neurons,called iNGNs,which are obtained by inducing Neurogenin-1 and Neurogenin-2 expression in induced pluripotent stem cells. We followed the functional development of iNGNs over months and they showed many hallmark properties for neuronal maturation,including robust electrical and synaptic activity. Using iNGNs expressing a variant of channelrhodopsin-2,called CatCh,we could control iNGN activity with blue light stimulation. In combination with optogenetic tools,iNGNs offer opportunities for studies that require precise spatial and temporal resolution. iNGNs developed spontaneous network activity,and these networks had excitatory glutamatergic synapses,which we characterized with single-cell synaptic recordings. AMPA glutamatergic receptor activity was especially dominant in postsynaptic recordings,whereas NMDA glutamatergic receptor activity was absent from postsynaptic recordings but present in extrasynaptic recordings. Our results on long-term cultures of iNGNs could help in future studies elucidating mechanisms of human synaptogenesis and neurotransmission,along with the ability to scale-up the size of the cultures. View Publication

Three different label-free,minimally invasive,live single cell analysis techniques were used to characterize embryonic stem cells,and the hepatocytes into which they were differentiated. Atomic Force Microscopy measures the cell's mechanical properties,Raman spectroscopy measures its chemical properties,and dielectrophoresis measures the membrane's capacitance. We were able to assign cell type of individual cells with accuracies of 96.5% (Atomic Force Microscopy),92.5 % (Raman spectroscopy),and *** % (Dielectrophoresis). These techniques,used either independently or in combination,offer label-free methods to study individual living cells. Although they can be applied to any phenotypical or environmental change,these techniques have most potential in human cell therapies where the use of biomarkers is best avoided. If all three properties are independent,then a combined accuracy of *** % can be achieved in cell characterization. We suggest how these methods could be combined into one microfluidic chip for cell sorting,and how they can be applied to cell culture. View Publication

During development,neural crest (NC) cells are induced by signaling events at the neural plate border of all vertebrate embryos. Initially arising within the central nervous system,NC cells subsequently undergo an epithelial to mesenchymal transition to migrate into the periphery,where they differentiate into diverse cell types. Here we provide evidence that postnatal human epidermal keratinocytes (KC),in response to fibroblast growth factor 2 and insulin like growth factor 1 signals,can be reprogrammed toward a NC fate. Genome-wide transcriptome analyses show that keratinocyte-derived NC cells are similar to those derived from human embryonic stem cells. Moreover,they give rise in vitro and in vivo to NC derivatives such as peripheral neurons,melanocytes,Schwann cells and mesenchymal cells (osteocytes,chondrocytes,adipocytes,and smooth muscle cells). By demonstrating that human keratin-14+ KC can form NC cells,even from clones of single cells,our results have important implications in stem cell biology and regenerative medicine. Stem Cells 2017. View Publication

In vitro models of the developing brain such as three-dimensional brain organoids offer an unprecedented opportunity to study aspects of human brain development and disease. However,the cells generated within organoids and the extent to which they recapitulate the regional complexity,cellular diversity and circuit functionality of the brain remain undefined. Here we analyse gene expression in over 80,000 individual cells isolated from 31 human brain organoids. We find that organoids can generate a broad diversity of cells,which are related to endogenous classes,including cells from the cerebral cortex and the retina. Organoids could be developed over extended periods (more than 9 months),allowing for the establishment of relatively mature features,including the formation of dendritic spines and spontaneously active neuronal networks. Finally,neuronal activity within organoids could be controlled using light stimulation of photosensitive cells,which may offer a way to probe the functionality of human neuronal circuits using physiological sensory stimuli. View Publication