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

Sodium butyrate,at millimolar concentrations,when added to cell cultures produces many morphological and biochemical modifications in a reversible manner. Some of them occur in all cell lines. They concern regulatory mechanisms of gene expression and cell growth: an hyperacetylation of histone resulting from an inhibition of histone deacetylase and an arrest of cell proliferation are almost constantly observed. Some other modifications vary from one cell type to another: induction of proteins,including enzymes,hormones,hemoglobin,inhibition of cell differentiation,reversion of transformed characteristics of cells to normal morphological and biochemical pattern,increase in interferon antiviral efficiency and induction of integrated viruses. Most if not all these effects of butyrate could result from histone hyperacetylation,from changes in chromatin structures as measured by accessibility to DNases and from modifications in cytoskeleton assembly. We do not know at the present time whether butyrate acts on a very specific target site in cell or if it acts on several cell components. View Publication

Retinal ganglion cells (RGCs) are the projection neurons of the retina and transmit visual information to postsynaptic targets in the brain. While this function is shared among nearly all RGCs,this class of cell is remarkably diverse,comprised of multiple subtypes. Previous efforts have identified numerous RGC subtypes in animal models,but less attention has been paid to human RGCs. Thus,efforts of this study examined the diversity of RGCs differentiated from human pluripotent stem cells (hPSCs) and characterized defined subtypes through the expression of subtype-specific markers. Further investigation of these subtypes was achieved using single-cell transcriptomics,confirming the combinatorial expression of molecular markers associated with these subtypes,and also provided insight into more subtype-specific markers. Thus,the results of this study describe the derivation of RGC subtypes from hPSCs and will support the future exploration of phenotypic and functional diversity within human RGCs. View Publication

There is increasing evidence that breast cancers contain tumor-initiating cells with stem cell properties. The importance of estrogen in the development of the mammary gland and in breast cancer is well known,but the influence of estrogen on the stem cell population has not been assessed. We show that estrogen reduces the proportion of stem cells in the normal human mammary gland and in breast cancer cells. The embryonic stem cell genes NANOG,OCT4,and SOX2 are expressed in normal breast stem cells and at higher levels in breast tumor cells and their expression decreases upon differentiation. Overexpression of each stem cell gene reduces estrogen receptor (ER) expression,and increases the number of stem cells and their capacity for invasion,properties associated with tumorigenesis and poor prognosis. These results indicate that estrogen reduces the size of the human breast stem cell pool and may provide an explanation for the better prognosis of ER-positive tumors. View Publication

Recent studies have demonstrated that cancer stem cells play an important role in the pathobiology of head and neck squamous cell carcinomas (HNSCC). However,little is known about functional interactions between head and neck cancer stem-like cells (CSC) and surrounding stromal cells. Here,we used aldehyde dehydrogenase activity and CD44 expression to sort putative stem cells from primary human HNSCC. Implantation of 1,000 CSC (ALDH+CD44+Lin-) led to tumors in 13 (out of 15) mice,whereas 10,000 noncancer stem cells (ALDH-CD44-Lin-) resulted in 2 tumors in 15 mice. These data demonstrated that ALDH and CD44 select a subpopulation of cells that are highly tumorigenic. The ability to self-renew was confirmed by the observation that ALDH+CD44+Lin- cells sorted from human HNSCC formed more spheroids (orospheres) in 3-D agarose matrices or ultra-low attachment plates than controls and were serially passaged in vivo. We observed that approximately 80% of the CSC were located in close proximity (within 100-μm radius) of blood vessels in human tumors,suggesting the existence of perivascular niches in HNSCC. In vitro studies demonstrated that endothelial cell-secreted factors promoted self-renewal of CSC,as demonstrated by the upregulation of Bmi-1 expression and the increase in the number of orospheres as compared with controls. Notably,selective ablation of tumor-associated endothelial cells stably transduced with a caspase-based artificial death switch (iCaspase-9) caused a marked reduction in the fraction of CSC in xenograft tumors. Collectively,these findings indicate that endothelial cell-initiated signaling can enhance the survival and self-renewal of head and neck CSC. View Publication

Studies in embryonic development have guided successful efforts to direct the differentiation of human embryonic and induced pluripotent stem cells (PSCs) into specific organ cell types in vitro. For example,human PSCs have been differentiated into monolayer cultures of liver hepatocytes and pancreatic endocrine cells that have therapeutic efficacy in animal models of liver disease and diabetes,respectively. However,the generation of complex three-dimensional organ tissues in vitro remains a major challenge for translational studies. Here we establish a robust and efficient process to direct the differentiation of human PSCs into intestinal tissue in vitro using a temporal series of growth factor manipulations to mimic embryonic intestinal development. This involved activin-induced definitive endoderm formation,FGF/Wnt-induced posterior endoderm pattering,hindgut specification and morphogenesis,and a pro-intestinal culture system to promote intestinal growth,morphogenesis and cytodifferentiation. The resulting three-dimensional intestinal 'organoids' consisted of a polarized,columnar epithelium that was patterned into villus-like structures and crypt-like proliferative zones that expressed intestinal stem cell markers. The epithelium contained functional enterocytes,as well as goblet,Paneth and enteroendocrine cells. Using this culture system as a model to study human intestinal development,we identified that the combined activity of WNT3A and FGF4 is required for hindgut specification whereas FGF4 alone is sufficient to promote hindgut morphogenesis. Our data indicate that human intestinal stem cells form de novo during development. We also determined that NEUROG3,a pro-endocrine transcription factor that is mutated in enteric anendocrinosis,is both necessary and sufficient for human enteroendocrine cell development in vitro. PSC-derived human intestinal tissue should allow for unprecedented studies of human intestinal development and disease. View Publication

Many human diseases share a developmental origin that manifests during childhood or maturity. Aneuploid syndromes are caused by supernumerary or reduced number of chromosomes and represent an extreme example of developmental disease,as they have devastating consequences before and after birth. Investigating how alterations in gene dosage drive these conditions is relevant because it might help treat some clinical aspects. It may also provide explanations as to how quantitative differences in gene expression determine phenotypic diversity and disease susceptibility among natural populations. Here,we aimed to produce induced pluripotent stem cell (iPSC) lines that can be used to improve our understanding of aneuploid syndromes. We have generated iPSCs from monosomy X [Turner syndrome (TS)],trisomy 8 (Warkany syndrome 2),trisomy 13 (Patau syndrome) and partial trisomy 11;22 (Emanuel syndrome),using either skin fibroblasts from affected individuals or amniocytes from antenatal diagnostic tests. These cell lines stably maintain the karyotype of the donors and behave like embryonic stem cells in all tested assays. TS iPSCs were used for further studies including global gene expression analysis and tissue-specific directed differentiation. Multiple clones displayed lower levels of the pseudoautosomal genes ASMTL and PPP2R3B than the controls. Moreover,they could be transformed into neural-like,hepatocyte-like and heart-like cells,but displayed insufficient up-regulation of the pseudoautosomal placental gene CSF2RA during embryoid body formation. These data support that abnormal organogenesis and early lethality in TS are not caused by a tissue-specific differentiation blockade,but rather involves other abnormalities including impaired placentation. View Publication

New drugs for preserving and restoring pancreatic β-cell function are critically needed for the worldwide epidemic of type 2 diabetes and the cure for type 1 diabetes. We previously identified a family of neurogenic 3,5-disubstituted isoxazoles (Isx) that increased expression of neurogenic differentiation 1 (NeuroD1,also known as BETA2); this transcription factor functions in neuronal and pancreatic β-cell differentiation and is essential for insulin gene transcription. Here,we probed effects of Isx on human cadaveric islets and MIN6 pancreatic β cells. Isx increased the expression and secretion of insulin in islets that made little insulin after prolonged ex vivo culture and increased expression of neurogenic differentiation 1 and other regulators of islet differentiation and insulin gene transcription. Within the first few hours of exposure,Isx caused biphasic activation of ERK1/2 and increased bulk histone acetylation. Although there was little effect on histone deacetylase activity,Isx increased histone acetyl transferase activity in nuclear extracts. Reconstitution assays indicated that Isx increased the activity of the histone acetyl transferase p300 through an ERK1/2-dependent mechanism. In summary,we have identified a small molecule with antidiabetic activity,providing a tool for exploring islet function and a possible lead for therapeutic intervention in diabetes. View Publication

Oligodendrocytes-the myelin-forming cells of the central nervous system-can be regenerated during adulthood. In adults,new oligodendrocytes originate from oligodendrocyte progenitor cells (OPCs),but also from neural stem cells (NSCs). Although several factors supporting oligodendrocyte production have been characterized,the mechanisms underlying the generation of adult oligodendrocytes are largely unknown. Here we show that genetic inactivation of SIRT1,a protein deacetylase implicated in energy metabolism,increases the production of new OPCs in the adult mouse brain,in part by acting in NSCs. New OPCs produced following SIRT1 inactivation differentiate normally,generating fully myelinating oligodendrocytes. Remarkably,SIRT1 inactivation ameliorates remyelination and delays paralysis in mouse models of demyelinating injuries. SIRT1 inactivation leads to the upregulation of genes involved in cell metabolism and growth factor signalling,in particular PDGF receptor α (PDGFRα). Oligodendrocyte expansion following SIRT1 inactivation is mediated at least in part by AKT and p38 MAPK-signalling molecules downstream of PDGFRα. The identification of drug-targetable enzymes that regulate oligodendrocyte regeneration in adults could facilitate the development of therapies for demyelinating injuries and diseases,such as multiple sclerosis. View Publication

It has been suggested that the isolation of scalable populations of limbal stem cells may lead to radical changes in ocular therapy. In particular,the derivation and transplantation of corneal stem cells from these populations may result in therapies providing clinical normality of the diseased or damaged cornea. Although feasible in theory,the lack of donor material in sufficient quantity and quality currently limits such a strategy. A potential scalable source of corneal cells could be derived from pluripotent stem cells (PSCs). We developed an in vitro and serum-free corneal differentiation model which displays significant promise. Our stepwise differentiation model was designed with reference to development and gave rise to cells which displayed similarities to epithelial progenitor cells which can be specified to cells displaying a corneal epithelial phenotype. We believe our approach is novel,provides a robust model of human development and in the future,may facilitate the generation of corneal epithelial cells that are suitable for clinical use. Additionally,we demonstrate that following continued cell culture,stem cell-derived corneal epithelial cells undergo transdifferentiation and exhibit squamous metaplasia and therefore,also offer an in vitro model of disease. View Publication

BACKGROUND: Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been successfully used to knock out endogenous genes in stem cell research. However,the deficiencies of current gene-based delivery systems may hamper the clinical application of these nucleases. A new delivery method that can improve the utility of these nucleases is needed.backslashnbackslashnRESULTS: In this study,we utilized a cell-penetrating peptide-based system for ZFN and TALEN delivery. Functional TAT-ZFN and TAT-TALEN proteins were generated by fusing the cell-penetrating TAT peptide to ZFN and TALEN,respectively. However,TAT-ZFN was difficult to purify in quantities sufficient for analysis in cell culture. Purified TAT-TALEN was able to penetrate cells and disrupt the gene encoding endogenous human chemokine (C-C motif) receptor 5 (CCR5,a co-receptor for HIV-1 entry into cells). Hypothermic treatment greatly enhanced the TAT-TALEN-mediated gene disruption efficiency. A 5% modification rate was observed in human induced pluripotent stem cells (hiPSCs) treated with TAT-TALEN as measured by the Surveyor assay.backslashnbackslashnCONCLUSIONS: TAT-TALEN protein-mediated gene disruption was applicable in hiPSCs and represents a promising technique for gene knockout in stem cells. This new technique may advance the clinical application of TALEN technology. View Publication

Alcohol consumption has long been associated with a majority of liver diseases and has been found to influence both fetal and adult liver functions. In spite of being one of the major causes of morbidity and mortality in the world,currently,there are no effective strategies that can prevent or treat alcoholic liver disease (ALD),due to a lack of human-relevant research models. Recent success in generation of functionally active mature hepatocyte-like cells from human-induced pluripotent cells (iPSCs) enables us to better understand the effects of alcohol on liver functions. Here,we describe the method and effect of alcohol exposure on multistage hepatic cell types derived from human iPSCs,in an attempt to recapitulate the early stages of liver tissue injury associated with ALD. We exposed different stages of iPSC-induced hepatic cells to ethanol at a pathophysiological concentration. In addition to stage-specific molecular markers,we measured several key cellular parameters of hepatocyte injury,including apoptosis,proliferation,and lipid accumulation. View Publication

The derivation of astrocytes from human pluripotent stem cells is currently slow and inefficient. We demonstrate that overexpression of the transcription factors SOX9 and NFIB in human pluripotent stem cells rapidly and efficiently yields homogeneous populations of induced astrocytes. In our study these cells exhibited molecular and functional properties resembling those of adult human astrocytes and were deemed suitable for disease modeling. Our method provides new possibilities for the study of human astrocytes in health and disease. View Publication