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

The longevity of organisms is maintained by stem cells. If an organism loses the ability to maintain a balance between quiescence and differentiation in the stem/progenitor cell compartment due to aging and/or stress,this may result in death or age-associated diseases,including cancer. Ewing sarcoma is the most lethal bone tumor in young patients and arises from primitive stem cells. Here,we demonstrated that endogenous Ewing sarcoma gene (Ews) is indispensable for stem cell quiescence,and that the ablation of Ews promotes the early onset of senescence in hematopoietic stem progenitor cells. The phenotypic and functional changes in Ews-deficient stem cells were accompanied by an increase in senescence-associated β-galactosidase staining and a marked induction of p16(INK4a) compared with wild-type counterparts. With its relevance to cancer and possibly aging,EWS is likely to play a significant role in maintaining the functional capacity of stem cells and may provide further insight into the complexity of Ewing sarcoma in the context of stem cells. View Publication

Amniogenesis-the development of amnion-is a critical developmental milestone for early human embryogenesis and successful pregnancy. However,human amniogenesis is poorly understood due to limited accessibility to peri-implantation embryos and a lack of in vitro models. Here we report an efficient biomaterial system to generate human amnion-like tissue in vitro through self-organized development of human pluripotent stem cells (hPSCs) in a bioengineered niche mimicking the in vivo implantation environment. We show that biophysical niche factors act as a switch to toggle hPSC self-renewal versus amniogenesis under self-renewal-permissive biochemical conditions. We identify a unique molecular signature of hPSC-derived amnion-like cells and show that endogenously activated BMP-SMAD signalling is required for the amnion-like tissue development by hPSCs. This study unveils the self-organizing and mechanosensitive nature of human amniogenesis and establishes the first hPSC-based model for investigating peri-implantation human amnion development,thereby helping advance human embryology and reproductive medicine. View Publication

Platelets are an abundant source of CD40 ligand (CD154),an immunomodulatory and proinflammatory molecule implicated in the onset and progression of several inflammatory diseases,including systemic lupus erythematosus (SLE),diabetes,and cardiovascular disease. Heretofore considered largely restricted to activated T cells,we initiated studies to investigate the source and regulation of platelet-associated CD154. We found that CD154 is abundantly expressed in platelet precursor cells,megakaryocytes. We show that CD154 is expressed in primary human CD34+ and murine hematopoietic precursor cells only after cytokine-driven megakaryocyte differentiation. Furthermore,using several established megakaryocyte-like cells lines,we performed promoter analysis of the CD154 gene and found that NFAT,a calcium-dependent transcriptional regulator associated with activated T cells,mediated both differentiation-dependent and inducible megakaryocyte-specific CD154 expression. Overall,these data represent the first investigation of the regulation of a novel source of CD154 and suggests that platelet-associated CD154 can be biochemically modulated. View Publication

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) eliminates many epigenetic modifications that characterize differentiated cells. In this study,we tested whether functional differences between chronic obstructive pulmonary disease (COPD) and non-COPD fibroblasts could be reduced utilizing this approach. Primary fibroblasts from non-COPD and COPD patients were reprogrammed to iPSCs. Reprogrammed iPSCs were positive for oct3/4,nanog,and sox2,formed embryoid bodies in vitro,and induced teratomas in nonobese diabetic/severe combined immunodeficient mice. Reprogrammed iPSCs were then differentiated into fibroblasts (non-COPD-i and COPD-i) and were assessed either functionally by chemotaxis and gel contraction or for gene expression by microarrays and compared with their corresponding primary fibroblasts. Primary COPD fibroblasts contracted three-dimensional collagen gels and migrated toward fibronectin less robustly than non-COPD fibroblasts. In contrast,redifferentiated fibroblasts from iPSCs derived from the non-COPD and COPD fibroblasts were similar in response in both functional assays. Microarray analysis identified 1,881 genes that were differentially expressed between primary COPD and non-COPD fibroblasts,with 605 genes differing by more than twofold. After redifferentiation,112 genes were differentially expressed between COPD-i and non-COPD-i with only three genes by more than twofold. Similar findings were observed with microRNA (miRNA) expression: 56 miRNAs were differentially expressed between non-COPD and COPD primary cells; after redifferentiation,only 3 miRNAs were differentially expressed between non-COPD-i and COPD-i fibroblasts. Interestingly,of the 605 genes that were differentially expressed between COPD and non-COPD fibroblasts,293 genes were changed toward control after redifferentiation. In conclusion,functional and epigenetic alterations of COPD fibroblasts can be reprogrammed through formation of iPSCs. View Publication

Induced pluripotent stem cells (IPS) are an artificially derived type of pluripotent stem cell,showing many of the same characteristics as natural pluripotent stem cells. IPS are a hopeful therapeutic model; however there is a critical need to determine their response to environmental toxins. Effects of arsenic on cells have been studied extensively; however,its effect on IPS is yet to be elucidated. Arsenic trioxide (ATO) has been shown to inhibit cell proliferation,induce apoptosis and genotoxicity in many cells. Based on ATOs action in other cells,we hypothesize that it will induce alterations in morphology,inhibit cell viability and induce a genotoxic effect on IPS. Cells were treated for 24 hours with ATO (0-9 µg/mL). Cell morphology,viability and DNA damage were documented. Results indicated sufficient changes in morphology of cell colonies mainly in cell ability to maintain grouping and ability to remain adherent. Cell viability decreased in a dose dependent manner. There were significant increases in tail length and moment as well as destruction of intact DNA as concentration increased. Exposure to ATO resulted in a reproducible dose dependent sequence of events marked by changes in morphology,decrease of cell viability,and induction of genotoxicity in IPS. View Publication

The molecular mechanism responsible for a decline of stem cell functioning after replicative stress remains unknown. We used mouse embryonic fibroblasts (MEFs) and hematopoietic stem cells (HSCs) to identify genes involved in the process of cellular aging. In proliferating and senescent MEFs one of the most differentially expressed transcripts was Enhancer of zeste homolog 2 (Ezh2),a Polycomb group protein (PcG) involved in histone methylation and deacetylation. Retroviral overexpression of Ezh2 in MEFs resulted in bypassing of the senescence program. More importantly,whereas normal HSCs were rapidly exhausted after serial transplantations,overexpression of Ezh2 completely conserved long-term repopulating potential. Animals that were reconstituted with 3 times serially transplanted control bone marrow cells all died due to hematopoietic failure. In contrast,similarly transplanted Ezh2-overexpressing stem cells restored stem cell quality to normal levels. In a genetic genomics" screen� View Publication

Haematopoietic stem cells (HSCs) regenerate blood cells throughout the lifespan of an organism. With age,the functional quality of HSCs declines,partly owing to the accumulation of damaged DNA. However,the factors that damage DNA and the protective mechanisms that operate in these cells are poorly understood. We have recently shown that the Fanconi anaemia DNA-repair pathway counteracts the genotoxic effects of reactive aldehydes. Mice with combined inactivation of aldehyde catabolism (through Aldh2 knockout) and the Fanconi anaemia DNA-repair pathway (Fancd2 knockout) display developmental defects,a predisposition to leukaemia,and are susceptible to the toxic effects of ethanol-an exogenous source of acetaldehyde. Here we report that aged Aldh2(-/-) Fancd2(-/-) mutant mice that do not develop leukaemia spontaneously develop aplastic anaemia,with the concomitant accumulation of damaged DNA within the haematopoietic stem and progenitor cell (HSPC) pool. Unexpectedly,we find that only HSPCs,and not more mature blood precursors,require Aldh2 for protection against acetaldehyde toxicity. Additionally,the aldehyde-oxidizing activity of HSPCs,as measured by Aldefluor stain,is due to Aldh2 and correlates with this protection. Finally,there is more than a 600-fold reduction in the HSC pool of mice deficient in both Fanconi anaemia pathway-mediated DNA repair and acetaldehyde detoxification. Therefore,the emergence of bone marrow failure in Fanconi anaemia is probably due to aldehyde-mediated genotoxicity restricted to the HSPC pool. These findings identify a new link between endogenous reactive metabolites and DNA damage in HSCs,and define the protective mechanisms that counteract this threat. View Publication

There is a foundational need for quality control tools in stem cell laboratories engaged in basic research,regenerative therapies,and toxicological studies. These tools require automated methods for evaluating cell processes and quality during in vitro passaging,expansion,maintenance,and differentiation. In this paper,an unbiased,automated high-content profiling toolkit,StemCellQC,is presented that non-invasively extracts information on cell quality and cellular processes from time-lapse phase-contrast videos. Twenty four (24) morphological and dynamic features were analyzed in healthy,unhealthy,and dying human embryonic stem cell (hESC) colonies to identify those features that were affected in each group. Multiple features differed in the healthy versus unhealthy/dying groups,and these features were linked to growth,motility,and death. Biomarkers were discovered that predicted cell processes before they were detectable by manual observation. StemCellQC distinguished healthy and unhealthy/dying hESC colonies with 96% accuracy by non-invasively measuring and tracking dynamic and morphological features over 48 hours. Changes in cellular processes can be monitored by StemCellQC and predictions can be made about the quality of pluripotent stem cell colonies. This toolkit reduced the time and resources required to track multiple pluripotent stem cell colonies and eliminated handling errors and false classifications due to human bias. StemCellQC provided both user-specified and classifier-determined analysis in cases where the affected features are not intuitive or anticipated. Video analysis algorithms allowed assessment of biological phenomena using automatic detection analysis,which can aid facilities where maintaining stem cell quality and/or monitoring changes in cellular processes are essential. In the future StemCellQC can be expanded to include other features,cell types,treatments,and differentiating cells. View Publication

BACKGROUND Efficient slow freezing protocols within serum-free and feeder-free culture systems are crucial for the clinical application of human embryonic stem (hES) cells. Frequently,however,hES cells must be cryopreserved as clumps when using conventional slow freezing protocols,leading to lower survival rates during freeze-thaw and limiting their recovery and growth efficiency after thawing,as well as limiting downstream applications that require single cell suspensions. We describe a novel method to increase freeze-thaw survival and proliferation rate of single hES cells in serum-free and feeder-free culture conditions. METHODS hES cells maintained on Matrigel-coated dishes were dissociated into single cells with Accutase and slow freezing. After thawing at 37 degrees C,cells were cultured in mTeSR medium supplemented with 10 microM of Rho-associated kinase inhibitor Y-27632 for 1 day. RESULTS The use of Y-27632 and Accutase significantly increases the survival of single hES cells after thawing compared with a control group (P textless 0.01). Furthermore,by treatment of hES cell aggregates with EGTA to disrupt cell-cell interaction,we show that Y-27632 treatment does not directly affect hES cell apoptosis. Even in the presence of Y-27632,hES cells deficient in cell-cell interaction undergo apoptosis. Y-27632-treated freeze-thawed hES cells retain typical morphology,stable karyotype,expression of pluripotency markers and the potential to differentiate into derivatives of all three germ layers after long-term culture. CONCLUSIONS The method described here allows for cryopreservation of single hES cells in serum-free and feeder-free conditions and therefore we believe this method will be ideal for current and future hES cell applications that are targeted towards a therapeutic end-point. View Publication

Mesenchymal stem cells (MSC) have been extensively studied and gained wide popularity due to their therapeutic potential. Spontaneous transformation of MSC,from both human and murine origin,has been reported in many studies. MSC transformation depends on the culture conditions,the origin of the cells and the time on culture; however,the precise biological characteristics involved in this process have not been fully defined yet. In this study,we investigated the role of p53 in the biology and transformation of murine bone marrow (BM)-derived MSC. We demonstrate that the MSC derived from p53KO mice showed an augmented proliferation rate,a shorter doubling time and also morphologic and phenotypic changes,as compared to MSC derived from wild-type animals. Furthermore,the MSC devoid of p53 had an increased number of cells able to generate colonies. In addition,not only proliferation but also MSC differentiation is controlled by p53 since its absence modifies the speed of the process. Moreover,genomic instability,changes in the expression of c-myc and anchorage independent growth were also observed in p53KO MSC. In addition,the absence of p53 implicates the spontaneous transformation of MSC in long-term cultures. Our results reveal that p53 plays a central role in the biology of MSC. View Publication

The identification of the trans-acting factors and cis-regulatory modules that are involved in human pluripotent stem cell (hPSC) maintenance and differentiation is necessary to dissect the operating regulatory networks in these processes and thereby identify nodes where signal input will direct desired cell fate decisions in vitro or in vivo. To deconvolute these networks,we established a method to influence the differentiation state of hPSCs with a CRISPR-associated catalytically inactive dCas9 fused to an effector domain. In human embryonic stem cells,we find that the dCas9 effectors can exert positive or negative regulation on the expression of developmentally relevant genes,which can influence cell differentiation status when impinging on a key node in the regulatory network that governs the cell state. This system provides a platform for the interrogation of the underlying regulators governing specific differentiation decisions,which can then be employed to direct cellular differentiation down desired pathways. View Publication

The development of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) facilitates in vitro studies of human disease mechanisms,speeds up the process of drug screening,and raises the feasibility of using cell replacement therapy in clinics. However,the study of genotype-phenotype relationships in ESCs or iPSCs is hampered by the low efficiency of site-specific gene editing. Transcription activator-like effector nucleases (TALENs) spurred interest due to the ease of assembly,high efficiency and faithful gene targeting. In this study,we optimized the TALEN design to maximize its genomic cutting efficiency. We showed that using optimized TALENs in conjunction with single-strand oligodeoxynucleotide (ssODN) allowed efficient gene editing in human cells. Gene mutations and gene deletions for up to 7.8 kb can be accomplished at high efficiencies. We established human tumor cell lines and H9 ESC lines with homozygous deletion of the microRNA-21 (miR-21) gene and miR-9-2 gene. These cell lines provide a robust platform to dissect the roles these genes play during cell differentiation and tumorigenesis. We also observed that the endogenous homologous chromosome can serve as a donor template for gene editing. Overall,our studies demonstrate the versatility of using ssODN and TALEN to establish genetically modified cells for research and therapeutic application. View Publication