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

Cryopreservation is the use of low temperatures to preserve structurally intact living cells. The cells that survive the thermodynamic journey from the 37 °C incubator to the -196 °C liquid nitrogen storage tank are free from the influences of time. Thus,cryopreservation is a critical component of cell culture and cell manufacturing protocols. Successful cryopreservation of human cells requires that the cells be derived from patient samples that are collected in a standardized manner,and carefully handled from blood draw through cell isolation. Furthermore,proper equipment must be in place to ensure consistency,reproducibility,and sterility. In addition,the correct choice and amount of cryoprotectant agent must be added at the correct temperature,and a controlled rate of freezing (most commonly 1 °C/min) must be applied prior to a standardized method of cryogenic storage. This appendix describes how human primary cells can be frozen for long-term storage and thawed for growth in a tissue culture vessel. View Publication

Neural cultures derived from Huntington's disease (HD) patient-derived induced pluripotent stem cells were used for 'omics' analyses to identify mechanisms underlying neurodegeneration. RNA-seq analysis identified genes in glutamate and GABA signaling,axonal guidance and calcium influx whose expression was decreased in HD cultures. One-third of gene changes were in pathways regulating neuronal development and maturation. When mapped to stages of mouse striatal development,the profiles aligned with earlier embryonic stages of neuronal differentiation. We observed a strong correlation between HD-related histone marks,gene expression and unique peak profiles associated with dysregulated genes,suggesting a coordinated epigenetic program. Treatment with isoxazole-9,which targets key dysregulated pathways,led to amelioration of expanded polyglutamine repeat-associated phenotypes in neural cells and of cognitive impairment and synaptic pathology in HD model R6/2 mice. These data suggest that mutant huntingtin impairs neurodevelopmental pathways that could disrupt synaptic homeostasis and increase vulnerability to the pathologic consequence of expanded polyglutamine repeats over time. View Publication

The epicardium contributes both multi-lineage descendants and paracrine factors to the heart during cardiogenesis and cardiac repair,underscoring its potential for cardiac regenerative medicine. Yet little is known about the cellular and molecular mechanisms that regulate human epicardial development and regeneration. Here,we show that the temporal modulation of canonical Wnt signaling is sufficient for epicardial induction from 6 different human pluripotent stem cell (hPSC) lines,including a WT1-2A-eGFP knock-in reporter line,under chemically-defined,xeno-free conditions. We also show that treatment with transforming growth factor beta (TGF-β)-signalling inhibitors permitted long-term expansion of the hPSC-derived epicardial cells,resulting in a more than 25 population doublings of WT1+ cells in homogenous monolayers. The hPSC-derived epicardial cells were similar to primary epicardial cells both in vitro and in vivo,as determined by morphological and functional assays,including RNA-seq. Our findings have implications for the understanding of self-renewal mechanisms of the epicardium and for epicardial regeneration using cellular or small-molecule therapies. View Publication

Neural crest (NC) cells contribute to the development of many complex tissues of all three germ layers during embryogenesis,and its abnormal development accounts for several congenital birth defects. Generating NC cells-including specific subpopulations such as cranial,cardiac,and trunk NC cells-from human pluripotent stem cells will provide a valuable model system to study human development and disease. Here,we describe a rapid and robust NC differentiation method called LSB-short" that is based on dual SMAD pathway inhibition. This protocol yields high percentages of NC cell populations from multiple human induced pluripotent stem and human embryonic stem cell lines in 8 days. The resulting cells can be propagated easily View Publication

Three-dimensional (3D) cell cultures produce more in vivo-like multicellular structures such as spheroids that cannot be obtained in two-dimensional (2D) cell cultures. Thus,they are increasingly employed as models for cancer and drug research,as well as tissue engineering. It has proven challenging to stabilize spheroid architectures for detailed morphological examination. Here we overcome this issue using a silica bioreplication (SBR) process employed on spheroids formed from human pluripotent stem cells (hPSCs) and hepatocellular carcinoma HepG2 cells cultured in the nanofibrillar cellulose (NFC) hydrogel. The cells in the spheroids are more round and tightly interacting with each other than those in 2D cultures,and they develop microvilli-like structures on the cell membranes as seen in 2D cultures. Furthermore,SBR preserves extracellular matrix-like materials and cellular proteins. These findings provide the first evidence of intact hPSC spheroid architectures and similar fine structures to 2D-cultured cells,providing a pathway to enable our understanding of morphogenesis in 3D cultures. View Publication

Mucosal-associated invariant T (MAIT) cells exhibit different characteristics from those of TCRalpha7.2- conventional T cells. They play important roles in various inflammatory diseases,including rheumatoid arthritis and inflammatory bowel disease. MAIT cells express a single T cell receptor alpha chain,TCRalpha7.2 segment associated with Jalpha33 and CDR3 with fixed length,which recognizes bacteria-derived vitamin B metabolites. However,the characteristics of MAIT cells and TCRalpha7.2+ CD161- T cells have never been compared. Here,we performed RNA sequencing to compare the properties of MAIT cells,TCRalpha7.2- conventional T cells and TCRalpha7.2+ CD161- T cells. Genome-wide transcriptomes of MAIT cells,TCRalpha7.2- conventional T cells,and TCRalpha7.2+ CD161- T cells were compared and analyzed using causal network analysis. This is the first report comparing the transcriptomes of MAIT cells,TCRalpha7.2- conventional T cells and TCRalpha7.2+ CD161- T cells. We also identified the predominant signaling pathways of MAIT cells,which differed from those of TCRalpha7.2- conventional T cells and TCRalpha7.2+ CD161- T cells,through a gene set enrichment test and upstream regulator analysis and identified the genes responsible for the characteristic MAIT cell phenotypes. Our study advances the complete understanding of MAIT biology. View Publication

The recent advances,offered by cell therapy in the regenerative medicine field,offer a revolutionary potential for the development of innovative cures to restore compromised physiological functions or organs. Adult myogenic precursors,such as myoblasts or satellite cells,possess a marked regenerative capacity,but the exploitation of this potential still encounters significant challenges in clinical application,due to low rate of proliferation in vitro,as well as a reduced self-renewal capacity. In this scenario,induced pluripotent stem cells (iPSCs) can offer not only an inexhaustible source of cells for regenerative therapeutic approaches,but also a valuable alternative for in vitro modeling of patient-specific diseases. In this study we established a reliable protocol to induce the myogenic differentiation of iPSCs,generated from pericytes and fibroblasts,exploiting skeletal muscle-derived extracellular vesicles (EVs),in combination with chemically defined factors. This genetic integration-free approach generates functional skeletal myotubes maintaining the engraftment ability in vivo. Our results demonstrate evidence that EVs can act as biological shuttles" to deliver specific bioactive molecules for a successful transgene-free differentiation offering new opportunities for disease modeling and regenerative approaches." View Publication

Over 45,000 new cases of oral and pharyngeal cancers are diagnosed and account for over 8,000 deaths a year in the United States. An environmental chemical receptor,the aryl hydrocarbon receptor (AHR),has previously been implicated in oral squamous cell carcinoma (OSCC) initiation as well as in normal tissue-specific stem cell self-renewal. These previous studies inspired the hypothesis that the AHR plays a role in both the acquisition and progression of OSCC,as well as in the formation and maintenance of cancer stem-like cells. To test this hypothesis,AHR activity in two oral squamous cell lines was modulated with AHR prototypic,environmental and bacterial AHR ligands,AHR-specific inhibitors,and phenotypic,genomic and functional characteristics were evaluated. The data demonstrate that: 1) primary OSCC tissue expresses elevated levels of nuclear AHR as compared to normal tissue,2) Ahr mRNA expression is up-regulated in 320 primary OSCC,3) AHR hyper-activation with several ligands,including environmental and bacterial ligands,significantly increases AHR activity,ALDH1 activity,and accelerates cell migration,4) AHR inhibition blocks the rapid migration of OSCC cells and reduces cell chemoresistance,5) AHR knockdown inhibits tumorsphere formation in low adherence conditions,and 6) AHR knockdown inhibits tumor growth and increases overall survival in vivo. These data demonstrate that the AHR plays an important role in development and progression of OSCC,and specifically cancer stem-like cells. Prototypic,environmental and bacterial AHR ligands may exacerbate OSCC by enhancing expression of these properties. IMPLICATIONS This study,for the first time,demonstrates the ability of diverse AHR ligands to regulate AHR activity in oral squamous cell carcinoma cells,as well as regulate several important characteristics of oral cancer stem cells,in vivo and in vitro. View Publication

The differentiation efficiency of human embryonic stem cells (hESCs) into heart muscle cells (cardiomyocytes) is highly sensitive to culture conditions. To elucidate the regulatory mechanisms involved,we investigated hESCs grown on three distinct culture platforms: feeder-free Matrigel,mouse embryonic fibroblast feeders,and Matrigel replated on feeders. At the outset,we profiled and quantified their differentiation efficiency,transcriptome,transcription factor binding sites and DNA-methylation. Subsequent genome-wide analyses allowed us to reconstruct the relevant interactome,thereby forming the regulatory basis for implicating the contrasting differentiation efficiency of the culture conditions. We hypothesized that the parental expressions of FOXC1,FOXD1 and FOXQ1 transcription factors (TFs) are correlative with eventual cardiomyogenic outcome. Through WNT induction of the FOX TFs,we observed the co-activation of WNT3 and EOMES which are potent inducers of mesoderm differentiation. The result strengthened our hypothesis on the regulatory role of the FOX TFs in enhancing mesoderm differentiation capacity of hESCs. Importantly,the final proportions of cells expressing cardiac markers were directly correlated to the strength of FOX inductions within 72 hours after initiation of differentiation across different cell lines and protocols. Thus,we affirmed the relationship between early FOX TF expressions and cardiomyogenesis efficiency. View Publication