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

The rapid generation of various species and strains of laboratory animals using CRISPR/Cas9 technology has dramatically accelerated the interrogation of gene function in vivo. So far,the dominant approach for genotyping of genome-modified animals has been the T7E1 endonuclease cleavage assay. Here,we present a polyacrylamide gel electrophoresis-based (PAGE) method to genotype mice harboring different types of indel mutations. We developed 6 strains of genome-modified mice using CRISPR/Cas9 system,and utilized this approach to genotype mice from F0 to F2 generation,which included single and multiplexed genome-modified mice. We also determined the maximal detection sensitivity for detecting mosaic DNA using PAGE-based assay as 0.5%. We further applied PAGE-based genotyping approach to detect CRISPR/Cas9-mediated on- and off-target effect in human 293T and induced pluripotent stem cells (iPSCs). Thus,PAGE-based genotyping approach meets the rapidly increasing demand for genotyping of the fast-growing number of genome-modified animals and human cell lines created using CRISPR/Cas9 system or other nuclease systems such as TALEN or ZFN. View Publication

Glioma tumour-initiating cells (GTICs) can originate upon the transformation of neural progenitor cells (NPCs). Studies on GTICs have focused on primary tumours from which GTICs could be isolated and the use of human embryonic material. Recently,the somatic genomic landscape of human gliomas has been reported. RTK (receptor tyrosine kinase) and p53 signalling were found dysregulated in ∼90% and 86% of all primary tumours analysed,respectively. Here we report on the use of human-induced pluripotent stem cells (hiPSCs) for modelling gliomagenesis. Dysregulation of RTK and p53 signalling in hiPSC-derived NPCs (iNPCs) recapitulates GTIC properties in vitro. In vivo transplantation of transformed iNPCs leads to highly aggressive tumours containing undifferentiated stem cells and their differentiated derivatives. Metabolic modulation compromises GTIC viability. Last,screening of 101 anti-cancer compounds identifies three molecules specifically targeting transformed iNPCs and primary GTICs. Together,our results highlight the potential of hiPSCs for studying human tumourigenesis. View Publication

Pluripotent stem cells can be genetically labeled to facilitate differentiation studies. In this paper,we describe a gene-targeting protocol to knock in a GFP cassette into key gene loci in human pluripotent stem cells (hPSCs),and then use the genetically tagged hPSCs to guide in vitro differentiation,immunocytochemical and electrophysiological profiling and in vivo characterization after cell transplantation. The Olig transcription factors have key roles in the transcription regulatory pathways for the genesis of motor neurons (MNs) and oligodendrocytes (OLs). We have generated OLIG2-GFP hPSC reporter lines that reliably mark MNs and OLs for monitoring their sequential differentiation from hPSCs. The expression of the GFP reporter recapitulates the endogenous expression of OLIG genes. The in vitro characterization of fluorescence-activated cell sorting-purified cells is consistent with cells of the MN or OL lineages,depending on the stages at which they are collected. This protocol is efficient and reliable and usually takes 5-7 months to complete. The genetic tagging-differentiation methodology used herein provides a general framework for similar work for differentiation of hPSCs into other lineages. View Publication

Nanog,Oct4,and Sox2 are the core regulators of mouse (m)ESC pluripotency. Although their basic importance in human (h)ESCs has been demonstrated,the mechanistic functions are not well defined. Here,we identify general and cell-line-specific requirements for NANOG,OCT4,and SOX2 in hESCs. We show that OCT4 regulates,and interacts with,the BMP4 pathway to specify four developmental fates. High levels of OCT4 enable self-renewal in the absence of BMP4 but specify mesendoderm in the presence of BMP4. Low levels of OCT4 induce embryonic ectoderm differentiation in the absence of BMP4 but specify extraembryonic lineages in the presence of BMP4. NANOG represses embryonic ectoderm differentiation but has little effect on other lineages,whereas SOX2 and SOX3 are redundant and repress mesendoderm differentiation. Thus,instead of being panrepressors of differentiation,each factor controls specific cell fates. Our study revises the view of how self-renewal is orchestrated in hESCs. View Publication

Mouse embryonic fibroblasts (MEFs) are commonly used as feeder cells for the generation of human induced pluripotent stem cells (hiPSCs). However,medical applications of cell derivatives of hiPSCs generated with a MEF feeder system run the risk of having xeno-factor contamination due to long-term cell culturing under an animal factor-containing environment. We developed a new method for the derivation of human fibroblast-like cells (FLCs) from a previously established hiPSC line in an FLC differentiation medium. The method was based on direct differentiation of hiPSCs seeded on Matrigel followed by expansion of differentiating cells on gelatin. Using inactivated FLCs as feeder layers,primary human foreskin fibroblasts were successfully reprogrammed into a state of pluripotency by Oct4,Sox2 Klf4,and c-Myc (OSKM) transcription factor genes,with a reprogramming efficiency under an optimized condition superior to that obtained on MEF feeder layers. Furthermore,the FLCs were more effective in supporting the growth of human pluripotent stem cells. The pluripotency and differentiation capability of the cells cultured on FLC feeder layers were well retained. Our results suggest that FLCs are a safe alternative to MEFs for hiPSC generation and expansion,especially in the clinical settings wherein hiPSC derivatives will be used for medical treatment. View Publication

Direct reprogramming of human fibroblasts to a pluripotent state has been achieved through ectopic expression of the transcription factors OCT4,SOX2,and either cMYC and KLF4 or NANOG and LIN28. Little is known,however,about the mechanisms by which reprogramming occurs,which is in part limited by the low efficiency of conversion. To this end,we sought to create a doxycycline-inducible lentiviral system to convert primary human fibroblasts and keratinocytes into human induced pluripotent stem cells (hiPSCs). hiPSCs generated with this system were molecularly and functionally similar to human embryonic stem cells (hESCs),demonstrated by gene expression profiles,DNA methylation status,and differentiation potential. While expression of the viral transgenes was required for several weeks in fibroblasts,we found that 10 days was sufficient for the reprogramming of keratinocytes. Using our inducible system,we developed a strategy to induce hiPSC formation at high frequency. Upon addition of doxycycline to hiPSC-derived differentiated cells,we obtained secondary" hiPSCs at a frequency at least 100-fold greater than the initial conversion. The ability to reprogram cells at high efficiency provides a unique platform to dissect the underlying molecular and biochemical processes that accompany nuclear reprogramming." View Publication

Human embryonic stem cells (hESCs),unlike mouse ones (mESCs),are vulnerable to apoptosis upon dissociation. Here,we show that the apoptosis,which is of a nonanoikis type,is caused by ROCK-dependent hyperactivation of actomyosin and efficiently suppressed by the myosin inhibitor Blebbistatin. The actomyosin hyperactivation is triggered by the loss of E-cadherin-dependent intercellular contact and also observed in dissociated mouse epiblast-derived pluripotent cells but not in mESCs. We reveal that Abr,a unique Rho-GEF family factor containing a functional Rac-GAP domain,is an indispensable upstream regulator of the apoptosis and ROCK/myosin hyperactivation. Rho activation coupled with Rac inhibition is induced in hESCs upon dissociation,but not in Abr-depleted hESCs or mESCs. Furthermore,artificial Rho or ROCK activation with Rac inhibition restores the vulnerability of Abr-depleted hESCs to dissociation-induced apoptosis. Thus,the Abr-dependent Rho-high/Rac-low" state plays a decisive role in initiating the dissociation-induced actomyosin hyperactivation and apoptosis in hESCs." View Publication

The amyloid-beta precursor protein (AbetaPP) is a ubiquitously expressed adhesion and neuritogenic protein whose processing has previously been shown to be regulated by reproductive hormones including the gonadotropin luteinizing hormone (LH) in human neuroblastoma cells. We report for the first time the expression of AbetaPP in human embryonic stem (hES) cells at the mRNA and protein levels. Using N- and C-terminal antibodies against AbetaPP,we detected both the mature and immature forms of AbetaPP as well as truncated variants ( approximately 53kDa,47kDa,and 29kDa) by immunoblot analyses. Expression of AbetaPP is regulated by both the stemness of the cells and pregnancy-associated hormones. Addition of human chorionic gonadotropin,the fetal equivalent of LH that is dramatically elevated during pregnancy,markedly increased the expression of all AbetaPP forms. These results indicate a critical molecular signaling link between the hormonal environment of pregnancy and the expression of AbetaPP in hES cells that is suggestive of an important function for this protein during early human embryogenesis prior to the formation of neural precursor cells. View Publication

Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs exhibit basal-apical polarity,junctional complexes,integrin-dependent matrix adhesion,and E-cadherin-dependent cell-cell adhesion,all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures,programmed cell death is commonly observed. If individualized human ESCs are prevented from reattaching and forming colonies,their viability is significantly reduced. Here,we show that actin-myosin contraction is a critical effector of the cell death response to human ESC dissociation. Inhibition of myosin heavy chain ATPase,downregulation of myosin heavy chain,and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ESCs. ROCK inhibition decreases phosphorylation of myosin light chain,suggesting that inhibition of actin-myosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ESCs. View Publication

Cultures of human embryonic stem cell typically rely on protein matrices or feeder cells to support attachment and growth,while mechanical,enzymatic or chemical cell dissociation methods are used for cellular passaging. However,these methods are ill defined,thus introducing variability into the system,and may damage cells. They also exert selective pressures favouring cell aneuploidy and loss of differentiation potential. Here we report the identification of a family of chemically defined thermoresponsive synthetic hydrogels based on 2-(diethylamino)ethyl acrylate,which support long-term human embryonic stem cell growth and pluripotency over a period of 2-6 months. The hydrogels permitted gentle,reagent-free cell passaging by virtue of transient modulation of the ambient temperature from 37 to 15 °C for 30 min. These chemically defined alternatives to currently used,undefined biological substrates represent a flexible and scalable approach for improving the definition,efficacy and safety of human embryonic stem cell culture systems for research,industrial and clinical applications. View Publication

The establishment of protocols to differentiate human pluripotent stem cells (hPSCs) including embryonic (ESC) and induced pluripotent (iPSC) stem cells into functional hepatocyte-like cells (HLCs) creates new opportunities to study liver metabolism,genetic diseases and infection of hepatotropic viruses (hepatitis B and C viruses) in the context of specific genetic background. While supporting efficient differentiation to HLCs,the published protocols are limited in terms of differentiation into fully mature hepatocytes and in a smaller-well format. This limitation handicaps the application of these cells to high-throughput assays. Here we describe a protocol allowing efficient and consistent hepatic differentiation of hPSCs in 384-well plates into functional hepatocyte-like cells,which remain differentiated for more than 3 weeks. This protocol affords the unique opportunity to miniaturize the hPSC-based differentiation technology and facilitates screening for molecules in modulating liver differentiation,metabolism,genetic network,and response to infection or other external stimuli. View Publication