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

Telomerase plays an important role in governing the life span of cells for its capacity to extend telomeres. As high activity of telomerase has been found in stem cells and cancer cells specifically,various methods have been developed for the evaluation of telomerase activity. To overcome the time-consuming procedures and complicated manipulations of existing methods,we developed a novel method named Telomeric Repeat Elongation Assay based on Quartz crystal microbalance (TREAQ) to monitor telomerase activity during the self-renewal and differentiation of human induced pluripotent stem cells (hiPSCs). TREAQ results indicated hiPSCs possess invariable telomerase activity for 11 passages on Matrigel and a steady decline of telomerase activity when differentiated for different periods,which is confirmed with existing golden standard method. The pluripotency of hiPSCs during differentiation could be estimated through monitoring telomerase activity and compared with the expression levels of markers of pluripotency gene via quantitative real time PCR. Regular assessment for factors associated with pluripotency or stemness was expensive and requires excessive sample consuming,thus TREAQ could be a promising alternative technology for routine monitoring of telomerase activity and estimate the pluripotency of stem cells. View Publication

Mutations in SCN1A,the gene encoding the α subunit of Nav1.1 channel,can cause epilepsies with wide ranges of clinical phenotypes,which are associated with the contrasting effects of channel loss-of-function or gain-of-function. In this project,CRISPR/Cas9- and TALEN-mediated genome-editing techniques were applied to induced pluripotent stem cell (iPSC)-based-disease model to explore the mechanism of epilepsy caused by SCN1A loss-of-function mutation. By fluorescently labeling GABAergic subtype in iPSC-derived neurons using CRISPR/Cas9,we for the first time performed electrophysiological studies on SCN1A-expressing neural subtype and monitored the postsynaptic activity of both inhibitory and excitatory types. We found that the mutation c.A5768G,which led to no current of Nav1.1 in exogenously transfected system,influenced the properties of not only Nav current amount,but also Nav activation in Nav1.1-expressing GABAergic neurons. The two alterations in Nav further reduced the amplitudes and enhanced the thresholds of action potential in patient-derived GABAergic neurons,and led to weakened spontaneous inhibitory postsynaptic currents (sIPSCs) in the patient-derived neuronal network. Although the spontaneous excitatory postsynaptic currents (sEPSCs) did not change significantly,when the frequencies of both sIPSCs and sEPSCs were further analyzed,we found the whole postsynaptic activity transferred from the inhibition-dominated state to excitation in patient-derived neuronal networks,suggesting that changes in sIPSCs alone were sufficient to significantly reverse the excitatory level of spontaneous postsynaptic activity. In summary,our findings fill the gap of our knowledge regarding the relationship between SCN1A mutation effect recorded on exogenously transfected cells and on Nav1.1-expressing neurons,and reveal the physiological basis underlying epileptogenesis caused by SCN1A loss-of-function mutation. View Publication

Evolutionary innovations can be driven by changes in the rates of RNA translation and the emergence of new genes and small open reading frames (sORFs). In this study,we characterized the transcriptional and translational landscape of the hearts of four primate and two rodent species through integrative ribosome and transcriptomic profiling,including adult left ventricle tissues and induced pluripotent stem cell-derived cardiomyocyte cell cultures. We show here that the translational efficiencies of subunits of the mitochondrial oxidative phosphorylation chain complexes IV and V evolved rapidly across mammalian evolution. Moreover,we discovered hundreds of species-specific and lineage-specific genomic innovations that emerged during primate evolution in the heart,including 551 genes,504 sORFs and 76 evolutionarily conserved genes displaying human-specific cardiac-enriched expression. Overall,our work describes the evolutionary processes and mechanisms that have shaped cardiac transcription and translation in recent primate evolution and sheds light on how these can contribute to cardiac development and disease. Ruiz-Orera et al. used comparative transcriptomics and translatomics to analyze the cardiac evolution in primates and discovered species-specific and lineage-specific genomic innovations that might contribute to cardiac development and disease. View Publication

Ceramides are bioactive sphingolipids that have physiological effects on inflammation,apoptosis,and mitochondrial dysfunction. They may play a critical role in the harm of ischemia/reperfusion (IR). Ceramides and IR injury are not well-studied,and there is a lack of human data. Current studies aimed to investigate the role of ceramide buildup in cardiomyocytes (CMs) death using CMs derived from human induced pluripotent stem cell (hiPSC) as a model for simulating IR injury in vitro. In our model,serum- and glucose-free media was used to expose hiPSC-derived CMs to hypoxia (3% O 2 ) for 6 h (hrs),followed by reoxygenation (20% O 2 ) for 16 h. In contrast to normoxia (control) or hypoxia (ischemia),our data showed that following IR,there was an increase in the formation of mitochondrial superoxide and the mRNA levels of genes regulating ceramide synthesis,such as CerS2 and CerS4 in CMs. Further,there was a considerable rise in the levels of total ceramide,long-chain (C16:0,C18:0,and C18:1),and very long-chain (C22:0 and C24:1) ceramide species in CMs following reperfusion in comparison to control or ischemic CMs. Interestingly,compared to CMs exposed to IR without inhibitor,our data showed that inhibition of ceramide formation with fumonisin B1 (FB1) significantly lowered ceramide levels,reduced apoptosis,improved mitochondrial function,and enhanced survival of CMs exposed to IR. Furthermore,we used a transgenic mouse model,in which the CerS2 gene was overexpressed in the CMs of α-MHC-CerS2 mice,to validate the basic idea that ceramide contributes to heart disease in vivo. Our results showed that the heart tissues of α-MHC-CerS2 mice had significant levels of long-chain and very long-chain ceramides,which causes increased apoptosis,proinflammatory cytokines,interstitial inflammatory cell infiltration,and collagen deposition. Results from both in vitro and in vivo experiments show that ceramides have a significant role in either mediating or inducing damage to CMs. Additionally,in vitro findings show that ceramide reduction improves the outcome of IR injury by lowering intracellular Ca 2+ [Ca 2+ ] i concentration and improves mitochondrial function changes during IR. The online version contains supplementary material available at 10.1186/s13287-025-04340-3. View Publication

How the neuroectoderm-derived eye field breaks symmetry to specify iris muscle is not well understood. Recent studies have begun to transcriptionally characterize mouse iris muscle; however,little is known about the transcriptional foundation of human iris development. Human pluripotent stem cells (hPSCs) enable the study of iris muscle specification. Here we compare iris smooth muscle from native adult iris tissues to evaluate successful specification of iris muscle from hPSC lines. We utilize a previously published eye-like organoid protocol that specified cells of the eye field to also generate iris muscle. We describe a population transcriptionally similar to native iris and describe an iris muscle gene signature. Human iris muscle not only contains pigment,but also expresses pigment synthesis genes and is responsive to acetylcholine. Integration of single-cell RNA-seq datasets confirm the similarity between the iris muscle to the adult iris,establishing the usefulness of the model in studying neuroectoderm-derived iris muscle specification,and related diseases. Single-cell RNA sequencing reveals that iris muscle,derived from neuroectoderm,can form in stem cell–derived eye organoids – enabling the modelling of iris muscle pathologies like aniridia and proliferative vitreoretinopathy. View Publication

SALL4,a human homolog to Drosophila spalt,is a novel zinc finger transcriptional factor essential for development. We cloned SALL4 and its isoforms (SALL4A and SALL4B). Through immunohistochemistry and real-time reverse-transcription-polymerase chain reaction (RT-PCR),we demonstrated that SALL4 was constitutively expressed in human primary acute myeloid leukemia (AML,n = 81),and directly tested the leukemogenic potential of constitutive expression of SALL4 in a murine model. SALL4B transgenic mice developed myelodysplastic syndrome (MDS)-like features and subsequently AML that was transplantable. Increased apoptosis associated with dysmyelopoiesis was evident in transgenic mouse marrow and colony-formation (CFU) assays. Both isoforms could bind to beta-catenin and synergistically enhanced the Wnt/beta-catenin signaling pathway. Our data suggest that the constitutive expression of SALL4 causes MDS/AML,most likely through the Wnt/beta-catenin pathway. Our murine model provides a useful platform to study human MDS/AML transformation,as well as the Wnt/beta-catenin pathway's role in the pathogenesis of leukemia stem cells. View Publication

BACKGROUND: Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are a promising source of cells for regenerating myocardium. However,several issues,especially the large-scale preparation of hiPS-CMs and elimination of undifferentiated iPS cells,must be resolved before hiPS cells can be used clinically. The cell-sheet technique is one of the useful methods for transplanting large numbers of cells. We hypothesized that hiPS-CM-sheet transplantation would be feasible,safe,and therapeutically effective for the treatment of ischemic cardiomyopathy.backslashnbackslashnMETHODS AND RESULTS: Human iPS cells were established by infecting human dermal fibroblasts with a retrovirus carrying Oct3/4,Sox2,Klf4,and c-Myc. Cardiomyogenic differentiation was induced by WNT signaling molecules,yielding hiPS-CMs that were almost 90% positive for α-actinin,Nkx2.5,and cardiac troponin T. hiPS-CM sheets were created using thermoresponsive dishes and transplanted over the myocardial infarcts in a porcine model of ischemic cardiomyopathy induced by ameroid constriction of the left anterior descending coronary artery (n=6 for the iPS group receiving sheet transplantation and the sham-operated group; both groups received tacrolimus daily). Transplantation significantly improved cardiac performance and attenuated left ventricular remodeling. hiPS-CMs were detectable 8 weeks after transplantation,but very few survived long term. No teratoma formation was observed in animals that received hiPS-CM sheets.backslashnbackslashnCONCLUSIONS: The culture system used yields a large number of highly pure hiPS-CMs,and hiPS-CM sheets could improve cardiac function after ischemic cardiomyopathy. This newly developed culture system and the hiPS-CM sheets may provide a basis for the clinical use of hiPS cells in cardiac regeneration therapy. View Publication

A portion of the genetic basis for many common autoimmune disorders has been uncovered by genome-wide association studies (GWAS),but GWAS do not reveal causal variants,effector genes,or the cell types impacted by disease-associated variation. We have generated 3D genomic datasets consisting of promoter-focused Capture-C,Hi-C,ATAC-seq,and RNA-seq and integrated these data with GWAS of 16 autoimmune traits to physically map disease-associated variants to the effector genes they likely regulate in 57 human cell types. These 3D maps of gene cis-regulatory architecture are highly powered to identify the cell types most likely impacted by disease-associated genetic variation compared to 1D genomic features,and tend to implicate different effector genes than eQTL approaches in the same cell types. Most of the variants implicated by these cis-regulatory architectures are highly trait-specific,but nearly half of the target genes connected to these variants are shared across multiple autoimmune disorders in multiple cell types,suggesting a high level of genetic diversity and complexity among autoimmune diseases that nonetheless converge at the level of target gene and cell type. Substantial effector gene sharing led to the common enrichment of similar biological networks across disease and cell types. However,trait-specific pathways representing potential areas for disease-specific intervention were identified. To test this,we pharmacologically validated squalene synthase,a cholesterol biosynthetic enzyme encoded by the FDFT1 gene implicated by our approach in MS and SLE,as a novel immunomodulatory drug target controlling inflammatory cytokine production by human T cells. These data represent a comprehensive resource for basic discovery of gene cis-regulatory mechanisms,and the analyses reported reveal mechanisms by which autoimmune-associated variants act to regulate gene expression,function,and pathology across multiple,distinct tissues and cell types. View Publication

Dax-1 (Nr0b1) is an orphan member of the nuclear hormone receptor superfamily that has a key role in adrenogonadal development and function. Recent studies have also implicated Dax-1 in the transcriptional network controlling embryonic stem (ES) cell pluripotency. Here,we show that Dax-1 expression is affected by differentiating treatments and pharmacological activation of beta-catenin-dependent transcription in mouse ES cells. Furthermore,Dax-1 knockdown induced upregulation of multilineage differentiation markers,and produced enhanced differentiation and defects in ES viability and proliferation. Through RNA interference and transcriptome analysis,we have identified genes regulated by Dax-1 in mouse ES cells at 24 and 48 hours after knockdown. Strikingly,the great majority of these genes are upregulated,showing that the prevalent function of Dax-1 is to act as a transcriptional repressor in mouse ES cells,as confirmed by experiments using the Gal4 system. Genes involved in tissue differentiation and control of proliferation are significantly enriched among Dax-1-regulated transcripts. These data show that Dax-1 is an essential element in the molecular circuit involved in the maintenance of ES cell pluripotency and have implications for the understanding of stem cell function in both physiological (adrenal gland) and clinical (Ewing tumors) settings where Dax-1 plays a pivotal role in development and pathogenesis,respectively. View Publication

Hepatocellular carcinoma (HCC) is associated with a high morbidity and mortality due to its high rate of recurrence. However,little is known about the biological characteristics of recurrent HCC cells. A single patient's primary and recurrent HCC-derived cell lines,Hep-11 and Hep-12,respectively,were established by primary culture. These two cell lines have the same hepatitis B virus integration site and share many common amplifications and deletions,which suggest that they have the same clonal origin. While Hep-11 cells were non-tumorigenic at 16 weeks following injection of up to 10 000 cells,injection of only 100 Hep-12 cells was sufficient to initiate tumor growth,and all single Hep-12 clones were tumorigenic in immunodeficient mice. Compared with Hep-11,Hep-12 cells expressed the oval cell markers AFP,NCAM/CD56,c-kit/CD117,as well as multiple stem cell markers such as Nanog,OCT4 and SOX2. In addition,textgreater90% of Hep-12 cells were aldehyde dehydrogenase positive. They were also less resistant to paclitaxel,but more resistant to doxorubicin,cisplatin and hydroxycamptothecin (HCPT),which had been administrated to the patient. Furthermore,Hep-12 cells expressed higher levels of poly (adenosine diphosphate-ribose) polymerase-1 (PARP-1) than Hep-11,and PARP-1 inhibition potentiated the sensitivity to HCPT in Hep-12 cells but not in Hep-11 cells. These results indicate that a large population of the recurrent HCC-derived Hep-12 cells were tumor-initiating cells and that elevated expression of PARP-1 was related to their resistance to HCPT. View Publication

Comprehensive in vitro and in vivo studies comparing EpCAM-based methods with other cytometric CTC enrichment technologies in metastatic colorectal cancer (mCRC) patients are lacking. We compare four manual cytometric methods to detect CTCs in vitro and in mCRC patients. The EpCAM-based technology,MACS HEA MicroBeads((R)),showed a significant better tumor cell recovery rate compared to other cytometric methods (p-valuetextless0.0001). CTCs of 38 mCRC patients were enriched with MACS HEA MicroBeads(R). Progression-free survival did significantly differ between mCRC patients without detectable and with textgreateror= 1 CTCs (p=0.007). CTC enrichment with EpCAM coupled antibodies is superior to other cytometric methods and is a feasible method for CTC detection in mCRC patients. View Publication