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

The limited proliferative capacity of erythroid precursors is a major obstacle to generate sufficient in vitro-derived red blood cells for clinical purposes. While BMI1,a Polycomb Repressive Complex 1 member,is both necessary and sufficient to drive extensive proliferation of self-renewing erythroblasts,its mechanism of action remains poorly understood. Here we report that BMI1 overexpression leads to 10 billion-fold increase in self-renewal of human erythroblasts,which can terminally mature and agglutinate with typing reagent monoclonal antibodies. BMI1 and RING1B occupancy,along with repressive histone marks,are present at known BMI1 target genes,including the INK-ARF locus,consistent with altered cell cycle kinetics following BMI1 inhibition. Upregulation of BMI1 target genes with low repressive histone modifications,including key regulators of cholesterol homeostasis,along with functional studies,suggest that both cholesterol import and synthesis are essential for BMI1-associated self-renewal. We conclude that BMI1 regulates erythroid self-renewal not only through gene repression but also through gene activation and offer a strategy to expand immature erythroid precursors for eventual clinical uses. Subject terms: Self-renewal,Cell growth,Stem-cell research View Publication

Exposure to thalidomide during a critical window of development results in limb defects in humans and non-human primates while mice and rats are refractory to these effects. Thalidomide-induced teratogenicity is dependent on its binding to cereblon (CRBN),the substrate receptor of the Cul4A-DDB1-CRBN-RBX1 E3 ubiquitin ligase complex. Thalidomide binding to CRBN elicits subsequent ubiquitination and proteasomal degradation of CRBN neosubstrates including SALL4,a transcription factor of which polymorphisms phenocopy thalidomide-induced limb defects in humans. Herein,thalidomide-induced degradation of SALL4 was examined in human induced pluripotent stem cells (hiPSCs) that were differentiated either to lateral plate mesoderm (LPM)-like cells,the developmental ontology of the limb bud,or definitive endoderm. Thalidomide and its immunomodulatory drug (IMiD) analogs,lenalidomide,and pomalidomide,dose-dependently inhibited hiPSC mesendoderm differentiation. Thalidomide- and IMiD-induced SALL4 degradation can be abrogated by CRBN V388I mutation or SALL4 G416A mutation in hiPSCs. Genetically modified hiPSCs expressing CRBN E377V/V388I mutant or SALL4 G416A mutant were insensitive to the inhibitory effects of thalidomide,lenalidomide,and pomalidomide on LPM differentiation while retaining sensitivity to another known limb teratogen,all-trans retinoic acid (atRA). Finally,disruption of LPM differentiation by atRA or thalidomide perturbed subsequent chondrogenic differentiation in vitro. The data here show that thalidomide,lenalidomide,and pomalidomide affect stem cell mesendoderm differentiation through CRBN-mediated degradation of SALL4 and highlight the utility of the LPM differentiation model for studying the teratogenicity of new CRBN modulating agents. View Publication

The combination of in vitro multi-electrode arrays (MEAs) and the neuronal differentiation of stem cells offers the capability to study human neuronal networks from patient or engineered human cell lines. Here,we use MEA-based assays to probe synaptic function and network interactions of hiPSC-derived neurons. Neuronal network behaviour first emerges at approximately 30 days of culture and is driven by glutamate neurotransmission. Over a further 30 days,inhibitory GABAergic signalling shapes network behaviour into a synchronous regular pattern of burst firing activity and low activity periods. Gene mutations in L-type voltage gated calcium channel subunit genes are strongly implicated as genetic risk factors for the development of schizophrenia and bipolar disorder. We find that,although basal neuronal firing rate is unaffected,there is a dose-dependent effect of L-type voltage gated calcium channel inhibitors on synchronous firing patterns of our hiPSC-derived neural networks. This demonstrates that MEA assays have sufficient sensitivity to detect changes in patterns of neuronal interaction that may arise from hypo-function of psychiatric risk genes. Our study highlights the utility of in vitro MEA based platforms for the study of hiPSC neural network activity and their potential use in novel compound screening. View Publication

In order to overcome the challenges associated with a limited number of airway epithelial cells that can be obtained from clinical sampling and their restrained capacity to divide ex vivo,miniaturization of respiratory drug discovery assays is of pivotal importance. Thus,a 96-well microplate system was developed where primary human small airway epithelial (hSAE) cells were cultured at an air-liquid interface (ALI). After four weeks of ALI culture,a pseudostratified epithelium containing basal,club,goblet and ciliated cells was produced. The 96-well ALI cultures displayed a cellular composition,ciliary beating frequency,and intercellular tight junctions similar to 24-well conditions. A novel custom-made device for 96-parallelized transepithelial electric resistance (TEER) measurements,together with dextran permeability measurements,confirmed that the 96-well culture developed a tight barrier function during ALI differentiation. 96-well hSAE cultures were responsive to transforming growth factor $\beta$1 (TGF-$\beta$1) and tumor necrosis factor $\alpha$ (TNF-$\alpha$) in a concentration dependent manner. Thus,the miniaturized cellular model system enables the recapitulation of a physiologically responsive,differentiated small airway epithelium,and a robotic integration provides a medium throughput approach towards pharmaceutical drug discovery,for instance,in respect of fibrotic distal airway/lung diseases. View Publication

Patient-derived pancreatic ductal adenocarcinoma (PDAC) organoid systems show great promise for understanding the biological underpinnings of disease and advancing therapeutic precision medicine. Despite the increased use of organoids,the fidelity of molecular features,genetic heterogeneity,and drug response to the tumor of origin remain important unanswered questions limiting their utility. To address this gap in knowledge,primary tumor- and patient-derived xenograft (PDX)-derived organoids,and 2D cultures for in-depth genomic and histopathologic comparisons with the primary tumor were created. Histopathologic features and PDAC representative protein markers (e.g.,claudin 4 and CA19-9) showed strong concordance. DNA- and RNA-sequencing (RNAseq) of single organoids revealed patient-specific genomic and transcriptomic consistency. Single-cell RNAseq demonstrated that organoids are primarily a clonal population. In drug response assays,organoids displayed patient-specific sensitivities. In addition,the in vivo PDX response to FOLFIRINOX and gemcitabine/abraxane treatments were examined,which was recapitulated in vitro with organoids. This study has demonstrated that organoids are potentially invaluable for precision medicine as well as preclinical drug treatment studies because they maintain distinct patient phenotypes and respond differently to drug combinations and dosage. IMPLICATIONS: The patient-specific molecular and histopathologic fidelity of organoids indicate that they can be used to understand the etiology of the patient's tumor and the differential response to therapies and suggests utility for predicting drug responses. View Publication

Under standard culture conditions,tumor cells are exposed to 20% O(2),whereas the mean tumor oxygen levels within the tumor are much lower. We demonstrate,using low-passaged human tumor cell cultures established from glioma,that a reduction in the oxygen level in these cell cultures dramatically increases the percentage of CD133 expressing cells. View Publication

Mature mammary epithelial cells are generated from undifferentiated precursors through a hierarchical process,but the molecular mechanisms involved,particularly in the human mammary gland,are poorly understood. To address this issue,we isolated highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells from normal human mammary tissue and compared their transcriptomes obtained using three different methods. Elements unique to each subset of mammary cells were identified,and changes that accompany their differentiation in vivo were shown to be recapitulated in vitro. These include a stage-specific change in NOTCH pathway gene expression during the commitment of bipotent progenitors to the luminal lineage. Functional studies further showed NOTCH3 signaling to be critical for this differentiation event to occur in vitro. Taken together,these findings provide an initial foundation for future delineation of mechanisms that perturb primitive human mammary cell growth and differentiation. View Publication

Consistent and robust manufacturing is essential for the translation of cell therapies,and the utilisation automation throughout the manufacturing process may allow for improvements in quality control,scalability,reproducibility and economics of the process. The aim of this study was to measure and establish the comparability between alternative process steps for the culture of hiPSCs. Consequently,the effects of manual centrifugation and automated non-centrifugation process steps,performed using TAP Biosystems' CompacT SelecT automated cell culture platform,upon the culture of a human induced pluripotent stem cell (hiPSC) line (VAX001024c07) were compared. This study,has demonstrated that comparable morphologies and cell diameters were observed in hiPSCs cultured using either manual or automated process steps. However,non-centrifugation hiPSC populations exhibited greater cell yields,greater aggregate rates,increased pluripotency marker expression,and decreased differentiation marker expression compared to centrifugation hiPSCs. A trend for decreased variability in cell yield was also observed after the utilisation of the automated process step. This study also highlights the detrimental effect of the cryopreservation and thawing processes upon the growth and characteristics of hiPSC cultures,and demonstrates that automated hiPSC manufacturing protocols can be successfully transferred between independent laboratories. View Publication

An increasing number of genetic variants have been implicated in autism spectrum disorders (ASDs),and the functional study of such variants will be critical for the elucidation of autism pathophysiology. Here,we report a de novo balanced translocation disruption of TRPC6,a cation channel,in a non-syndromic autistic individual. Using multiple models,such as dental pulp cells,induced pluripotent stem cell (iPSC)-derived neuronal cells and mouse models,we demonstrate that TRPC6 reduction or haploinsufficiency leads to altered neuronal development,morphology and function. The observed neuronal phenotypes could then be rescued by TRPC6 complementation and by treatment with insulin-like growth factor-1 or hyperforin,a TRPC6-specific agonist,suggesting that ASD individuals with alterations in this pathway may benefit from these drugs. We also demonstrate that methyl CpG binding protein-2 (MeCP2) levels affect TRPC6 expression. Mutations in MeCP2 cause Rett syndrome,revealing common pathways among ASDs. Genetic sequencing of TRPC6 in 1041 ASD individuals and 2872 controls revealed significantly more nonsynonymous mutations in the ASD population,and identified loss-of-function mutations with incomplete penetrance in two patients. Taken together,these findings suggest that TRPC6 is a novel predisposing gene for ASD that may act in a multiple-hit model. This is the first study to use iPSC-derived human neurons to model non-syndromic ASD and illustrate the potential of modeling genetically complex sporadic diseases using such cells.Molecular Psychiatry advance online publication,11 November 2014; doi:10.1038/mp.2014.141. View Publication