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

The ability to analyze protein function in a native context is central to understanding cellular physiology. This study explores whether tagging endogenous proteins with a reporter is a scalable strategy for generating cell models that accurately quantitate protein dynamics. Specifically,it investigates whether CRISPR-mediated integration of the HiBiT luminescent peptide tag can easily be accomplished on a large-scale and whether integrated reporter faithfully represents target biology. For this purpose,a large set of proteins representing diverse structures and functions,some of which are known or potential drug targets,were targeted for tagging with HiBiT in multiple cell lines. Successful insertion was detected for 86{\%} of the targets,as determined by luminescence-based plate assays,blotting,and imaging. In order to determine whether endogenously tagged proteins yield more representative models,cells expressing HiBiT protein fusions either from endogenous loci or plasmids were directly compared in functional assays. In the tested cases,only the edited lines were capable of accurately reproducing the anticipated biology. This study provides evidence that cell lines expressing HiBiT fusions from endogenous loci can be rapidly generated for many different proteins and that these cellular models provide insight into protein function that may be unobtainable using overexpression-based approaches. View Publication

Cited2 (cAMP-responsive elementbinding protein [CBP]/p300-interacting transactivators with glutamic acid [E] and aspartic acid [D]-rich tail 2) is a newly identified transcriptional modulator. Knockout of the Cited2 gene results in embryonic lethality with embryos manifesting heart and neural tube defects. Cited2-/- fetal liver displayed significant reduction in the numbers of Lin(-)c-Kit+Sca-1+ cells,Lin(-)c-Kit+ cells,and progenitor cells of different lineages. Fetal liver cells from Cited2-/- embryos gave rise to markedly reduced number of colonies in the colony-forming unit assay. Primary and secondary transplantation studies showed significantly compromised reconstitution of T-lymphoid,B-lymphoid,and myeloid lineages in mice that received a transplant of Cited2-/- fetal liver cells. Competitive reconstitution experiments further showed that fetal liver hematopoietic stem cell (HSC) function is severely impaired due to Cited2 deficiency. Microarray analysis showed decreased expression of Wnt5a and a panel of myeloid molecular markers such as PRTN3,MPO,Neutrophil elastase,Cathepsin G,and Eosinophil peroxidase in Cited2-/- fetal livers. Decreased expression of Bmi-1,Notch1,LEF-1,Mcl-1,and GATA2 was also observed in Cited2-/- Lin(-)c-Kit+ cells. The present study uncovers for the first time a novel role of Cited2 in the maintenance of hematopoietic homeostasis during embryogenesis and thus provides new insights into the molecular regulation of hematopoietic development. View Publication

With their unique ability to differentiate into all cell types,embryonic stem (ES) cells hold great therapeutic promise. To improve the efficiency of embryoid body (EB)-mediated ES cell differentiation,we studied murine EBs on the basis of their size and found that EBs with an intermediate size (diameter 100-300 microm) are the most proliferative,hold the greatest differentiation potential,and have the lowest rate of cell death. In an attempt to promote the formation of this subpopulation,we surveyed several biocompatible substrates with different surface chemical parameters and identified a strong correlation between hydrophobicity and EB development. Using self-assembled monolayers of various lengths of alkanethiolates on gold substrates,we directly tested this correlation and found that surfaces that exhibit increasing hydrophobicity enrich for the intermediate-size EBs. When this approach was applied to the human ES cell system,similar phenomena were observed. Our data demonstrate that hydrophobic surfaces serve as a platform to deliver uniform EB populations and may significantly improve the efficiency of ES cell differentiation. View Publication

Reported here is a piggyBac transposon-based expression system for the generation of doxycycline-inducible,stably transfected mammalian cell cultures for large-scale protein production. The system works with commonly used adherent and suspension-adapted mammalian cell lines and requires only a single transfection step. Moreover,the high uniform expression levels observed among clones allow for the use of stable bulk cell cultures,thereby eliminating time-consuming cloning steps. Under continuous doxycycline induction,protein expression levels have been shown to be stable for at least 2 mo in the absence of drug selection. The high efficiency of the system also allows for the generation of stable bulk cell cultures in 96-well format,a capability leading to the possibility of generating stable cell cultures for entire families of membrane or secreted proteins. Finally,we demonstrate the utility of the system through the large-scale production (140-750 mg scale) of an endoplasmic reticulum-resident fucosyltransferase and two potential anticancer protein therapeutic agents. View Publication

This paper presents a 360°,size-adjustable microelectrode array (MEA) system for the long-term electrophysiological monitoring of cerebral organoids derived from human pluripotent stem cells. The system consists of eight independently positionable multielectrode probes,each carrying eight electrodes arranged vertically. This configuration resulted in 64 recording channels surrounding the organoid. The multielectrode probes were mounted on custom-designed miniature manipulators with three degrees of freedom. This setup enabled positioning and contact with organoids of varying sizes (approximately 1–3.7 mm in diameter). The design allowed circumferential access and facilitated standard incubator-based cultivation without disrupting the recording setup. Fabricated using flexible printed circuit technology,this MEA system offers relatively low production costs. It is also amenable to widespread implementation in laboratory settings. Experimental results demonstrated the successful recording of neuronal activity,including spike detection and signal stability,over 2 weeks of continuous organoid culture. These results suggests that the three-dimensional system provides broad spatial coverage and supports long-term monitoring for basic biomedical research. It also holds potential for future applications such as biohybrid computing. View Publication

The advancement of Long-Read Sequencing (LRS) techniques has significantly increased the length of sequencing to several kilobases,thereby facilitating the identification of alternative splicing events and isoform expressions. Recently,numerous computational tools for isoform detection using long-read sequencing data have been developed. Nevertheless,there remains a deficiency in comparative studies that systemically evaluate the performance of these tools,which are implemented with different algorithms,under various simulations that encompass potential influencing factors. In this study,we conducted a benchmark analysis of thirteen methods implemented in nine tools capable of identifying isoform structures from long-read RNA-seq data. We evaluated their performances using simulated data,which represented diverse sequencing platforms generated by an in-house simulator,RNA sequins (sequencing spike-ins) data,as well as experimental data. Our findings demonstrate IsoQuant as a highly effective tool for isoform detection with LRS,with Bambu and StringTie2 also exhibiting strong performance. These results offer valuable guidance for future research on alternative splicing analysis and the ongoing improvement of tools for isoform detection using LRS data. Recently,various computational tools have emerged for detecting mRNA isoforms using long-read sequencing data. Here,the authors systemically evaluate and compare the performance of these tools. View Publication

The Smoothened receptor (Smo) mediates hedgehog (Hh) signaling critical for development,cell growth,and migration,as well as stem cell maintenance. Aberrant Hh signaling pathway activation has been implicated in a variety of cancers,and small-molecule antagonists of Smo have entered human clinical trials for the treatment of cancer. Here,we report the biochemical characterization of allosteric interactions of agonists and antagonists for Smo. Binding of two radioligands,[(3)H]3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-{\{}[3-(4-pyridinyl)-phenyl]methyl{\}}-1-benzothiophene-2-carboxamide (SAG-1.3) (agonist) and [(3)H]cyclopamine (antagonist),was characterized using human Smo expressed in human embryonic kidney 293F membranes. We observed full displacement of [(3)H]cyclopamine by all Smo agonist and antagonist ligands examined. N-[(1E)-(3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)methylidene]-4-(phenylmethyl)-1-piperazinamine (SANT-1),an antagonist,did not fully inhibit the binding of [(3)H]SAG-1.3. In a functional cell-based beta-lactamase reporter gene assay,SANT-1 and N-[3-(1H-benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-tris(ethyloxy)-benzamide (SANT-2) fully inhibited 3-chloro-4,7-difluoro-N-[trans-4-(methylamino)cyclohexyl]-N-{\{}[3-(4-pyridinyl)phenyl]methyl{\}}-1-benzothiophene-2-carboxamide (SAG-1.5)-induced Hh pathway activation. Detailed Schild-type" radioligand binding analysis with [(3)H]SAG-1.3 revealed that two structurally distinct Smoothened receptor antagonists SANT-1 and SANT-2 bound in a manner consistent with that of allosteric modulation. Our mechanism of action characterization of radioligand binding to Smo combined with functional data provides a better understanding of small-molecule interactions with Smo and their influence on the Hh pathway." View Publication

Recent advances in the differentiation and production of human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) have stimulated development of strategies to use these cells in human cardiac regenerative therapies. A prerequisite for clinical trials and translational implementation of hPSC-derived CMs is the ability to manufacture safe and potent cells on the scale needed to replace cells lost during heart disease. Current differentiation protocols generate fetal-like CMs that exhibit proarrhythmogenic potential. Sufficient maturation of these hPSC-derived CMs has yet to be achieved to allow these cells to be used as a regenerative medicine therapy. Insights into the native cardiac environment during heart development may enable engineering of strategies that guide hPSC-derived CMs to mature. Specifically,considerations must be made in regard to developing methods to incorporate the native intercellular interactions and biomechanical cues into hPSC-derived CM production that are conducive to scale-up. View Publication

Thrombopoietin (TPO) is a potent regulator of megakaryopoiesis and stimulates megakaryocyte (MK) progenitor expansion and MK differentiation. In this study,we show that TPO induces activation of the mammalian target of rapamycin (mTOR) signaling pathway,which plays a central role in translational regulation and is required for proliferation of MO7e cells and primary human MK progenitors. Treatment of MO7e cells,human CD34+,and primary MK cells with the mTOR inhibitor rapamycin inhibits TPO-induced cell cycling by reducing cells in S phase and blocking cells in G0/G1. Rapamycin markedly inhibits the clonogenic growth of MK progenitors with high proliferative capacity but does not reduce the formation of small MK colonies. Addition of rapamycin to MK suspension cultures reduces the number of MK cells,but inhibition of mTOR does not significantly affect expression of glycoproteins IIb/IIIa (CD41) and glycoprotein Ib (CD42),nuclear polyploidization levels,cell size,or cell survival. The downstream effectors of mTOR,p70 S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1),are phosphorylated by TPO in a rapamycin- and LY294002-sensitive manner. Part of the effect of the phosphatidyl inositol 3-kinase pathway in regulating megakaryopoiesis may be mediated by the mTOR/S6K/4E-BP1 pathway. In conclusion,these data demonstrate that the mTOR pathway is activated by TPO and plays a critical role in regulating proliferation of MK progenitors,without affecting differentiation or cell survival. View Publication