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

Numerous studies have shown that somite development is a necessary stage of myogenesis chondrogenesis and osteogenesis. Our previous study has established a stable presomitic mesoderm progenitor cell line (UiPSM) in vitro. Naturally,we wanted to explore whether UiPSM cell can develop bone and myogenic differentiation. Selective culture conditions yielded PAX3 and PAX7 positive skeletal muscle precursors from UiPSM cells. The skeletal muscle precursors undergo in vitro maturation resulting in myotube formation. MYOD effectively promoted the maturity of the skeletal myocytes in a short time. We found that UiPSM and MYOD mediated UiPSM cell-derived skeletal myocytes were viable after transplantation into the tibialis anterior muscle of MITRG mice,as assessed by bioluminescence imaging and scRNA-seq. Lack of teratoma formation and evidence of long-term myocytes engraftment suggests considerable potential for future therapeutic applications. Moreover,UiPSM cells can differentiate into osteoblast and chondroblast cells in vitro. UiPSM differentiation has potential as a developmental model for musculoskeletal development research and treatment of musculoskeletal disorders. The online version contains supplementary material available at 10.1186/s13578-024-01274-w. View Publication

A cDNA clone coding for a functional human prostanoid FP receptor has been isolated from a uterus cDNA library. The human FP receptor consists of 359 amino acid residues with a predicted molecular mass of 40,060,and has the seven putative transmembrane domains characteristic of G-protein-coupled receptors. Challenge of Xenopus oocytes expressing the FP receptor with 10 nM of either prostaglandin (PG) F2 alpha or the selective FP-receptor agonist fluprostenol resulted in an elevation in intracellular Ca2+. Radioreceptor binding studies using membranes prepared from mammalian COS cells transfected with the FP receptor cDNA showed that the rank order of potency for prostaglandins and prostaglandin analogs in competition for [3H]PGF2 alpha specific binding sites was as predicted for the FP receptor,with PGF2 alpha approximately fluprostenol textgreater PGD2 textgreater PGE2 textgreater U46619 textgreater iloprost. In summary,we have cloned the human prostanoid FP receptor which is functionally coupled to the Ca2+ signalling pathway. View Publication

BACKGROUND The ability to site-specifically conjugate a protein to a payload of interest (e.g.,a fluorophore,small molecule pharmacophore,oligonucleotide,or other protein) has found widespread application in basic research and drug development. For example,antibody-drug conjugates represent a class of biotherapeutics that couple the targeting specificity of an antibody with the chemotherapeutic potency of a small molecule drug. While first generation antibody-drug conjugates (ADCs) used random conjugation approaches,next-generation ADCs are employing site-specific conjugation. A facile way to generate site-specific protein conjugates is via the aldehyde tag technology,where a five amino acid consensus sequence (CXPXR) is genetically encoded into the protein of interest at the desired location. During protein expression,the Cys residue within this consensus sequence can be recognized by ectopically-expressed formylglycine generating enzyme (FGE),which converts the Cys to a formylglycine (fGly) residue. The latter bears an aldehyde functional group that serves as a chemical handle for subsequent conjugation. RESULTS The yield of Cys conversion to fGly during protein production can be variable and is highly dependent on culture conditions. We set out to achieve consistently high yields by modulating culture conditions to maximize FGE activity within the cell. We recently showed that FGE is a copper-dependent oxidase that binds copper in a stoichiometric fashion and uses it to activate oxygen,driving enzymatic turnover. Building upon that work,here we show that by supplementing cell culture media with copper we can routinely reach high yields of highly converted protein. We demonstrate that cells incorporate copper from the media into FGE,which results in increased specific activity of the enzyme. The amount of copper required is compatible with large scale cell culture,as demonstrated in fed-batch cell cultures with antibody titers of 5 g textperiodcentered L(-1),specific cellular production rates of 75 pg textperiodcentered cell(-1) textperiodcentered d(-1),and fGly conversion yields of 95-98 %. CONCLUSIONS We describe a process with a high yield of site-specific formylglycine (fGly) generation during monoclonal antibody production in CHO cells. The conversion of Cys to fGly depends upon the activity of FGE,which can be ensured by supplementing the culture media with 50 uM copper(II) sulfate. View Publication

The generation of human induced pluripotent stem cells (hiPSC) from somatic cells has enabled the possibility to provide patient-specific hiPSC for cell-based therapy,drug discovery,and other translational applications. Two major obstacles in using hiPSC for clinical application reside in the risk of genomic modification when they are derived with viral transgenes and risk of teratoma formation if undifferentiated cells are engrafted. In this study,we report the generation of footprint-free" hiPSC-derived astrocytes. These are efficiently generated� View Publication

Clinical trials are currently used to test therapeutic efficacies for lung cancer,infections and diseases. Animal models are also used as surrogates for human disease. Both approaches are expensive and time-consuming. The utility of human biospecimens as models is limited by specialized tissue processing methods that preserve subclasses of analytes (e.g. RNA,protein,morphology) at the expense of others. We present a rapid and reproducible method for the cryopreservation of viable lung tissue from patients undergoing lobectomy or transplant. This method involves the pseudo-diaphragmatic expansion of pieces of fresh lung tissue with cryoprotectant formulation (pseudo-diaphragmatic expansion-cryoprotectant perfusion or PDX-CP) followed by controlled-rate freezing in cryovials. Expansion-perfusion rates,volumes and cryoprotectant formulation were optimized to maintain tissue architecture,decrease crystal formation and increase long-term cell viability. Rates of expansion of 4 cc/min or less and volumes ranging from 0.8-1.2 × tissue volume were well-tolerated by lung tissue obtained from patients with chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis,showing minimal differences compared to standard histopathology. Morphology was greatly improved by the PDX-CP procedure compared to simple fixation. Fresh versus post-thawed lung tissue showed minimal differences in histology,RNA integrity numbers and post-translational modified protein integrity (2-dimensional differential gel electrophoresis). It was possible to derive numerous cell types,including alveolar epithelial cells,fibroblasts and stem cells,from the tissue for at least three months after cryopreservation. This new method should provide a uniform,cost-effective approach to the banking of biospecimens,with versatility to be amenable to any post-acquisition process applicable to fresh tissue samples. View Publication

Predicting the absorption of orally administered drugs is crucial to drug development. Current in vitro models lack physiological relevance,robustness,and reproducibility,thus hindering reliable predictions. In this study,we developed a reproducible and robust culture method to generate a human intestinal organoid-derived monolayer model that can be applied to study drug absorption through a step-by-step approach. Our model showed similarity to primary enterocytes in terms of the drug absorption-related gene expression profile,tight barrier function,tolerability toward artificial bile juice,drug transporter and metabolizing enzyme function,and nuclear receptor activity. This method can be applied to organoids derived from multiple donors. The permeability of launched 19 drugs in our model demonstrated a correlation with human Fa values,with an R 2 value of 0.88. Additionally,by combining the modeling and simulation approaches,the estimated FaFg values for seven out of nine drugs,including CYP3A substrates,fell within 1.5 times the range of the human FaFg values. Applying this method to the drug discovery process might bridge the gap between preclinical and clinical research and increase the success rates of drug development. View Publication

Cardiovascular progenitor cells (CPCs) expressing the ISL1-LIM-homeodomain transcription factor contribute developmentally to cardiomyocytes in all 4 chambers of the heart. Here,we show that ISL1-CPCs can be applied to myocardial regeneration following injury. We used a rapid 3D methylcellulose approach to form murine and human ISL1-CPC spheroids that engrafted after myocardial infarction in murine hearts,where they differentiated into cardiomyocytes and endothelial cells,integrating into the myocardium and forming new blood vessels. ISL1-CPC spheroid-treated mice exhibited reduced infarct area and increased blood vessel formation compared with control animals. Moreover,left ventricular (LV) contractile function was significantly better in mice transplanted with ISL1-CPCs 4 weeks after injury than that in control animals. These results provide proof-of-concept of a cardiac repair strategy employing ISL1-CPCs that,based on our previous lineage-tracing studies,are committed to forming heart tissue,in combination with a robust methylcellulose spheroid-based delivery approach. View Publication

Directed evolution is a process of mutation and artificial selection to breed biomolecules with new or improved activity. Directed evolution platforms are primarily prokaryotic or yeast-based,and stable mammalian systems have been challenging to establish and apply. To this end,we develop PROTein Evolution Using Selection (PROTEUS),a platform that uses chimeric virus-like vesicles to enable extended mammalian directed evolution campaigns without loss of system integrity. This platform is stable and can generate sufficient diversity for directed evolution in mammalian systems. Using PROTEUS,we alter the doxycycline responsiveness of tetracycline-controlled transactivators,generating a more sensitive TetON-4G tool for gene regulation with mammalian-specific adaptations. PROTEUS is also compatible with intracellular nanobody evolution,and we use it to evolve a DNA damage-responsive anti-p53 nanobody. Overall,PROTEUS is an efficient and stable platform to direct evolution of biomolecules within mammalian cells. Subject terms: Synthetic biology,Synthetic biology,Molecular evolution,Next-generation sequencing View Publication

There are several different technical approaches to the isolation of hematopoietic stem cells (HSCs) with long-term repopulating ability,but these have problems in terms of yield,complexity,or cell viability. Simpler strategies for HSC isolation are needed. We have enriched primitive hematopoietic progenitors from murine bone marrow of mice from different genetic backgrounds by lineage depletion followed by selection of cells with high aldehyde dehydrogenase activity using the Aldefluor reagent (BD Biosciences,Oxford,U.K.). Lin- ALDH(bright) cells comprised 26.8 +/- 1.0% of the total Lin- population of C57BL6 mice,and 23.5 +/- 1.0% of the Lin- population of BALB/c mice expressed certain cell-surface markers typical of primitive hematopoietic progenitors. In vitro hematopoietic progenitor function was substantially higher in the Lin- ALDH(bright) population compared with the Lin- ALDH(low) cells. These cells have higher telomerase activity and the lowest percentage of cells in S phase. These data strongly suggest that progenitor enrichment from Lin- cells on the basis of ALDH is a valid method whose simplicity of application makes it advantageous over conventional separations. View Publication