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

The production of red blood cells is tightly regulated by erythropoietin (Epo). The phosphoinositide 3-kinase (PI 3-kinase) pathway was previously shown to be activated in response to Epo. We studied the role of this pathway in the control of Epo-induced survival and proliferation of primary human erythroid progenitors. We show that phosphoinositide 3 (PI 3)-kinase associates with 4 tyrosine-phosphorylated proteins in primary human erythroid progenitors,namely insulin receptor substrate-2 (IRS2),Src homology 2 domain-containing inositol 5'-phosphatase (SHIP),Grb2-associated binder-1 (Gab1),and the Epo receptor (EpoR). Using different in vitro systems,we demonstrate that 3 alternative pathways independently lead to Epo-induced activation of PI 3-kinase and phosphorylation of its downstream effectors,Akt,FKHRL1,and P70S6 kinase: through direct association of PI 3-kinase with the last tyrosine residue (Tyr479) of the Epo receptor (EpoR),through recruitment and phosphorylation of Gab proteins via either Tyr343 or Tyr401 of the EpoR,or through phosphorylation of IRS2 adaptor protein. The mitogen-activated protein (MAP) kinase pathway was also activated by Epo in erythroid progenitors,but we found that this process is independent of PI 3-kinase activation. In erythroid progenitors,the functional role of PI 3-kinase was both to prevent apoptosis and to stimulate cell proliferation in response to Epo stimulation. Finally,our results show that PI 3-kinase-mediated proliferation of erythroid progenitors in response to Epo occurs mainly through modulation of the E3 ligase SCF(SKP2),which,in turn,down-regulates p27(Kip1) cyclin-dependent kinase (CDK) inhibitor via proteasome degradation. View Publication

Recombinant human GM-CSF has been expressed as a fusion protein in E. coli in the form of inclusion bodies. Using denaturing agents,acid cleavage and sulfitolysis,the biologically inactive GM-CSF protein could be highly purified and additionally renaturated under suitable reoxidizing conditions. The thorough repair of the two disulfide bridges could be confirmed by sequencing fragments obtained by tryptic digestion. Refolding of the molecule has been studied by CD spectrometry and identity by Western blotting and SDS-PAGE analysis. As could be demonstrated,full biological activity (colony-forming assay with fresh human bone marrow cells) was restored during renaturation of the GM-CSF protein. Further proof of biological equivalence of the E. coli-derived protein with a yeast-derived biologically active rh GM-CSF has been published elsewhere. View Publication

Modeling tissues and organs using conventional 2D cell cultures is problematic as the cells rapidly lose their in vivo phenotype. In microfluidic bioreactors the cells reside in microstructures that are continuously perfused with cell culture medium to provide a dynamic environment mimicking the cells natural habitat. These micro scale bioreactors are sometimes referred to as organs-on-chips and are developed in order to improve and extend cell culture experiments. Here,we describe the two manufacturing techniques photolithography and soft lithography that are used in order to easily produce microfluidic bioreactors. The use of these bioreactors is exemplified by a toxicity assessment on 3D clustered human pluripotent stem cells (hPSC)-derived cardiomyocytes by beating frequency imaging. View Publication

Current Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using short-read sequencing strategies resolve expressed Ab transcripts with limited resolution of the C region. In this article,we present the near-full-length AIRR-seq (FLAIRR-seq) method that uses targeted amplification by 5' RACE,combined with single-molecule,real-time sequencing to generate highly accurate (99.99%) human Ab H chain transcripts. FLAIRR-seq was benchmarked by comparing H chain V (IGHV),D (IGHD),and J (IGHJ) gene usage,complementarity-determining region 3 length,and somatic hypermutation to matched datasets generated with standard 5' RACE AIRR-seq using short-read sequencing and full-length isoform sequencing. Together,these data demonstrate robust FLAIRR-seq performance using RNA samples derived from PBMCs,purified B cells,and whole blood,which recapitulated results generated by commonly used methods,while additionally resolving H chain gene features not documented in IMGT at the time of submission. FLAIRR-seq data provide,for the first time,to our knowledge,simultaneous single-molecule characterization of IGHV,IGHD,IGHJ,and IGHC region genes and alleles,allele-resolved subisotype definition,and high-resolution identification of class switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes,FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles,28 (87%) of which were previously uncharacterized. Together,these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV,IGHD,IGHJ,and IGHC gene diversity for the most comprehensive view of bulk-expressed Ab repertoires to date. View Publication

Mesenchymal stem cells (MSCs) are promising for cell-based therapies targeting a wide range of diseases. However,challenges in translating MSC-based therapies to clinical applications necessitate standardized protocols following Good Manufacturing Practices (GMP) guidelines. This study aimed at developing GMP-complained protocols for FPMSCs isolation and manipulation,necessary for translational research,by (1) optimize culture of MSCs derived from an infrapatellar fat pad (FPMSC) condition through animal-free media comparison and (2) establish feasibility of MSC isolation,manufacturing and storage under GMP-compliance (GMP-FPMSC). FPMSCs from three different patients were isolated following established protocols and the efficacy of two animal component-free media formulations in the culturing media were evaluated. The impact of different media formulations on cell proliferation,purity,and potency of MSCs was evaluated through doubling time,colony forming unit assay,and percentage of MSCs,respectively. Furthermore,the isolation and expansion of GMP-FPMSCs from four additional donors were optimized and characterized at each stage according to GMP requirements. Viability and sterility were checked using Trypan Blue and Bact/Alert,respectively,while purity and identity were confirmed using Endotoxin,Mycoplasma assays,and Flow Cytometry. The study also included stability assessments post-thaw and viability assessment to determine the shelf-life of the final GMP-FPMSC product. Statistical analyses were conducted using one-way ANOVA with Tukey’s Multiple Comparisons. The study demonstrated that FPMSCs exhibited enhanced proliferation rates when cultured in MSC-Brew GMP Medium compared to standard MSC media. Cells cultured in this media showed lower doubling times across passages,indicating increased proliferation. Additionally,higher colony formation in FPMSCs cultured in MSC-Brew GMP Medium were observed,supporting enhanced potency. Data from our GMP validation,including cells from 4 different donors,showed post-thaw GMP-FPMSC maintained stem cell marker expression and all the specifications required for product release,including > 95% viability (> 70% is required) and sterility,even after extended storage (up to 180 days),demonstrating the reproducibility and potential of GMP-FPMSCs for clinical use as well as the robustness of the isolation and storage protocols. The study underscores the feasibility of FPMSCs for clinical uses under GMP conditions and emphasizes the importance of optimized culture protocols to improve cell proliferation and potency in MSC-based therapies. The online version contains supplementary material available at 10.1186/s12860-025-00539-7. View Publication

Tumor complexity and intratumor heterogeneity contribute to subclonal diversity. Despite advances in next-generation sequencing (NGS) and bioinformatics,detecting rare mutations in primary tumors and metastases contributing to subclonal diversity is a challenge for precision genomics. Here,in order to identify rare mutations,we adapted a recently described epithelial reprograming assay for short-term propagation of epithelial cells from primary and metastatic tumors. Using this approach,we expanded minor clones and obtained epithelial cell-specific DNA/RNA for quantitative NGS analysis. Comparative Ampliseq Comprehensive Cancer Panel sequence analyses were performed on DNA from unprocessed breast tumor and tumor cells propagated from the same tumor. We identified previously uncharacterized mutations present only in the cultured tumor cells,a subset of which has been reported in brain metastatic but not primary breast tumors. In addition,whole-genome sequencing identified mutations enriched in liver metastases of various cancers,including Notch pathway mutations/chromosomal inversions in 5/5 liver metastases,irrespective of cancer types. Mutations/rearrangements in FHIT,involved in purine metabolism,were detected in 4/5 liver metastases,and the same four liver metastases shared mutations in 32 genes,including mutations of different HLA-DR family members affecting OX40 signaling pathway,which could impact the immune response to metastatic cells. Pathway analyses of all mutated genes in liver metastases showed aberrant tumor necrosis factor and transforming growth factor signaling in metastatic cells. Epigenetic regulators including KMT2C/MLL3 and ARID1B,which are mutated in {\textgreater}50{\%} of hepatocellular carcinomas,were also mutated in liver metastases. Thus,irrespective of cancer types,organ-specific metastases may share common genomic aberrations. Since recent studies show independent evolution of primary tumors and metastases and in most cases mutation burden is higher in metastases than primary tumors,the method described here may allow early detection of subclonal somatic alterations associated with metastatic progression and potentially identify therapeutically actionable,metastasis-specific genomic aberrations. View Publication

BACKGROUND Mammalian target of rapamycin complex 1 (mTORC1) is a protein kinase that relays nutrient availability signals to control numerous cellular functions including autophagy,a process of cellular self-eating activated by nutrient depletion. Addressing the therapeutic potential of modulating mTORC1 signaling and autophagy in human disease requires active chemicals with pharmacologically desirable properties. METHODOLOGY/PRINCIPAL FINDINGS Using an automated cell-based assay,we screened a collection of {\textgreater}3,500 chemicals and identified three approved drugs (perhexiline,niclosamide,amiodarone) and one pharmacological reagent (rottlerin) capable of rapidly increasing autophagosome content. Biochemical assays showed that the four compounds stimulate autophagy and inhibit mTORC1 signaling in cells maintained in nutrient-rich conditions. The compounds did not inhibit mTORC2,which also contains mTOR as a catalytic subunit,suggesting that they do not inhibit mTOR catalytic activity but rather inhibit signaling to mTORC1. mTORC1 inhibition and autophagosome accumulation induced by perhexiline,niclosamide or rottlerin were rapidly reversed upon drug withdrawal whereas amiodarone inhibited mTORC1 essentially irreversibly. TSC2,a negative regulator of mTORC1,was required for inhibition of mTORC1 signaling by rottlerin but not for mTORC1 inhibition by perhexiline,niclosamide and amiodarone. Transient exposure of immortalized mouse embryo fibroblasts to these drugs was not toxic in nutrient-rich conditions but led to rapid cell death by apoptosis in starvation conditions,by a mechanism determined in large part by the tuberous sclerosis complex protein TSC2,an upstream regulator of mTORC1. By contrast,transient exposure to the mTORC1 inhibitor rapamycin caused essentially irreversible mTORC1 inhibition,sustained inhibition of cell growth and no selective cell killing in starvation. CONCLUSION/SIGNIFICANCE The observation that drugs already approved for human use can reversibly inhibit mTORC1 and stimulate autophagy should greatly facilitate the preclinical and clinical testing of mTORC1 inhibition for indications such as tuberous sclerosis,diabetes,cardiovascular disease and cancer. View Publication

Genetic variants in the X‐chromosomal gene coding for the calcium−/calmodulin‐dependent serine protein kinase (CASK) are associated with a neurodevelopmental disorder. CASK is a member of the membrane‐associated guanylate kinase (MAGUK) family of proteins. It acts as a scaffold at presynaptic sites,as a regulator of the transport of glutamate receptors,and as a transcriptional regulator. The PDZ domain of CASK has been reported to bind to presynaptic cell adhesion molecules such as Neurexin1‐3,CNTNAP2,SynCAM and SALM1. Structural analyses of related MAGUKs indicate that the canonical SH3 and GK domains combine with the PDZ domain to form the so‐called PSG supramodule. Conserved aromatic residues (Y723 and W914) flanking the GK domain contribute to the formation of a dimeric structure of two PSG modules,which is required for high‐affinity binding to the type 2 PDZ ligand motif of,for example,Neurexin. Here we identify previously uncharacterized patient variants in the SH3 domain of CASK (I672V; P673L),which alter the intermolecular binding pocket for Y723. Both variants interfere with the binding of Neurexin‐1β,in a manner similar to the previously reported Y723C variant. Intriguingly,binding to the type 1 PDZ ligand of the cell adhesion molecule SALM1 is not altered. Using a set of highly selective patient variants,we show that the binding of SALM1 to CASK is actually not mediated by the CASK PDZ domain or the PSG supramodule,but depends on other type 1 PDZ domain‐containing proteins such as SAP97 and Veli,which associate with CASK through its L27 domains. Our data underline the relevance of an intact PSG tandem of CASK for human health. View Publication

A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens,or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here,to yield functional human haematopoietic stem cells,we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG,HOXA5,HOXA9,HOXA10,LCOR,RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid,B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders. View Publication

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage embryos. They can maintain an undifferentiated state indefinitely and can differentiate into derivatives of all three germ layers,namely ectoderm,endoderm and mesoderm. Although much progress has been made in the propagation and differentiation of ES cells,induction of photoreceptors has generally required coculture with or transplantation into developing retinal tissue. Here,we describe a protocol for generating retinal cells from ES cells by stepwise treatment with defined factors. This method preferentially induces photoreceptor and retinal pigment epithelium (RPE) cells from mouse and human ES cells. In our protocol,differentiation of RPE and photoreceptors from mouse ES cells requires 28 d and the differentiation of human ES cells into mature RPE and photoreceptors requires 120 and 150 d,respectively. This differentiation system and the resulting pluripotent stem cell-derived retinal cells will facilitate the development of transplantation therapies for retinal diseases,drug testing and in vitro disease modeling. It will also improve our understanding of the development of the central nervous system,especially the eye. View Publication

The extrafollicular (EF) plasmablast response to self-antigens that contain Toll-like receptor (TLR) ligands is prominent in murine lupus models and some bacterial infections,but the inhibitors and activators involved have not been fully delineated. Here,we used two conventional dendritic cell (cDC) depletion systems to investigate the role of cDCs on a classical TLR-dependent autoreactive EF response elicited in rheumatoid-factor B cells by DNA-containing immune complexes. Contrary to our hypothesis,cDC depletion amplified rather than dampened the EF response in Fas-intact but not Fas-deficient mice. Further,we demonstrated that cDC-dependent regulation requires Fas and Fas ligand (FasL) expression by T cells,but not Fas expression by B cells. Thus,cDCs activate FasL-expressing T cells that regulate Fas-expressing extrafollicular helper T (Tefh) cells. These studies reveal a regulatory role for cDCs in B cell plasmablast responses and provide a mechanistic explanation for the excess autoantibody production observed in Fas deficiency. View Publication

Acute myeloid leukemia (AML) is a heterogeneous group of aggressive bone marrow cancers arising from transformed hematopoietic stem and progenitor cells (HSPC). Therapy-related AML and MDS (t-AML/MDS) comprise a subset of AML cases occurring after exposure to alkylating chemotherapy and/or radiation and are associated with a very poor prognosis. Less is known about the pathogenesis and disease-initiating/leukemia stem cell (LSC) subpopulations of t-AML/MDS compared to their de novo counterparts. Here,we report the development of mouse models of t-AML/MDS. First,we modeled alkylator-induced t-AML/MDS by exposing wild type adult mice to N-ethyl-N-nitrosurea (ENU),resulting in several models of AML and MDS that have clinical and pathologic characteristics consistent with human t-AML/MDS including cytopenia,myelodysplasia,and shortened overall survival. These models were limited by their inability to transplant clinically aggressive disease. Second,we established three patient-derived xenograft models of human t-AML. These models led to rapidly fatal disease in recipient immunodeficient xenografted mice. LSC activity was identified in multiple HSPC subpopulations suggesting there is no canonical LSC immunophenotype in human t-AML. Overall,we report several new t-AML/MDS mouse models that could potentially be used to further define disease pathogenesis and test novel therapeutics. View Publication