Biology subjects

Magdy, T.

Publications and source records attributed to Magdy, T..

3 recordsLinked to original sources

bioRxiv2024

CD14 Blockade Does Not Improve Outcomes of Deep Vein Thrombosis Following Inferior Vena Cava Stenosis in Mice

BackgroundNeutrophil-mediated persistent inflammation and neutrophil extracellular trap formation (NETosis) promote deep vein thrombosis (DVT). CD14, a co-receptor for toll-like receptor 4 (TLR4), is actively synthesized by neutrophils, and the CD14/TLR4 signaling pathway has been implicated in proinflammatory cytokine overproduction and several aspects of thromboinflammation. The role of CD14 in the pathogenesis of DVT remains unclear. ObjectiveTo determine whether CD14 blockade improves DVT outcomes. MethodsBulk RNA sequencing and proteomic analyses were performed using isolated neutrophils following inferior vena cava (IVC) stenosis in mice. DVT outcomes (IVC thrombus weight and length, thrombosis incidence, neutrophil recruitment, and NETosis) were evaluated following IVC stenosis in mice treated with a specific anti-CD14 antibody, 4C1, or control antibody. ResultsMice with IVC stenosis exhibited increased plasma levels of granulocyte colony-stimulating factor (G-CSF) along with a higher neutrophil-to-lymphocyte ratio and increased plasma levels of cell-free DNA, elastase, and myeloperoxidase. Quantitative measurement of total neutrophil mRNA and protein expression revealed distinct profiles in mice with IVC stenosis compared to mice with sham surgery. Neutrophils of mice with IVC stenosis exhibited increased inflammatory transcriptional and proteomic responses, along with increased expression of CD14. Treatment with a specific anti-CD14 antibody, 4C1, did not result in any significant changes in the IVC thrombus weight, thrombosis incidence, or neutrophil recruitment to the thrombus. ConclusionThe results of the current study are important for understanding the role of CD14 in the regulation of DVT and suggest that CD14 lacks an essential role in the pathogenesis of DVT following IVC stenosis.

pathology↗

bioRxiv2023

A Novel Transcription Factor Combination for Direct Reprogramming to a Spontaneously Contracting Human Cardiomyocyte-like State

The reprogramming of somatic cells to a spontaneously contracting cardiomyocyte-like state using defined transcription factors has proven successful in mouse fibroblasts. However, this process has been less successful in human cells, thus limiting the potential clinical applicability of this technology in regenerative medicine. We hypothesized that this issue is due to a lack of cross-species concordance between the required transcription factor combinations for mouse and human cells. To address this issue, we identified novel transcription factor candidates to induce cell conversion between human fibroblasts and cardiomyocytes, using the network-based algorithm Mogrify. We developed an automated, high-throughput method for screening transcription factor, small molecule, and growth factor combinations, utilizing acoustic liquid handling and high-content kinetic imaging cytometry. Using this high-throughput platform, we screened the effect of 4,960 unique transcription factor combinations on direct conversion of 24 patient-specific primary human cardiac fibroblast samples to cardiomyocytes. Our screen revealed the combination of MYOCD, SMAD6, and TBX20 (MST) as the most successful direct reprogramming combination, which consistently produced up to 40% TNNT2+ cells in just 25 days. Addition of FGF2 and XAV939 to the MST cocktail resulted in reprogrammed cells with spontaneous contraction and cardiomyocyte-like calcium transients. Gene expression profiling of the reprogrammed cells also revealed the expression of cardiomyocyte associated genes. Together, these findings indicate that cardiac direct reprogramming in human cells can be achieved at similar levels to those attained in mouse fibroblasts. This progress represents a step forward towards the clinical application of the cardiac direct reprogramming approach. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/532629v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@17d6539org.highwire.dtl.DTLVardef@180a0b8org.highwire.dtl.DTLVardef@1b7cb46org.highwire.dtl.DTLVardef@182618b_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIUsing network-based algorithm Mogrify, acoustic liquid handling, and high-content kinetic imaging cytometry we screened the effect of 4,960 unique transcription factor combinations. C_LIO_LIUsing 24 patient-specific human fibroblast samples we identified the combination of MYOCD, SMAD6, and TBX20 (MST) as the most successful direct reprogramming combination. C_LIO_LIMST cocktail results in reprogrammed cells with spontaneous contraction, cardiomyocyte-like calcium transients, and expression of cardiomyocyte associated genes. C_LI

cell biology↗

bioRxiv2019

Negligible-Cost and Weekend-Free Chemically Defined Human iPSC Culture

Human induced pluripotent stem cell (hiPSC) culture has become routine, yet pluripotent cell media costs, frequent media changes, and reproducibility of differentiation have remained restrictive, limiting the potential for large-scale projects. Here, we describe the formulation of a novel hiPSC culture medium (B8) as a result of the exhaustive optimization of medium constituents and concentrations, establishing the necessity and relative contributions of each component to the pluripotent state and cell proliferation. B8 eliminates 97% of the costs of commercial media, made possible primarily by the in-lab generation of three E. coli-expressed, codon-optimized recombinant proteins: an engineered form of fibroblast growth factor 2 (FGF2) with improved thermostability (FGF2-G3); transforming growth factor {beta}3 (TGF{beta}3) - a more potent TGF{beta} able to be expressed in E. coli; and a derivative of neuregulin 1 (NRG1) containing the EGF-like domain. The B8 formula is specifically optimized for fast growth and robustness at low seeding densities. We demonstrated the derivation and culture of 34 hiPSC lines in B8 as well as maintenance of pluripotency long-term (over 100 passages). This formula also allows a weekend-free feeding schedule without sacrificing growth rate or capacity for differentiation. Thus, this simple, cost-effective, and open source B8 media, will enable large hiPSC disease modeling projects such as those being performed in pharmacogenomics and large-scale cell production required for regenerative medicine.

cell biology↗