Project description:We report a novel technique to reprogram human fibroblasts into endothelial and smooth muscle cells using partial iPSC reprogramming and chemically defined media. Using appropriate media conditions for differentiation of human pluripotent cells to CD34+ vascular progenitor cells, we show that temporary expression of pluripotent transcription factors and treatment with chemically-defined media, will induce differentiation of human fibroblasts to CD34+ vascular progenitor cells. Sorted CD34+ cells can then be directed to differentiate into vascular endothelial cells expressing a variety of smooth muscle markers. We have assessed the global DNA methylation (Illumina Infinium HD 450K DNA methylationBeadChips) and transcriptional (Illumina HT12v4 Gene Expression Bead Array) profiles of transdifferentiated endothelial cells and smooth muscle, human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) differentiated CD34+ angioblasts, hESCs, hiPSC, primary smooth muscle and primary human umbilical vein endothelial cells using microarrays.
Project description:We report a novel technique to reprogram human fibroblasts into endothelial and smooth muscle cells using partial iPSC reprogramming and chemically defined media. Using appropriate media conditions for differentiation of human pluripotent cells to CD34+ vascular progenitor cells, we show that temporary expression of pluripotent transcription factors and treatment with chemically-defined media, will induce differentiation of human fibroblasts to CD34+ vascular progenitor cells. Sorted CD34+ cells can then be directed to differentiate into vascular endothelial cells expressing a variety of smooth muscle markers. We have assessed the global DNA methylation (Illumina Infinium HD 450K DNA methylationBeadChips) and transcriptional (Illumina HT12v3 and HT12v4 Gene Expression Bead Array) profiles of transdifferentiated endothelial cells and smooth muscle, human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) differentiated CD34+ angioblasts, hESCs, hiPSC, primary smooth muscle and primary human umbilical vein endothelial cells using microarrays.
Project description:Smooth muscle cells and pericytes are mural cells. Pericytes can differentiate into myofibroblasts, chondrocytes, vascular smooth muscle cells, and adipocytes, marking them as a distinct progenitor population. Our goal was to molecularly define the progenitor cell populations in human adipose tissues and test the adipogenic potential of human mural cells. Single-cell transcriptomic profiling of primary human cultured vascular smooth muscle cells (VSMCs) treated with either lipogenic media or VSMC differentiation media was conducted to elucidate signals associated with the lipogenic phenotype observed in these primary vascular cells.
Project description:The response of primary human endothelial (ECs) and vascular smooth muscle cells (VSMCs) to TiO2 nanotube arrays is studied through gene expression analysis. Microarrays revealed that nanotubes enhanced EC proliferation and motility, decreased VSMC proliferation, and decreased expression of molecules involved in inflammation and coagulation in both cell types. Network generated from significantly affected genes suggests that cells may be sensing nanotopographical cues via pathways previously implicated in sensing shear stress. DNA microarrays were used to analyze the response of primary human endothelial (ECs) and vascular smooth muscle cells (VSMCs) to nanotopographical TiO2 surfaces. The experiment incorporated a 1 color design and used Agilent arrays that contained roughly 44,00 60mer probes that provide complete coverage of the human genome. 19 arrays were hybridized and represent 4 biological replicates for each group ( with the exception of group VSMC-NT30, which has 3 biological replicates). Data was analyzed separately for each cell type ( EC or VSMC). Gene expression comparisons were made between control cells grown on flat titanium (Ti) and cells grown on either 30nm nanotubes (NT30) or 100nm nantubes ( NT-100).
Project description:To identify the origin of circular RNAs (circRNAs) in human atherosclerotic plaques, we analyzed the expression levels of circRNAs identified during the differentiation of human pluripotent stem cells (hiPSCs) into vascular endothelial cells (ECs) and smooth muscle cells (SMCs) in carotid biopsies by single-cell RNA sequencing.
Project description:Nearly 20% of hospitalized patients diagnosed with severe SARS-CoV-2 infection are at risk for thromboembolic events. We developed a model of SARS-CoV-2 infection using human-induced pluripotent stem cell-derived endothelial cells, pericytes, and smooth muscle cells to recapitulate the vascular pathology associated with SARS-CoV-2 exposure. Our results demonstrate that perivascular cells, particularly smooth muscle cells (SMCs), are a specifically susceptible vascular target for SARS-CoV-2 infection. Utilizing RNA sequencing, we characterized the transcriptomic changes accompanying SARS-CoV-2 infection of SMCs, and endothelial cells (ECs). We observed that infected human SMCs shift to a pro-inflammatory state and increase the expression of key mediators of the coagulation cascade. Further, we showed human ECs exposed to the secretome of infected SMCs produce hemostatic factors that can contribute to vascular dysfunction, despite not being susceptible to direct infection. The findings here recapitulate observations from patient sera in human COVID-19 patients and provide mechanistic insight into the unique vascular implications of SARS-CoV-2 infection at a cellular level.
Project description:Nearly 20% of hospitalized patients diagnosed with severe SARS-CoV-2 infection are at risk for thromboembolic events. We developed a model of SARS-CoV-2 infection using human-induced pluripotent stem cell-derived endothelial cells, pericytes, and smooth muscle cells to recapitulate the vascular pathology associated with SARS-CoV-2 exposure. Our results demonstrate that perivascular cells, particularly smooth muscle cells (SMCs), are a specifically susceptible vascular target for SARS-CoV-2 infection. Utilizing RNA sequencing, we characterized the transcriptomic changes accompanying SARS-CoV-2 infection of SMCs, and endothelial cells (ECs). We observed that infected human SMCs shift to a pro-inflammatory state and increase the expression of key mediators of the coagulation cascade. Further, we showed human ECs exposed to the secretome of infected SMCs produce hemostatic factors that can contribute to vascular dysfunction, despite not being susceptible to direct infection. The findings here recapitulate observations from patient sera in human COVID-19 patients and provide mechanistic insight into the unique vascular implications of SARS-CoV-2 infection at a cellular level.
Project description:Platelet-derived growth factor (PDGF) signalling and the subsequent activation of the calcium ion channel, ORAI1 are critical drivers of pathological remodelling of native vascular smooth muscle cells to proliferative state, which is a process associated with various vascular diseases. This study aims to reveal transcriptional networks altered following ORAI1 inhibition in vascular smooth muscle cells. To study the effect of ORAI1 inhibition on VSMC biology, we performed RNA-Seq analysis of PDGF-stimulated primary human aortic smooth muscle cells treated with either ORAI1 inhibitor, (n=4) or with vehicle (n=4), and investigated the effect of ORAI1 inhibition on the transcriptional response of cells.
Project description:The prevalence of cardiovascular disease varies with sex, and the impact of intrinsic sex-based differences on vasculature is not well understood. Animal models can provide important insight into some aspects of human biology, however not all discoveries in animal systems translate well to humans. To explore the impact of chromosomal sex on proteomic phenotypes, we used iPSC-derived vascular smooth muscle cells from healthy donors of both sexes to identify sex-based proteomic differences and their possible effects on cardiovascular pathophysiology. Our analysis confirmed that differentiated cells have a proteomic profile more similar to healthy primary aortic smooth muscle than iPSCs. We also identified sex-based differences in iPSC-derived vascular smooth muscle in pathways related to ATP binding, glycogen metabolic process, and cadherin binding as well as multiple proteins relevant to cardiovascular pathophysiology and disease. Additionally, we explored the role of autosomal and sex chromosomes in protein regulation, identifying that proteins on autosomal chromosomes also show sex-based regulation that may affect the protein expression of proteins from autosomal chromosomes. This work supports the biological relevance of iPSC-derived vascular smooth muscle cells as a model for disease, and further exploration of the pathways identified here can lead to the discovery of sex-specific pharmacological targets for cardiovascular disease.