ABSTRACT
The use of human pluripotent stem cells for in vitro disease modelling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF-A or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies exceeding 80% within six days. On purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease.
Subject(s)
Cell Differentiation , Cell Lineage , Endothelial Cells/physiology , Induced Pluripotent Stem Cells/physiology , Muscle, Smooth, Vascular/physiology , Myocytes, Smooth Muscle/physiology , Animals , Becaplermin , Biomarkers/metabolism , Bone Morphogenetic Protein 4/pharmacology , Cell Differentiation/drug effects , Cell Line , Cell Lineage/drug effects , Coculture Techniques , Dose-Response Relationship, Drug , Endothelial Cells/drug effects , Endothelial Cells/enzymology , Endothelial Cells/transplantation , Gene Expression Profiling/methods , Gene Expression Regulation, Developmental , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Human Umbilical Vein Endothelial Cells/physiology , Humans , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/enzymology , Induced Pluripotent Stem Cells/transplantation , Metabolomics/methods , Mice, Inbred NOD , Mice, SCID , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/enzymology , Muscle, Smooth, Vascular/transplantation , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/enzymology , Myocytes, Smooth Muscle/transplantation , Neovascularization, Physiologic , Phenotype , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-sis/pharmacology , Time Factors , Transcription, Genetic , Transfection , Vascular Endothelial Growth Factor A/pharmacology , Wnt Signaling Pathway/drug effectsABSTRACT
Inclacumab, a novel monoclonal antibody against P-selectin in development for the treatment and prevention of atherosclerotic cardiovascular diseases, was administered in an ascending single-dose study as intravenous infusion to evaluate safety, pharmacokinetics, and pharmacodynamics. Fifty-six healthy subjects were enrolled in this randomized, double-blind placebo-controlled study. Each dose level (0.03-20 mg/kg) was investigated in separate groups of 8 subjects (6 on inclacumab, 2 on placebo). Platelet-leukocyte aggregates, free/total soluble P-selectin concentration ratio, drug concentrations, bleeding time, platelet aggregation, antibody formation, and routine laboratory parameters were measured frequently until 32 weeks. Pharmacokinetic profiles were indicative of target-mediated drug disposition. Platelet-leukocyte aggregate inhibition and soluble P-selectin occupancy showed dose dependency and were strongly correlated to inclacumab plasma concentrations, with IC50 of 740 and 4600 ng/mL, respectively. Inclacumab was well tolerated by the majority of subjects and did neither affect bleeding time nor platelet aggregation. These findings allowed the investigation of the potential beneficial therapeutic use of inclacumab in patient study.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Cardiovascular Agents/administration & dosage , P-Selectin/antagonists & inhibitors , Adult , Antibodies, Monoclonal/adverse effects , Antibodies, Monoclonal/blood , Antibodies, Monoclonal/pharmacokinetics , Bleeding Time , Cardiovascular Agents/adverse effects , Cardiovascular Agents/blood , Cardiovascular Agents/pharmacokinetics , Double-Blind Method , England , Female , Healthy Volunteers , Hemorrhage/chemically induced , Humans , Infusions, Intravenous , Male , Middle Aged , P-Selectin/immunology , Platelet Aggregation/drug effects , Predictive Value of Tests , Risk Assessment , Young AdultABSTRACT
We demonstrate the random motility (RAMOT) assay based on image correlation spectroscopy for the automated, label-free, high-throughput characterization of random cell migration. The approach is complementary to traditional migration assays, which determine only the collective net motility in a particular direction. The RAMOT assay is less demanding on image quality compared to single-cell tracking, does not require cell identification or trajectory reconstruction, and performs well on live-cell, time-lapse, phase contrast video microscopy of hundreds of cells in parallel. Effective diffusion coefficients derived from the RAMOT analysis are in quantitative agreement with Monte Carlo simulations and allowed for the detection of pharmacological effects on macrophage-like cells migrating on a planar collagen matrix. These results expand the application range of image correlation spectroscopy to multicellular systems and demonstrate a novel, to our knowledge, migration assay with little preparative effort.
Subject(s)
Cell Movement , Microscopy, Phase-Contrast/methods , Animals , Cell Line, Tumor , Cell Movement/drug effects , Collagen/pharmacology , Humans , Macrophages/cytology , Monte Carlo Method , Rats , Spectrometry, Fluorescence , Stochastic ProcessesABSTRACT
BACKGROUND AND OBJECTIVE: Elevated levels of platelet-leukocyte aggregates (PLAs) have been reported in several cardiovascular diseases and suggested to contribute to disease pathology. Our aim was to characterize the effects of inclacumab, a novel human anti-P-selectin antibody, on the interactions between leukocytes and platelets in preclinical and clinical studies. EXPERIMENTAL APPROACHES: Dual-label flow cytometry was used to detect the effect of inclacumab on agonist-induced platelet-leukocyte/platelet-monocyte aggregates in cynomolgus monkeys and humans, following ex vivo and in vivo administration. Platelet-dependent leukocyte activation and leukocyte adhesion to a platelet monolayer were also investigated after ex vivo administration of inclacumab to human blood. RESULTS: Treatment of cynomolgus monkeys with inclacumab profoundly inhibited thrombin receptor-activating peptide (TRAP) or adenosine diphosphate (ADP)-induced PLAs with an IC50 (<2 µg/mL) similar to the in vitro spiking experiments. Maximal inhibition of PLAs persisted for ≥28 days following single dose of inclacumab. In human blood, inclacumab was about 2-fold more potent in inhibiting TRAP-induced PLAs (IC50: 0.7 µg/mL) compared to monkeys. PLA formation was suppressed independently of the inducing platelet agonist. Inclacumab also inhibited the activation of the leukocyte integrin Mac-1 and leukocyte adhesion to a platelet monolayer under flow conditions. In clinical studies, inclacumab inhibited TRAP-induced PLA formation in a dose-dependent manner following single and multiple dose administration to healthy volunteers. It also reduced elevated circulating PLA levels in patients with peripheral arterial disease. CONCLUSION: By inhibiting platelet-leukocyte interactions, demonstrated in multiple preclinical and clinical studies, inclacumab may provide an effective treatment for cardiovascular diseases.
