The transcriptional regulator Runx2 (runt-related transcription factor 2) has essential but distinct roles in osteoblasts and chondrocytes in skeletal development. However, Runx2-mediated regulatory mechanisms underlying the distinctive programming of osteoblasts and chondrocytes are not well understood. Here, we perform an integrative analysis to investigate Runx2-DNA binding and chromatin accessibility ex vivo using neonatal osteoblasts and chondrocytes. We find that Runx2 engages with cell-type-distinct chromatin-accessible regions, potentially interacting with different combinations of transcriptional regulators, forming cell-type-specific hotspots, and potentiating chromatin accessibility. Genetic analysis and direct cellular reprogramming studies suggest that Runx2 is essential for establishment of chromatin accessibility in osteoblasts. Functional enhancer studies identify an Sp7 distal enhancer driven by Runx2-dependent binding and osteoblast-specific chromatin accessibility, contributing to normal osteoblast differentiation. Our findings provide a framework for understanding the regulatory landscape encompassing Runx2-mediated and cell-type-distinct enhancer networks that underlie the specification of osteoblasts.

Accurate EV quantitation is essential for ensuring quality, consistency, and safety during large-scale extracellular vesicle (EV) manufacturing. In the upstream phase, EV quantitation allows for the monitoring of cell culture conditions that impact EV yield and quality, while in the downstream phase, it helps to control the efficiency and purity of EV purification. Nanoparticle Tracking Analysis is the most commonly used method for EV quantification, while it faces significant limitations due to interference from nanosize contaminants like protein aggregates, especially in crude samples (e.g. cell culture media). To address this issue, we developed a highly specific and accurate ELISA assay that quantifies EVs even in crude samples. With ultra-pure EV standard samples, this assay showed reliable quantitative result of EV detection to support method development as well as in-process control of large-scale EV manufacture. The detection range of this assay is from 4.1E7 to 3E10 EVs/mL, with an LOD of 1.04E7 EVs/mL and an LOQ of 3.21E7 EVs/mL. We therefore developed this assay into a testing kit and demonstrated that this EV quantification ELISA kit is capable of ensuring minimal interference from impurities and supporting the process development and in-process control in EV production.

1. Extracellular Vesicles (EVs) are nanoscale to microscale membranous vesicles released by cells. They are vital for intercellular communications in physiological and pathological conditions, offering immense potential in diagnostics and therapeutical applications due to their ability to transfer diverse biological cargos between cells. While research of EVs is continuously gaining popularity in academia, large-scale manufacturing aiming for clinical applications remains challenging. Herein, we introduce a novel large-scale EV production strategy, including an upstream process with fed-batch suspension HEK293 cell culture, and a downstream process with chromatographic purification for ultra-pure EVs. Such promising EV production strategy enables the potential capability of large scale GMP manufacture of EVs for clinical applications.