miR-126 binds to the 3'UTR of EGFL7

Endothelial development is an important process for generating new blood vessels during embryonic development as well as in the adult life. During embryogenesis, the endothelial network is established and this network is then remodelled in wound repair and cancer progression, e.g. Development of the endothelium is a closely controlled process, regulated by many different factors and pathways that work together to form the complex endothelial network. By identifying these factors and the interplay between them we not only shed light on this fundamental developmental process but also open possibilities that this knowledge could be used for therapeutic purposes. Most notably, the progression of cancer and metastasis is dependent on the development of new blood vessels and controlling this process could be vital in cancer treatments. Preliminary studies of the research group have suggested a connection between the BMP-Smad1/5 signaling pathway, EGFL7 and miR-126 in endothelial development. The aim of this project was to determine whether miR-126 could bind to the 3’UTR sequences of EGFL7, TMEM100 and Smad6 and therefore demonstrate a connection between BMP-Smad1/5 signaling pathway, EGFL7 and miR-126. For this purpose we created a pISO reporter plasmid that contained the 3’UTR sequences of EGFL7, TMEM100 and Smad6, respectively, behind a luciferase reporter gene. HEK cells that overexpressed miR-126 were then transfected with the reporter plasmids and binding was assessed according to luciferase activity. The results of this study provide evidence that miR-126 does bind to the 3’UTR sequences of EGFL7 and Smad6 but does not bind to the 3’UTR sequence of TMEM100

A protocol for the differentiation of human embryonic stem cells into midbrain dopaminergic neurons

Summary: Here, we present a protocol for the generation of functional midbrain dopaminergic (mDA) neurons from human embryonic stem cells (hESCs), which mimics the development of the human ventral midbrain. We describe steps for hESC proliferation, induction of mDA progenitors, freezing stocks of mDA progenitors as an intermediate starting point to reduce the time to make mDA neurons, and maturation of mDA neurons. The entire protocol is feeder-free and uses chemically defined materials.For complete details on the use and execution of this protocol, please refer to Nishimura et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics