<em>In Vitro</em> Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

Abstract

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides transporting oxygen and nutrients to ensure fetal growth, fetal circulation controls many crucial developmental events taking place within the endothelial layer through mechanical cues. Biomechanical signals induce blood vessel structural changes, establish arteriovenous specification, and control the development of hematopoietic stem cells.The inaccessibility of the developing tissues limits the understanding of the role of circulation in early human development; therefore, in vitro models are pivotal tools for the study of vessel mechanobiology. This paper describes a protocol to differentiate endothelial cells from human induced pluripotent stem cells and their subsequent seeding into a fluidic device to study their response to mechanical cues. This approach allows for long-term culture of endothelial cells under mechanical stimulation followed by retrieval of the endothelial cells for phenotypical and functional characterization.The in vitro model established here will be instrumental to elucidate the intracellular molecular mechanisms that transduce the signaling mediated by mechanical cues, which ultimately orchestrate vessel development during human fetal life.

A novel human pluripotent stem cell-based gene activation system identifies IGFBP2 as a mediator in the production of hematopoietic progenitors in vitro

“…Following the observation that IGFBP2 supports haematopoietic progenitor differentiation in vitro from human iPSCs we performed a time course single-cell RNA experiment of SFCi55 iPSCs differentiated in its presence. We isolated CD34 + cells and plated them in EHT culture on laminin 28 , rather than on OP9 support, to assess exclusively the specific effects of IGFBP2- on differentiating iPSCs. FAC-sorted single/live cells from day 10 and 13 in the presence and absence of IGFBP2 were subjected to single-cell RNAseq.…”

“…Laminin EHT culture was performed as previously described 28 . Briefly, 24 well plates were coated for at least 2 hours with recombinant human Laminin-521 (Thermo-Fisher).…”