Name: Video: Seeding CD34+ Cells into Fluidic Chips
Uploaded: 2023-07-28T12:30:00
Duration: 1 min 16 s
Description: 271 Views. Seeding CD34+ Cells into Fluidic Chips

seeding cd34+ cells into fluidic chips

Differentiation and Mechanical Stimulation of hiPSC-Derived Endothelial Cells

During embryonic development, the heart is the first organ to establish functionality1, with detectable contractions from the earliest stage of endocardial tube formation2. Circulation, along with the mechanical cues mediated by the flow of blood within the vessel, controls many crucial aspects of early development. Prior to fetal circulation establishment, the vasculature is organized into a primary capillary plexus; upon cardiac functioning, this plexus reorganizes into venous and arterial vasculature3. The role of mechanical cues in arteriovenous specification is reflected by the pan-endothelial expression of arterial and venous markers before blood flow initiation4.
Hemodynamic forces not only control the development of the vasculature itself but also play a fundamental role in the control of blood cell formation. Hematopoietic stem and progenitor cells (HSPCs) emerge from specialized endothelial cells called hemogenic endothelium5,6,7,8, present in different anatomical regions of the embryos exclusively in the early stage of development. Heart-deficient models, together with in vitro models, have demonstrated that mechanical cues instruct and increase HSPC production from the hemogenic endothelium9,10,11,12,13,14.
Different types of flow dynamics have been shown to differentially control the cell cycle15, known to be important in both hemogenic endothelium16,17 and arterial cell specification18. Altogether, mechanical cues are critical determinants of cell identity and function during development. Novel in vitro fluidic devices allow us to overcome the limitations involved with studying developmental mechanobiology during human blood development in vivo.
The overall goal of the protocol in this manuscript is to describe, step-by-step, the experimental pipeline to study the effect of shear stress on human endothelial cells derived in vitro from human induced pluripotent stem cells (hiPSCs). This protocol contains detailed instructions on the differentiation of hiPSCs into endothelial cells and their subsequent seeding into fluidic chips for the stimulation protocol. Using this, different in vitro-derived endothelial cells can be tested for their ability to sense the shear stress by analyzing their orientation in response to the flow. This will allow other laboratories to address questions about the response to shear stress and its functional consequences on different endothelial cell identities.

Author Spotlight: Studying hiPSC-Derived Endothelial Cells Cultured Under Fluidic-Mediated Mechanical Stimulation 

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

Watch this Scientific Journal Video about Seeding CD34+ Cells into Fluidic Chips at JoVE.com

Seeding CD34+ Cells into Fluidic Chips  

Seeding CD34+ Cells into Fluidic Chips

Research

JoVE Journal

Developmental Biology

271 Views. Seeding CD34+ Cells into Fluidic Chips

Video: Seeding CD34+ Cells into Fluidic Chips  

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&#160;retrieval of the endothelial cells for phenotypical and functional characterization. The in vitro model established here will be instrumental to elucidate&#160;the intracellular molecular mechanisms that transduce the signaling mediated by mechanical cues, which ultimately orchestrate vessel development during human fetal life.

Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation.