This work was supported by the Research Advanced Grant 2021 from the European Hematology Association, the Global Research Award 2021 from the American Society of Hematology, and the Internal Strategy Support Fund ISSF3 funded by the Welcome Trust and the University of Edinburgh. We thank Fiona Rossi from the Flow Cytometry Facility for support in the flow cytometry analysis. For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) license to any Author Accepted Manuscript version arising from this submission.

Differentiation and Mechanical Stimulation of hiPSC-Derived Endothelial Cells

<ol>
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The authors have no conflicts of interest to disclose.

The protocol that we describe here allows for the generation of mechanosensitive endothelial cells from human pluripotent stem cells and the study of their response to mechanical stimulation mediated by controlled shear stress. This protocol is entirely cytokine-based and fully compatible with GMP reagents for potential translation into the production of cells for cell therapy.
The derivation of hiPSCs provides scientists with an instrumental model for the early stages of embryonic development that enables the study of processes that are otherwise difficult to study in vivo24. In fact, human embryonic tissues available for research are collected from embryos lacking circulation, and this might have a significant impact on the molecular signature controlled by mechanical cues. The approach described here enables live-imaging and real-time study of cell response to shear stress. The combination of hiPSCs with fluidics provides a model of study that overcomes the limited availability and the inaccessibility of the developing fetal tissues when the initiation of circulation remodels and controls the establishment of the cardiovascular and blood system3,9,10,25.
A limitation of the protocol is that the endothelial cells derived from this protocol might not reflect the various identities of different endothelial cells present in the developing tissues. To overcome this limitation, a specific combination of cytokines might be needed during the differentiation process preceding the fluidic stimulation to obtain the desired identity or tissue-specific phenotype26. The isolation of endothelial subsets can be obtained using a more refined immunophenotype during the isolation step. This protocol isolates endothelial cells based only on the expression of CD34, thereby allowing for column isolation instead of fluorescence-activated cell sorting (FACS); this reduces cell death and the risk of contamination. Furthermore, this protocol is specifically designed to study the role of shear stress mediated by laminar flow. Alternative fluidic approaches will have to be employed to study the effect of other mechanical cues, such as stretching or compression, or other types of flow such as perturbed or disturbed flow.
We have previously shown that iPSC-derived endothelial cells mimic the heterogeneous arteriovenous cellular identities27 similar to that observed in the fetal dorsal aorta28,29,30. This is of particular importance in the context of vessel development and cellular specification, known to be controlled by blood circulation. Studies in different models showed that lack of circulation results in altered arteriovenous specification11,14,31. The mechanisms that connect mechanical cues with cell specification are still unknown and the pipeline described here allows for refined functional studies that could not be tested in vivo.
This pipeline describes the production and the stimulation of endothelial cells derived from hiPSCs using commercially available fluidic channels, avoiding the need for casting the devices as for the widely used polydimethylsiloxane (PDMS) devices12. Furthermore, the use of PDMS chips makes the collection of the stimulated cells particularly challenging, while with this protocol, the cells can be easily retrieved from the channel. This significantly improves the analytic power allowing for subsequent analysis such as proteomic and transcriptomic analyses, flow cytometry, and functional assays, which might need further culture or in vivo assays.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.6 Luer uncoated slide</td>
			<td>ibidi</td>
			<td>IB-80186</td>
			<td></td>
		</tr>
		<tr>
			<td>25% BSA</td>
			<td>Life Technologies</td>
			<td>A10008-01</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Greiner Bio-one</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Life Technologies</td>
			<td>A1110501</td>
			<td>Cited as Dissociation reagent</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Merck</td>
			<td>A4544-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspiration pipette</td>
			<td>Sardtedt</td>
			<td>86.1252.011</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>Cited as Neuronal cell culture supplement (50x)</td>
		</tr>
		<tr>
			<td>BD FACS DIVA</td>
			<td>BD Biosciences</td>
			<td>Version 8.0.1</td>
			<td>Cited as flow cytometry software</td>
		</tr>
		<tr>
			<td>BD LSR Fortessa 5 Laser</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Life Technologies</td>
			<td>PHG0021</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 Microbead kit</td>
			<td>Miltenyil Biotec</td>
			<td>30-046-702</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 PE clone 4H11</td>
			<td>Invitrogen</td>
			<td>12-0349-42</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 PerCP-eFluor 710 clone 4H11</td>
			<td>Invitrogen</td>
			<td>44-0349-42</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellstar cell-repellent surface 6-well plates</td>
			<td>Greiner Bio-one</td>
			<td>657970</td>
			<td>Cited as cell-repellent plate</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Cayman Chemicals&#160;</td>
			<td>13122-1mg-CAY</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostor CS10&#160; cell cryopreservation</td>
			<td>Merck</td>
			<td>C2874-100ML</td>
			<td>Cited as Cryopreservation solution</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>VWR</td>
			<td>200-664-3</td>
			<td>Cited as DMSO</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Life Technologies</td>
			<td>10565-018</td>
			<td></td>
		</tr>
		<tr>
			<td>DPB Ca2+ Mg2+</td>
			<td>Life Technologies</td>
			<td>14080055</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Life Technologies</td>
			<td>14200075</td>
			<td></td>
		</tr>
		<tr>
			<td>EASY Strainer 40 &#956;m</td>
			<td>Greiner Bio-one</td>
			<td>542040</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Life technologies</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>FcR Blocking Reagent</td>
			<td>Miltenyil Biotec</td>
			<td>130-059-901</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td></td>
			<td>Version 1.53c</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Jo</td>
			<td></td>
			<td>Version 10.7.1</td>
			<td>Cited as flow cytometry sanalysis oftware</td>
		</tr>
		<tr>
			<td>FLT3L</td>
			<td>Peprotech</td>
			<td>300-19-10uG</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluidic unit</td>
			<td>ibidi</td>
			<td>10903</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Life Technologies</td>
			<td>35050038</td>
			<td>Cited as L-glutamine supplement</td>
		</tr>
		<tr>
			<td>Ham F-12&#160;</td>
			<td>Life Technologies</td>
			<td>11765054</td>
			<td></td>
		</tr>
		<tr>
			<td>Holo-transferrin</td>
			<td>Merk</td>
			<td>T0665-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Serum Albumin</td>
			<td>Fujifilm UK LTD</td>
			<td>9988</td>
			<td></td>
		</tr>
		<tr>
			<td>Ibidi Pump system</td>
			<td>ibidi</td>
			<td>10902</td>
			<td>Cited as Pump system</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Life Technologies</td>
			<td>12440053</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>ioLight/Thisle Scientific</td>
			<td>IOL-IO-INVERT</td>
			<td>Cited as inverted in-incubator microscope</td>
		</tr>
		<tr>
			<td>Lyophilised BSA&#160;</td>
			<td>Merck</td>
			<td>A2153-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS Separator</td>
			<td>Miltenyil Biotec</td>
			<td>130-042-102</td>
			<td>Cited as Magnetic separator</td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>Miltenyil Biotec</td>
			<td>130-042-201</td>
			<td>Cited as Magnetic column</td>
		</tr>
		<tr>
			<td>MTG</td>
			<td>Merck</td>
			<td>M6145-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Life Technologies</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Notebook for pump system</td>
			<td>ibidi</td>
			<td>10908</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 37-41%</td>
			<td>Fisher Chemicals</td>
			<td>F/1501/PB15</td>
			<td></td>
		</tr>
		<tr>
			<td>Pastette</td>
			<td>Greiner Bio-one</td>
			<td>612398</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion Set YELLOW/GREEN: 50 cm, ID 1.6 mm, 10 mL reservoirs</td>
			<td>Ibidi</td>
			<td>IB-10964</td>
			<td>Cited as Perfusion set</td>
		</tr>
		<tr>
			<td>Polystyrene Round Bottom Tubes</td>
			<td>Falcon</td>
			<td>352008</td>
			<td>Cited as Flow cytometry tubes</td>
		</tr>
		<tr>
			<td>Prism 9</td>
			<td></td>
			<td>Verison 9.4.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Pump control software</td>
			<td>ibidi</td>
			<td>version 1.6.1</td>
			<td>Cited as Pump software</td>
		</tr>
		<tr>
			<td>ReLeSR</td>
			<td>Stem cell tecchonologies</td>
			<td>5872</td>
			<td>Cited as Detaching solution</td>
		</tr>
		<tr>
			<td>rhBMP4</td>
			<td>R&#38;D</td>
			<td>314-BP-010</td>
			<td></td>
		</tr>
		<tr>
			<td>rhEPO</td>
			<td>R&#38;D</td>
			<td>287-TC-500</td>
			<td></td>
		</tr>
		<tr>
			<td>rhIGF1</td>
			<td>Peprotech</td>
			<td>100-11-100uG</td>
			<td></td>
		</tr>
		<tr>
			<td>rhIL11</td>
			<td>Peprotech</td>
			<td>200-11-10uG</td>
			<td></td>
		</tr>
		<tr>
			<td>rhIL3</td>
			<td>Peprotech</td>
			<td>200-03-10uG</td>
			<td></td>
		</tr>
		<tr>
			<td>rhIL6</td>
			<td>R&#38;D</td>
			<td>206-IL-010</td>
			<td></td>
		</tr>
		<tr>
			<td>rhLaminin-521</td>
			<td>Life technologies</td>
			<td>A29248</td>
			<td>Cited as Laminin</td>
		</tr>
		<tr>
			<td>rhSCF</td>
			<td>Life Technologies</td>
			<td>PHC2111</td>
			<td></td>
		</tr>
		<tr>
			<td>rhTPO</td>
			<td>R&#38;D</td>
			<td>288-TPN-025</td>
			<td></td>
		</tr>
		<tr>
			<td>rhVEGF</td>
			<td>R&#38;D</td>
			<td>293-VE-010</td>
			<td></td>
		</tr>
		<tr>
			<td>RLT Lysis Buffer</td>
			<td>Qiagen</td>
			<td>79216</td>
			<td></td>
		</tr>
		<tr>
			<td>Serial Connector for &#181;-Slides: Sterile, Sterile</td>
			<td>ibidi</td>
			<td>IB-10830</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro-CD34 SFM media</td>
			<td>Life Technologies</td>
			<td>10639011</td>
			<td>Cited as Serum-Free media for CD34+ cells (SFM-34)</td>
		</tr>
		<tr>
			<td>StemPro-CD34 Nutrient Supplement&#160;</td>
			<td>Life Technologies</td>
			<td>10641-025</td>
			<td>Cited as 34 nutrient supplement</td>
		</tr>
		<tr>
			<td>StemPro hESC SFM</td>
			<td>Life Technologies</td>
			<td>A1000701</td>
			<td>Cited as Culture media&#160;</td>
		</tr>
		<tr>
			<td>StemPro supplement</td>
			<td>Life Technologies</td>
			<td>A10006-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitronectin (VTN-N) recombinant human protein, truncated</td>
			<td>Invitrogen</td>
			<td>A31804</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>Cited as iRock</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro model of fetal human vessel on-chip to study developmental mechanobiology

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.

We describe here a protocol for the differentiation and mechano-stimulation of endothelial cells derived from hiPSCs that allows the study of their response to mechanical cues (Figure 1). This protocol results in the production of functionally mechanosensitive endothelial cells. We provide here representative results and describe the expected phenotype to assess how the cells respond to the cytokine stimulation during the differentiation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65492/65492fig01v2.jpg" /> 
Figure 1: Schematic of the differentiation and mechanical stimulation protocol. Schematic of the differentiation protocol showing the timing of the different mixes of cytokines, the CD34+ cell isolation, fluidic chip seeding, and final analysis of the mechanically stimulated cells. <a href="https://www.jove.com/files/ftp_upload/65492/65492fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Culture of hiPSCs 
It is important to start the protocol from hiPSCs that are growing correctly in self-renewal conditions. A good indication of the quality of the culture is the speed of their growth. After thawing, the cells might need 2-3 weeks to reach the correct phase of growth that will ensure good differentiation. When the cells can be passaged twice a week at the ratio of 1:6 reaching almost full confluency, this is the time that they are ready to be differentiated on the same day they need to be passaged.
Differentiation of hiPSCs into endothelial cells 
The first step of the differentiation, consisting of the formation of embryoid bodies (EBs), is cell line-dependent and may need some optimization for the specific cell line in use. The dissociation described in protocol steps 1.3.2.2-1.3.2.4 can be modified by either reducing or extending the incubation with the dissociation reagent and the subsequent dissociation with the Pasteur pipette. Furthermore, other dissociation reagents can be used for this step in addition to the physical dissociation of the colonies with a cutting tool or a P100 pipette tip. EBs of good quality show a defined edge by day 2 of the differentiation and appear clear and bright when observed using a microscope; darker areas might indicate cell death within the EBs (Figure 2).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65492/65492fig2v3.jpg" /> 
Figure 2: Embryoid bodies&#160;morphology. (A) Day 2 embryoid bodies showing well-defined outer edges and consistent size. (B) Day 2 embryoid bodies of poor quality showing extensive cell death leading to disaggregation of the structure. Scale bar = 500 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65492/65492fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
At day 2, the addition of CHIR99021 to the EBs inhibits the GSK-3 protein resulting in the activation of the Wnt pathway. Different cell lines have different responses to CHIR treatment, and this should be tested by quantifying the number of CD34+ cells obtained at day 8 by using different concentrations (Figure 3).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65492/65492fig3v3.jpg" /> 
Figure 3: Endothelial cell differentiation with different CHIR treatments. Endothelial cell commitment quantified by flow cytometry at day 8 of differentiation by CD34 membrane expression, following CHIR treatment at day 2 at (A) 3 &#181;M, (B) 5 &#181;M, and (C) 7 &#181;M. Flow cytometry data were obtained using five-laser cytometers and dedicated software (see Table of Materials). <a href="https://www.jove.com/files/ftp_upload/65492/65492fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
CD34+ cell isolation 
It is important to validate that the CD34+ enrichment using the magnetic beads provides at least 80% CD34+ after elution of the column. To ensure sufficient purity, an aliquot of cells obtained from the magnetic isolation can be analyzed by flow cytometry making sure to use a different antibody clone than the one used for the magnetic enrichment. Here, the 4H11 clone was used and ~85% purity was achieved post enrichment (Figure 4).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65492/65492fig4v3.jpg" /> 
Figure 4: Membrane expression of CD34 before and after enrichment by magnetic sorting. Day 8 dissociated embryoid bodies (grey) and cells after magnetic enrichment (green) were stained for CD34 expression and analyzed by flow cytometry, showing successful enrichment post sorting. Flow cytometry data were obtained using five-laser cytometers and dedicated software (see Table of Materials). <a href="https://www.jove.com/files/ftp_upload/65492/65492fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Seeding cells into the fluidic channel 
When seeding the cells in the fluidic channel, it is crucial to track the adhesion and proliferation of the endothelial cells. After seeding, the cells take ~5 h to fully adhere to the channel (Figure 5A). An alternative coating solution may also be tested to improve adhesion at this stage. To confirm that the tested cells are mechanosensitive and thus, able to respond to mechanical stimulation, the cell orientation can be tested over time. Cells before the stimulation show random orientation (Figure 5A and Figure 5C) and they reorient parallel to the direction of the flow (Figure 5B,C). The protocol described here allows for the collection of the cells from the channel to perform downstream analysis, for example, flow cytometry, for the study of their membrane immunophenotype, providing the endothelial identity of the stimulated cells (Figure 5D,E).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65492/65492fig5v3.jpg" /> 
Figure 5: Mechanoresponsiveness of hiPSCs-derived endothelial cells. (A) Confluent layer of isolated CD34+ cells 48 h post seeding. (B) Reoriented layer of endothelial cells 3 days under dynamic culture. (C) Orientation analysis of the endothelial cells after 5 days of dynamic culture. (D) CD34 expression profile of cells cultured under flow for 5 days. (E) Percentage of CD34+ cells of cell population retrieved from the fluidic channel. Images were taken using an inverted in-incubator microscope; flow cytometry data were obtained using five-laser cytometers and dedicated software (see Table of Materials). Scale bars = 100 &#181;m (A,B). <a href="https://www.jove.com/files/ftp_upload/65492/65492fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td>Stock concentration</td>
			<td>Volume added</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Iscove&#8217;s Modified Dulbecco&#8217;s Medium (IMDM)</td>
			<td>-</td>
			<td>333 mL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ham&#8217;s F-12 Nutrient mixture (F-12)</td>
			<td>-</td>
			<td>167 mL</td>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;</td>
		</tr>
		<tr>
			<td>N-2 supplement (100x)</td>
			<td>100 x</td>
			<td>5 mL</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>B-27 supplement (50x)&#160;</td>
			<td>50 x</td>
			<td>10 mL</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>10 mg/mL</td>
			<td>1.25 mL</td>
			<td>25 &#181;g/mL</td>
		</tr>
		<tr>
			<td>&#945;-Monothioglycerol (MTG)</td>
			<td>11.5 M</td>
			<td>19.5 &#181;L</td>
			<td>448.5 &#181;M</td>
		</tr>
		<tr>
			<td>Human Serum Albumin</td>
			<td>100 mg/mL</td>
			<td>2.5 mL</td>
			<td>0.5 mg/mL</td>
		</tr>
		<tr>
			<td>Holo-Transferrin</td>
			<td>100 mg/mL</td>
			<td>0.75 mL</td>
			<td>150 &#181;g/mL</td>
		</tr>
	</tbody>
</table>
Table 1: Composition and recipe for 500 mL of Serum-free Differentiation (SFD) medium.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Days of differentiation</td>
			<td>Cytokine Mix</td>
			<td>Cytokine</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Day 0 - 2</td>
			<td>Mix 1</td>
			<td>BMP4</td>
			<td>20 ng/mL</td>
		</tr>
		<tr>
			<td>Day 2</td>
			<td>Mix 2</td>
			<td>CHIR99021</td>
			<td>7 &#956;M</td>
		</tr>
		<tr>
			<td>From day 3 onward</td>
			<td rowspan="2">Mix 3 and 4</td>
			<td>VEGF</td>
			<td>15 ng/mL</td>
		</tr>
		<tr>
			<td></td>
			<td>bFGF</td>
			<td>5 ng/mL</td>
		</tr>
		<tr>
			<td>From day 6 onward</td>
			<td rowspan="8">Mix 4</td>
			<td>IL6</td>
			<td>10 ng/mL</td>
		</tr>
		<tr>
			<td></td>
			<td>FLT3L</td>
			<td>10 ng/mL</td>
		</tr>
		<tr>
			<td></td>
			<td>IGF1</td>
			<td>25 ng/mL</td>
		</tr>
		<tr>
			<td></td>
			<td>IL11</td>
			<td>5 ng/mL</td>
		</tr>
		<tr>
			<td></td>
			<td>SCF</td>
			<td>50 ng/mL</td>
		</tr>
		<tr>
			<td></td>
			<td>EPO</td>
			<td>3 U/mL</td>
		</tr>
		<tr>
			<td></td>
			<td>TPO</td>
			<td>30 ng/mL</td>
		</tr>
		<tr>
			<td></td>
			<td>IL3</td>
			<td>30 ng/mL</td>
		</tr>
	</tbody>
</table>
Table 2: Mixes of cytokines used for endothelial cell differentiation, days in which they are added to the SFD medium, and final concentration.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Shear Stress (dyn/cm2)&#160;</td>
			<td>Time (h)</td>
		</tr>
		<tr>
			<td>0.5</td>
			<td>1</td>
		</tr>
		<tr>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>1.5</td>
			<td>1</td>
		</tr>
		<tr>
			<td>2</td>
			<td>1</td>
		</tr>
		<tr>
			<td>2.5</td>
			<td>1</td>
		</tr>
		<tr>
			<td>3</td>
			<td>1</td>
		</tr>
		<tr>
			<td>3.5</td>
			<td>1</td>
		</tr>
		<tr>
			<td>4</td>
			<td>1</td>
		</tr>
		<tr>
			<td>4.5</td>
			<td>1</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Until end of the experiment</td>
		</tr>
	</tbody>
</table>
Table 3: Shear stress values for the dynamic culture and length of their application.
Supplementary Figure S1: Geometry and dimensions of the chip and tubing used for this protocol. <a href="https://www.jove.com/files/ftp_upload/65492/SF1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure S2: Step-by-step guide for the software controlling the air pump with a description of each step. <a href="https://www.jove.com/files/ftp_upload/65492/65492_Supp Figure S2 FINAL.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure S3: Guide for the orientation analysis using FIJI showing the drawing of the cell shape, elliptic fitting, and final measurement. <a href="https://www.jove.com/files/ftp_upload/65492/65492_SF3.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Table S1: Unit size, resuspension volume, and stock concentrations for cytokines used in differentiation protocol. <a href="https://www.jove.com/files/ftp_upload/65492/Ventura et al, 2023 - Supplementary Table 1.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Studying hiPSC-Derived Endothelial Cells Cultured Under Fluidic-Mediated Mechanical Stimulation at JoVE.com

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

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.

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

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides ...

in-vitro-model-fetal-human-vessel-on-chip-to-study-developmental

Research

JoVE Journal

Developmental Biology

1.5K Views.  University of Edinburgh. 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.

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

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

This manuscript presents a unique in vitro model of immunopurified human villous cytotrophoblast cells cultured under hypoxia/reoxygenation. This model is suitable to study the protective effects of promising treatments, such as melatonin, on pregnancy complications associated with increased oxidative stress and altered placental function.

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density ...

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide suffering from corneal vision loss1. To address the demand for viable human corneas, significant progress in three-dimensional (3D) tissue engineering has been made2,3,4. These cornea models range from simple monolayer systems to multilayered models, leading to 3D full-thickness corneal equivalents2.
However, the use of a 3D tissue-engineered cornea in the context of in vitro disease models studied to date lacks resemblance to the multilayered 3D corneal tissue structure, function, and the networking of different cell types (i.e., nerve, epithelium, stroma, and endothelium)2,3. In addition, the demand for in vitro cornea tissue models has increased in an attempt to reduce animal testing for pharmaceutical products. Thus, more sophisticated models are required to better match systems to human physiological requirements, and the development of a model that is more relevant to the patient population is absolutely necessary. Given that multiple cell types in the cornea are affected by diseases and dystrophies, such as Keratoconus, Diabetic Keratopathy, and Fuchs, this model includes a 3D co-culture model of primary human corneal fibroblasts (HCFs) from healthy donors and neurons from the SH-SY5Y cell line. This allows us for the first time to investigate the interactions between the two cell types within the human corneal tissue. We believe that this model could potentially dissect the underlying mechanisms associated with the stromal-nerve interactions of corneal diseases that exhibit nerve damages. This 3D model mirrors the basic anatomical and physiological nature of the corneal tissue in vivo and can be used in the future as a tool for investigating corneal defects as well as screening the efficacy of various agents before animal testing.

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

This protocol describes a novel three-dimensional in vitro model, where corneal stromal cells and differentiated neuronal cells are cultured together to assist in the examination and understanding of the interactions of the two cell types.

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...