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><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&amp;nbsp;</td><td>13122-1mg-CAY</td><td></td></tr><tr><td>Cryostor CS10&amp;nbsp; 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 Ca&lt;sup&gt;2+&lt;/sup&gt; Mg&lt;sup&gt;2+&lt;/sup&gt;</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 &amp;mu;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&amp;nbsp;</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&amp;nbsp;</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&amp;amp;D</td><td>314-BP-010</td><td></td></tr><tr><td>rhEPO</td><td>R&amp;amp;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&amp;amp;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&amp;amp;D</td><td>288-TPN-025</td><td></td></tr><tr><td>rhVEGF</td><td>R&amp;amp;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 &amp;micro;-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&amp;nbsp;</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&amp;nbsp;</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>&amp;beta;-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 

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

Bioengineering

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection (TAAD). However, human TAAD sometimes cannot be characterized by animal models. There is an apparent species gap between clinical human studies and animal experiments that may hinder the discovery of therapeutic drugs. In contrast, the conventional cell culture model is unable to simulate in vivo biomechanical stimuli. To this end, microfabrication and microfluidic techniques have developed greatly in recent years, providing novel techniques for establishing organoids-on-a-chip models that replicate the biomechanical microenvironment. In this study, a human aorta smooth muscle cell organ-on-a-chip (HASMC-OOC) model was developed to simulate the pathophysiological parameters of aortic biomechanics, including the amplitude and frequency of cyclic strain experienced by human aortic smooth muscle cells (HASMCs) that play a vital role in TAAD. In this model, the morphology of HASMCs became elongated in shape, aligned perpendicularly to the strain direction, and presented a more contractile phenotype under strain conditions than under static conventional conditions. This was consistent with the cell orientation and phenotype in native human aortic walls. Additionally, using bicuspid aortic valve-related TAAD (BAV-TAAD) and tricuspid aortic valve-related TAAD (TAV-TAAD) patient-derived primary HASMCs, we established BAV-TAAD and TAV-TAAD disease models, which replicate HASMC characteristics in TAAD. The HASMC-OOC model provides a novel in vitro platform that is complementary to animal models for further exploring the pathogenesis of TAAD and discovering therapeutic targets.

Here, we developed a human aorta smooth muscle cell organ-on-a-chip model to replicate the in vivo biomechanical strain of smooth muscle cells in the human aortic wall.

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection ...

Studying Tissue Rigidity and Shear Stress Effects on Endothelial Cells

In Vitro Model Integrating Substrate Stiffness and Flow to Study Endothelial Cell Responses

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, which are crucial for understanding vascular health and the onset of diseases such as atherosclerosis. Traditionally, studies have explored the impacts of shear stress and substrate stiffness on ECs, independently. However, this integrated system combines these factors to provide a more precise simulation of the mechanical environment of the vasculature. The objective is to examine EC mechanotransduction across various tissue stiffness levels and flow conditions using human ECs. We detail the protocol for synthesizing gelatin methacrylate (GelMA) hydrogels with tunable stiffness and seeding them with ECs to achieve confluency. Additionally, we describe the design and assembly of a cost-effective flow chamber, supplemented by computational fluid dynamics simulations, to generate physiological flow conditions characterized by laminar flow and appropriate shear stress levels. The protocol also incorporates fluorescence labeling for confocal microscopy, enabling the assessment of EC responses to both tissue compliance and flow conditions. By subjecting cultured ECs to multiple integrated mechanical stimuli, this model enables comprehensive investigations into how factors such as hypertension and aging may affect EC function and EC-mediated vascular diseases. The insights gained from these investigations will be instrumental in elucidating the mechanisms underlying vascular diseases and in developing effective treatment strategies.

We synthesized and characterized a tunable gelatin-based substrate for culturing vascular endothelial cells (ECs) under relevant vascular flow conditions. This biomimetic surface replicates both physiological and pathological conditions, enabling the study of mechanical forces on EC behavior and advancing our understanding of vascular health and disease mechanisms.

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Biology

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...

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

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Chapters in this video

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Micropatterning and Assembly of 3D Microvessels

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

Microfluidic Model to Mimic Initial Event of Neovascularization

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall

In Vitro Model Integrating Substrate Stiffness and Flow to Study Endothelial Cell Responses

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca²⁺]_iand Nitric Oxide Production

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing