generating brain endothelial cells from induced pluripotent stem cells

Generating Brain Endothelial Cells from Induced Pluripotent Stem Cells

Resuspend 7.5 x 105 cells in 12 milliliters of stem cell maintenance medium and 10 micromolar ROCK inhibitor. Aspirate matrix from a T75 flask and transfer the cell suspension to the flask. Shake the flask to distribute the cells, and place it in the incubator.
Refresh the media every day for the next two days to remove ROCK inhibitor, and support the growth of stem cell colonies. On the third day, begin differentiation by changing the media to an unconditioned medium.
Change the medium daily for the next five days. Six days after starting differentiation, selectively expand the endothelial cell population by switching to endothelial cell or EC medium with 20 nanograms per milliliter bFGF and 10 micromolar retinoic acid. Incubate the cells for two days.
Prepare collagen IV and Fibronectin solution according to manuscript directions, and use it to coat cell culture plates. Aspirate the EC medium from the cells, and add 12 milliliters of cell dissociation reagent. Incubate the flask at 37 degrees Celsius until 90% of the cells have detached.
During the incubation time, remove the coating solution from the previously prepared plates, and let them dry in a sterile hood. Once the cells have detached, use a 10-milliliter pipette to create a single-cell suspension.
Transfer the cells to a 50-milliliter tube, and dilute them with an equal volume of human endothelial serum-free free medium. Then, count the cells with a hemocytometer. Pellet the cells at 1,500 g for 10 minutes. Then resuspend them in freshly prepared EC medium with bFGF and retinoic acid for a concentration of 2 million cells per milliliter.
Add 500 microliters of the cell suspension to the top of a 12-well insert and 1.5 milliliters of medium to the bottom. Distribute the cells evenly across the insert, and incubate the plate at 37 degrees Celsius and 5% carbon dioxide. On the next day, change the media to EC without bFGF or retinoic acid.

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1. Preparation of materials required for iPSC culture and BEC differentiation.
<ol>
	<li>Matrix coating of tissue culture (TC) plastic for IMR90-4 iPSC culture
	<ol>
		<li>Aliquot basement membrane matrix gel (e.g., Matrigel) into 2.5 mg aliquots and store at -20 &#176;C. 
		NOTE: Work quickly when handling the matrix gel and aliquot on ice, as it forms a gel above 4 &#176;C and cannot be aliquoted once it has solidified.</li>
		<li>For coating TC plastic, quickly add one aliquot of matrix gel to 30 mL of Dulbecco&#39;s Modified Eagle Medium (DMEM)/F12 medium in a 50 mL conical tube. Add 1 mL of the medium onto the frozen matrix gel, pipette up and down until it is thawed, and transfer immediately to the 50 mL conical tube with the remaining medium.</li>
		<li>Use 1 mL of this matrix gel-coating solution per well of a 6-well plate and 12 mL per T75 flask. 
		NOTE: Matrix gel-coated TC plastic can be prepared up to two weeks before it is used. It is, however, critical to avoid that, the matrix gel solution dries out, which may require the occasional addition of more DMEM/F12 on top of the wells.</li>
	</ol>
	</li>
	<li>Prepare stem-cell maintenance medium by adding 50 mL of 50x supplement to 450 mL of stem-cell maintenance basal medium in the sterile environment of a biosafety cabinet.</li>
	<li>To prepare 500 mL of unconditioned medium (UM), combine 392.5 mL of DMEM/F12 with 100 mL of knock out serum replacement (KOSR), 5 mL of non-essential amino acids, L-glutamine at a final concentration of 1 mM, and 3.5 &#181;L of &#946;-mercaptoethanol. Filter-sterilize and store at 4 &#176;C for up to 2 weeks.</li>
	<li>To prepare 200 mL of endothelial cell (EC) medium plus retinoic acid (RA) plus bFGF, combine 198 mL of human endothelial serum free medium (hESFM), 2 mL of filter-sterilized platelet-poor derived serum (PDS), and 20 ng/mL bFGF. Filter sterilize and store for up to 2 weeks at 4 &#176;C. Just before addition to cells, add 10 &#956;M of RA to EC medium. 
	NOTE: As PDS has been discontinued and may therefore be limited, this protocol has been successfully conducted using B27 in place of PDS.</li>
	<li>To prepare 200 mL of EC medium without RA or bFGF, combine 198 mL of hESFM and 2 mL of filter sterilized PDS and filter sterilize. Store for up to 4 weeks at 4 &#176;C.</li>
</ol>
2. Differentiation of brain endothelial cells from human iPSCs
<ol>
	<li>Plating of iPSCs for differentiation (day -3 of the differentiation protocol)
	<ol>
		<li>Per IMR90-4 maintenance culture well used to plate for differentiation, aspirate the culture medium, add 1 mL of enzymatic cell dissociation reagent per well, and incubate at 37 &#176;C for 7 min.</li>
		<li>Inactivate the enzymatic cell dissociation reagent by transferring the 1 mL of dissociated cell suspension into a 15 mL conical tube with at least 2 mL of fresh stem-cell maintenance medium per 1 mL of cells. Spin down the cell suspension at 1,500 x g for 5 min.</li>
		<li>Resuspend the cell pellet in 1 mL of stem-cell maintenance medium per well of IMR90-4 cells used, and count the cells using a hemocytometer. 
		NOTE: It may be helpful to dilute 1:1 with 0.4% trypan blue to distinguish between live and dead cells when counting. Depending on the density of iPSCs, one well of a 6-well plate usually yields 1&#8210;2 x 106 cells.</li>
		<li>For differentiation in a T75 flask, add 7.5 x 105 cells to 12 mL of stem-cell maintenance medium and ROCK inhibitor (Y27632 dihydrochloride) at a final concentration of 10 &#181;M. Aspirate matrix gel-coating solution from a T75 flask and transfer the cell suspension to the flask. Distribute the cells equally by shaking the flask back and forth and left to right and incubate at 37 &#176;C and 5% CO2. 
		NOTE: It is critical to add ROCK inhibitor at this step to enhance survival of the dissociated single stem cells. Cells should be evenly distributed across the flask and in singlets exhibiting a spread, mesenchymal-like morphology due to the ROCK inhibitor treatment.</li>
	</ol>
	</li>
	<li>On day -2 and day -1, change media to fresh stem-cell maintenance medium; 12 mL per T75.</li>
	<li>On day 0, start differentiation by changing media to UM; 12 mL per T75.</li>
	<li>Change UM daily (day 1 &#8211; day 5). 
	NOTE: The cells typically reach confluence after 2 to 3 days in UM, which can be observed with the naked eye or through an inverted bright field microscope. As the differentiation progresses, nestin+ &#34;neural tracts&#34; become visible with PECAM-1+ cells in between.</li>
	<li>Selectively expand the endothelial cell population by switching to EC medium with 20 ng/mL bFGF and 10 &#181;M retinoic acid (RA) (day 6) and incubating for two days. EC medium with bFGF can be prepared up to two weeks prior. RA is added from frozen stock on the day of use (e.g., 1 &#181;L of 10 mM RA stock per 1 mL of EC + bFGF). 
	NOTE: Successful differentiation can also be achieved without supplementation of RA at days 6 and 8. Omission of RA will, however, yield BECs with reduced TEER.</li>
	<li>Coat cell culture plates and membrane inserts (e.g., Transwell) with collagen IV and fibronectin (day 7), for purification of the BECs and following experiments.
	<ol>
		<li>For coating of membrane inserts, combine 4 parts collagen IV (1 mg/mL in 0.5 mg/mL acetic acid), 1 part fibronectin (1 mg/mL) and 5 parts sterile tissue-grade water. ECM solution can be diluted 1:5 for coating cell culture plates (i.e., 4 parts collagen IV, 1 part fibronectin, 45 parts water). Incubate with coating solution at 37 &#176;C overnight. 
		&#8203;NOTE: Plates and membrane inserts can also be coated for at least 4 h prior to subculturing on the same day of the purification step.</li>
	</ol>
	</li>
	<li>Purify BECs by subculturing the differentiated cells on collagen IV and fibronectin-coated plates or membrane inserts (day 8).
	<ol>
		<li>Aspirate EC medium and add enzymatic cell dissociation reagent (12 mL per T75). Incubate at 37 &#176;C until 90% of the cells have detached from the flask. 
		NOTE: Cell dissociation can take up to 1 h.</li>
		<li>During the incubation time, remove the collagen IV/fibronectin coating solution from previously prepared plates/inserts and let them dry in a sterile hood. It takes approximately 20 min for the inserts to dry.</li>
		<li>Once the cells have detached, wash them off the flask using a 10 mL pipette. Pipette up and down to achieve a single cell suspension. 
		NOTE: Single cell suspension is important for reliable cell counting and to achieve solid monolayers.</li>
		<li>Dilute with at least equal volume of fresh hESFM in a 50 mL conical tube and count cells using a hemocytometer.</li>
		<li>Pellet the cells at 1,500 x g for 10 min.</li>
		<li>Resuspend cells in appropriate volume of freshly prepared EC + bFGF + RA to achieve a suspension of 2 x 106 cells/mL for seeding on membrane inserts. Add 500 &#181;L (1 x 106 cells) on top of a 12-well insert and 1.5 mL of EC + bFGF + RA medium on the bottom. For seeding on 24 and 48-well plates, dilute cell suspension 1:2 and add 500 &#181;L (5 x 105 cells) and 250 &#181;L (2.5 x 105 cells) per well, respectively. Distribute cells evenly across the well/insert and incubate at 37 &#176;C under 5% CO2.</li>
	</ol>
	</li>
	<li>Change media on plates/transwells to EC without bFGF or RA (day 9).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accutase (1x)</td>
			<td>Sigma</td>
			<td>A6964</td>
			<td></td>
		</tr>
		<tr>
			<td>All-trans retinoic acid (RA)</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td></td>
		</tr>
		<tr>
			<td>b-Mercaptoethanol</td>
			<td>Merck (Sigma-Aldrich)</td>
			<td>805740</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates and flasks</td>
			<td>Sarstedt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge (Heraeus Megafuge 1.0R)</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Class II biosafety cabinet</td>
			<td>Nuaire</td>
			<td>NU-437-400E</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide incubator (DHD Autoflow Carbon dioxide Air-Jacketed Incubator)</td>
			<td>Nuaire</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Sigma</td>
			<td>C5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar Transwell polyester filters (12- or 24-well)</td>
			<td>Corning</td>
			<td>3460, 3470</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma</td>
			<td>F1141</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer (Neubauer)</td>
			<td>A. Hartenstein</td>
			<td>ZK06</td>
			<td></td>
		</tr>
		<tr>
			<td>Human basic fibroblast growth factor (bFGF)</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Endothelial Serum Free Medium (hESFM)</td>
			<td>Gibco</td>
			<td>11111-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope (Wilovert)</td>
			<td>Hund (Will Wetzlar)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iPS(IMR90)-4 cells</td>
			<td>WiCell</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout serum replacement (KOSR)</td>
			<td>Gibco</td>
			<td>10828-028</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine (GlutaMAX)</td>
			<td>Invitrogen</td>
			<td>35050-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Matrix</td>
			<td>Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids (NEAA)</td>
			<td>Gibco</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr>
			<td>StemFlex basal medium + 50x StemFlex supplement</td>
			<td>Gibco</td>
			<td>A3349401</td>
			<td>Stem-cell maintenance medium</td>
		</tr>
		<tr>
			<td>Swinging Bucket Rotor (Heraeus #2704)</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Endres, L. M., et al., <a href="https://app.jove.com/v/61400">Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells.</a> J. Vis. Exp. (2020)
This video demonstrates a protocol to induce the differentiation of induced pluripotent stem cells into brain endothelial cells.

1. Preparation of materials required for iPSC culture and BEC differentiation.

	Matrix coating of tissue culture (TC) plastic for IMR90-4 iPSC culture
	
	...

Take an induced pluripotent stem cell, or iPSC, culture in maintenance media containing rho-kinase inhibitors.
Growth factors keep cells undifferentiated, while rho-kinase inhibitors enhance cell survival.
Replace the media with fresh maintenance media, removing rho-kinase inhibitors. Incubate, promoting colony formation.
Regularly replace with unconditioned media, which provides a controlled environment, triggering iPSC differentiation into neural progenitors and then brain endothelial cells, followed by cell proliferation.
Replace with endothelial cell media containing retinoic acid and fibroblast growth factors. Incubate.
Fibroblast growth factors facilitate cell proliferation, while retinoic acid promotes brain endothelial cell maturation.
Add proteolytic enzymes to detach the cells. Collect the cells and centrifuge. Discard the supernatant.
Resuspend the cells in endothelial media and seed them onto a multi-well membrane insert coated with collagen and fibronectin.
The well contains endothelial media. Incubate for selective brain endothelial cell adhesion.
Replace the media with non-supplemented endothelial media for further experiments.

12 조회수. 유도 만능 줄기 세포에서 뇌 내피 세포 생성

비디오: 유도 만능 줄기 세포에서 뇌 내피 세포 생성 - 실험

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

인간 만능 줄기 세포로부터 혈구 내피 세포의 유도 분화

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

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JoVE Journal

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Differentiation of Human Induced Pluripotent Stem Cells to Brain Microvascular Endothelial Cell-Like Cells with a Mature Immune Phenotype

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous system (CNS). Due to the limited access to disease-related BBB samples, it is still not well understood whether BBB malfunction is causative for disease development or rather a consequence of the neuroinflammatory or neurodegenerative process. Human induced pluripotent stem cells (hiPSCs) therefore provide a novel opportunity to establish in vitro BBB models from healthy donors and patients, and thus to study disease-specific BBB characteristics from individual patients. Several differentiation protocols have been established for deriving brain microvascular endothelial cell (BMEC)-like cells from hiPSCs. Consideration of the specific research question is mandatory for the correct choice of the respective BMEC-differentiation protocol. Here, we describe the extended endothelial cell culture method (EECM), which is optimized to differentiate hiPSCs into BMEC-like cells with a mature immune phenotype, allowing the study of immune cell-BBB interactions. In this protocol, hiPSCs are first differentiated into endothelial progenitor cells (EPCs) by activating Wnt/&#946;-catenin signaling. The resulting culture, which contains smooth muscle-like cells (SMLCs), is then sequentially passaged to increase the purity of endothelial cells (ECs) and to induce BBB-specific properties. Co-culture of EECM-BMECs with these SMLCs or conditioned medium from SMLCs allows for the reproducible, constitutive, and cytokine-regulated expression of EC adhesion molecules. Importantly, EECM-BMEC-like cells establish barrier properties comparable to primary human BMECs, and due to their expression of all EC adhesion molecules, EECM-BMEC-like cells are different from other hiPSC-derived in vitro BBB models. EECM-BMEC-like cells are thus the model of choice for investigating the potential impact of disease processes at the level of the BBB, with an impact on immune cell interaction in a personalized fashion.

Here, we describe a protocol, the extended endothelial cell culture method (EECM), that allows differentiation of pluripotent stem cells to brain microvascular endothelial cell (BMEC)-like cells. These cells show endothelial cell adhesion molecule expression and are thus a human blood-brain barrier model suitable to study immune cell interactions in vitro.

인간 유도 만능 줄기 세포를 성숙한 면역 표현형을 가진 뇌 미세혈관 내피 세포 유사 세포로 분화

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous ...

Generating Brain Endothelial Cells from Induced Pluripotent Stem Cells

내레이션 대본

Generating Brain Endothelial Cells from Induced Pluripotent Stem Cells