Name: 视频: 将人诱导的多能干细胞分化为脑微血管内皮细胞 - 概念
Uploaded: 2024-09-27T15:58:22.000+00:00
Duration: 1 min 22 s
Description: 22 次观看. 来源:Pong， S. et al.， 来自人诱导多能干细胞的脑微血管内皮细胞的衍生、扩增、冷冻保存和表征。J. Vis. Exp. （2020 年） 该视频演示了将人 iPSC 分化为脑微血管内皮细胞的多步骤过程。所得的分化细胞与脑微血管内皮细胞非常相似，其特征是紧密连接，使其成为脑部疾病研究的理想选择。

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
NOTE: Briefly, fibroblasts were reprogrammed to iPSC via mRNA-based genetic reprogramming. The iPSCs were maintained in stem cell medium (SCM) (see material list) and stored at a density of ~1.2 x 102 cells/mL with 1 mL of SCM, 10 &#956;M with rho-associated protein kinase inhibitor (ROCKi) Y-27632, and 10% (v/v) dimethyl sulfide (DMSO), in cryopreserved vials in liquid nitrogen at -160 &#176;C. All of the following procedures below are carried out in a biosafety cabinet unless stated otherwise.
1. Basement membrane matrix dilution and plate coating
<ol>
	<li>Dilute (1:50) growth factor reduced basement membrane matrix purified from Engelbreth-Holm-Swarm tumor in Dulbecco&#39;s Modified Eagle Medium (DMEM) without phenol red.</li>
	<li>Coat cell culture plates with the appropriate amount of diluted basement membrane matrix (i.e., 6-well plate = 1mL, 12-well plate = 0.5 mL) and incubate these plates at 37 &#176;C for at least 1 hour.</li>
</ol>
2. Induced pluripotent stem cells (iPSC) maintenance
NOTE: The maximum confluency per well in a 6-well flat-bottom plate is ~1.2 x 106 cells.
<ol>
	<li>Thaw cryopreserved iPSCs into SCM with 10 &#956;M Y-27632 and plate onto a 6-well plate coated with diluted growth factor reduced basement membrane matrix.</li>
	<li>Maintain iPSCs in SCM with 10 &#956;M Y-27632 for the first 24 hours after thawing. Switch to fresh medium after 24 hours.</li>
	<li>Maintain iPSCs in SCM until cells reach 80-90% confluency before passaging.
	<ol>
		<li>Calculate how many iPSCs will be needed for differentiation by multiplying desired density for differentiation (15,600 cells/cm2) by the surface area of the well. For a 6-well flat-bottom plate, multiply 15,600 cells/cm2 by 9.6 cm2 for a total of 149,760 cells/well.</li>
	</ol>
	</li>
	<li>To passage, wash the cells with Hanks&#39; Balanced Salt Solution (HBBS). Then, incubate the cells with non-enzymatic ethylenediaminetetraacetic acid (EDTA) (see material list) for 5 minutes at 37 &#176;C.
	<ol>
		<li>Use a cell scraper to gently lift off the cells. Collect cells in fresh SCM.</li>
		<li>Plate cells onto cell culture plates coated with diluted SCM and maintain cells as described in step 2.3 or store them at ~1.2 x 106 cells/mL in 1 mL of SCM, 10 &#956;M Y-27632, and 10% DMSO (v/v) in cryopreserved vials in liquid nitrogen at temperature of -160 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Differentiation of iPSCs to BMECs
NOTE: Non-enzymatic EDTA separates cells into clumps. Enzymatic EDTA (see Table of Materials) separates cells into single cell suspension. Retinoic acid (RA) should be protected from light.
<ol>
	<li>Wash iPSCs once with Dulbecco&#39;s Phosphate Buffer Saline (DPBS). Incubate with enzymatic EDTA (1 mL for 6-well plate, 0.5 mL for 12-well plate, and 0.25 mL for 24-well plate) for approximately 5 minutes at 37 &#176;C to yield a single cell suspension.</li>
	<li>Collect cells and centrifuge at 300 x g (relative centrifugal force) for 5 minutes at room temperature. Resuspend cell pellets in SCM containing 10 &#956;M Y-27632.</li>
	<li>Determine cell density using Trypan Blue and automated cell counter or a hemocytometer device. Plate cells at a density of 15,600 cells/cm2 or 149,760 cells/well of a 6-well flat-bottom plate (with a surface area of 9.6 cm2/well) in SCM containing 10 &#956;M Y-27632 for 24 hours.</li>
	<li>Initiate differentiation after 24 hours by changing SCM to E6 medium. Change E6 medium daily for the next 4 days.</li>
	<li>On day 4 of differentiation, replace E6 medium with human endothelial serum-free medium (hESFM) supplemented with diluted (1:200) B27 supplement, 20 ng/mL basic fibroblast growth factor (bFGF), and 10 &#956;M RA. Do not change this medium for the next 48 hours.</li>
	<li>Prepare 200 mL of hESFM with diluted (1:200) B27, mix 1 mL of 50x concentrated B27 supplement to 199 mL of hESFM.</li>
	<li>Prepare 20 ng/mL of bFGF by reconstituting 50 &#956;g of bFGF in 250 &#956;L of Tris buffer (5 mM Tris, pH 7.6, 150 mMsodium chloride, NaCl) to make 200 &#956;g/mL stock solution. Prepare 200 mL of hESFM containing 20 ng/mL bFGF by mixing 20 &#956;L of 200 &#956;g/mL bFGF with 200 mL of hESFM.</li>
	<li>Prepare 10 &#956;M RA by first making a 40 mg/mL of RA stock solution by adding 2.5 mL of DMSO to 100 mg of RA powder. Dilute this concentration to 3 mg/mL to make a 10 mM stock solution. Prepare 200 mL of hESFM containing 10 &#956;M RA by mixing 200 &#956;L of 10&#956;M RA in 200 mL hESFM.</li>
</ol>
4. Coating collagen IV (COL4) and fibronectin (FN) Matrix for Purification of iPSC-Derived BMEC
<ol>
	<li>Add 2 mL of sterile water to 2 mg of FN to make 1 mg/mL FN stock solution. Add 5 mL of sterile water to 5 mg of COL4 to make a 1 mg/mL COL4 stock solution.
	<ol>
		<li>Allow FN to dissolve for at least 30 minutes at 37 &#176;C and the COL4 to dissolve at room temperature.</li>
	</ol>
	</li>
	<li>Dilute FN stock solution in sterile water to a final concentration of 100 &#956;g/mL and COL4 stock solution to a final concentration of 400 &#956;g/mL.</li>
	<li>Coat the desired plates (6-well plate = 1 mL of COL4/FN solution, 12-well plate = 0.5 mL, 24-well plate= 0.25 mL, and 12-transwell filtered plate = 0.25 mL) with the mixture of 400 &#956;g/mL COL4 and 100 &#956;g/mL FN.</li>
	<li>Incubate plates for a minimum of 2 hours or overnight at 37&#176;C; for Transwell filtered plates, a minimum of 4 hours is recommended.</li>
</ol>
5. Sub-culture and purification of iPSC-Derived BMECs
NOTE: Incubation with enzymatic EDTA may take longer than 15 minutes depending on the confluency of the cells on day 6 of differentiation.
<ol>
	<li>On day 6 of differentiation, wash cells twice with DPBS. Incubate with 1 mL of enzymatic EDTA for at least 15 minutes at 37&#176;C until a single cell suspension is obtained.</li>
	<li>Collect cells via centrifugation at 300 x g for 5 minutes at room temperature. Resuspend cell pellets with fresh hESFM with diluted (1:200) B27 supplement, 20 ng/mL bFGF, and 10 &#956;M RA.</li>
	<li>Seed cells onto plates coated with a mixture of 400 &#956;g/mL COL4 and 100 &#956;g/mL FN. Seed cells using a ratio of 1 well of a 6-well plate to 3 wells of a 12-well plate, 3 wells of a 12-transwell filtered plate, or 6 wells of a 24-well plate.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accutase</td>
			<td>Sigma Aldrich</td>
			<td>A6964-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV from human placenta</td>
			<td>Sigma Aldrich</td>
			<td>C5533-5mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 2 mL Internal Threaded Polypropylene Cryogenic Vial</td>
			<td>Corning</td>
			<td>8670</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Flat Bottom Cell Culture Plates (6-wells)</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 (without phenol red)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1413202</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D2438-50mL</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (+/+)</td>
			<td>Gibco/Thermo Fisher Scientific</td>
			<td>14040-117</td>
			<td></td>
		</tr>
		<tr>
			<td>Essential 6 Medium (Thermo Fisher)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1516401</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma Aldrich</td>
			<td>F2006-2mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1413202</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; Balance Salt Solution with calcium and magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>24020-117</td>
			<td></td>
		</tr>
		<tr>
			<td>Human endothelial serum-free medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11111044</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen Countess Automated Cell Counter</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma Aldrich</td>
			<td>R2625-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride (ROCK inhibitor)</td>
			<td>Tocris/Thermo Fisher Scientific</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%) for use with the Countess Automated Cell Counter</td>
			<td>Thermo Fisher Scientific</td>
			<td>T10282</td>
			<td></td>
		</tr>
	</tbody>
</table>

differentiating human-induced pluripotent stem cells into brain microvascular endothelial cells

Source: Pong, S. et al., <a href="https://app.jove.com/v/61629">Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells.</a> J. Vis. Exp. (2020)
This video demonstrates a multi-step process for differentiating human iPSCs into brain microvascular endothelial cells. The resulting differentiated cells closely mimic brain microvascular endothelial cells, characterized by tight junctions, making them ideal for research on brain diseases.

Differentiating Human-Induced Pluripotent Stem Cells into Brain Microvascular Endothelial Cells

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
NOTE: Briefly, fibroblasts were ...

Begin with a multi-well plate containing human-induced pluripotent stem cells, or iPSCs, in a medium with a Rho-kinase inhibitor, which promotes cell survival.
Replace the medium with an essential medium containing nutrients and growth factors.
Incubate to allow the proliferation and differentiation of iPSCs into precursor cells.
Replace this medium with a differentiation medium supplemented with fibroblast growth factors and retinoic acid, then incubate.
The growth factors facilitate the differentiation of precursor cells into microvascular endothelial cells, while retinoic acid promotes the synthesis of tight junction proteins.
Wash and treat the cells with an enzyme-EDTA solution to detach the cells, creating a single-cell suspension.
Centrifuge to collect the cells and remove the supernatant. Then, resuspend the cells in a fresh differentiation medium.
Seed this cell suspension into a multi-well plate coated with collagen and adhesion proteins.
Incubate to allow cell adhesion, forming tight junctions between the cells, resembling brain microvascular endothelial cells.

differentiating-human-induced-pluripotent-stem-cells-into-brain

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22 次观看. 来源:Pong， S. et al.， 来自人诱导多能干细胞的脑微血管内皮细胞的衍生、扩增、冷冻保存和表征。J. Vis. Exp. （2020 年） 该视频演示了将人 iPSC 分化为脑微血管内皮细胞的多步骤过程。所得的分化细胞与脑微血管内皮细胞非常相似，其特征是紧密连接，使其成为脑部疾病研究的理想选择。 

视频: 将人诱导的多能干细胞分化为脑微血管内皮细胞 - 概念

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.

血源性内皮细胞与人多能干细胞的定向分化

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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Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central nervous system (CNS) by penetrating highly specialized brain endothelial cells (BECs). As Nm is a human-specific pathogen, the lack of robust in vivo model systems makes study of the host-pathogen interactions between Nm and BECs challenging and establishes a need for a human based model that mimics native BECs. BECs possess tighter barrier properties when compared to peripheral endothelial cells characterized by complex tight junctions and elevated trans-endothelial electrical resistance (TEER). However, many in vitro models, such as primary BECs and immortalized BECs, either lack or rapidly lose their barrier properties after removal from the native neural microenvironment. Recent advances in human stem-cell technologies have developed methods for deriving brain-like endothelial cells from induced pluripotent stem-cells (iPSCs) that better phenocopy BECs when compared to other in vitro human models. The use of iPSC-derived BECs (iPSC-BECs) to model Nm-BEC interaction has the benefit of using human cells that possess BEC barrier properties, and can be used to examine barrier destruction, innate immune activation, and bacterial interaction. Here we demonstrate how to derive iPSC-BECs from iPSCs in addition to bacterial preparation, infection, and sample collection for analysis.

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses.

脑膜炎奈瑟菌 诱导多能干细胞来源的脑内皮细胞的感染

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central ...

免疫与感染

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.

人诱导多能干细胞分化为具有成熟免疫表型的脑微血管内皮细胞样细胞

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

Differentiating Human-Induced Pluripotent Stem Cells into Brain Microvascular Endothelial Cells

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Differentiating Human-Induced Pluripotent Stem Cells into Brain Microvascular Endothelial Cells