HN was supported by the Uehara Memorial Foundation, an ECTRIMS Postdoctoral Research Exchange Fellowship, JSPS under the Joint Research Program implemented in association with SNSF (JRPs) Grant No. JPJSJRP20221507 and KAKENHI Grant No. 22K15711, JST FOREST Program (Grant Number JPMJFR2269, Japan), YOKOYAMA Foundation for Clinical Pharmacology Grant No. YRY-2217, the ICHIRO KANEHARA FOUNDATION, the Narishige Neuroscience Research Foundation, the NOVARTIS Foundation (Japan) for the Promotion of Science, and the YAMAGUCHI UNIVERSITY FUND. BE was supported by the Swiss MS Society and the Swiss National Science Foundation (grants 310030_189080 and ZLJZ3_214086)&#160;and&#160;Strategic Japanese-Swiss Science and Technology Programme (SJSSTP)&#160;grant IZLJZ3_214086.

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+and+characterization+of+human+pluripotent+stem+cell-derived+brain+microvascular+endothelial+cells.">Differentiation and characterization of human pluripotent stem cell-derived brain microvascular endothelial cells.</a> Methods. 101, 93-102 (2016).</li><li>Praca, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+brain+capillary-like+endothelial+cells+from+human+pluripotent+stem+cell-derived+endothelial+progenitor+cells.">Derivation of brain capillary-like endothelial cells from human pluripotent stem cell-derived endothelial progenitor cells.</a> Stem Cell Reports. 13 (4), 599-611 (2019).</li><li>Al-Ahmad, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+expression+and+activity+of+glucose+transporters+between+stem+cell-derived+brain+microvascular+endothelial+cells+and+hCMEC/D3+cells.">Comparative study of expression and activity of glucose transporters between stem cell-derived brain microvascular endothelial cells and hCMEC/D3 cells.</a> American Journal of Physiology. Cell Physiology. 313 (4), C421-C429 (2017).</li><li>Canfield, S. 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BE received a grant from Biogen to study extended dosing of Natalizumab on T-cell migration across the blood-brain barrier and a grant from CSL Behring to investigate the molecular underpinnings of blood-brain barrier dysfunction in neurological disorders. HN and BE are inventors of the provisional US patent applications related to the EECM-BMEC-like cells (63/084980 and 63/185815).

Critical points and troubleshooting 
Before starting EPC differentiation, researchers should ensure that no spontaneous cell differentiation events have occurred in the hiPSC cultures. The absence of spontaneously differentiated cells and the use of pure hiPSC colonies is critical for obtaining reproducible results. The hiPSC seeding density on Day -3 is important for obtaining a high purity of CD31+ EPCs after MACS. The seeding density for each hiPSC line and each passage may require optimization. Depending on the hiPSC line and passage number, the seeding density can range between 75 x 103 to 400 x 103 hiPSCs per well of a 12-well plate (20-100 x 103/cm2). The minimum density checkpoint of hiPSCs is the cell density on Day 2. The hiPSCs should reach 100% confluency by Day 2 at the latest. If the hiPSCs are not confluent by Day 2, the purity of CD31+ EPCs after MACS will usually be quite low. In this case, the hiPSC seeding density can be increased. If large numbers of differentiating cells detach from the plate around Day 3 to Day 5, the initial hiPSC seeding density can be decreased. The 7-8 &#181;M CHIR99021 in our experience is the optimal concentration for the hiPSCs lines used here, but the concentration may need to be optimized for other hiPSC lines that may respond differently to the inhibitor treatment. The purity of CD31+ EPCs should be confirmed before and after MACS. Before continuing MACS, the pre-sorted cell mixture should be &#62;10% CD31+ cells. CD31+ cell percentages of &#60;6% typically result in &#60;80% EPCs after MACS. Optimization of the initial seeding density and/or the CHIR99021 concentration is needed in this situation.
For successful selective passaging and generation of pure EC monolayers, the post-MACS purity of CD31+ EPCs is critical. If the purity after MACS is &#60;90%, one or two additional washes are recommended (steps 1.4.11-1.4.12). Ideally, the post-MACS purity should be &#62;95%. The EPC seeding density on collagen-coated plates should be optimized according to the hiPSC line to achieve 100% confluency within 3-7 days. Waiting until the ECs are 100% confluent will usually lead to successful selective passage. However, even for 100% confluent ECs, some hiPSC lines&#39; SMLCs also detach early. In this case, selective passaging at lower confluency (e.g., &#8804;80%) may be effective. If some SMLCs detach earlier than ECs, the ECs often cannot be rescued from the mixed EC-SMLC population. In this case, shortening the activation time for the dissociation reagent in passaging ECs and repetitive selective passaging may be helpful. The use of a commercial dissociation reagent rather than trypsin as a dissociation reagent is beneficial for selective passaging, as trypsin does not allow the separate detachment of ECs and SMLCs. Our permeability assays using small molecule tracers and testing of tight junction and adhesion molecule expression levels indicate that EPCs, EECM-BMEC-like cells, and SMLCs can be stored in liquid nitrogen for at least 2 years.
Significance and limitations of the method 
The method differentiates CD31+ EPCs from hiPSCs through the use of chemical GSK-3 inhibitors to activate Wnt/&#946;-catenin signaling. After positive selection of CD31+ EPCs by MACS, EPCs are cultured in a defined endothelial medium that promotes differentiation into mixed endothelial and SMLC populations. Selective passaging of these mixed populations with different adhesive properties allows the separation of ECs from SMLCs. After one or two passages, EECM-BMEC-like cells exhibit barrier properties and the expression of endothelial adhesion molecules that recapitulate those of primary human BMECs. Co-culture with SMLCs or their supernatants induces the cytokine-induced expression of VCAM-1.
In vivo, the BBB maintains CNS homeostasis by establishing low paracellular and transcellular permeability of molecules, through the transport of nutrients via specific transporters and the control of immune cell trafficking into the CNS. For studies of the BBB, a suitable model that displays the respective molecules and functions of interest is essential. Production of EECM-BMEC-like cells using defined reagents and samples from patients or healthy subjects provides a scalable human BBB model. The advantages of a model using EECM-BMEC-like cells over other BBB models are: 1) a morphology and endothelial transcriptome profile30 that resembles that of primary human BMECs; 2) the presence of mature tight junctions; 3) desirable barrier properties; and 4) the robust expression of endothelial adhesion molecules, including ICAM-1, ICAM-2, VCAM-1, E- and P-selectin, CD99, melanoma cell adhesion molecule (MCAM), and activated leukocyte cell adhesion molecule (ALCAM)22. Thus, this model is particularly useful for studying interactions between immune cells and BMECs. Although the permeability of small molecule tracers is higher for EECM-BMEC-like cells than that previously reported for iPSC-derived BMEC-like cells14,15, the barrier properties do compare quite well to those described for primary human BMECs. This similarity indicates that EECM-BMEC-like cells are likely to be a good in vitro model of the BBB. E-selectin expression on EECM-BMEC-like cells under physiological conditions must be taken into account when using this model to study non-inflamed BBBs that lack constitutive E-selectin expression in vivo31. In our previous study, we demonstrated that EECM-BMEC-like cells could phenocopy the BBB, as observed in the brains of MS patients with respect to disrupted tight junctions. This results in a higher permeability of small molecules and increased expression of functional adhesion molecule, mediating the increased adhesion and transmigration of immune cells across the BMEC-like cells32. Furthermore, we showed that the activation of Wnt/&#946;-catenin signaling can ameliorate the disruption of tight junctions and increased VCAM-1 expression in MS-derived EECM-BMEC-like cells32. These results indicate that the model is indeed useful to study the role of the BBB in neuroimmunological diseases, such as MS.
Taken together, EECM-BMEC-like cells are a promising tool for in-depth understanding of pathophysiological mechanisms at the level of the BBB and as a tool to develop new therapeutic targets for BBB stabilization. In the future, the model can be applied to study BBB dysfunction in a wider spectrum of diseases and could open avenues for novel therapeutic approaches.

The neurovascular unit (NVU) in the central nerve system (CNS) consists of the highly specialized microvascular endothelial cells (ECs), pericytes embedded in the endothelial basement membrane as well as the parenchymal basement membrane and astrocyte end-feet1. Within the NVU, brain microvascular endothelial cells (BMECs) are the key components that form the blood-brain barrier (BBB). BMECs form complex and continuous tight junctions and have extremely low pinocytotic activity compared to microvascular ECs in peripheral organs, which allow the BBB to inhibit the free paracellular diffusion of water-soluble molecules into the CNS. The expression of specific influx transporters and efflux pumps by BMECs ensures the uptake and export of nutrients and harmful molecules, respectively, from the CNS2. In addition, the BBB strictly controls immune cell entry into the CNS by expressing low levels of endothelial adhesion molecules crucial for immune cell trafficking into the CNS3. Under physiological conditions, the expression levels of adhesion molecules on the surface of BMECs, such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), are low, but these levels increase in some neurological disorders2. Morphological and functional breakdown of the BBB is reported in many neurological diseases, such as stroke4, multiple sclerosis (MS)5, and several neurodegenerative diseases6,7,8. Detailed investigation of the cellular and molecular characteristics of BMECs under both physiological and pathological conditions is an approach to identifying novel therapeutic strategies that target the BBB.
Until recently, primary or immortalized human and rodent BMECs were used to study the BBB. However, whether conclusions based on animal models of the BBB are readily applicable to the human BBB is unclear, since the expression of several important molecules, including adhesion molecules and solute carrier proteins, differs between humans and rodents9,10. Although human BMEC lines like hCMEC/D3 express appropriate levels of adhesion molecules11, these immortalized BMECs generally do not have complex tight junctions and robust barrier properties12. Primary human BMECs are useful to study barrier functions13, but they are not readily available to all researchers. Further, primary BMECs from patients can be difficult to obtain since they must be collected through a brain biopsy or surgery that is only performed under specific clinical conditions.
Recent advances in stem cell technology have allowed the differentiation of various human cell types, arising from stem cell sources like human induced pluripotent stem cells (hiPSCs). The hiPSC-derived models allow us to establish pathophysiological models using patient-derived samples. Several hiPSC-derived cell types can be combined to establish autologous co-cultures or organoids that better mimic physiological conditions. Several widely-used protocols14,15,16,17,18,19 may be used to differentiate hiPSC-derived BMEC-like cells that have robust diffusion barrier properties with the expression of BBB-specific transporters and efflux pumps, and are useful to study the paracellular diffusion of small molecules, molecular transport mechanisms, and drug delivery to the brain20,21. However, previous studies have shown that widely used hiPSC-derived BMEC-like cells lack the expression of key endothelial adhesion molecules, including VCAM-1, selectins, and ICAM-2, which are responsible for mediating interactions between immune cells and the BBB22. Furthermore, previous hiPSC-derived BMECs have been reported to display mixed endothelial and epithelial characteristics at the transcriptional level23. Therefore, we developed the extended endothelial cell culture method (EECM), a novel protocol that allows the differentiation of hiPSCs into BMEC-like cells that resemble primary human BMECs with respect to morphology, barrier characteristics, and endothelial adhesion molecule expression. This protocol describes the detailed methodological procedures to differentiate hiPSCs to BMEC-like cells displaying a mature immune phenotype.

<table><tbody><tr><td>0.22 mm Syringe filter</td><td>TPP</td><td>99722</td><td></td></tr><tr><td>15 mL Centrifuge tube</td><td>Falcon</td><td>352196</td><td></td></tr><tr><td>40 &amp;mu;m Falcon cell strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>5 mL Round-bottom tube</td><td>SPL</td><td>40005</td><td></td></tr><tr><td>50 mL Centrifuge tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>96-Well plate, round bottom</td><td>SPL</td><td>34096</td><td></td></tr><tr><td>Accutase</td><td>Sigma-Aldrich</td><td>A6964-500ml</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>695092</td><td></td></tr><tr><td>Advanced DMEM/F12</td><td>Life Technologies</td><td>12634</td><td></td></tr><tr><td>All-in-One Fluorescence Microscope</td><td>Keyence</td><td>BZ-X810</td><td></td></tr><tr><td>B-27 Supplement (503), serum free</td><td>Thermo Fischer Scientific</td><td>17504044</td><td></td></tr><tr><td>Bovine serum albumin (BSA), 7.5% in dPBS</td><td>Sigma-Aldrich</td><td>A8412</td><td></td></tr><tr><td>CellAdhere Dilution Buffer</td><td>STEMCELL Technologies</td><td>ST-07183</td><td></td></tr><tr><td>CellTracker Green CMFDA Dye</td><td>Invitrogen</td><td>C7025</td><td></td></tr><tr><td>Chamber slides</td><td>Thermo Fischer Scientific</td><td>178599</td><td></td></tr><tr><td>CHIR99021</td><td>Selleck Chemicals</td><td>S1263</td><td></td></tr><tr><td>Collagen IV from human placenta</td><td>Sigma-Aldrich</td><td>C5533</td><td></td></tr><tr><td>Corning tissue culture plates (12-well)</td><td>Corning</td><td>3512</td><td></td></tr><tr><td>Corning tissue culture plates (6-well)</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>Cryo tube innercap 2.0 mL</td><td>Watson</td><td>1396-201S</td><td></td></tr><tr><td>Dimethylsulfoxide (DMSO), sterile</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>DMEM (13), [+] 4.5 g/L D-glucose, [-] L-glutamine, [-] pyruvate</td><td>Thermo Fischer Scientific</td><td>31053-028</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s (d) PBS (without calcium, magnesium)</td><td>Thermo Fisher</td><td>14190250</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium/nutrient mixture F-12 (DMEM-F12)</td><td>Thermo Fischer Scientific</td><td>11320074</td><td></td></tr><tr><td>EasySepFITC Positive Selection Kit II</td><td>STEMCELL Technologies</td><td>18558</td><td></td></tr><tr><td>EasySepMagnet</td><td>Stemcell Technologies</td><td>18000</td><td></td></tr><tr><td>Ethylenediaminetetraacetic Acid Solution0.02% in DPBS</td><td>Sigma</td><td>E8008-100ML</td><td></td></tr><tr><td>Fetal Bovine Serum, qualified</td><td>Thermo Fischer Scientific</td><td>10270106</td><td></td></tr><tr><td>Fibronectin from bovine plasma</td><td>Sigma-Aldrich</td><td>F1141</td><td></td></tr><tr><td>Ficoll-Paque PLUS</td><td>Sigma-Aldrich</td><td>GE17144002</td><td></td></tr><tr><td>FIJI software (Version 2.0.0)</td><td>Image J, USA</td><td></td><td></td></tr><tr><td>FlowJo version10</td><td>BD</td><td></td><td></td></tr><tr><td>Fluorescein sodium salt</td><td>Sigma-Aldrich</td><td>F6377</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Thermo Fischer Scientific</td><td>35050-061</td><td></td></tr><tr><td>Glutaraldehyde solution</td><td>Sigma-Aldrich</td><td>G6257</td><td></td></tr><tr><td>HCL</td><td>Sigma-Aldrich</td><td>H1758</td><td></td></tr><tr><td>HEPES buffer solution</td><td>Thermo Fischer Scientific</td><td>15630-056</td><td></td></tr><tr><td>Human Endothelial Serum Free Medium (hESFM)</td><td>Thermo Fischer Scientific</td><td>11111-044</td><td></td></tr><tr><td>Human fibroblast growth factor 2 (FGF2)</td><td>Tocris</td><td>233-FB-500</td><td></td></tr><tr><td>iPS human induced pluripotent stem cells</td><td>Riken RBC</td><td>HPS1006</td><td></td></tr><tr><td>Kanamycin Sulfate (100x)</td><td>Thermo Fischer Scientific</td><td>15160-047</td><td></td></tr><tr><td>L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate</td><td>Sigma-Aldrich</td><td>A8960-5G</td><td></td></tr><tr><td>L-Glutamine 200 mM (1003)</td><td>Thermo Fischer Scientific</td><td>25030-024</td><td></td></tr><tr><td>Matrigel, growth factor reduced</td><td>Corning</td><td>354230</td><td></td></tr><tr><td>MEM NEAA (1003)</td><td>Thermo Fischer Scientific</td><td>11140-035</td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>32213</td><td></td></tr><tr><td>Mowiol</td><td>Sigma-Aldrich</td><td>81381</td><td></td></tr><tr><td>mTeSR Plus-cGMP</td><td>STEMCELL Technologies</td><td>ST-100-0276</td><td></td></tr><tr><td>mTeSR1 complete kit (basal medium plus 53 supplement)</td><td>STEMCELL Technologies</td><td>85850</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>71376</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Millipore</td><td>104005</td><td></td></tr><tr><td>Pen Strep</td><td>Thermo Fischer Scientific</td><td>15140-122</td><td></td></tr><tr><td>Recombinant Human IFN-gamma Protein</td><td>R&amp;amp;D Systems</td><td>285-IF-100</td><td></td></tr><tr><td>Recombinant Human IL-2</td><td>BD Biosciences</td><td>554603</td><td></td></tr><tr><td>Recombinant Human TNF-alpha Protein</td><td>R&amp;amp;D Systems</td><td>210-TA-020</td><td></td></tr><tr><td>ROCK inhibitor Y-27632</td><td>Tocris</td><td>1254</td><td></td></tr><tr><td>RPMI medium 1640</td><td>Thermo Fischer Scientific</td><td>21875-034</td><td></td></tr><tr><td>Scalpel</td><td>FEATHER</td><td>2975-11</td><td></td></tr><tr><td>Skim milk</td><td>BD Biosciences</td><td>232100</td><td></td></tr><tr><td>Sodium azide (NaN3)</td><td>Sigma-Aldrich</td><td>71290</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Thermo Fischer Scientific</td><td>11360-039</td><td></td></tr><tr><td>Transwells, PC Membrane, 0.4 mm, 12 mm, TC-Treated</td><td>Corning</td><td>3401</td><td></td></tr><tr><td>Tris base</td><td>Sigma-Aldrich</td><td>93362</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Vitronectin XF</td><td>STEMCELL Technologies</td><td>078180</td><td></td></tr><tr><td>Water, sterile, cell culture</td><td>Sigma-Aldrich</td><td>W3500</td><td></td></tr></tbody></table>

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.

Permeability assay 
The permeability of sodium fluorescein was calculated by measuring the fluorescence intensity of the medium collected from the lower chamber at 15, 30, 45, and 60 min. A total of 150 &#181;L of medium is sampled at each time point and the missing volume of 150 &#181;L is replaced with hECSR medium. the fluorescence intensity is read using a fluorescent plate reader (485 nm excitation/530 nm emission) and the correct signals, clearance volumes, and permeabilities are calculated using a previously described formula18 (Table 2). It is recommended to confirm whether the fluorescence intensity of sodium fluorescein increases over time. Multiple filters-at least triplicates-should be used for one assay to ensure reproducibility. For healthy control-derived EECM-BMEC-like cells, the sodium fluorescein (376 Da) permeability should be below 0.3 x 10-3 cm/min. To confirm the formation of a confluent EECM-BMEC-like cell monolayer, immunofluorescence staining for junctional proteins of the EECM-BMEC-like cells of each filter used in the permeability assays should be performed following this assay.
Immunofluorescence staining 
Immunofluorescence staining of EECM-BMEC-like cell junctional molecules, including claudin-5, occludin, and VE-cadherin1, was used to assess cell morphology and the presence of continuous and mature junctions (Figure 4). The monolayers of EECM-BMEC-like cells on the membranes of the filter inserts were fixed with cold methanol (-20 &#176;C) for 20 s, blocked with blocking buffer (Table 1), and then incubated with primary and secondary antibodies. The EECM-BMEC-like cells exhibited spindle like shapes and zigzag shaped junctions, both of which are characteristic morphological features of BMECs27. Stimulation of the EECM-BMEC-like cells seeded on chamber slides with pro-inflammatory cytokines, such as tumor necrosis factor-&#945; (TNF-&#945;) and interferon-&#947; (INF-&#947;) (0.1 ng/mL TNF-&#945; + 2 IU/mL IFN- &#947;) diluted in SMLC-derived conditioned medium, upregulated the expression of adhesion molecules, such as ICAM-1 and VCAM-128 (Figure 5). Representative images of smooth muscle cell markers, including &#945;-smooth muscle actin (SMA), calponin, and smooth muscle protein 22-Alpha (SM22a)29, are shown in Figure 6. SMLCs seeded on the chamber slide were fixed with 4% paraformaldehyde for 10 min, blocked with blocking buffer, and then incubated with primary and secondary antibodies.
Flow cytometry analysis of cell surface adhesion molecule expression by EECM-BMEC-like cells 
Representative results for cell surface expression of endothelial adhesion molecules on EECM-BMEC like cells are displayed in Figure 7. Stimulation with pro-inflammatory cytokines, like TNF-&#945; and INF-&#947;, upregulated the cell surface expression of several adhesion molecules, including ICAM-1, VCAM-1, and P-selectin. Cultivating EECM-BMEC-like cells with SMLC-conditioned medium enhanced endothelial VCAM-1 cell surface expression. The effect of the induction of VCAM-1 cell surface expression may vary between batches of SMLC-conditioned medium. It is recommended that several batches of conditioned medium harvested from SMLCs, derived from the same hiPSC source, be stored when differentiating SMLCs, in order to verify which batch induces the appropriate expression of VCAM-1.
Immune cell adhesion assay under static conditions 
The number of attached immune cells correlated to the expression level of functional adhesion molecules on the surface of EECM-BMEC-like cells. Stimulation with inflammatory cytokines upregulated the expression of endothelial adhesion molecules and promoted the increased number of immune cells that adhered to EECM-BMEC-like cell monolayers (Figure 8). The current experiment demonstrated the functionality of adhesion molecules on EECM-BMEC-like cells, making this model suitable for studying immune cell-EC interactions.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65134/65134fig02.jpg" /> 
Figure 2: Purification of CD31+ ECs. Dot plots of representative flow cytometry data from scatter gating of ECs and FITC-labeled CD31 staining of cell populations before (step 1.4.7) and after (step 1.4.14) MACS. MACS improves the purity of CD31+ EPCs in the population. Abbreviations: SSC = side scatter; FSC = forward scatter; FITC = fluorescein isothiocyanate; MACS = magnetic activated cell sorting. <a href="https://www.jove.com/files/ftp_upload/65134/65134fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65134/65134fig03.jpg" /> 
Figure 3: EECM-BMEC-monolayer permeability (10-3 cm/min) calculated from the raw fluorescence intensity of sodium fluorescein. The linear slope of clearance volume is calculated using linear regression for each filter (Figure 3A). The permeability of sodium fluorescein is calculated using two formulas (Figure 3B). (A) The linear slope of clearance volume versus time was calculated using linear regression for filter 1 (mc1) and the blank filter (mf). The mc1 and mf are coefficients of Xc1 and Xf, respectively. (B) Formula for calculating fluorescein permeability (Pe) using mc and mf (Formula 1). Pe units were converted using the surface area of a filter (Formula 2). <a href="https://www.jove.com/files/ftp_upload/65134/65134fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65134/65134fig04.jpg" /> 
Figure 4: EECM-BMEC-like cells display mature cellular junctions. Immunofluorescence staining for claudin-5, occludin, or VE-cadherin (red) in EECM-BMEC-like cells grown on membranes of insert filters. Nuclei were stained using 4&#8242;,6-diamidino-2-phenylindole (DAPI) (blue). Staining was performed on the exact same filter inserts used for the permeability assays. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65134/65134fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65134/65134fig05.jpg" /> 
Figure 5: Expression of endothelial adhesion molecules by EECM-BMEC-like cells. Immunofluorescence staining was performed on EECM-BMEC-like cells grown on membranes of filter inserts in the presence of SMLC-derived CM. Immunostaining for ICAM-1 or VCAM-1 (red) is shown for non-stimulated and 1 ng/mL TNF-&#945; + 20 IU/mL IFN- &#947; stimulated EECM-BMEC-like cells. Nuclei were stained with DAPI (blue). Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65134/65134fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65134/65134fig06.jpg" /> 
Figure 6: Characterization of SMLCs. Immunocytochemistry of &#945;-smooth muscle actin (SMA), calponin, or smooth muscle protein 22-Alpha (SM22a) (red) for SMLCs grown on chamber slides is shown. Nuclei were stained with DAPI (blue). Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65134/65134fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65134/65134fig07.jpg" /> 
Figure 7: Endothelial cell surface expression of adhesion molecules on EECM-BMEC-like cells. Results of flow cytometry analysis of EC surface adhesion molecule expression on EECM-BMEC-like cells is shown. EECM-BMEC-like cells were cultured using SMLC-derived conditioned medium. Blue, red, and gray lines of the histogram overlays show the non-stimulated (NS) condition, 1 ng/mL TNF-&#945; + 20 IU/mL IFN-&#947;-stimulated condition, and isotype control, respectively. The cell surface expression of endothelial adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1), ICAM-2, vascular cell adhesion molecule 1 (VCAM-1), P-selectin, E-selectin, CD99, and platelet endothelial cell adhesion molecule-1 (PECAM-1) were assessed. <a href="https://www.jove.com/files/ftp_upload/65134/65134fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65134/65134fig08.jpg" /> 
Figure 8: Adhesion of immune cells on EECM-BMEC-like cells. (A) Images of fluorescently labeled adherent immune cells on non-stimulated (NS) and 0.1 ng/mL TNF-&#945; + 2 IU/mL IFN-&#947;-stimulated (TNF-&#945; + IFN-&#947;) EECM-BMEC-like cell monolayers. The images correspond to the centers of the wells. Scale bar = 50 &#181;m. (B) The number of fluorescently labeled immune cells on monolayers of NS and TNF-&#945; + IFN-&#947;-stimulated EECM-BMEC-like cells. Adherent immune cells/fields of view (FOVs) were automatically counted using FIJI software. Dots represent the number of attached T cells. Bars show the mean value, and error bars show the standard deviation (SD) of eight trials. <a href="https://www.jove.com/files/ftp_upload/65134/65134fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Details of specific reagents for the assays. The name and exact amount of ingredients for each specific reagent are described. <a href="https://www.jove.com/files/ftp_upload/65134/Table1_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Example of raw data of the fluorescence plate reader for Pe calculation. Numbers in boldface type are the raw fluorescence intensity of sodium fluorescein measured by a plate reader. In order to accurately analyze the data, it is necessary to remove the background signal from the raw values and account for any signal loss resulting from sampling the bottom chamber, and subsequently correct the signal. For example, after subtracting the background, the 15 min sample exhibits a signal of 100 relative fluorescence units (RFU), and the 30 min sample exhibits a signal of 150 RFU. The corrected signal at 30 min is (150 RFU + the missing values at 15 min [100 RFU x 150 &#181;L/1,500 &#181;L]), which is 150 RFU + 10 RFU = 160 RFU. The clearance volume = (1,500 x [SB,t])/(ST,60 min), where 1,500 is the volume of the bottom chamber (1,500 &#181;L), SB,t is the corrected signal at time t, and ST,60 min is the signal of the top chamber at 60 min. <a href="https://www.jove.com/files/ftp_upload/65134/Table2_RE.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Differentiation of Human Induced Pluripotent Stem Cells to Brain Microvascular Endothelial Cell-Like Cells with a Mature Immune Phenotype at JoVE.com

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

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.

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

differentiation-human-induced-pluripotent-stem-cells-to-brain

Research

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Bioengineering

2.0K Views.  Yamaguchi University. 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.

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

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Developmental Biology

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

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.

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

Generating Human Brain Organoids with Resident Microglia Using iPSC-Derived HPCs

Derivation of a Human Brain Organoid with Microglia Development

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for studying human brain development and pathogenesis of neurological diseases. However, the lack of incorporation of microglia in the human brain organoids is still a major hurdle for 3D models of neuroinflammation. Current approaches include either the incorporation of fully differentiated microglia into mature brain organoids or the induction of microglial differentiation from the early stage of iPSC-derived embryoid bodies (EBs). The first approach misses the stage when microglial differentiation interacts with the adjacent neural environment, and the later approach is technically challenging, resulting in inconsistency among the final organoids in terms of the quantity and quality of microglia. To model brain organoids with microglia to study the early interactions between microglial and neuronal development, highly pure hematopoietic progenitor cells (HPC) differentiated from human iPSCs were incorporated&#160;into iPSC-derived EBs to make brain organoids. Using immunostaining and single-cell RNA sequencing (sc-RNA-seq) analysis, we confirmed that HPCs were incorporated into the 3D organoids, which eventually developed into brain organoids with both microglia and neurons. Compared to brain organoids without HPCs, this approach produces significant microglial incorporation in the brain organoids. This novel 3D organoid model, which consists of both microglial and neural development properties, can be used to study the early interactions between innate immune and nervous system development and potentially as a model for neuroinflammation and neuroinfectious disorders.

We present a protocol to generate a human brain organoid with resident microglia by incorporating Induced pluripotent stem cell (iPSC)-derived hematopoietic progenitor cells (HPCs) into organoid development.

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool&#160;for ...

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.

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

Immunology and Infection

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Neuroscience

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia in health and disease. Furthermore, iPSC-derived microglia can be maintained in co-culture with iPSC-derived cortical neurons, which enable investigations of microglia-neuron interactions that are hypothesized to be dysregulated in a number of neuropsychiatric disorders. Human iPSCs were differentiated to generate microglia using an adapted version of a protocol developed by the Fossati group, and the iPSC-derived microglia were validated with marker analysis and real-time PCR. Human microglia generated using this protocol were positive for the markers CD11C, IBA1, P2RY12, and TMEM119, and expressed the microglial-related genes AIF1, CX3CR1, ITGAM, ITGAX, P2RY12, and TMEM119. Human iPSC-derived cortical neurons that had been differentiated for 30 days were plated with microglia and maintained in co-culture until day 60, when experiments were undertaken. The density of dendritic spines in cortical neurons in co-culture with microglia was quantified under baseline conditions and in the presence of pro-inflammatory cytokines. In order to examine how microglia modulate neuronal function, calcium imaging experiments of the cortical neurons were undertaken using the calcium indicator Fluo-4 AM. Live calcium activity of cortical neurons was obtained using a confocal microscope, and fluorescence intensity was quantified using ImageJ. This report describes how co-culturing human iPSC-derived microglia and cortical neurons provide new approaches to interrogate the effects of microglia on cortical neurons.

This protocol describes a methodology to differentiate microglia from human iPSCs and maintain them in co-culture with iPSC-derived cortical neurons in order to study mechanistic underpinnings of neuroimmune interactions using human neurons and microglia.

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...

Differentiating Human-Induced Pluripotent Stem Cells into Microglia-Like Cells

Source: Funes, S. et al., <a href="https://app.jove.com/v/64323">Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes.</a>&#160;J. Vis. Exp. (2022)
This video demonstrates the procedure of the differentiation of human pluripotent cells into microglia-like cells. The process involves forming embryoid bodies, transferring them to coated wells, and using specific growth factors to guide differentiation into primitive macrophage precursors, which finally differentiate into microglia-like cells.

1. Microglia differentiation
NOTE: An overview of the protocol is summarized in&#160;Figure 1.

	Induced pluripotent stem cell (iPSC) culture
	NOTE: ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Advanced Neuronal Models

Generating Brain Endothelial Cells from Induced Pluripotent Stem Cells

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
	
	...

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

Summary

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Chapters in this video

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Derivation of a Human Brain Organoid with Microglia Development

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

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Differentiating Human-Induced Pluripotent Stem Cells into Microglia-Like Cells

Generating Brain Endothelial Cells from Induced Pluripotent Stem Cells