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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S., 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+blood-brain+barrier+endothelial+cells+from+human+pluripotent+stem+cells.">Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells.</a> Nature Biotechnology. 30 (8), 783-791 (2012).</li><li>Qian, T., 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=Directed+differentiation+of+human+pluripotent+stem+cells+to+blood-brain+barrier+endothelial+cells.">Directed differentiation of human pluripotent stem cells to blood-brain barrier endothelial cells.</a> Science Advances. 3 (11), e1701679 (2017).</li><li>Cecchelli, R., 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=A+stable+and+reproducible+human+blood-brain+barrier+model+derived+from+hematopoietic+stem+cells.">A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells.</a> PLoS One. 9 (6), e99733 (2014).</li><li>Hollmann, E. K., 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=Accelerated+differentiation+of+human+induced+pluripotent+stem+cells+to+blood-brain+barrier+endothelial+cells.">Accelerated differentiation of human induced pluripotent stem cells to blood-brain barrier endothelial cells.</a> Fluids and Barriers of the CNS. 14 (1), 9 (2017).</li><li>Stebbins, M. 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. G., 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=An+isogenic+neurovascular+unit+model+comprised+of+human+induced+pluripotent+stem+cell-derived+brain+microvascular+endothelial+cells,+pericytes,+astrocytes,+and+neurons.">An isogenic neurovascular unit model comprised of human induced pluripotent stem cell-derived brain microvascular endothelial cells, pericytes, astrocytes, and neurons.</a> Fluids and Barriers of the CNS. 16 (1), 25 (2019).</li><li>Nishihara, H., 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=Advancing+human+induced+pluripotent+stem+cell-derived+blood-brain+barrier+models+for+studying+immune+cell+interactions.">Advancing human induced pluripotent stem cell-derived blood-brain barrier models for studying immune cell interactions.</a> The FASEB Journal. 34 (12), 16693-16715 (2020).</li><li>Lu, T. M., 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=Pluripotent+stem+cell-derived+epithelium+misidentified+as+brain+microvascular+endothelium+requires+ETS+factors+to+acquire+vascular+fate.">Pluripotent stem cell-derived epithelium misidentified as brain microvascular endothelium requires ETS factors to acquire vascular fate.</a> Proceedings of the National Academy of Sciences. 118 (8), e2016950118 (2021).</li><li>Lian, X., 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=Efficient+differentiation+of+human+pluripotent+stem+cells+to+endothelial+progenitors+via+small-molecule+activation+of+WNT+signaling.">Efficient differentiation of human pluripotent stem cells to endothelial progenitors via small-molecule activation of WNT signaling.</a> Stem Cell Reports. 3 (5), 804-816 (2014).</li><li>Aisenbrey, E. A., Murphy, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+alternatives+to+Matrigel.">Synthetic alternatives to Matrigel.</a> Nature Reviews Materials. 5 (7), 539-551 (2020).</li><li>Nishihara, H., 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+of+human+pluripotent+stem+cells+to+brain+microvascular+endothelial+cell-like+cells+suitable+to+study+immune+cell+interactions.">Differentiation of human pluripotent stem cells to brain microvascular endothelial cell-like cells suitable to study immune cell interactions.</a> STAR Protocols. 2 (2), 100563 (2021).</li><li>Calabria, A. R., Weidenfeller, C., Jones, A. R., de Vries, H. E., Shusta, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Puromycin-purified+rat+brain+microvascular+endothelial+cell+cultures+exhibit+improved+barrier+properties+in+response+to+glucocorticoid+induction.">Puromycin-purified rat brain microvascular endothelial cell cultures exhibit improved barrier properties in response to glucocorticoid induction.</a> Journal of Neurochemistry. 97 (4), 922-933 (2006).</li><li>Engelhardt, B., Capture Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=crawl,+cross:+the+T+cell+code+to+breach+the+blood-brain+barriers.">crawl, cross: the T cell code to breach the blood-brain barriers.</a> Trends in Immunology. 33 (12), 579-589 (2012).</li><li>Duband, J. L., Gimona, M., Scatena, M., Sartore, S., Small, J. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#956;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&#8217;s (d) PBS (without calcium, magnesium)</td>
			<td>Thermo Fisher</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;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&#38;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&#38;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 ...

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1.9K 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

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. As an alternative, an increasing number of these patients are choosing to cryopreserve ovarian tissue for autograft following recovery and remission. Despite improvements in outcomes among patients undergoing auto-transplantation of cryopreserved ovarian tissue, efficient revascularization of grafted tissue remains a major obstacle. To mitigate ischemia and thus improve outcomes in patients undergoing auto-transplantation, we developed a vascular cell-based strategy for accelerating perfusion of ovarian tissue. We describe a method for co-transplantation of exogenous endothelial cells (ExECs) with cryopreserved ovarian tissue in a mouse xenograft model. We extend this approach to employ ExECs that have been engineered to constitutively express Anti-Mullerian hormone (AMH), thus enabling sustained paracrine signaling input to ovarian grafts. Co-transplantation with ExECs increased follicular volume and improved antral follicle development, and AMH-expressing ExECs promoted retention of quiescent primordial follicles. This combined strategy may be a useful tool for mitigating ischemia and modulating follicular activation in the context of fertility preservation and/or infertility at large.

For some patients, the only option for fertility preservation is cryopreservation of ovarian tissue. Unfortunately, delayed revascularization undermines follicular viability. Here, we present a protocol to co-transplant human ovarian tissue with endothelial cells for utilization as a cell-based strategy combining accelerated perfusion with a direct paracrine delivery of bioactive molecules.

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney&#8217;s filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm&#8217;s Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (&#62;90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...