This work is supported by NIH 3-UG3-NS115064-01, R01NS14239, Cure Alzheimer&#39;s Fund, NASA 80ARCO22CA004, Chan-Zuckerberg Initiative, MJFF/ASAP Foundation, and Brain Injury Association of America. C.G. is supported by NIH F31NS130909. Figure 1A was created with BioRender.com.

1. Differentiating iPSCs into iBBB cells
NOTE: These differentiation protocols have been previously described in Mesentier-Louro et al.34.
<ol>
	<li>Coating cell culture plates
	<ol>
		<li>Thaw reduced growth factor (GF) membrane matrix overnight at 4 &#176;C. Dilute 500 &#181;L of basement membrane matrix in 49.5 mL of DMEM. Keep this solution cold to prevent premature polymerization of the coating solution.</li>
		<li>Add 1-2 mL per well of a 6-well tissue culture-treated plate. Leave the coating solution on for at least 20 min at 37 &#176;C before replacing it with the appropriate warmed culture medium.</li>
	</ol>
	</li>
	<li>Differentiation of Human iPSCs into ETV2-inducible endothelial cells 
	Timing: 8 days 
	NOTE: This protocol is adapted from Blanchard et al.24 and Qian et al.35. We use the doxycycline-inducible expression of the transcription factor ETV2, as described by Wang et al.36 to improve the yield. ETV2 was introduced via PiggyBac plasmid and the cells were then transfected.
	<ol>
		<li>Culture iPSCs on reduced GF basement membrane matrix-coated plates in feeder-free pluripotent stem cell medium until the cells reach ~70% confluency. 
		NOTE: We recommend the inducible iPSCs be maintained with selection for the PiggyBac plasmid (i.e., puromycin)</li>
		<li>Day 0: Dissociate the cells with a protease-collagenase mixture for 5 min. Transfer the dissociated cells into a conical tube with a 1:3 ratio of the protease-collagenase mixture to stem cell medium. Spin down cells at 300 &#215; g for 3 min. Resuspend the cell pellet with stem cell medium and plate the cells at 20,800 cells/cm2 on reduced GF basement membrane matrix-coated plates in stem cell medium supplemented with 10 &#181;M Y27632.</li>
		<li>Day 1: Replace the medium with DeSR1 [DMEM/F12 + glutamine supplement, 1x Minimum Essential Medium Non-Essential Amino Acids (MEM-NEAA), 1% penicillin-streptomycin (P/S)], supplemented with 10 ng/mL BMP4, 6 &#181;M CHIR99021, and 5 &#181;g/mL doxycycline.</li>
		<li>Day 3: Replace the medium with DeSR2 [DMEM/F12 + glutamine supplement, 1x MEM-NEAA, 1x N-2, 1x B-27, 1% P/S] supplemented with 5 &#181;g/mL doxycycline.</li>
		<li>Day 5: Replace medium with hECSR [Human Endothelial Serum-Free Medium, 1x MEM-NEAA, 1x B-27, 1% P/S] supplemented with 50 ng/mL VEGF-A, 2 &#181;M forskolin, and 5 &#181;g/mL doxycycline.</li>
		<li>Day 7: Replace medium with hECSR supplemented with 50 ng/mL VEGF-A, 2 &#181;M forskolin, and 5 &#181;g/mL doxycycline.</li>
		<li>Day 8: Split ECs 1:3 with a protease-collagenase mixture and re-seed onto fresh reduced GF basement membrane matrix-coated plates in hECSR supplemented with 50 ng/mL VEGF-A and 5 &#181;g/mL doxycycline. 
		NOTE: This is the best time point to encapsulate ECs into iBBBs for uniform vasculature formation.</li>
		<li>Day 10+: Feed the cells every 2-3 days with hECSR supplemented with 50 ng/mL VEGF-A and 5 &#181;g/mL doxycycline. Continue to split the cells 1:3 with a protease-collagenase mixture when the plates are confluent. 
		NOTE: iPSC-derived ECs can be frozen at any point starting Day 8. Lift the cells, as if to passage. Resuspend the pellet in EC freezing medium [60% knockout serum replacement (KSR), 30% hECSR, 10% DMSO,&#160;50 ng/mL VEGF-A,&#160;and 10 &#181;M Y27632], splitting the cells 1:3, and freeze using a freezing container at -80 &#176;C. Thaw onto reduced GF basement membrane matrix-coated plates in hECSR supplemented with&#160;50 ng/mL VEGF-A, 5 &#181;g/mL doxycycline.</li>
		<li>Confirm brain microvascular endothelial cell differentiation by immunocytochemistry using antibodies against CD31/PECAM1 or VE-cadherin (Figure 2A).</li>
	</ol>
	</li>
	<li>Differentiation of Human iPSCs into pericytes 
	Timing: 6 days 
	NOTE: This protocol was adapted from Patsch et al.36.
	<ol>
		<li>Culture iPSCs on reduced GF basement membrane matrix-coated plates in feeder-free conditions in stem cell medium until the cells reach ~70% confluency.</li>
		<li>Day 0: Dissociate the cells with a protease-collagenase mixture for 5 min. Transfer the dissociated cells into a conical tube with a 1:3 ratio of the protease-collagenase mixture to stem cell medium. Spin down cells at 300 &#215; g for 3 min. Resuspend the cell pellet with stem cell medium and plate the cells at 37,000-40,000 cells/cm2 (depending on the cell line) on reduced GF basement membrane matrix-coated plates in stem cell medium supplemented with 10 &#181;M Y27632.</li>
		<li>Day 1: Replace the media with N2B27 [50% DMEM/F12 + glutamine supplement, 50% Neurobasal, 1x MEM-NEAA, 0.5x, glutamine supplement, 1x N-2, 1x- B-27, 1% P/S] supplemented with 25 ng/mL BMP4 and 8 uM CHIR99021.</li>
		<li>Days 3-4: Replace the media with N2B27 supplemented with 2 ng/mL Activin A and 10 ng/mL PDGF-BB, and feed daily.</li>
		<li>Day 5: Split PCs with a protease-collagenase mixture and seed onto 0.1% gelatin-coated plates at 35,000 cells/cm2 in N2B27 supplemented with 10 ng/mL PDGF-BB. 
		NOTE: Only split cells once confluent. Some lines might require a few more days before they are ready to split; continue to change the media daily until cells are confluent.</li>
		<li>Expand PCs in N2B27 supplemented with 10 ng/mL PDGF-BB, changing media every 2-3 days. Remove PDGF-BB from the media after the cells once again reach confluency and undergo a second passage. 
		NOTE: Pericytes are ready to be used in iBBBs after the second passage. Once expanded, PCs can be frozen. Lift the cells as if to passage, resuspend the cell pellet in freezing media [90% KSR and 10% DMSO] and freeze using a freezing container at -80 &#176;C. Thaw onto 0.1% gelatin-coated plates at 35,000 cells/cm2 in N2B27.</li>
		<li>Confirm pericyte differentiation by immunocytochemistry using antibodies against PDGFRB and NG2 (Figure 2B).</li>
	</ol>
	</li>
	<li>Differentiation of Human iPSCs into neural progenitor cells (NPCs) and astrocytes 
	Timing: 45 days total (15 days for NPCs followed by 30 days for astrocytes) 
	&#8203;NOTE: The NPC differentiation protocol was adapted from Chambers et al.38, and the astrocyte differentiation is as described in TCW et al.39.
	<ol>
		<li>Culture iPSCs on reduced GF basement membrane matrix-coated plates in feeder-free conditions in stem cell medium until the cells reach ~70% confluency.</li>
		<li>Dissociate the cells with a protease-collagenase mixture for 5 min. Transfer the dissociated cells into a conical tube with a 1:3 ratio of the protease-collagenase mixture to stem cell medium. Spin down cells at 300 &#215; g for 3 min. Resuspend the cell pellet with stem cell medium and plate the cells at 100,000 cells/cm2 on reduced GF basement membrane matrix-coated plates in stem cell medium supplemented with 10 &#181;M Y27632.</li>
		<li>Replace the stem cell medium the following day. Continue to feed the cells every other day until they reach &#62;95% confluency (3-4 days depending on the cell line).</li>
		<li>NPC Day 0: Replace the media with N2B27 [50% DMEM/F12 + glutamine supplement, 50% Neurobasal, 1x MEM-NEAA, 0.5x, glutamine supplement, 1x N-2, 1X- B-27, 1% P/S] supplemented with 10 &#181;M SB43152 and 100 nM LDN193189.</li>
		<li>NPC Days 1-9: Feed the cells daily with N2B27 supplemented with 10 &#181;M SB43152 and 100 nM LDN193189.</li>
		<li>NPC Day 10: Split NPCs 1:3 with the protease-collagenase mixture and seed onto fresh reduced GF basement membrane matrix-coated plates in N2B27 supplemented with 20 ng/mL FGF-basic and 10 &#181;M Y27632.</li>
		<li>NPC Days 11 - 13: Feed NPCs daily with N2B27 supplemented with 20 ng/mL FGF-basic.</li>
		<li>NPC Day 14: Split NPCs 1:3 with the protease-collagenase mixture and reseed onto fresh reduced GF basement membrane matrix-coated plates in N2B27 supplemented with 20 ng/mL FGF-basic and 10 &#181;M Y27632.</li>
		<li>NPC Day 15/Astrocyte Day 0: This is Day 0 of the astrocyte differentiation. Feed the cells with complete Astrocyte medium (AM). 
		NOTE: NPCs can be maintained in N2B27 supplemented with 20 ng/mL FGF-basic and passaged when confluent. To freeze NPCs, resuspend the cell pellet in freezing media [90% KSR and 10% DMSO], split the cells 1:3, and freeze using a freezing container at -80 &#176;C. Thaw onto reduced GF basement membrane matrix-coated plates in N2B27 supplemented with 20 ng/mL bFGF and 10 &#181;M Y27632.</li>
		<li>Astrocyte Days 1-30: Feed the cells every 2-3 days with AM. When cells are &#62;90% confluent, passage using the protease-collagenase mixture and plate at 15,000 cells/cm2 on fresh reduced GF basement membrane matrix-coated plates in AM. 
		NOTE: Astrocytes should be fully differentiated in 30 days. Astrocytes can be frozen in freezing media [90% KSR and 10% DMSO] in a freezing container at -80 &#176;C. Thaw onto reduced GF basement membrane matrix-coated plates in AM at 25,000-35,000 cells/cm2.</li>
		<li>Confirm NPC differentiation by immunocytochemistry using antibodies against Nestin and SOX2. Confirm astrocyte differentiation by immunocytochemistry using antibodies against S100B and CD44 (Figure 2C).</li>
	</ol>
	</li>
</ol>
2. Assembling the iBBB
<ol>
	<li>Thaw reduced growth factor (GF) basement membrane matrix overnight at 4 &#176;C. Do not dilute this matrix in DMEM as the iBBBs are encapsulated in 100% reduced GF basement membrane matrix. Each iBBB requires 50 &#181;L of the matrix.</li>
	<li>Dissociate the differentiated ECs with a protease-collagenase mixture at 37 &#176;C for 5 min. Transfer the dissociated cells into a conical tube with a 1:3 ratio of the protease-collagenase mixture to hECSR (Human Endothelial Serum-Free Medium, 1x MEM-NEAA, 1x B-27, 1% P/S). Spin down cells at 300 &#215; g for 3 min. Resuspend the cell pellet in hECSR. 
	NOTE: Use a conical tube, either 15 mL or 50 mL, that is large enough to accommodate the volume of dissociated cells plus media. Cells can also be divided among multiple tubes and then recombined upon resuspension.</li>
	<li>Dissociate the differentiated PCs with the protease-collagenase mixture at 37 &#176;C for 5 min. Transfer the dissociated cells into a conical tube with a 1:3 ratio of the protease-collagenase mixture to N2B27 (50% DMEM/F12 + glutamine supplement, 50% Neurobasal, 1x MEM-NEAA, 0.5x glutamine supplement, 1x N-2, 1x B-27, 1% penicillin-streptomycin [P/S]). Spin down cells at 300 &#215; g for 3 min. Resuspend the cell pellet in N2B27.</li>
	<li>Dissociate the differentiated astrocytes with the protease-collagenase mixture at 37 &#176;C for 5 min. Transfer the dissociated cells into a conical tube with a 1:3 ratio of the protease-collagenase mixture to complete Astrocyte Medium (AM). Spin down cells at 300 &#215; g for 3 min. Resuspend the cell pellet in AM.</li>
	<li>Using a hemocytometer, count each cell type. Dilute each type of cell suspension to a final concentration of 1 &#215; 106 cells/mL in the appropriate medium.</li>
	<li>Combine the calculated number of cells for each cell type in a conical tube: 50 &#181;L of iBBB requires 2.5 &#215; 105 ECs, 5 &#215; 104 PCs, and 5 &#215; 104 astrocytes. Include 10% more than the calculated number of cells to account for pipetting errors. Spin down the cell mixture at 300 &#215; g for 3 min. 
	NOTE: It is highly recommended to plate some leftover cells of each cell type in 2D monocultures to fix and stain for quality control of the input cells. Plate each cell type at 35,000 cells/cm2 on previously prepared plates coated with reduced GF basement membrane matrix and fix after 3-4 days. See Table 1 for cell-type specific antibodies.</li>
	<li>Aspirate medium from the cell pellet leaving approximately 50 &#181;L of the supernatant above the cell pellet. Using a P200, gently resuspend the cell pellet in residual medium to create a single-cell slurry.</li>
	<li>Mix the cell slurry in enough reduced GF basement membrane matrix supplemented with 10 ng/mL VEGF-A for the desired number of iBBBs at 50 &#181;L of reduced GF basement membrane matrix per iBBB, taking care to mix the cells homogeneously while not introducing any air bubbles. 
	NOTE: It is critical to keep the cells and reduced GF basement membrane matrix on ice at this stage to prevent premature polymerization.</li>
	<li>Pipette 50 &#181;L of reduced GF basement membrane matrix mixture into the well of a 48-well glass-bottom culture dish. Distribute the volume evenly throughout the bottom of the dish (Figure 1C).</li>
	<li>Incubate the iBBBs at 37 &#176;C for 30-40 min to allow the reduced GF basement membrane matrix to polymerize, encapsulating the cells in the matrix.</li>
	<li>Add 500 &#181;L of AM supplemented with 10 ng/mL VEGF-A, 5 &#181;g/mL doxycycline, and 10 &#181;M Y27632.</li>
	<li>Change the medium the following day to AM supplemented with 10 ng/mL VEGF-A. Continue to change the medium every 2-3 days. After 2 weeks, stop supplementing with VEGF-A; iBBBs are ready for downstream assays after 2 weeks.</li>
</ol>
3. Inducing cerebral amyloid angiopathy (CAA) with A&#946; fibrils
<ol>
	<li>Prepare stock solutions of amyloid-&#946;1-40 and amyloid-&#946;1-42 at 200 &#181;M in PBS.</li>
	<li>Add A&#946; to iBBB medium on 2-week iBBBs to a final concentration of 20 nM. Incubate the cells in A&#946;-supplemented medium for 96 h (4 days).</li>
	<li>After 96 h, remove the medium and continue with fixing (section 4).</li>
</ol>
4. Fixing and staining the iBBB
<ol>
	<li>To fix the iBBB, remove the medium, wash in 1 mL of PBS, and incubate in 500 &#181;L of 4% paraformaldehyde overnight at 4 &#176;C.</li>
	<li>Remove the 4% paraformaldehyde solution, and rinse for 4 x (at least) 15 min in 1 mL of PBS.</li>
	<li>Permeabilize the samples in 0.3% PBST (PBS with 0.3% Triton X-100) for 1 h at room temperature with gentle shaking.</li>
	<li>Incubate the cultures in blocking buffer (5% Normal Serum in 0.3% PBST) at room temperature for at least 1 h with gentle shaking. 
	NOTE: The type of serum used is dependent on the host species for the secondary antibodies used. For example, if the host species for the secondary antibodies is&#160;goat, use normal goat serum; if the host species is&#160;donkey, use normal donkey serum. This can also be done overnight at 4 &#176;C with gentle shaking.</li>
	<li>Dilute primary antibodies to the recommended or determined concentration in blocking buffer.</li>
	<li>Replace the blocking solution with the primary antibody dilution. Make sure that the iBBBs are fully submerged in antibody solution for even incubation; 100-150 &#181;L is enough to cover a 50 &#181;L iBBB in a 48-well glass-bottom culture dish. Incubate overnight at 4 &#176;C with gentle shaking.</li>
	<li>Remove the primary antibody. Wash iBBBs for 3 x 10-20 min in 0.3%&#160;PBS.</li>
	<li>Dilute the secondary antibody to the recommended or determined concentration in blocking buffer. Additionally, add a nuclear stain, such as Hoechst, to the secondary antibody solution at the recommended or determined concentration.</li>
	<li>Replace the last PBS wash with the secondary antibody dilution. Make sure that the iBBBs are fully submerged in antibody solution for even incubation; 100-150 &#181;L is enough to cover a 50 &#181;L iBBB in a 48-well glass-bottom culture dish. Incubate at room temperature for 2 h with gentle shaking. 
	NOTE: This can also be done overnight at 4 &#176;C with gentle shaking.</li>
	<li>Wash iBBBs 4-5x for at least 1 h per wash in&#160;PBS. Store in PBS or anti-fading mounting media at 4 &#176;C until the samples are imaged.</li>
	<li>To image on an inverted microscope, keep iBBBs in glass-bottom plates or dishes. Place a glass coverslip over the glass well to keep the iBBBs in place (Figure 1D).</li>
</ol>

A 3D Blood-Brain Barrier Model Derived from Human Pluripotent Stem-Cells

The authors report no conflicts of interest.

BBB dysfunction is a co-morbidity, and potentially, a cause or exacerbating factor in numerous neurological diseases7,40,41. However, it is nearly impossible to study the molecular and cell biology underlying the dysfunction and breakdown of the BBB in humans with neurovascular disease. The inducible-BBB (iBBB) presented in this protocol provides an in vitro system that recapitulates important cell interactions of the B...

The blood-brain barrier (BBB) is a key microvascular network separating the central nervous system (CNS) from the periphery to maintain an ideal environment for proper neuronal function. It has a critical role in regulating the influx and efflux of substances into the CNS by maintaining metabolic homeostasis1,2,3,4, clearing waste4,5,6, and protecting the brain from pathogens and toxins7,8.
The primary cell type of the BBB is the endothelial cell (EC). Endothelial cells, derived from the mesoderm lineage, form the walls of the vasculature1,9. Microvascular ECs form tight junctions with each other to greatly decrease the permeability of their membrane10,11,12,13,14 while expressing transporters to facilitate the movement of nutrients into and out of the CNS1,4,12,14. Microvascular ECs are encircled by pericytes (PCs)-mural cells that regulate microvascular function and homeostasis and are critical for regulating the permeability of the BBB to molecules and immune cells15,16,17. The astrocyte, a major glial cell type, is the final cell type comprising the BBB. Astrocyte end-feet wrap around the EC-PC vascular tubes while the cell bodies extend into the brain parenchyma, forming a connection between neurons and vasculature1. Distinct solute and substrate transporters are localized on astrocyte&#160;end-feet (e.g., aquaporin 4 [AQP-4]) that have a critical role in BBB function18,19,20,21.
The BBB is critical in maintaining proper brain health function, and dysfunction of the BBB has been reported in many neurological diseases, including Alzheimer&#39;s disease (AD)22,23,24,25, multiple sclerosis7,26,27,28, epilepsy29,30, and stroke31,32. It is increasingly recognized that cerebrovascular abnormalities play a central role in neurodegeneration, contributing to increased susceptibility to ischemic and hemorrhagic events. For example, more than 90% of AD patients have cerebral amyloid angiopathy (CAA), a condition characterized by the deposition of amyloid &#946; (A&#946;) along the cerebral vasculature. CAA increases BBB permeability and decreases BBB function, leaving the CNS vulnerable to ischemia, hemorrhagic events, and accelerated cognitive decline33.
We recently developed an in vitro model of the human BBB, derived from&#160;patient-induced pluripotent stem cells, which incorporates ECs, PCs, and astrocytes encapsulated in a 3D matrix (Figure 1A). The iBBB recapitulates physiologically relevant interactions, including vascular tube formation and localization of astrocyte end-feet with vasculature24. We applied the iBBB to model the susceptibility of CAA mediated by APOE4 (Figure 1B). This enabled us to identify the causal cellular and molecular mechanisms by which APOE4 promotes CAA, and leverage these insights to develop therapeutic strategies that reduce CAA pathology and improve learning and memory in vivo in APOE4 mice24. Here, we provide a detailed protocol and video tutorial for reconstructing the BBB from human iPSCs and modeling CAA in vitro.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6e10 amyloid-&#946; antibody</td>
			<td>Biolegend</td>
			<td>SIG-39320</td>
			<td>Used at 1:1000</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>Peprotech</td>
			<td>20-14E</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488, 555, 647 secondary antibodies</td>
			<td>Invitrogen</td>
			<td>Various</td>
			<td>Used at 1:1000</td>
		</tr>
		<tr>
			<td>Amyloid-beta 40 fibril</td>
			<td>AnaSpec</td>
			<td>AS-24235</td>
			<td></td>
		</tr>
		<tr>
			<td>Amyloid-beta 42 fibril</td>
			<td>AnaSpec</td>
			<td>AS-20276</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquaporin-4 antibody</td>
			<td>Invitrogen</td>
			<td>PA5-53234</td>
			<td>Used at 1:300</td>
		</tr>
		<tr>
			<td>Astrocyte basal media and supplements</td>
			<td>ScienCell</td>
			<td>1801</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 serum-free supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>Peprotech</td>
			<td>120-05ET</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Cyamn Chemical</td>
			<td>13112</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 with GlutaMAX medium</td>
			<td>Gibco</td>
			<td>10565018</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Millipore-Sigma</td>
			<td>D3072-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-basic</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G slide mounting medium</td>
			<td>VWR</td>
			<td>100502-406</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>R&#38;D Systems</td>
			<td>1099/10</td>
			<td></td>
		</tr>
		<tr>
			<td>GeltrexTM LDEV-Free hESC-qualified Reduced Growth Factor Basement&#160;</td>
			<td>Gibco</td>
			<td>A1413302</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom 48-well Culture Dishes</td>
			<td>Mattek Corporation</td>
			<td>P48G-1.5-6-F</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342&#160;</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Endothelial Serum-free medium</td>
			<td>Gibco</td>
			<td>11111044</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN193189</td>
			<td>Tocris</td>
			<td>6053</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium Non-essential Amino Acid Solution (MEM-NEAA)&#160;</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Millipore-Sigma</td>
			<td>S30-100mL</td>
			<td>Use serum to match secondary antibody host</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)&#160;</td>
			<td>ThermoFisher</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF-BB</td>
			<td>Peprotech</td>
			<td>100-14B</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGFRB (Platelet-derived growth factor receptor beta) antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF385</td>
			<td>Used at 1:500</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS), pH 7.4</td>
			<td>Gibco</td>
			<td>10010031</td>
			<td></td>
		</tr>
		<tr>
			<td>Pecam1 (Platelet endothelial cell adhesion molecule 1) antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF806</td>
			<td>Used at 1:500</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>PiggyBac plasmid (PB_iETV2_P2A_GFP_Puro)</td>
			<td>AddGene</td>
			<td>&#160;Catalog #168805</td>
			<td></td>
		</tr>
		<tr>
			<td>S100B antibody</td>
			<td>Sigma-Aldrich</td>
			<td>S2532-100uL</td>
			<td>Used at 1:500</td>
		</tr>
		<tr>
			<td>SB43152</td>
			<td>Reprocell</td>
			<td>04-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioflavin T</td>
			<td>Chem Impex</td>
			<td>22870</td>
			<td>Used at 25uM</td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T8787-250mL</td>
			<td></td>
		</tr>
		<tr>
			<td>VE-cadherin (CD144) antibody</td>
			<td>R&#38;D systems</td>
			<td>AF938</td>
			<td>Used at 1:500</td>
		</tr>
		<tr>
			<td>VEGF-A</td>
			<td>Peprotech</td>
			<td>100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632</td>
			<td>R&#38;D Systems</td>
			<td>1254/10</td>
			<td></td>
		</tr>
		<tr>
			<td>ZO-1</td>
			<td>Invitrogen</td>
			<td>MA3-39100-A488</td>
			<td>Dilution = 1:500</td>
		</tr>
	</tbody>
</table>

reconstruction of the blood-brain barrier in vitro to model and therapeutically target neurological disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer&#39;s disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic&#160;delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.

Author Spotlight: A Personalized Approach Towards Investigating Alzheimer&#039;s Disease Using an In Vitro Blood-Brain Barrier Model 

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

A properly formed iBBB solidifies into a single translucent disc (Figure 3A). It is normal for the iBBB to detach from the surface onto which it was first pipetted after a few days. This cannot be avoided, but is not a major concern to the proper formation of the iBBB if care is taken when changing media to not accidentally aspirate the iBBB. After 24 h, evenly distributed, single cells can be identified under a brightfield microscope (Figure 3B). After 2 weeks,...

Watch this Scientific Journal Video about A Personalized Approach Towards Investigating Alzheimer's Disease Using an In Vitro Blood-Brain Barrier Model at JoVE.com

The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease.

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...

reconstruction-blood-brain-barrier-vitro-to-model-therapeutically

Research

Education

JoVE Core

JoVE Journal

Pharmacokinetics and Pharmacodynamics

Neuroscience

Unit 1

Pharmacokinetics: Drug Distribution and Protein Binding

SCIENTISTS IN ACTION

Preparing Cells to Assemble a 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier

Begin by preparing the differentiated endothelial cells, parasites, and astrocytes for assembling the iBBB model. After aspirating the cell culture medium from the differentiated endothelial cells, wash the cells with PBS once, then add a protease collagenase mixture to the washed cells and dissociate the cells at 37 degrees Celsius for five minutes. Transfer the protease collagenase mix with the dissociated cells into a conical tube containing hECSR, maintaining a 1:3 ratio of the mix to hECSR, then centrifuge the cells at 300 G for three minutes and aspirate the supernatant before resuspending the resultant cell pellet in the hECSR.Dissociate the differentiated parasites with the protease collagenase mixture at 37 degrees Celsius for five minutes. Add the appropriate amount of N2B27 in a conical tube and transfer the dissociated cells in the protease collagenase mix to it, maintaining a 1:3 ratio of the mix to N2B27. Spin down the cells at 300 G for three minutes.After aspirating the supernatant, resuspend the cell pellet in N2B27. Dissociate the differentiated astrocytes with the protease collagenase mixture at 37 degrees Celsius for five minutes. Transfer the dissociated cells in the protease collagenase mix to a conical tube containing complete astrocyte medium at a 1:3 ratio of the mix to the medium.After centrifuging the cells at 300 G for three minutes, aspirate the supernatant and resuspend the cell pellet in complete astrocyte medium. The cells are now ready for assembling the iBBB.

Constructing the 3D Human-Stem-Cell-Derived In Vitro Blood Brain Barrier

Assemble the human pluripotent stem cell derived blood-brain barrier, or iBBB, using the dissociated endothelial cells, parasites, and astrocytes, Combine the three types of cells in a conical tube while including 10%more than the calculated number of cells to account for pipetting errors. Spin down the cell mixture at 300 g for three minutes. Aspirate the supernatant, leaving the cell pellet with approximately 50 microliters of the medium.Using a P200 pipette, gently resuspend the cell pellet in the residual medium to create a single cell slurry. Place the tube containing the resultant cell slurry on ice and add a sufficient amount of reduced GF Basement Membrane Matrix to it per the desired number of iBBBs. Mix the cells homogenously while not introducing any air bubbles.Pipette 50 microliters of the resultant cell matrix mixture into a well of a 48-well or 96-well glass-bottom culture dish and distribute it evenly throughout the bottom of the dish. Incubate the dish at 37 degrees Celsius for 30 to 40 minutes to polymerize the matrix while encapsulating the cells. Finally, add 500 microliters of supplemented astrocyte medium to the well and maintain the iBBBs in culture.For a 96-well plate, add 100 to 150 microliters of medium to the well until they are sufficiently developed for downstream assays, which typically takes two weeks. The properly formed iBBB appeared as a solidified, single translucent disk under a brightfield microscope. After 24 hours, evenly distributed single cells were identified.After two weeks, more distinct structures, although difficult to define, were visible.

1.4K Views.  Icahn School of Medicine at Mount Sinai.  The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease.

A 3D Blood-Brain Barrier Model Derived from Human Pluripotent Stem-Cells

Summary