TABLE 1.

Major iterations of hPSC-derived iBMEC protocols.

Year	References	Protocol	Changes vs. 2012	Cell line/Maintenance	Differentiation media	QPCR	Antibodies	Barrier assays	Transporters/transcytosis
2012	Lippmann et al. (2012). Nature biotechnology	(1) Prior diff cells are passaged on Matrigel (mTESR1 for 2–3 days. (2). Media is switched to lack of FGF (UM) for 5–7 days. (3). Switch to EC media human Endothelial Serum-Free Medium (Invitrogen) supplemented with 20 ng/mL bFGF and 1% platelet-poor plasma derived bovine serum32 (PDS; Biomedical Technologies, Inc.). (4) 1–2 days of EC medium treatment, cells were dissociated with dispase (2 mg/mL; Invitrogen) and plated onto 12-well tissue culture polystyrene plates and maintained in EC media.	NA	ES line: H9, IPS: IMR90-4, iPS-DF19-9-11T33, iPS-DF6-9-9T. Irradiated MEFS, DMEMF12 20%KOSR, 1xMEM, 1mM-lglutamine, 4ng.ml bFGF	UM = lack of FGF and EC = Endothelial Serum-Free Medium (Invitrogen) supplemented with 20 ng/mL bFGF and 1% platelet-poor plasma derived bovine serum32 (PDS; Biomedical Technologies, Inc.)	PECAM1, CDH5, vWF, LDLR, LRP1, INSR, LEPR, BCAM, TFRC, AGER, STRA6, SLC7A5, SLC1A1, SLC38A5, SLC16A1, SLC2A1, ABCB1, ABCG2, ABCC1, ABCC2, ABCC4, and ABCC5. PLVAP, SLC21A14, FST, FZD7, FZD4, FZD6, STRA6, LEF1, APCDD1, SLC2A1, ABCB1 control: GAPDH, NO EC CONTROL	PECAM-1 (Rabbit, Thermo Fisher) CLAUDIN-5 (Mouse, Invitrogen) Occludin (Mouse, Invitrogen) P-glycoprotein (Mouse, Thermo Fisher) GLUT-1a (Rabbit antiserum) VE-Cadherin (Mouse, SCBT) Nestin (Rabbit, Millipore) βIII tubulin (Rabbit, Sigma) βcatenin (FITC-conjugated Mouse, BD Biosciences) Wnt7a FISH Wnt7b FISH GFAP (Polyclonal Rabbit, Dako) aSMA (Mouse, American Research Products)	TEER, coculture with rat astrocytes	Inulin, sucrose, glucose, vincristine, colchicine, prazosin, diazepam, rodhamine 123 ((cyclosporine). No EC control
2014	Lippmann et al. (2014)Lippmann et al., . Scientific reports	(1) Prior diff cells are passaged on Matrigel (mTESR1 for 2–3 days. (2). Media is switched to lack of FGF (UM) for 5–7 days. (3). Switch to EC media human Endothelial Serum-Free Medium (Invitrogen) supplemented with 20 ng/mL bFGF and 1% platelet-poor plasma derived bovine serum32 (PDS; Biomedical Technologies, Inc.). (4). 1–2 days of EC medium treatment, cells were dissociated with dispase (2 mg/mL; Invitrogen) and plated onto 12-well tissue culture polystyrene plates and maintained in EC media (RA	Addition of Retinoic Acid on day 6 use of versene to dissociate the cells instead of dispase, results in less debris	IMR90-4 and DF19-9-11T iPSCs and H9 hESCs in mTESR or 2012	UM = lack of FGF and EC = Endothelial Serum-Free Medium (Invitrogen) supplemented with 20 ng/mL bFGF and 1% platelet-poor plasma derived bovine serum32 (PDS; Biomedical Technologies, Inc.) + RA	ABCB1, ABCG2, ABCC1, ABCC2, ABCC5, and STRA6	PECAM-1 (Rabbit, Thermo Scientific) GLUT-1 (Mouse, Thermo Scientific) Occludin (Mouse, Life Technologies) CLAUDIN-5 (Mouse, Life Technologies) VE-Cadherin (Mouse, SCBT) E-Cadherin (Goat, R&D Systems) P-glycoprotein (Mouse, Life Technologies) BCRP (Mouse, Millipore) MRP1 (Mouse, Millipore) GFAP (Rabbit, Dako) βIII tubulin (Rabbit, Sigma) Nestin (Mouse, Millipore) αSMA (Mouse, American Research Products) PDGFRβ (Rabbit, Cell Signaling)	TEER, coculture with NPC astrocytes, neurons and primary pericytes	DOXO, rhodamine DCFDA
2017	Qian et al. (2017). Science advances	6 (μM of CHIR on D0-1, he medium was removed and cells were transitioned to DeSR2 (DeSR1 plus B27 supplement) for another 5 days with daily medium changes. At day 6, cells were switched to hECSR1 medium [human endothelial serum-free medium (hESFM) supplemented with basic fibroblast growth factor (bFGF, 20 ng/ml), 10 (M RA, and B27] to induce RA signaling in the hPSC-derived endothelial progenitors in an attempt to drive the specification to BMECs. Cells were maintained in this medium for 2 days. At day 8, cells were replated onto a Matrigel-coated substrate in hECSR1, and at day 9, the medium was switched to hECSR2 (hECSR1 lacking RA and bFGF).	Accutase instead of Versene	Human iPSCs [iPS(IMR90)-4 (72), iPS-DF 19-9-11T (73), and hESCs (H9) (29)] were maintained on Matrigel (Corning)–coated surfaces in mTeSR1	6 μM CHIR99021 (Selleckchem) in DeSR1: DMEM/Ham’s F12 (Thermo Fisher Scientific), 1× MEM-NEAA (Thermo Fisher Scientific), 0.5× GlutaMAX (Thermo Fisher Scientific), and 0.1 mM β-mercaptoethanol (Sigma). After 24 h, the medium was changed to DeSR2: DeSR1 plus 1× B27 (Thermo Fisher Scientific) every day for another 5 days. At day 6, the medium was switched to hECSR1: hESFM (Thermo Fisher Scientific) supplemented with bFGF (20 ng/ml), 10 μM RA, and 1× B27		Brachyury (R&D Systems) PAX2 (SCBT) CD31 (Thermo Fisher) VE-Cadherin (SCBT) vWF (Dako) VEGFR2 (SCBT) CLAUDIN-5 (Invitrogen) Occludin (Invitrogen) ZO-1 (Invitrogen) GLUT-1 (Thermo Fisher) PGP (Thermo Fisher) BCRP (Millipore) MRP1 (Millipore) OCT3/4 (SCBT) TRA-1-60 (SCBT) NANOG (SCBT) ICAM-1 (R&D Systems)	“We also compared the differentiation reproducibility with that of the previously reported UM protocol (33). Although both methods produce BMECs capable of substantial barrier formation from multiple hPSC lines, BMECs differentiated from H9 hESCs and 19-9-11 iPSCs using the defined method exhibited higher TEERs and lower batch-to-batch variation.”	Efflux transporter activities were measured by the intracellular accumulation of (G) rhodamine 123, (H) Hoechst, and (I) DCFDA, substrates for Pgp, BCRP, and MRP, respectively. CsA, Ko143, and MK571 were used as specific inhibitors of Pgp, BCRP, and MRP, respectively
2017	Hollmann et al. (2017). Fluids barriers CNS	Modified 2014 protocol	E8 and E6 media, E6 for 4 days then continued as Lippmann et al. (2014) protocol.	MR90-4 iPSCs CC3 iPSCs, CD12 iPSCs, and SM14 iPSCs in growth factor-reduced Matrigel (VWR) in E8 medium	E8 medium was prepared by adding 100 μL of human insulin solution (Sigma-Aldrich), 500 μL of 10 mg/mL of human holo-transferrin (R&D Systems), 500 μL of 100 μg/mL human basic fibroblast growth factor (bFGF; PeproTech), and 500 μL of 2 μg/mL TGFβ1 (PeproTech) to 500 mL of E4. The final concentrations are 2.14 mg/L insulin, 100 μg/L bFGF, 2 μg/L TGFβ1, and 10.7 mg/L holo-transferrin E6 medium was prepared by adding 100 μL of human insulin solution and 500 μL of 10 mg/mL of human holo-transferrin to 500 mL of E4. The final concentrations are 2.14 mg/L insulin and 10.7 mg/L holo-transferrin UM and EC same as 2–14		PECAM-1 (Rabbit, Thermo Scientific) GLUT-1 (Mouse, Thermo Scientific) OCCLUDIN (Mouse, Thermo Scientific) CLAUDIN-5 (Mouse, Thermo Scientific) VE-Cadherin (Goat, R&D Systems) GFAP (Rabbit, Dako) PDGFR-B (Rabbit, SCBT) NG2 (Mouse, SCBT) αSMA (Mouse, SCBT)	TEER	Intracellular accumulation of rhodamine 123 (a Pgp substrate) was evaluated in the absence of bFGF and RA. Cells were incubated with 10 μM PSC833 or 10 μM MK-571 for 1 h at 37°C. They were then incubated for an additional house with 10 μM rhodamine 123 or 10 μM H2DCFDA.

A summary of the original hPSC-derived iBMEC protocol (Lippmann et al., 2012) as well as the major adaptations made in subsequent studies (Lippmann et al., 2014; Hollmann et al., 2017; Qian et al., 2017) highlighting the key changes and experimental conditions used in each study.