Supplementary Figure 1. Examination of neural cells and Wnt … · 2012-08-17 · Supplementary...

Supplementary Figure 1. Examination of neural cells and Wnt signaling in differentiating

H9 hESC cultures at day 4 of UM treatment.

(a) Flow cytometry distribution of differentiating H9 hESCs at day 4 of UM treatment. Green dots

indicate nestin+/βIII tubulin- events, red dots indicate nestin+/βIII tubulin+ events, blue dots

indicate nestin-/βIII tubulin+ events, and black dots indicate nestin-/βIII tubulin- events. (b)

Expression of WNT7A and WNT7B detected by RT-PCR at 4 days of UM treatment. (c-d)

Expression of nestin (c) and WNT7A (d) were detected by combined fluorescence in situ

Nature Biotechnology: doi:10.1038/nbt.2247

hybridization/immunocytochemistry. (e-f) Expression of βIII tubulin (e) and WNT7A (f). (g-h)

Expression of nestin (g) and WNT7B (h). (i-j) Expression of βIII tubulin (i) and WNT7B (j). Scale

bars indicate 50 µm. Adjacent horizontal panels represent the same field.


Supplementary Figure 2. Identification of GFAP+ and α-SMA+ cells in the differentiating

IMR90-4 cultures.

(a) Glial fibrillary acidic protein-positive (GFAP+) cells (red) could be identified among the βIII

tubulin+ cells (green) by immunocytochemistry at day 6 of UM treatment. (b) α-smooth muscle

actin (α-SMA+) cells (green) were also identified and are shown overlaid with DAPI nuclear stain

(blue). Scale bars indicate 50 µm. Neither cell type was present in significant quantities.


Supplementary Figure 3. IMR90-4-derived BMECs express requisite BBB markers after

continued differentiation in EC medium.

IMR90-4-derived BMECs were cultured for 6 days in UM and 2 days in EC medium prior to

immunolabeling. (a-b) PECAM-1 (a) co-expressed with claudin-5 (b). (c) Expression of occludin.

(d) Expression of GLUT-1. (e) Expression of p-glycoprotein. All scale bars indicate 50 µm.


Supplementary Figure 4. DF19-9-11T-derived BMECs express requisite BBB markers.

DF19-9-11T-derived BMECs were cultured for 6 days in UM and 2 days in EC medium prior to

immunolabeling. (a-b) PECAM-1 (a) is shown co-expressed with claudin-5 (b). (c) Expression

of GLUT-1. (d) Expression of p-glycoprotein. All scale bars indicate 50 µm.


Supplementary Figure 5. DF6-9-9T-derived BMECs express requisite BBB markers.

DF6-9-9T-derived BMECs were cultured for 6 days in UM and 2 days in EC medium prior to

immunolabeling. (a-b) PECAM-1 (a) is shown co-expressed with occludin (b). (c-d) GLUT-1 (c)

is shown co-expressed with claudin-5 (d). (e) Expression of p-glycoprotein. All scale bars

indicate 50 µm.


Supplementary Figure 6. H9-derived BMECs express requisite BBB markers.

H9-derived BMECs were cultured for 7 days in UM and 6 days in EC medium prior to

immunolabeling. (a-b) PECAM-1 (a) is shown co-expressed with claudin-5 (b). (c) Expression of

occludin. (d) Expression of GLUT-1. (e) Expression of p-glycoprotein. All scale bars indicate 50

µm.


Supplementary Figure 7. Examination of neural cells and Wnt signaling in differentiating

IMR90-4 iPSC cultures.

(a) Expression of WNT7A and WNT7B detected by RT-PCR at 4 days of UM treatment. (b-c)

Expression of nestin (b) and WNT7A (c) were detected by combined fluorescence in situ

hybridization/immunocytochemistry at day 6 of UM treatment. (d-e) Expression of βIII tubulin (d)

and WNT7A (e) were detected at day 6 of UM treatment. Arrowheads indicate a cell with

positive expression of βIII tubulin that lacks WNT7A expression. Panels (b) and (c), and panels

(d) and (e), are the same field. Scale bars indicate 50 µm.


Supplementary Figure 8. DF19-9-11T, DF6-9-9T, and H9 cell lines demonstrate β-catenin

nuclear localization during BMEC specification.

(a) After 6 days in UM and 2 days in EC medium, DF19-9-11T-derived BMECs were

immunolabeled for PECAM-1 expression (red) and β-catenin (green). (b) After 6 days in UM and

2 days in EC medium, DF6-9-9T-derived BMECs were immunolabeled for GLUT-1 (red) and β-

catenin (green). (c) After 7 days in UM and 3 days in EC medium, H9-derived BMECs were

immunolabeled for GLUT-1 (red) and β-catenin (green). H9-derived ECs required a longer time

frame to acquire nuclear β-catenin, which may explain the mixture of BBB and non-BBB ECs.

Scale bars represent 50 µm.


Supplementary Figure 9. DF19-9-11-derived BMECs subcultured onto

collagen/fibronectin-coated surface maintain BBB markers.

(a) vWF (red) is shown co-localized with occludin (green) by immunofluorescence. DAPI (blue)

is overlaid. (b) Expression of PECAM-1 (red) overlaid with DAPI (blue). (c) Expression of

claudin-5. (d) Expression of GLUT-1. (e) Expression of p-glycoprotein. All scale bars indicate 50

µm.


Supplementary Figure 10. DF6-9-9-derived BMECs subcultured onto collagen/fibronectin-

coated surfaces maintain BBB markers.

(a-b) PECAM-1 (a) is shown co-expressed with claudin-5 by immunofluorescence (b). DAPI

(blue) is shown overlaid with PECAM-1. (c) Expression of occludin. (d) Expression of GLUT-1.

(e) Expression of p-glycoprotein. All scale bars indicate 50 µm.


Supplementary Figure 11. Continuous tight junction expression is maintained by IMR90-

4-derived BMECs after subculture onto collagen/fibronectin-coated Transwell® filters.

IMR90-4-derived BMECs were differentiated for 6 days in UM and 2 days in EC medium prior to

subculture onto collagen/fibronectin-coated filters. Immunofluorescence images were recorded

once cells reached confluence after 2 days. (a) claudin-5. (b) occludin. (c) ZO-1. All scale bars

indicate 50 µm.


Supplementary Figure 12. Stability of the IMR90-4-derived BMEC phenotype over

extended culture times

IMR90-4-derived BMECs were co-cultured with rat astrocytes and TEER was measured

approximately every 24 hours. TEER values are normalized to the maximum value within each

experiment. The 1% PDS time course is the same as that documented in Figure 4b, and the

10% FBS time courses are demonstrating the consistent maximum and plateau time course

profiles for three independent biological replicates.


Supplementary Figure 13. Basolateral to apical transport of rhodamine 123 decreases in

the presence of cyclosporin A.

Basolateral to apical transport of rhodamine 123 decreases in the presence of cyclosporin A

(80±5%) compared to the control (100±4%). Statistical significance was calculated by the

Student’s t-test; *, p<0.07.


Supplementary Table 1. In vitro permeability of the IMR90-4-derived BMECs.

Compound Molecular Weight

(g/mol) Octanol/water partition

coefficient (logD)1-6 Transporter

recognition7-10 Average Pe

(10-3 cm/min)b

Inulin Variablea -0.25 None 0.029 ± 0.02

Sucrose 342 -3.3 None 0.034 ± 0.015

Glucose 180 -2.8 GLUT-1 (influx) 0.22 ± 0.07

Vincristine 825 2.8 MDR1, MRP1,

MRP2 0.062 ± 0.02

Colchicine 399 1.03 MDR1, MRP1 0.092 ± 0.1

Prazosin 383 2.16 BCRP (efflux), Organic cation

transporters (influx) 0.29 ± 0.04

Diazepam 285 2.82 None 1.1 ± 0.5 aInulin is a polysaccharide polymer with an unspecified chain length. bMean ± standard deviation was calculated from three completely independent biological replicates except sucrose (eighteen replicates).


Supplementary Table 2. Efflux transporter substrates and inhibitors.

Compounds11-13 Transporter recognition11-13

Substrates

Rhodamine 123 MDR1

Doxorubicin MDR1, BCRP, MRP1, MRP2

Inhibitors

Cyclosporin A MDR1

Ko143 BCRP

MK 571 MRP family


Supplementary Table 3. Antibodies used for immunofluorescence and flow cytometry.

Targeted antigen Antibody description Vendor Dilution

PECAM-1 Polyclonal rabbit Thermo Fisher 1:25

von Willebrand Factor Polyclonal rabbit Dako 1:100

VE-cadherin Mouse monoclonal SCBT 1:100

ZO-1 Polyclonal rabbit Invitrogen 1:100

Occludin Mouse monoclonal Invitrogen 1:50

Claudin-5 Mouse monoclonal Invitrogen 1:200

GLUT-1a Polyclonal rabbit antiserum N/A 1:500

GLUT-1a Mouse monoclonal Thermo Fisher 1:100

p-glycoprotein Mouse monoclonal Thermo Fisher 1:25

β-catenin FITC-conjugated mouse

monoclonal BD Biosciences 1:150

βIII tubulin Polyclonal rabbit Sigma 1:500

Nestinb Polyclonal rabbit Millipore 1:1000

Nestinb Mouse monoclonal Millipore 1:500

GFAP Polyclonal rabbit Dako 1:500

α-smooth muscle actin Mouse monoclonal ARP 1:100

Digoxigenin Mouse monoclonal Sigma 1:500

K14 Rabbit polyclonal Thermo Fisher 1:100

K18 Mouse monoclonal Thermo Fisher 1:100

p63 Mouse monoclonal SCBT 1:33 aThe polyclonal GLUT-1 antiserum7 was used for immunofluorescence only. The monoclonal GLUT-1 antibody was used for immunofluorescence and flow cytometry. bThe polyclonal nestin antibody was used for immunofluorescence and the monoclonal nestin antibody was used for immunofluorescence and flow cytometry.


Supplementary Table 4. Primers used for RT-PCR and qPCR. Genea Forward sequence Reverse sequence GAPDH CACCGTCAAGGCTGAGAACG GCCCCACTTGATTTTGGAGG SLC2A1 ACGCTCTGATCCCTCTCAGT GCAGTACACACCGATGATGAAG ABCB1 TGAATCTGGAGGAAGACATGAC CCAGGCACCAAAATGAAACC LEF1 CAGATGTCAACTCCAAACAAGG GATGGGATATACAGGCTGACC

STRA6 TTTGGAATCGTGCTCTCCG AAGGTGAGTAAGCAGGACAAG FZD4 TACCTCACAAAACCCCCATCC GGCTGTATAAGCCAGCATCAT FZD6 TCGTCAGTACCATATCCCATG CCCATTCTGTGCATGTCTTTT FZD7 GATGATAACGGCGATGTGA AACAAAGCAGCCACCGCAGAC FST GTTCATGGAGGACCGCAGTG TCTTCTTGTTCATTCGGCATT

APCDD1 GGAGTCACAGTGCCATCACAT CCTGACCTTACTTCACAGCCT PECAM1 GAGTATTACTGCACAGCCTTCA AACCACTGCAATAAGTCCTTTC

CDH5 CGCAATAGACAAGGACATAACAC GGTCAAACTGCCCATACTTG VWF CCCGAAAGGCCAGGTGTA AGCAAGCTTCCGGGGACT LDLR GCCATTGTCGTCTTTATGTC AAACACATACCCATCAACGA

SLC7A5 TTAAAGTAGATCACCTCCTCGA GGATGAGATTCGTACCAGAG SLC16A1 GGTGTTTCTTAGTAGTTATGGG TCTTATTGGCTTTGTGTTGG

INSR TGTTCATCCTCTGATTCTCTG GCTTAGATGTTCCCAAAGTC LEPR GGAAATCACACGAAATTCAC GCACGATATTTACTTTGCTC BCAM GCTTTCCTTACCTCTAAACAG GAAGGTGATAGAACTGAGCG

SLC38A5 TGTCAGTGTTCAACCTCAG GTGGATGGAGTAGGACGA SLC1A1 GTTATTCTAGGTATTGTGCTGG CTGATGAGATCTAACATGGC ABCG2 TCAGGTCTGTTGGTCAATCTC GTTTCCTGTTGCATTGAGTCC ABCC1 AATAGAAGTGTTGGGCTGAG CGAGACACCTTAAAGAACAG ABCC2 ATATAAGAAGGCATTGACCC ATCTGTAGAACACTTGACCA ABCC4 AATCTACAACTCGGAGTCCA CAAGCCTCTGAATGTAAATCC ABCC5 TCACTACATTAAGACTCTGTCC GGATACTTTCTTTAGGACGAGAG AGER GTAGATTCTGCCTCTGAACTC CTTCACAGATACTCCCTTCTC

SLC21A14b AAAGATGTGGAAGTAGAGGA ATGCTTAGGAGAATTGACAC PLVAPb CAATGCAGAGATCAATTCAAGG ACGCTTTCCTTATCCTTAGTG TFRC GCACAGCTCTCCTATTGAAAC GGTATCCCTCTAGCCATTCAG LRP1 GACTACATTGAATTTGCCAGCC TCTTGTGGGCTCGGTTAATG

WNT7A CGGGAGATCAAGCAGAATG CGTGGCACTTACATTCCAG WNT7B GCTTCGTCAAGTGCAACA GGAGTGGATGTGCAAAATG WNT7Ac TGGAACAGAATAGTTGAGGGCT N/A WNT7Bc AGCCAAGGGACAGTGCGAGTGT N/A

aFor genes with multiple transcript variants (e.g. LEPR, BCAM), the primers were designed to encompass all transcript variants and not one single transcript isoform. bFidelity of SLC21A14 and PLVAP primers was confirmed using HUVECs because these genes were not expressed in the IMR90-4-derived BMECs. cDIG-labeled locked nucleic acid probe sequences used for fluorescence in situ hybridization. DIG-labeled locked nucleic acid sense probes were used as controls.


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Supplementary Figure 1. Examination of neural cells and Wnt … · 2012-08-17 · Supplementary...

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