Cell Stem Cell, Volume 8

Supplemental Information

LRRK2 Mutant iPSC-Derived DA Neurons

Demonstrate Increased Susceptibility

to Oxidative Stress Ha Nam Nguyen, Blake Byers, Branden Cord, Aleksandr Shcheglovitov, James Byrne, Prachi Gujar, Kehkooi Kee, Birgitt Schüle, Ricardo E. Dolmetsch, William Langston, Theo D. Palmer, and Renee Reijo Pera

Inventory of Supplemental Information

1. Supplemental Figures and Legends

Figure S1, related to Figure 1

Figure S2, related to Figure 2

Figure S3, related to Figure 4

Figure S4, related to Figure 5

Figure S5, related to Figure 6

2. Table S1, related to Figure 3

3. Table S2 (Cell Counts), related to Figure 5

4. Table S3 (Cell Counts), related to Figure 6

5. Table S4 (Cell Counts), related to Figure 7

Supplemental Tables S2, S3 and S4 are provided in MS Excel spreadsheets.

6. Movie S1 (Beating Cardiomyocytes), related to Figure 1

7. Supplemental Experimental Procedures 8. Supplemental References

Figure S1. Characterization of Human-Derived iPSCs, related to Figure 1.

(A) Cluster analysis of normal HUF5 and PD G2019S human dermal fibroblasts (HDF), HUF5-iPSC, PD

G2019S-iPSC , H9-hESC, and their derivatives after 20 and 35 days of spontaneous in vitro differentiation.

Gene expression was normalized to GAPDH, B-Actin, CTNNB1, EEF1α and RPLPO by using qBasePlus. Red

= High expression; Green = Low; Black = Zero.

(B) HUF5-iPSCs were derived from a 46-year-old healthy female dermal fibroblasts. These iPSCs express

pluripotency markers, SSEA3, TRA1-60, TRA1-81, NANOG, and SSEA4, but not SSEA1. They generated

teratoma in vivo and differentiated into cells representative of the three germ layer in vitro. Bar = 100 um.

(C) HUF5-iPSCs have a normal karyotype, based on at least 20 metaphase spreads analyzed.

(D) HUF5-iPSCs Nanog and Oct4 promoters are hypomethylated, compared to dermal fibroblast (HDF).

Closed and open circles represent methylated and unmethylated CpG sites, respectively.

Figure S2. Directed Differentiation of iPSCs into Midbrain Dopaminergic (mDA) Neurons, related to

Figure 2.

(A) A schematic diagram showing the overview of DA neuronal differentiation protocol (Chambers et al., 2009)

and representative cell culture morphology during the course of differentiation. SRM = standard hESC medium

without FGF2; N2 = DMEM:F12 with N2 supplement.

(B) Differentiation of iPSCs into DA neurons. Expression of neuronal-specific markers, including OTX2, Nestin,

Tyrosine Hydroxylase (TH), and Calbindin (CALB), in iPSC-derived neurons. Note that most OTX2-positive

cells do not co-localize with TH-positive cells and that some CALB-positive cells co-localize with TH-positive

cells. Scale bar = 50 µm.

Figure S3. Characterization of Human iPSC-Derived Neurons, related to Figure 4.

(A-C) Quantiative RT-PCR analysis showing the average expression of pluripotency, neuronal and stress-

response genes in undifferentiated human iPSC lines (normal HUF5-iPSC (5c2) and PD G2019S-iPSC (6c3))

and iPSC-derived neurons at various stages of maturation. Gene expression was normalized to GAPDH,

CTNNB1, EEF1α and RPLPO by using qBasePlus. Data represent mean ± SEM (n = 3 to 7 biological

replicates per clone).

(D) A representative of a single neuron after separated from a group of neurons. Scale bar = 10 µm.

(E) Single-cell qRT-PCR analysis from 35-day iPSC-derived neurons. Gene expression was normalized to

GAPDH, CENTB3 and CNNTB1 by using qBasePlus. Each number on the X-axis indicates a single neuron,

not corresponding to each other.

Figure S4. Effect of Hydrogen Peroxide on iPSC-derived Neurons, related to Figure 5.

(A) Quantification of cell death in DA neurons was measured by laser confocal imaging and co-labeling of

activated (or cleaved) CASP3 with TH. Arrows indicate cells double positive for CASP3 and TH. Scale bar =

10 µm.

(C) Quantification of TH-negative (TUJ+) neurons that are also positive for CASP3 after H2O2 treatments. Data

represent mean ± SEM (n = 3 to 4 wells per clone).

(D) RT-PCR analysis showing the average gene expression of HUF5-, G2019S- and H9-derived neurons after

48 hours of H2O2 treatment (25 µM). Error bars represent standard deviation (n = 4). * p < 0.05

Figure S5. Effect of 6-Hydroxydopamine on iPSC-derived DA Neurons, related to Figure 6.

RT-PCR analysis showing the average gene expression of HUF5-, G2019S- and H9-derived neurons after 24

hours of 6-OHDA treatment (10 µM). Data represent mean ± SEM (*p < 0.05; n = 4 biological replicates).

Table S1. Electrical properties of iPSC-derived neurons, related to Figure 3

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Small-Scale and Single Cell RT-PCR Analysis

Small aggregate of cells (~100 cells) were manually picked and, for single cells, digested with 0.05% trypsin,

transferred directly into RT-PreAmp master mix, as above, with addition of SuperRase-In (Ambion) and

SuperScriptt III (Invitrogen), and pre-amped: 1 cycle at 50ºC, 15 minutes; 1 cycle at 70ºC, 2 minutes; and 18

cycles at 95ºC, 15 seconds and at 60ºC, 4 minutes. Reactions were processed as above. Ct values for each

sample and gene were normalized to GAPDH, CNNTB1, CENTB3 and EEF1a by qBasePlus (Biogazelle).

Gene cluster analysis was via Gene Cluster 3.0 and visualized with TreeView (Eisen et al., 1998). Genes were

filtered for ≥ 80% response, normalized, mean centered, grouped by an un-centered correlation similarity

metric and clustered via complete linkage prior to visualization.

Electrophysiology

Electrophysiological experiments were performed on 35-day neurons growing on glass coverslips coated with

Matrigel, without co-culturing with astrocytes. Neurons were visualized on an inverted Nikon (Elipse TE2000)

microscope and recorded using EPC 10 amplifier (HEKA). Neuronal activity was measured using following

extracellular and intracellular solutions in mM: 129 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 30 Glucose, 25 HEPES, pH

7.4 – extracellular; 120 KGlu, 20 KCl, 4 NaCl, 4 Mg2ATP, 0.3 NaGTP, 10 Na2PCr, 0.5 EGTA, 10 HEPES, pH

7.25 – intracellular. Correspondingly, spontaneous postsynaptic currents, measured under this experimental

condition at -70 mV, are a superposition of EPSCs and IPSCs (ECl- ~ –46 mV). Data were collected and initially

analyzed with the Patchmaster software (HEKA). Further analysis and statistics were performed using

IgorPRO and MS Excel.

Karyotyping and Teratoma Assay

Spectral karyotyping (SKY) was as described (Nguyen and Reijo Pera, 2008). For teratoma formation, iPSCs

from a confluent 10-cm dish were harvested by Collagenase IV treatment and injected subcutaneously into

female SCID mice (BL17SCID, Charles River). After 5-8 weeks post-transplantation, tumors were dissected

from the host, fixed in 4% paraformaldehyde/PBS solution, paraffin embedded, sectioned and stained with

Hematoxylin and Eosin.

REFERENCES Chambers, S.M., Fasano, C.A., Papapetrou, E.P., Tomishima, M., Sadelain, M., and Studer, L. (2009). Highly efficient neural conversion of human es and ips cells by dual inhibition of smad signaling. Nat. Biotechnol. 27, 275-280.

Eisen, M.B., Spellman, P.T., Brown, P.O., and Botstein, D. (1998). Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. 95, 14863-14868.

Nguyen, H.N., and Reijo Pera, R.A. (2008). Metaphase spreads and spectral karyotyping of human embryonic stem cells. CSH Protocols doi:10.1101/pdb.prot5047.