Cell Stem Cell, Volume 14

Supplemental Information

Pathways Disrupted in Human ALS

Motor Neurons Identified

through Genetic Correction of Mutant SOD1

Evangelos Kiskinis, Jackson Sandoe, Luis A. Williams, Gabriella L. Boulting, Rob

Moccia, Brian J. Wainger, Steve Han, Theodore Peng, Sebastian Thams, Shravani

Mikkilineni, Cassidy Mellin, Florian T. Merkle, Brandi N. Davis-Dusenbery, Michael

Ziller, Derek Oakley, Justin Ichida, Stefania Dicostanza, Nick Atwater, Morgan L.

Maeder, Mathew J. Goodwin, James Nemesh, Robert E. Handsaker, Daniel Paull, Scott

Noggle, Steven A. McCarroll, J. Keith Joung, Clifford J. Woolf, Robert H Brown, and

Kevin Eggan

D

1 nA

40 ms

20 mV

100 ms 100 pA5 s

100 pA

5 s

DNA ISL TUJ1

DNA FOXP1

MAP2DNA HB9

MAP2 DNA MAP2 ChAT

Phase

A

B

C

Hb9::RFP

F

MAP2 ISLHOXA5 DNA DNAISLHOXA5

G

iPSCs Embryoid Bodies

NIM

+AA, BDNF RA, SAG

NIMmTeSR mTeSR mTeSR: KOSR (1:1)

mTeSR+SB431542 Dorsomorphin

KOSR

Single Cellsplated on glia for co-culture or alone on PDLLtime (days) -24 -23 -20 -19 -17 0-21

24 day motor neuron differentiation protocol39

b-SO

D1+/

A4V

RB9

d-SO

D1+/

A4V

39b-

SOD

1+/A4

V

0.0

0.2

0.4

0.6

0.8

RFPpositive

Hb9

Rel

ativ

e E

xpre

ssio

n

qRT-PCR of Hb9::RFP motor neurons

E

RFPnegative

Figure S1, related to Figure 1, Differentiation and Characterization of Patient-Specific iPS cell-derived Motor Neurons. (A) Schematic of differentiation protocol (see methods for details). (B-C) Differentiations typically yielded neurons that expressed TUJ1, the MN transcription factors ISL and HB9, as well as MAP2. Many ISL+ MNs were FOXP1high+ and all were HOXA5+ indicating their acquisition of a lateral motor column identity with a rostral phenotype, at 30 days post-dissociation, ISL+ MNs were ChAT-positive. (D-G) Hb9::RFP positive MNs are electrophysiologicaly active. RFP+ cells show a 6-fold enrich-ment in Hb9 expression levels than the RFP negative fraction. In whole-cell patch clamping recordings of day 15 neurons in co-culture with glia, MNs exhibited initial inward sodium currents followed by outward potassium currents upon depolarization in voltage-clamp mode, as well as repetitive action potential firing in current-clamp mode, as well as kainate and GABA responses (all scale bars=50µm).

A

B

CNumber of neurons on day 3

0

1000

TUJ1 positive, ISL negative

DNAhNUC

TUJ1hNUC TUJ1 DNA

cont

rol 1

1a

Cell line HOXA5/MAP2 HOXA5/ISL TUJ1/hNUC11a 95.3 98.718a 97.0 100.0 98.939b 94.0 97.7 99.0

RB9d 95.0 97.7 97.5

Percentage of neurons (%)

N.D.

2000

3000

4000

5000

Cel

l num

ber

TUJ1/ISL postive TUJ1 positive

D

39bSOD1+/A4V

RB9dSOD1+/A4V

11aControl

18aControl

Neural tube

LMC

MMC

Limb

Axial

E

0.00

1.00

2.00

3.00

4.00

5.00vOCT4 eOCT4

39b

39b-

Cor

r.

RB9

d

25b

27d

MG

H5b

RB2

1b 19f

RB8

B

Posi

tive

cont

rol

HU

ES-6

Hb9::RFP motor neurons

1.00

2.00

3.00

4.00eSOX2vSOX2

39b

39b-

Cor

r.

RB9

d

25b

27d

MG

H5b

RB2

1b 19f

RB8

B

Posi

tive

cont

rol

HU

ES-6

Hb9::RFP motor neurons

0.00

Rel

ativ

e Ex

pres

sion

Rel

ativ

e Ex

pres

sion

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

4.00

39b

39b-

Cor

r.

RB9

d

25b

27d

MG

H5b

RB2

1b 19f

RB8

B

Posi

tive

cont

rol

HU

ES-6

Hb9::RFP motor neurons

eKLF4

vKLF4

Rel

ativ

e Ex

pres

sion

FqRT-PCR analysis of reprogramming transgenes in iPSC-derived Hb9::RFP motor neurons

Figure S2, related to Figure 1, Characterization of Patient-Specific iPS cell-derived Motor Neurons. (A) Quantifications of efficiencies of differentiation for each of the 4 iPS cell lines. (B) More than 97% of TUJ1+ve neurons are iPSC-derived as they stain positive for the human nuclear antigen (hNUC). (C) Quantifications of HOXA5 and human (hNUC) MNs and neurons. N.D: Not determined. (D) Transplantation of Hb9::GFP+ MNs into chick embryo spinal cords showed MNs settling into the ventral root and extending axons (black arrow heads) towards the axial and limp musculature. (E) Graphic representation of panel J, black arrow heads match MN axons from (D), MMC: Median motor column, LMC: Lateral motor column. (F) Transgene expression in Hb9::RFP purified MNs. Endogenous levels of OCT4, SOX2 and KLF4 in HUES-6 ES cells are shown for com-parison. An iPSC line that shows residual expression of the reprogramming transgenes was used as a positive control.

Ventral horn

11a 18aControl

A

0

20

40

60

80

100

120

39bSOD1+/A4V

RB9d

day 30day 15day 0

Neu

ron

num

ber r

elat

ive

to d

3 (%

)Relative percentage of

ISL+/TUJ1+ motor neurons over time

0

20

40

60

80

100

Neu

ron

num

ber r

elat

ive

to d

3 (%

)

11a 18aControl

39bSOD1 +/A4V

RB9d

on SOD1G93A gliaon NT glia

Relative percentage of ISL+/TUJ1+ motor neurons over time B

**

**

C D

0

1

2

3

4

5

ISL+/TUJ1+ motor neurons

d30

Average number of primary processes (#/cell)

E

2

4

6

8

Average density of processes (#/cell)

40

60

80

Average length of processes (µm)

20

00

d30

d30

ISL+/TUJ1+ motor neurons

ISL+/TUJ1+ motor neurons

39b

SOD1+/A4V

RB9d11a 18a

Control39b

SOD1+/A4V

RB9d11a 18a

Control

39b RB9d11a 18a

Control

n.s. .

n.s.*

Figure S3, related to Figure 1, Survival and Morphological Alterations of SOD1+/A4V MNs. (A) SOD1+/A4V MNs exhibit reduced survival over time in culture (n=3, +/-SEM, P<0.05). (B) MNs are sensi-tive to the toxic effects of primary glia from SOD1G93A mice. (C) Quantifications of MN processes, evalu-ated as total number of TUJ1+ve processes per cell showed that SOD1+/A4V MNs are less elaborate after 30 days in culture; (D) and have shorter processes (n=3,+/-SEM, P<0.05), (E) but not fewer primary processes. (F) Soma size of ISL+/TUJ1+ MNs (µm2) measured at day 30. SOD1+/A4V MNs exhibit reduced size than controls (n=3,+/-SEM, P<0.05).

50

100

150

200

Average soma size ofISL+/TUJ1+ motor neurons

039b

SOD1+/A4V

RB9d11a 18a

Control

Som

a si

ze (µ

m2 )

F

d30

d30

*

*

*

*

SOD1+/A4V

Fixed/Stained on d3 and d30 after plating0 3 30

24 day differentiation

D

time (days)

% BrdU MNs (ISL+/TUJ1+)

0

20

40

day 3

day 30

0.2 0.4 0.9 0.42.2 2.6 2.7 2.9

11a control

18a control

39bSOD1+/A4V

RB9dSOD1+/A4V

ALS

RB

9d d

3 ne

uron

sdi

ffern

tiatie

d w

/ Brd

UCA

BiPSCs

Embryoid Bodies Neurons plated on glia

time (days) -24 -17 0

+BrdU

% BrdU+ MNs (ISL+/TUJ1+)

E

*

* ** **

ISLBrdUTUJ1DNA ISL

BrdU TUJ1

DNAday30

* *

* *

**

*

**

ISLBrdUTUJ1DNA day3 ISL

BrdU TUJ1

DNA

F

*

* *

*

*

* *

*

BrdUBrdU

BrdU

Neurons plated on glia

+

ALS

RB

9d d

3 ne

uron

s pl

ated

on

glia

and

cul

ture

d w

ith B

rdU

ALS

RB

9d d

30 n

euro

ns p

late

d on

gl

ia a

nd c

ultu

red

with

Brd

U

ISL DNA TUJ1 BrdUISL BrdU TUJ1 DNA

+BrdU

0

50

100

day 3

HUES-3control

G

Figure S4, related to Figure 1, Neuronal Proliferation Does not Contribute to the Difference in Motor Neuron Survival Between Control and SOD1+/A4V motor neurons. (A-C) Testing the ability of BrdU to label MNs. Differentiation media was supplemented with 1µM BrdU between days -17 to 0. Nearly 100% of neurons were labeled by day 3 after dissociation. Importantly the mitoticly active, supporting glial cells were never exposed to BrdU in this experiment. (D-E) Schematic of experimental strategy to evaluate the level of MN proliferation after dissociation; 1µM BrdU was added in plated co-cultures from days 0-30 and percentage of positive cells was analyzed. Between 0.2-0.8% of all MNs incorporate BrdU between days 0-3 while 2-2.8% between days 0-30 across all the iPSC lines. Importantly the mitoticly active supporting glial cells were exposed to BrdU in this experiment and many are BrdU+ (large DAPI nuclei). A representative image of BrdU incorporation after the addition of 1µM BrdU to dissociated MN cultures after 3 (F) and 30 (G) days. Top right inserts exhibit an ISL+/BrdU negative MN, bottom inserts exhibit two ISL+ MNs, with only one being postive for BrdU also. Single arrows indicates BrdU+/ISL+ MNs; Double arrows indicate BrdU+/ISL negative cells; Asterisks indicate BrdU negative/ISL+ MNs, (scale bar=30µm).

Primer-F2

Primer-R2

Primer-F4

Primer-R6

FRT site

TOPO plasmid pCR-4

SOD1 corrected allele 5’ Homology arm 3’ Homology armExon 1

A

B

Chromosome 1

Chromosome 2

Chromosome 3

Chromosome 4

Chromosome 5

Chromosome 6

Chromosome 7

Chromosome 8

Chromosome 9

Chromosome 10

Chromosome 11

Chromosome 12

Chromosome 13

Chromosome 14

Chromosome 15

Chromosome 16

Chromosome 17

Chromosome 18

Chromosome 19

Chromosome 20

Chromosome 21

Chromosome 22

Chromosome X

C

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r03ch

r04ch

r05ch

r06ch

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r08ch

r09ch

r10ch

r11ch

r12ch

r13ch

r14ch

r15ch

r16ch

r17ch

r18ch

r19ch

r20ch

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Figure S5, related to Figure 2, Quality Control Assays for the Isogenic Control iPSC line 39b-corrected cell line. (A) In order to confirm proper targeting and correction of the SOD1A4V mutant allele, a region of the SOD1 locus from iPSC line 39b-corrected was PCR-amplified in a single reaction with primers beyond the 5’ and 3’ homology arms and cloned into a TOPO vector. This was sequenced and compared to the SOD1 genomic sequence. The FRT footprint is the only site that does not match to the control sequence. (B) To evaluate whether large-scale copy number altera-tions had arisen during gene-targeting of the SOD1 locus, we measured sequencing depth of coverage across the genome in 10kb bins for the paren-tal 39b and the corrected 39b control line. Sequencing read depth for the two samples closely matched each other throughout the genome. (C) To confirm the absence of large-scale copy-number alterations, the Nanostring nCounter human Karyotype panel was used to measure the genomic content of each cell line across more than 6 loci per chromosome. This data was compared to a reference genome which harbored no duplications or deletions.

Day 30

Day 30

Day 30

Day 30

DAPI MAP2 ISL SOD1

DAPI MAP2 ISL SOD1+MG132, 1µM

Control

39b-

SO

D1+

/+39

b-S

OD

1+/

A4V

39b-

SO

D1+

/+39

b-S

OD

1+/

A4V

0

1000

2000

3000

4000

5000

6000

7000

8000

Baseline +MG132

39b-SOD1+/A4V

39b-SOD1+/+

Fluo

resc

ence

inte

nsity

SOD1 staining

**

ns

C

D

E

Figure S6, related to Figures 2-3, Differential Gene Expression in non-MNs and SOD1 Protein Accumulation. (A) qRT-PCR analysis in 39b and 39b corrected iPSCs. Genes identified as being differentially regulated as a result of the SOD1 mutation in Hb9::RFP MNs were evaluated in the parental iPSCs. 19% of the genes tested were found as also being significantly different in iPSCs. (B) Cluster dendogram of RNA sequencing from SOD1A4V and healthy sex-matched fibroblast samples. The A4V fibroblasts do not segregate from the healthy controls. (C-D) SOD1+/A4V MNs accumulate significantly more SOD1 protein after treatment with MG132 (1µM, 48hrs) as measured by immuno-cytochemistry relative to isogenic control MNs. (E) Quantification of the fluorescent intensity of SOD1 protein (n=25, P<0.05).

AR

elat

ive

expr

essi

on

39b-SOD1+/A4V 39b-SOD1+/+

iPS cell lines

Eif2

B3

**

Cluster dendogram of SOD1+/A4V and healthy control fibroblasts

RB

9-S

OD

1+/A

4V

39-S

OD

1+/A

4V

15-S

OD

1+/+

MG

H16

-SO

D1+/

+

MG

H7-

SO

D1+/

+

18-S

OD

1+/+

MG

H14

-SO

D1+/

+

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

AC

OT2

CA

T

S10

0A6

CO

XA

2

GLR

X

DN

AJC

12

CO

X7C

DY

NLL

1

EE

FIA

2

EIF

2AK

2

LRR

C7

LYR

MA

MD

H1

ND

UFA

B1

ND

UFB

7

NU

DC

D3

PA

M16

PIK

3C2A

TIM

M8B

UQ

CR

C1

US

P40

*

**

B

Control

500 ms

20 μ

v

+TTX, (0.5µM) 1.5

1.0

0.5

0.0

Linop

irdine

Saline

C D E F

TTXSali

ne0.00

0.02

0.04

0.06

Rat

io o

f spl

iced

/uns

plic

ed X

BP

1

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Bra

in p

ool #

2

Fibs

+ D

TT

US

Ant

erio

r neu

rons

HB

sN

M +9

0.00

0.04

0.08

0.12

0.16

0.20 136.3

XB

P1

splic

ing

ratio

: S/U

A B

0.00

0.40

0.80

1.20siSCR siATF4

0.00

0.40

0.80

1.20

Rel

ativ

e ex

pres

sion

ATF4 RNA levels

Rel

ativ

e ex

pres

sion

siSCR siXBP1

XBP1 RNA levels

39b-SOD1+/+39b-SOD1+/A4V 39b-SOD1+/+39b-SOD1+/A4V

Figure S7, related to Figures 5-6, ER Stress and Electrical Activity are Connected. (A) ES-derived MNs and spinal cord RNA show higher levels of XBP1 splicing relative to ES-derived anterior neurons and brain RNA. Quantification of gel bands of spliced/unspliced XBP1 transcript. (B) Knockdown of XBP1 and ATF4 by repeated siRNA treatment after 30 days in culture. (C-E) TTX and linopirdine can be used to lower and increase the electri-cal activity of MNs respectively, as measured by multi-electrode array recording. (F) DTT treatment leads to a strong increase in the levels of XBP1 splicing. (G) Effects on expression of UPR genes after dosing motor neurons with salubrinal.

0.0

0.4

0.8

1.2

0.0 1.0 2.0 3.0

Control

15µM

30µM

ATF4

Relative Expression0.0 1.0 2.0 3.0

Relative Expression

EDEM1

0.0 1.0 2.0 3.0Relative Expression

PDI

Control

15µM

30µM

Control

15µM

30µM

0.0 0.5 1.0 1.0 2.0Relative Expression

CHOP

Control

15µM

30µM

0.0 0.5 1.0 1.5 2.0Control

15µM

30µM

Relative Expression

PUMA

Sal

ubrin

alS

alub

rinal

G

Action potential firing

Action potential firing XBP1 splicing ratio: S/U

DTT

Vehicle

Nor

mal

ized

spi

kes/

min

Nor

mal

ized

spi

kes/

min

Spi

nal c

ord

pool

#2

Patient iPSC Diagnosis Genotype Age of

onset Sex Age of

Biopsy Reprog. factors Karyotype Scorecard

Assay

Silencing of Reprogramming

factors

OCT4/ TRA-1-81 ICC

Reference

39 39b FALS SOD1 +/A4V 43y F 44y O,S,K Normal Normal Yes Normal This report RB9 RB9d FALS SOD1 +/A4V 45y F 50y O,S,K Normal Normal Yes Normal This report

19 19f FALS C9orf72 (GGGGCC)>50

53y F 54y O,S,K Normal Normal Yes Normal This report

RB8 RB8B FALS C9orf72 (GGGGCC)>50

49y M 51y O,S,K,cM Normal Normal Yes Normal This report

15 15b Healthy N.A. N.A. F 49y O,S,K Normal Normal No Normal Boulting et al., 2011

17 17a Healthy N.A. N.A. F 72y O,S,K Normal Normal Yes Normal Boulting et al., 2011

20 20b Healthy N.A. N.A. M 56y O,S,K Normal Normal Yes Normal Boulting et al., 2011

11 11a Healthy N.A. N.A. M 37y O,S,K Normal Normal Yes Normal Boulting et al., 2011

18 18a, 18b Healthy N.A. N.A. F 48y O,S,K Normal Normal Yes Normal Boulting et al., 2011

Table S1, related to Figure 1, List of Patient-Specific iPSCs Used in This Study. All iPSCs were characterized by immunocytochemistry (ICC) for NANOG and TRA-1-81 and the pluripotency Scorecard assay (Bock et al., 2011), and had normal karyotypes. The expression of the reprogramming transgenes was evaluated by qRT-PCR. All data are available upon request. O: OCT4, S: SOX2, K: KLF4, cM: cMYC. N.A.: Non-applicable.

Table S2, related to Figure 3, List of genes found to be significantly altered in their expression levels in 39b-SOD1+/A4V relative to isogenic control motor neurons by RNA sequencing at an FDR 5%. (A) Upregulated in SOD1+/A4V. (B) Downregulated in SOD1+/A4V

Table S3, related to Figure 3, Gene enrichment analysis done by DAVID and GSET for upregulated and downregulated genes in 39b-SOD1+/A4V relative to isogenic control motor neurons. (A) DAVID, Upregulated in SOD1+/A4V. (B) DAVID, Downregulated in SOD1+/A4V. (C) GSET, Upregulated in SOD1+/A4V. (B) GSET, Downregulated in SOD1+/A4V

EXPERIMENTAL PROCEDURES

Cell culture

Cell cultures were maintained at 37C, 5% CO

2. Human fibroblasts were cultured in KO-DMEM

(Invitrogen), supplemented with 20% Earl’s salts 199 (Gibco), 10% Hyclone (Gibco), 1x

GlutaMax, (Invitrogen), and 100μM 2-mercaptoethanol and passaged by trypsinization (0.25%

trypsin EDTA, Invitrogen). HuES and iPS cells were maintained on Matrigel (BD Biosciences)

with mTeSR1 media (Stem Cell Technologies). Media was changed every 24 hours and lines

were passaged by dispase (Gibco, 1mg/mL in hES media for 30min at 37C). Human primary

astrocytes were purchased from Sciencell Research Laboratories and grown according to

supplier instructions.

Derivation of human fibroblasts and iPS cell generation

Human fibroblasts were generated from 3mm forearm dermal biopsies following informed

consent as described previously (Dimos et al., 2008). Generation of iPS cells was done

essentially as reported previously by retroviral transduction of KLF4, SOX2, OCT4 and (cMYC)

(Boulting et al., 2011). Retrovirus preparations were done at the Harvard Gene Therapy Initiative

at Harvard Medical School (Boston, MA). For iPS cell derivation, 30,000 human fibroblasts

were transduced at an MOI of 3-5 per gene, with viruses containing all three genes.

Scorecard assay

RNA samples were produced and analyzed as described previously (Bock et al., 2011) with

minor modifications. Whole iPS and ES cell colonies were isolated by dispase treatment and

plated in suspension in the presence of mTeSR1 media, cell aggregates (EBs) were allowed to

form and 48hrs later EBs were switched to KOSR media without FGF (DMEM/F12, 10%

KOSR, NEAA, Glutamax, and 100μM 2-mercaptoethanol). EBs were grown for a total of 16

days, at the end of which, cells were lysed and total RNA was extracted using Trizol

(Invitrogen). Subsequently, RNA was analyzed on the NanoString nCounter using a custom

codeset. The calculation of the iPS lines’ lineage scores were performed according to our

previously published protocol (Bock et al., 2011) using our published dataset for 20 human

embryonic stem cell lines as a reference.

Motor neuron differentiation

MN differentiation was carried out as previously described (Boulting et al., 2011) with

modifications (see also Figure S2). Briefly, pluripotent stem cell colonies were dissociated with

accutase and single cells were plated in suspension in low-adherence dishes, at a 400K/ml

density with 10M ROCK inhibitor (Sigma, Y-27632) in mTeSR media for 24hrs. Embryoid

bodies (EBs) were formed and media was gradually diluted (50% on day 3 and 100% on day 4)

to KOSR (DMEM/F12, 10% KOSR) between days 1-4 and to a neural induction medium (NIM:

DMEM/F12 with L-glutamine, NEAA, Heparin (2g/ml), N2 supplement (Invitrogen) for days

5-24. From days 1-6 cells were cultured in the presence of SB431542 (10M, Sigma Aldrich) and

Dorsmorphin (1M, Stemgent), and from days 5-24 with BDNF (10g/ml, R&D), ascorbic acid

(AA, 0.4g/ml, Sigma), Retinoic Acid (RA, 0.1M, Sigma) and Smoothened Agonist 1.3 (SAG

1.3, 1M, Calbiochem). At day 24 EBs were dissociated to single cells with Papain/DNase

(Worthington Bio) and plated onto poly-lysine laminin-coated chamber slides/plates/coverslips

Motor neuron survival assay

After 24 days of differentiation, neuronal EBs were dissociated and 20K were plated on poly-D-

lysine/Laminin coated 8-well chamber slides (BD biosciences) containing a confluent monolayer

of primary cortical mouse glia. Primary glial preparations from P0-P2 mouse pups were

generated as described previously (Boulting et al., 2011; Di Giorgio et al., 2008). Fresh glial

preparations (<1 month, <2 passages) were used. Co-cultures were maintained in Neurobasal

media (NB, Invitrogen), supplemented with B27 and N2 supplement (Invitrogen), 10g/mL each

of BDNF, GDNF, CNTF (R&D) and ascorbic acid (AA, 0.4g/ml, Sigma) and fed every 2-3

days. Slides were fixed at various time points (3, 15, 20, 30 days), cultures were stained and cell

numbers assessed. Whole-well images were quantified in a manner blinded to the genotype and

condition of the experiment. Neuronal numbers on day 3 were set as 100% and numbers on

subsequent time points were expressed as a percentage of day 3. To evaluate cell death, neuronal

cultures were plated without glia on coverslips and live cells were assayed using the In Situ Cell

Death Kit (Roche Diagnostics) according to manufacturer’s instructions.

Electrophysiology recordings

MNs were plated at 20K cells/cm2 on lysine/laminin-coated coverslips, in the presence of

primary mouse glial monolayers and allowed to mature for 2-4 weeks. MNs were identified by

RFP fluorescence, after transduction with the HB9::RFP lentivirus (Marchetto et al., 2008).

Whole-cell voltage-clamp or current-clamp recordings were made using a Multiclamp 700B

(Molecular Devices) at room temperature (21-23C). Data were digitized with a Digidata 1440A

A/D interface and recorded using pCLAMP 10_software (Molecular Devices). Data were

sampled at 20 kHz and low-pass filtered at 2 kHz. Patch pipettes were pulled from borosilicate

glass capillaries on a Sutter Instruments P-97 puller and had resistances of 2-4 M. The pipette

capacitance was reduced by wrapping the shank with Parafilm and compensated for using the

amplifier circuitry. Series resistance was typically 5-10 M, always less than 15 M, and

compensated by at least 80%. Linear leakage currents were digitally subtracted using a P/4

protocol. Voltages were elicited from a holding potential of -80 mV to test potentials ranging

from -80 mV to 30 mV in 10 mV increments. The intracellular solution was a potassium-based

solution and contained K gluconate, 135; MgCl2, 2; KCl, 6; HEPES, 10; Mg ATP, 5; 0.5 (pH 7.4

with KOH). The extracellular was sodium-based and contained NaCl, 135; KCl, 5; CaCl2, 2;

MgCl2, 1; glucose, 10; HEPES, 10, pH 7.4 with NaOH). Kainate and GABA were purchased

from Sigma. For MEA recordings after 24 days of differentiation, equal numbers of MN cultures

were plated on poly-D-lysine/laminin coated M768-GLx 12-well plates (Axion BioSystems) at

typical densities of 40,000-80,000/ well, recorded after approximately 14 days using an Axion

Maestro (Axion BioSystems) MEA device and analyzed using Axion Integrated Studio software

as described in the companion manuscript, Wainger et al. Total spike counts were determined by

summing total spikes recorded over one minute from the 64 electrodes in each well. Spike counts

were determined at baseline and at the listed time points after the application of 0.5 µM TTX

(Sigma), 2mM DTT (Sigma), salubrinal 15 M (Santa Cruz).

RNA preparation, RT-PCR and RNA sequencing

Total RNA was isolated from relevant cell types using Trizol LS (Invitrogen) according to

manufacturer’s instructions. MNs were differentiated as described above, MN cultures were

plated on glial monolayers, infected with the Hb9::RFP virus on days 5-7 and FACS was used

for purification of RFP MNs on day 15. A total of 300-1000ng was used to synthesize cDNA by

reverse transcription according to the iSCRIPT kit (Bio-rad).Quantitative RT-PCR was then

performed using SYBR green (Bio-Rad) and the iCycler system (Bio-rad). Quantitative levels

for all genes were normalized to the average levels of 3 housekeeping genes: GAPDH/b-

Actin/YWHAZ and expressed relative to the relevant control samples or the lowest expressing

sample in the experiment (see figure legends). QPCR for retroviral and endogenous

reprogramming genes was carried out as previously reported (Boulting et al., 2011). All primer

sequences are available upon request. Human spinal cord RNA was purchased from Clonetech

(#636530) and total human brain RNA was purchased from Applied Biosystems (#AM6050).

Spinal cord RNA was pooled from 22 male/female Caucasians aged between 22-69, while total

brain RNA was pooled from 23 male/female Caucasians aged between 23-86. For TTX (48hrs),

kainate (48hrs) and DTT (2hrs) treatments, co-cultures were treated, RFP positive neurons were

purified and RNA was subsequently isolated. For next-generation RNA sequencing, RNA

integrity numbers (RIN) above 7.5, determined by bioAnalyzer, were used for library

preparation. In brief, RNA sequencing libraries were generated from ~250ng total RNA using

the illumina TruSeq RNA kit v2, according to the manufacturer’s directions. Libraries were

sequenced at the Harvard Bauer Core Sequencing facility on a HiSeq 2000. All FASTQ files

were analyzed using FastQC software (v 0.10.1) to confirm that Phred scores were acceptable at

all read positions (median Phred score>25 and lower quartile>20). The FASTQ files were

aligned to the GRCh37/hg19 reference genome using Tophat (v 2.0.7). Duplicated reads were

removed using Picard Tools MarkDuplicates (v 1.44). Differential expression testing was

performed independently using two separate analysis packages: Cufflinks (v 2.1.1) and DESeq.

The Cufflinks output was visualized with the cummeRbund R package using a false discovery

rate of 0.05. For DESeq analysis, gene level annotation count files were first generated using the

HTseq count Python script (v 0.5.4). DESeq analysis was performed using the methods

recommended by the package authors. Gene Ontology term enrichment was determined for

significantly differentially expressed genes at a false discovery rate cutoff of 0.05 using the

Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7. Gene Set

Enrichment Analysis (GSEA, Broad Institute) was performed by first creating a pre-ranked gene

list of all genes included in differential expression testing ordered by log2 fold change. Analysis

was performed using the GSEA preranked tool with the REACTOME and KEGG Pathway

MSigDB collections.

Mitochondrial transport assays and EM analysis

After 24 days of differentiation, neuronal EBs were dissociated and 20K were plated on poly-D-

lysine/laminin-coated 35 mm glass bottom culture dishes (MatTek Corporation) or coverslips

(BD-Biosciences). Cultures were infected with Hb9::RFP lentivirus 5 days after dissociation and

MNs were selected based on expression of RFP. On days 23-26, MNs were stained with

MitoTracker® Green FM (50nM, Invitrogen) and transferred to a custom observation chamber

mounted on the stage of the microscope. Live microscopy of mitochondrial transport was

performed with a Nikon Eclipse Ti equipped with an automated stage and In Vivo Scientific

incubator. Mitochondrial movements were recorded for 5 minutes with 4-second time-lapse

intervals using NIS-Elements (Nikon) using a 63x lens. Kymographs were generated from each

video using NIS-Elements Analyzing Software (Nikon). Mitochondria were considered motile if

they traveled faster than 0.017 µm/second. The average distance between mitochondria was

calculated excluding motile mitochondria, and the total unoccupied space was divided by total

process length analyzed to yield the proportion of processes unoccupied by mitochondria.

Average distance was measured using the top portion of each kymograph using NIS-Elements

Analyzing Software (Nikon). For Electron Microscopy analysis, MN cultures were fixed with

2.5% glutaraldehyde-2% paraformaldehyde in 0.1M sodium cacodylate buffer (pH 7.4) and

maintained at 4°C O/N. Cultures were then postfixed in 1% OsO4-1.5% KFeCN6 for 30 min,

washed in water 3x and incubated in 1% aqueous uranyl acetate for 30mn followed by 2x washes

in water and subsequent dehydration in grades of alcohol (5min each; 50%, 70%, 95%, 2x

100%). Cells were then embedded in plastic and ~60nm thick sections were cut, picked up onto

copper grids, stained with lead citrate and analyzed in a JEOL 1200EX Transmission Electron

Microscope. At least 3 independent differentiation experiments were analyzed in each case and

pictures were taken by a technician blinded for sample IDs.

XBP1 splicing assay

300ng of RNA was used to generate cDNA. PCR reactions were set up using 2l of cDNA and

premixed Ampligold Taq Polymerase (Applied. Biosystems), at 66C annealing. PCR products

were analyzed on a 2% low-melting agarose gel. The ratio of spliced/unspliced bands was

quantified using Image J software. Primers available upon request.

Gene targeting

Zinc finger nucleases (ZFNs) were constructed using either the OPEN method as described

previously (Maeder et al., 2008) or a modified version of OPEN that uses antibiotic resistance

for the selection as previously described (Sander et al., 2011). Briefly, pools of ZF pre-selected

zinc finger (ZF) domains were ligated together to create a combinatorial library of three-3 finger

proteins. A bacterial two-hybrid-based selection system was used to interrogate the ZF library for

proteins that could bind the appropriate target sequences of interest. ZF proteins that bound the

target sequence were cloned into a mammalian expression vector and fused to heterodimeric

FokI nuclease protein domains to construct ZFNs. Active ZFNs capable of inducing a double

strand break at the desired locus were identified by screening pairs of nucleases for the capability

to induce characteristic indel mutations at the SOD1 target site in sequencing the locus of ZFN-

treated HEK 293s. 2.5 million iPS cells of the 39b cell line were accutased and nucleofected,

using Human Stem Cell Kit II and program A-023, with 1g of ZFN plasmid and 5g of

targeting plasmid. After nucleofection the cells were plated on matrigel with mTesr and ROCK

inhibitor. After 48hrs puromycin selection was applied for 1 week after which surviving colonies

were passaged and gDNA was extractedPCR was used to confirm proper targeting of the

cassette. To remove the puromycin cassette 2.5 million cells were nucleofected with 1g of a

mammalian expression plasmid containing hygromycin and 5g of a mammalian expression

plasmid containing the FLPo recombinase and plated on matrigel with mTeSR and ROCK

inhibitor. Twenty four hours after nucleofection, hygromycin was added for 48 hours. Colonies

were allowed to expand for 1 week then picked and genomic DNA was extracted. Sequencing of

the genomic DNA was used to confirm removal of the puromycin cassette. SOD1 expression

was verified by qPCR after RNA extraction and cDNA synthesis. PshAI digestion along with

sequencing of the qPCR product demonstrated loss of expression of the mutant allele. Copy

number qPCR using primer SOD1cnF and SOD1cnR was performed as described previously

(D'Haene et al., 2010) to rule out random integration events. Primer sequences are available upon

request.

Genome sequencing and analysis.

DNA samples were derived from the parental 39b cell line and the gene corrected clone using

phenol chloroform extraction. The sequencing libraries were made with 50ng genomic DNA

using the Illumina Nexterra DNA kit. Deep (30×) WGS was performed using the Illumina

HiSeq 2500 Platform (500 bp library, 101 bp reads). All subsequent alignments and analysis

were performed with hg19 as a reference. To investigate whether there were changes in copy

number due to the cell line transformation, we used Genome STRiP (Handsaker et al., NG, 2011)

to extract and process the read depth signal from the aligned sequencing data. Using a genome

alignability mask of size 101, we segmented the genome into non-overlapping windows each

containing 100Kb of uniquely alignable base positions (based on a read length of 101bp). In each

window, we computed normalized read depth by counting the read fragments aligned within that

window with a minimum mapping quality of 10 and normalized the counts based on genome-

wide sequencing depth and correcting for sequencing coverage bias due to local G+C content.

This was done separately for the parental and derived cell lines. To look for regions of copy

number change, we evaluated the ratio of normalized read depth in the derived cell line

compared to the parental cell line in each window. To find rare coding SNPs in ALS genes, we

annotated coding variants called by Haplotype caller with SNPeff (Abecasis et al., 2012). SNPs

classified as missense, silent, or nonsense were retained. We then integrated allele frequencies

for the European population from the thousand genomes project (Cingolani et al., 2012).

Variants were selected that overlapped target genes for ALS. To find variants that differed

between cell lines, we compared the genotypes of both lines in a stringent manner similar to the

methodologies used to discover de novo mutations. For a variant to be confidently different

between cell lines, we required a read depth of at least 2 and a likelihood score (PL) of at least

30 across both lines. Homozygous variants were required to have no more than 5% of

the reads observed from the alternate allele, while heterozygous variants were required to have at

least 30% of reads observed from the less frequent allele and at most 70% of the reads from the

more frequent allele. To examine the off target effects of the designed nuclease, variants within

the top 12,000 potential off target nuclease cut sites were selected from this filtered set of

confident variants.

Nanostring karyotyping

Karyotyping was undertaken using the Nanostring nCounter Human Karyotype Panel

(Nanostring Technologies, USA) and performed as per the manufacturers instructions. In brief,

the protocol is as follows: 600 ng of genomic DNA was Alu1 digested at 37 °C for 2 hours,

before being denatured at 95 °C for 5 minutes. To prevent renaturing samples were kept on ice.

A total of 300 ng of Alu1-digested DNA per sample was mixed with hybridization buffer,

capture and reporter codes. Following a 16 hour incubation at 65 °C, samples were transferred to

a Nanostring Prep station where hybridized DNA was bound to an imaging cartridge before

imaging. Using reference samples, a copy number was calculated for each chromosome

following normalization of the data using nSolver (Nanostring Technologies, USA) and

Microsoft Excel.

Treatments with small molecules and siRNAs.

For ER stress induction, human fibroblasts and human astrocytes were treated with Dithiothreitol

(DTT) (2mM) (Bio-Rad Laboratories) for 2hrs washed with PBS and either fixed, stained and

evaluated or trypsinized for RNA/protein collection. For analysis of SOD1 protein, MN cultures

were treated with vehicle or 1M MG132 for 48hrs. For assessing the role of XBP1 (s14913)

and ATF4 (s1702) 40nM of siRNA (Ambion) was transfected into MN cultures using siRNA-

Select in Optimem on days 10/20 and knockdown levels and survival were assessed on day 30.

Immunocytochemistry

Cell cultures were fixed in 4% PFA for 15minutes at 4°C, permeabilized with 0.2% Triton-X in

PBS for 45 minutes and blocked with 10% donkey serum in PBS-T (Triton 0.1%). Cells were

then incubated in primary antibody overnight and secondary antibodies for 1 hour in 2% donkey

serum in PBS-T after several washes in between. DNA was visualized by a Hoechst stain. The

following antibodies were used: Primary antibodies used in this study are TRA1-81 (1:500,

Chemicon, MAB4381), Nanog (1:500, R&D, AF 1997), Islet1 (1:200, DSHB, 40.2D6), HB9

(1:100, DSHB, MNR2 81.5C10-c), ChAT (1:100, Chemicon, AB144P), TUJ1 (1:1000, Sigma,

T2200), MAP2 (1:10000, Abcam ab5392), BrdU (3H579, Santa Cruz Biotechnology, sc-70441),

Ki67 (1:400, Abcam, ab833), GFP (1:500, Life Technologies, A10262), SOD1 (1:2000, Agrisera

#AS09 540), Hoxa5 and FOXP1 (courtesy of Susan Morton, Jessell lab). Secondary antibodies

used (488, 555, 594, and 647) were AlexaFluor (1:1000, Life Technologies) and DyLight (1:500,

Jackson ImmunoResearch Laboratories).

Chick embryo transplants

HUES3 Hb9::GFP+ MNs were differentiated and one EB was placed into the neural tube of a

stage 15 chick embryo using a tungsten needle. Embryos were harvested after 5 days, fixed and

sectioned in paraffin. Sections were immunostained with antibodies specific for GFP (rabbit,

Abcam) and an HRP-conjugated anti-rabbit antibody (Vector labs) and counterstained with

hematoxylin.

Western Blots

For analysis of Phospho-eIF2 protein, cells were lysed in RIPA buffer (150mM Sodium

Chloride; 1% Triton X-100; 0.5% sodium deoxycholate; 0.1% SDS; 50 mM Tris pH 8.0)

containing protease and phosphatase inhibitors (Roche) for 20 min on ice, and centrifuged at

high speed. Samples containing 20g of protein were separated by SDS-PAGE (NuPAGE®, Life

Technologies) and transferred to nitrocellulose membranes. Membranes were probed with anti-

Phospho-eIF2 antibody (#3597, Cell Signaling Technology), and anti-Tubulin (abcam,

ab4074) and anti-eIF2 (Cell Signaling Technology, #9722) antibodies were used as loading and

normalizing controls, respectively. For analysis of SOD1 protein, detergent-soluble fractions

were prepared using RIPA buffer and detergent-insoluble fractions were obtained using UREA

buffer (8M UREA; 4% CHAPS; 40 mM Tris; 0.2% Bio-Lyte® 3/10 ampholyte). 5g of

detergent-soluble and equivalent volumes of detergent-insoluble protein samples were separated

by SDS-PAGE (Bio Rad Laboratories), transferred to PDVF membranes and probed with anti-

SOD1 antibody (Agrisera #AS09 540) and anti-Tubulin (Sigma Aldrich # T6199). For

mitochondrial biogenesis analysis, 6ug of detergent soluble protein samples were analyzed using

the MitoBiogenesisTM

Western Blot Cocktail (ab123545).

Statistical analysis

Statistical significance was assessed by a standard Students T test (1 tail & 2 tail); P<0.05 was

considered significant. Two-tailed, unpaired tests were used except to confirm specific

hypotheses, in which case one-tailed, unpaired tests were used. Error bars represent ±s.e.m,

unless otherwise stated.

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