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0. 52. 105. 157. 209. 98.2 %. M1. HT140-E. Nanog-FITC-H. Co u n t. 102. 103. 104. 105. 106. 107. 0. 52. 105. 157. 209. M1. 97.2 %. HT146-B. Nanog-FITC-H.

Supporting Information for Neural stem cells for disease modeling and evaluation of therapeutics for infantile (CLN1/PPT1) and late infantile (CLN2/TPP1) neuronal ceroid lipofuscinoses

Ni Sima1,2, Rong Li1, Wei Huang1,2, Miao Xu1, Jeanette Beers3, Jizhong Zou3, Steven Titus1, Elizabeth A. Ottinger1, Juan J. Marugan1, Xing Xie2, Wei Zheng1†

1National

Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA. 2Department of Gynecologic Oncology, Women’s Reproductive Health Laboratory of Zhejiang Province, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, P. R. China. 3Center for Molecular Medicine, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA

†Corresponding author: [email protected]

Fig. S1

A

B

Fib GM20389

iPS HT146B

iPS HT146F

iPS HT146B; 46, XY

iPS HT146F; 46, XY

Fib GM16485

iPS HT140A 

iPS HT140E 

iPS HT140A; 46, XX

iPS HT140E; 46, XX (iPS HT140E; 46, XX)

Fib GM16486

HT143D (iPS HT140A) 

(iPS HT140E) 

iPS HT264A

iPS HT264B

iPS HT264A; 46, XY

iPS HT264B; 46, XY

Supplementary Fig. 1. Generation of Neuronal Ceroid Lipofuscinosis (NCL) induced pluripotent stem cells (iPSCs). (A) Generation of NCL patient iPS cell clones from patient skin fibroblast cells. Phase contrast images of patient fibroblasts and iPS cells. (B) iPSCs derived from three NCL patient fibroblasts displayed normal karyotype.

Fig. S2

HT146-F

98.2 %

105

52 0 2

3

10

4

10

5

6

10

Nanog-FITC-H

10

7

2

10

10

3

4

10

5

10

6

10

10

Nanog-FITC-H

7

0 3

10

4

10

5

6

10

Nanog-FITC-H

10

7

10

3

4

10

5

10

10

Nanog-FITC-H

M1

6

10

7

10

Count

98.57%

72 HT145F

60.16%

97

4

10

5

10

Nanog-FITC-H

6

10

7

10

6

10

6

10

10

7

HT140-E 209

M1

M1

157

99.5%

99.7%

105

52

52 0

2

10

3

10

4

10

5

10

Tra1-60-FITC-H

6

10

7

2

10

10

3

4

10

5

10

10

Tra1-60-FITC-H

10

7

M1 93.83%

97 49

0 3

5

10

146

49

0 10

4

10

Tra1-60-FITC-H

194

M1

146

2

3

10

HT264-B

194

215

10

2

10

HT264-B

HT264-A 287

144

7

10

0 2

10

10

Count

2

6

Tra1-60-FITC-H

105

52 0

10

5

10

157

97.2 %

105

52

4

10

HT140-A

Count

98.9 %

105

3

10

209

M1

157

Count

157

52 0

10

HT140-E 209

M1

99.5%

105

52 2

10

M1

157

0

HT140-A 209

Count

105

52 0

10

M1 99.7%

Count

105

209

157

Count

99.5 %

HT146-F

209

M1

157

Count

Count

157

Count

HT146-B

209

M1

Count

HT146-B 209

0 3

10

4

10

5

10

Nanog-FITC-H

6

10

10

7

3

10

4

10

5

10

Tra1-60-FITC-H

6

10

Supplementary Fig. 2. Pluripotent stem cell protein markers analyzed by flow cytometry. Cytometric analysis showed that these iPS cells expressed the pluripotency protein markers of Nanog and Tra-1-60.

10

7

Fig. S3

iPS HT146B

iPS HT146F

iPS HT140A

iPS HT140E

iPS HT264A

iPS HT264B

Oct4

Nanog

TRA-1-60

SSEA4

SOX2

iPS HT268A (WT)

Supplementary Fig. 3. Immunofluorescence staining of iPSC pluripotent stem cell protein markers. The iPSCs derived from wild-type (WT) control and NCL patient fibroblasts were stained for the pluripotency markers of SOX2, SSEA4, TRA-1-60, Nanog and Oct4. The images were taken with 20X objective lens.

Fig. S4

A

B Overlay

SOX2

NESTIN

Overlay

TPP1E4/IVS5

TPP1E4/E6

PPT1E8/E1

PAX6

Wild‐type

Wild‐type

TPP1E4/IVS5

TPP1E4/E6

PPT1E8/E1

SOX1

Supplementary Fig. 4. Protein marker expression in NSCs derived from WT control and NCL patient iPSCs. Representative images showed positivity for NSC marker proteins of Nestin, Sox1, Sox2 and PAX6. These NCL NSCs showed no morphological differences compared to WT control NSC cells. The images were taken with 20X objective lens.

Fig. S5 A PPT1E8/E1

TPP1E4/E6

TPP1E4/IVS5

FIB

WT

100 μM

Lysotracker: red; Nuclei: blue

B PPT1E8/E1

TPP1E4/E6

TPP1E4/IVS5

FIB

WT

100 μM

Nile Red: green; Nuclei: blue

D Relative Lysotracker Intensity

C

Supplementary Fig. 5. Enlarged lysosomes and lipid accumulation in NCL patient fibroblasts. Increased LysoTracker dye staining (A) indicates enlarged lysosomes and a strong Nile Red staining (B) indicates lipid accumulation. Enlarged lysosomes and lipid accumulation were observed in parental fibroblasts (FIB) of NCL patients compared to wild type (WT) control. Representative images of LysoTracker dye and Nile Red staining were shown with 20X objective lens. Quantification of the lysoTracker staining(C) and Nile Red staining (D) revealed significantly enlarged lysosomes and lipid accumulation in NCL patient fibroblasts. Data are displayed as mean ± SD. ** P < 0.01 vs. WT control.

Fig. S6

WT

PPT1E8/E1

NPC1

TPP1E4/E6

TPP1E4/IVS5

NSC

FIB

A

100 μM

Fillipin: green; Nuclei: red

B

WT NPC1

Relative Filipin Intensity

2.0

PPT1E8/E1

**

TPP1E4/E6 TPP1E4/IVS5

**

1.5 1.0 0.5 0.0 FIB

NSC

Supplementary Fig. 6. Filipin staining in NCL fibroblasts and NSCs. Representative images of Fillipin staining (A) showed no free cholesterol accumulation in patient NCL fibroblast and NSCs. A quantification of cholesterol accumulation in fibroblasts and NSCs was found no statistically significant (B). NPC1 patient fibroblasts and NSCs were used to serve as the positive control for Filipin staining. Data are displayed as mean ± SD. ** P < 0.01 vs. WT control.

Fig. S7

% Cell viability

A

% Cell viability

B

Supplementary Fig. 7. Cytotoxicity of δ-tocopherol (DT) and HPBCD on NCL patient NSCs. Cytotoxicity of DT (A) and HPBCD (B) was measured by an ATP content assay. DT lower than 20 uM concentration has no cytotoxic effect on all four NSCs cell lines. HPBCD showed no significant cytotoxic effect up to 1 mM concentration after a 3-day treatment period in those cells except for one of the NCL patient cell line, TPP1E4/IVS5, which decreased the cell viability by 64.2%

Fig. S8 A

DT 20 μM

DT 40 μM

HPBCD 250 μM HPBCD 500 μM

TPP1E4/IVS5

PPT1E8/E1

CTR

100 μM

pHrodo dextran / Nuclei B PPTI E8/E1

300

TPPI E4/IVS5

*** 200

***

100

20 CTR uM 40  DT 25 u 0u M  50 mH DT 0u PB m CD  H PB CD

20 CTR uM   4 25 0u DT 0u m   50 m H DT 0u PB m CD  H PB CD

0

Supplementary Fig. 8. Lysosomal pH indicated by a pHrodoTM PH sensor dye in NCL patient NSCs. Representative images of pHrodoTM dextran staining (A) after 3-day treatment of DT and HPBCD. A quantification of pHrodoTM dextran fluorescence signal showed no significant lysosomal pH changes except for 40 μM of DT treatment, which showed small decrease of fluorescence intensity (B), possibly caused by the cytotoxic effect of high concentration of DT as indicated by the cell cytotoxicity assay. Data are displayed as mean ± SD. *** P < 0.001, compared to the untreated control.