CRISPR Gene Editing in Stem Cells

cells (Figures 5C) in a dose-dependent manner with a concentra-

tion for 50% of maximum effect (EC

) of 16.2

M. In addition,

T5224 rescued the increased cell death rate in

CDKAL1

insulin

cells when cultured with high glucose or high palmitate

(Figures 5D and 5E). As measured using the annexin V assay

for apoptosis, T5224 also rescued the increased apoptotic rate

CDKAL1

insulin

cells under conditions of high fatty acid

concentration without affecting the rate in wild-type insulin

cells

(Figures 5F, 5G, and S5B), thus blunting hypersensitivity to

glucolipotoxicity.

The

CDKAL1

cells were treated with 30

M T5224 for 48 hr

and examined for impaired response to forskolin or glucose-stim-

ulated insulin secretion (FSIS and GSIS). Remarkably, the mutant

cells treated with T5224 showed increased insulin secretion in

response to forskolin treatment (Figures 5HandS5C), significantly

elevated compared to cells treated with DMSO and at a level

of insulin secretion comparable to wild-type cells (Figure 5H).

Similarly, T5224 treatment also rescued the impaired GSIS of

CDKAL1

cells (Figures 5I and S5D). Notably, T5224 treatment

did not significantly affect FSIS or GSIS in wild-type cells.

T5224 Rescues

CDKAL1

–/–

-Induced Beta Cell Defects

through Inhibition of the

FOS/JUN

Pathway

T5224 was reported to be an inhibitor of FOS/JUN activator pro-

tein-1 (AP-1) (Aikawa et al., 2008). To explore this potential mech-

anism of action, RNA-seq was used to compare the global gene

expression profiles in

CDKAL1

and wild-type insulin-GFP

cells. Pathway enrichment analysis highlighted the

FOS/JUN

and focal adhesion pathways as highly changed in

CDKAL1

insulin-GFP

cells (Figure 6A). Genes associated with the focal

adhesion Gene Ontology (GO) termwere consistently downregu-

lated (Figures 6B and S6A) whereas the

FOS/JUN

pathway (Fig-

ure 6C) was consistently upregulated in

CDKAL1

insulin-GFP

cells. Among the top 20 genes showing relatively increased

expression in

CDKAL1

insulin-GFP

cells are

FOSB

(6.3-

fold),

FOS

(3.5-fold), and

JUNB

(2.4-fold; Figure 6D), which was

confirmed by qRT-PCR (Figure 6E). Finally, western blotting ex-

periments validated the relatively increased expression of FOS

protein in mutant cells (Figure 6F).

To determine whether the mutation of

CDKAL1

induces

pancreatic beta cell defects through activation of the

FOS/JUN

pathway, two sgRNAs and two scrambled sgRNAs were de-

signed to knock out human

FOS

(Table S6). Wild-type and

CDKAL1

hESC-derived day 10 PPs were infected with lenti-

virus expressing either sgFOS or a scrambled sgRNA, and

following 4–6 days selection with puromycin, the cells were

differentiated to beta-like cells for an additional 16–20 days. In

cells expressing sgFOS, the expression of FOS was decreased

by more than 99% based on western blotting experiments, vali-

dating the targeting efficiency (Figure S6B). The cells were

cultured in the absence or presence of 35 mM D-glucose or

1 mM palmitate and analyzed with respect to the rates of cell

death and apoptosis by PI staining and annexin V, respectively.

Mutation of

FOS

using sgRNA rescues the increased cell death

rate of

CDKAL1

insulin-GFP

cells (Figure 6G) and cell

apoptotic rate of

CDKAL1

insulin-GFP

cells (Figures 6H, 6I,

and S6C). In contrast, mutation of

FOS

does not affect cell death

(Figure 6G) or apoptosis in wild-type insulin-GFP

cells (Figures

6H and 6I). In addition to sgRNA, two short hairpin RNAs

(shRNAs) against

FOS

were cloned into a lentiviral vector and

used to knock down

FOS

. The knockdown efficiency in day 10

PPs is more than 50% based on western blotting experiments

(Figure S6D). Consistent with the KO using sgFOS, knockdown

of FOS using shRNAs rescued the increased cell apoptotic

rate of

CDKAL1

insulin-GFP

cells when cultured in high

fatty acid condition (Figures S6E and S6F).

Likewise, wild-type and

CDKAL1

hESC-derived PPs in-

fected with lentivirus expressing sgFOS or a scrambled sgRNA

were differentiated for 20 days and measured for FSIS

and GSIS.

CDKAL1

cells infected with lentivirus containing

scrambled sgRNA showed impaired FSIS and GSIS compared

to wild-type cells. Transfection with lentivirus expressing sgFOS

rescued those phenotypes (Figures 6J, 6K, S6G, and S6H). How-

ever, KO of FOS did not affect FSIS (Figure 6J) or GSIS (Fig-

ure 6K) in wild-type cells. Consistently, knockdown of

FOS

using

shRNAs rescued the impaired FSIS (Figures S6I and S6K) and

GSIS (Figures S6J and S6L) in

CDKAL1

cells without affecting

wild-type cells. Together, this suggests that loss of

CDKAL1

causes hypersensitivity to glucolipotoxicity and impairs FSIS

and GSIS through the

FOS/JUN

pathway.

T5224 and Loss of

FOS

Rescues the Function of

CDKAL1

–/–

Cells In Vivo

To examine the effect of T5224 on

CDKAL1

cells in vivo, mice

transplanted with wild-type and

CDKAL1

cells were exam-

ined for GSIS at 10 weeks after transplantation. Consistent

with the 6-week results reported above, mice transplanted with

wild-type cells respond well to glucose stimulation. In contrast,

mice transplanted with

CDKAL1

cells showed impaired

GSIS (Figures 7A and S7A). After glucose stimulation, the insulin

level of mice transplanted with

CDKAL1

cells was signifi-

cantly lower than for mice transplanted with wild-type cells.

Subsequently, mice were treated with 300 mg/kg T5224 orally

and measured for GSIS 48 hr after treatment. Mice treated with

Figure 5. A High-Content Chemical Screen Identifies a Drug Candidate that Rescues Glucolipotoxicity Caused Specifically by Mutations in

CDKAL1

(A) Chemical structure of T5224.

(B and C) Efficacy curve of T5224 on the number of insulin

cells (B) and the percentage of PI

INS

cells (C).

(D and E) Immunocytochemistry analysis (D) and quantification of the percentage (E) of PI

/insulin

cells in wt and

CDKAL1

, insulin

cells treated with 30

T5224 when cultured in the presence of 2 mM D-glucose (ctrl-g), 35 mM D-glucose (glu), no palmitate (ctrl-p), or 1 mM palmitate (palm). PI

/insulin

cells are

highlighted by arrows.

(F and G) Flow cytometry analysis (F) and quantification (G) of apoptotic rate for WT or

CDKAL1

insulin-GFP

cells treated with DMSO or T5224.

(H and I) T5224 also rescues the impaired forskolin-induced (H) and glucose-induced insulin secretion (I).

Experiments in (A)–(C) were performed using cells derived from protocol 1. Experiments in (D)–(I) were performed using cells derived from protocol 2. n = 3

independent biological replicates for each condition. n.s. indicates a non-significant difference. p values calculated by unpaired two-tailed Student’s t test were

*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. The scale bar represents 50

m. See also Figure S5.

334

Cell Stem Cell

, 326–340, September 1, 2016