CRISPR Gene Editing in Stem Cells

T5224 restored the capacity to respond to glucose stimulation

(Figures 7B and S7B). T5224 treatment significantly increased

the level of insulin secretion after glucose stimulation. In addition,

the mice carrying

CDKAL1

cells showed glucose intolerance.

T5224 treatment restored the capacity of the SCID-beige

mice carrying human cells to maintain glucose homeostasis (Fig-

ures 7C and S7C). The AUC for mice after T5224 treatment was

significantly lower than for mice treated with control vehicle (Fig-

ures 7D and S7D). T5224 treatment was also examined using

mice carrying wild-type cells. Consistent with the in vitro results

(Figure 5), T5224 treatment affects neither GSIS (Figures S7E

and S7F) nor glucose tolerance of mice carrying wild-type

cells (Figures S7G–S7J). To determine the long-term effect

of T5224, mice carrying

CDKAL1

cells were treated with

300 mg/kg T5224 orally twice a week and measured for GSIS

and glucose tolerance 4 weeks after treatment. The long-term

treatment of T5224 restored both GSIS (Figures 7E and S7K)

and glucose tolerance (Figures 7F, 7G, S7L, and S7M) for mice

carrying

CDKAL1

cells. Finally, D30

CDKAL1

cells carrying

scrambled sgRNA and D30

CDKAL1

cells carrying sgFOS

were transplanted into mice that were than measured for func-

tion in vivo 6 weeks after transplantation. Consistent with

in vitro results (Figure 6), mice with

CDKAL1

cells carrying

sgFOS showed improved GSIS (Figures 7H and S7N) and a

stronger ability to maintain glucose homeostasis (Figures 7I,

7J, S7O, and S7P) than mice transplanted with

CDKAL1

cells

carrying scrambled sgRNA. Together, these data suggest that

T5224 or loss of FOS rescues the function of

CDKAL1

cells

in vivo.

DISCUSSION

With more than 80 loci associated with T2DM identified by

GWASs, a robust platform to evaluate the role of these loci using

disease-relevant cells is urgently needed. Here, we report proof

of principle for using isogenic hESC-derived glucose-responding

cells to evaluate the role of these loci in the function and survival

of human pancreatic beta cells under conditions mimicking both

health and disease. The derived glucose-responding cells share

the same genetic background, providing a unique resource to

determine the precise role of genes or loci in human pancreatic

beta cells independent of complications from genetic heteroge-

neity implied by other approaches, such as patient-derived

iPSCs.

We found that mutation of

KCNJ11

resulted in impaired insulin

secretion upon KCl, arginine, forskolin, IBMX, and glucose

stimulation, suggesting that

KCNJ11

plays an essential role in

insulin secretion, which is consistent with results in homozygous

Kcnj11

KO mice, as well as in homozygous

Kcnj11

-null

mice (Remedi et al., 2006; Boini et al., 2009). In the context of

reports that forced expression of

KCNQ1

in a mouse beta cell

line results in impairment of insulin secretion (Yamagata et al.,

2011) and islets isolated from

Kcnq1

mice reveal no differ-

ence in the extent of basal or stimulated insulin secretion

compared to islet from wild-type mice (Asahara et al., 2015),

we were surprised to find impaired insulin secretion in

KCNQ1

insulin-secreting cells. This apparent discrepancy

may suggest dose- and/or species-specific roles in pancreatic

beta cell function, highlighting the importance of using human-

relevant cell types.

An ultimate goal of exploring loci or genetic variants associ-

ated with disease through GWASs is to identify locus-/variant-

specific treatments. Risk alleles of SNPs at the

CDKAL1

locus

associated with diabetes are thought to be loss-of-function

alleles, which we modeled, generating null mutations. We found

that

CDKAL1

insulin

cells showed impaired FSIS and GSIS,

which is consistent with

Cdkal1

mice showing reduced

first-phase insulin exocytosis (Ohara-Imaizumi et al., 2010).

CDKAL1

insulin

cells also show increased ER stress, cell

apoptosis, and death when cultured in high-glucose and high-

fatty-acid conditions. Although there are papers describing the

potential contribution of lipotoxicity in T2DM, direct evidence

that lipotoxicity affects pancreatic beta cell death in vivo under

normal physiological and pathological conditions needs to be

further explored. Here, we found that

CDKAL1

insulin

cells

are hypersensitive to both high-glucose- and high-fatty-acid-

induced pancreatic beta-like cell death. Moreover,

CDKAL1

insulin

cells display defective GSIS and impaired ability to

maintain glucose homeostasis following transplantation into

STZ-treated mice. This is consistent with the in vitro functional

defects of

CDKAL1

insulin

cells. Because the mice are hy-

perglycemic after STZ treatment, the observed glucotoxicity

may further worsen the defects of

CDKAL1

insulin

cells.

From a high-content chemical screen, T5224 was found to

rescue the

CDKAL1

mutation-mediated pancreatic beta cell de-

fects. T5224 has been investigated in clinical trials for patients

with rheumatoid arthritis (Pharmaceutical Medicine, 2014) and

may have the potential to be repurposed for

CDKAL1

-specific

Figure 6. T5224 Rescues Beta Cell Defects Caused by

CDKAL1

Mutation through Inhibiting the

FOS/JUN

Pathway

(A) Pathway enrichment analysis on up/downregulated genes in

CDKAL1

insulin-GFP

cells using the DAVID function annotation tool.

(B) Heatmap of focal-adhesion-pathway-associated genes comparing wt and

CDKAL1

insulin-GFP

cells.

FOS/JUN

-pathway-associated genes comparing WT and

CDKAL1

insulin-GFP

cells.

(D) Top 20 upregulated genes in

CDKAL1

insulin-GFP

cells as compared to wild-type cells.

(E) qRT-PCR analysis of

JUNB

FOS

, and

FOSB

expression in wt and

CDKAL1

insulin-GFP

cells.

(F) Western blotting analysis of FOS protein in wt and

CDKAL1

cells at D30 of differentiation.

(G) Targeted mutation of

FOS

rescues the high death rate in

CDKAL1

insulin-GFP

cells in the presence of 35 mM D-glucose or 1 mM palmitate.

(H and I) Flow cytometry analysis (H) and quantification of apoptotic rate (I) of

CDKAL1

insulin-GFP

cells expressing Cas9 and either scrambled sgRNA or

sgFOS.

(J and K) Mutation of

FOS

rescues the impaired forskolin-induced (J) and glucose-induced (K) insulin secretion that is caused by mutation of

CDKAL1

sgFOS no. 1 and no. 2 represent two independent sgRNAs targeting different locations of exon1 of

c-FOS

. Scramble sgRNA no. 1 and scramble no. 2 ‘‘target’’

controls were designed to have low homology to the human genome and are used as non-targeting controls. hESCs were differentiated using protocol 2. The data

are presented as mean ± SD. 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, and

***p < 0.001. See also Figure S6.

336

Cell Stem Cell

, 326–340, September 1, 2016