CRISPR Gene Editing in Stem Cells

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percentage of insulin-GFP+ cells in D30 populations to be indis-
tinguishable between wild-type and the isogenic mutant lines
(Figures 1C and S1I). Together, these data suggest that biallelic
mutation of CDKAL1, KCNQ1, or KCNJ11 does not affect the
stepwise differentiation of insulin+ cells.
The expression of pancreatic beta cell makers in D30 hESC-
derived insulin+ cells was analyzed by immunocytochemistry,
and all cells, regardless of genotype, were found to express
markers indicative of mature pancreatic beta cells, including
PDX1, NKX6.1, and NKX2.2 (Figure 1D). Intracellular fluores-
cence-activated cell sorting (FACS) analysis showed that most
hESC-derived insulin + cells express the mature beta cell marker
NKX6.1, but not the alpha cell marker glucagon (Figures 1E and
S1J). Wild-type and isogenic mutant cell lines did not differ with
respect to the NKX6.1+/insulin+ cell or insulin+/glucagon cell
fractions (Figures 1E and S1J). Next, insulin-GFP+ cells were
purified by cell sorting and analyzed for transcript expression
levels with qRT-PCR (Figure S1K). Undifferentiated hESCs
served as a negative control and primary human islets as a pos-
itive control. Transcripts encoding mature pancreatic beta cells
markers, including NKX6.1, NKX2.2, PDX1, ISLET1, PAX6,
NEUROD1, GCK, G6PC2, UCN3 , and MAFA are highly ex-
pressed at levels comparable to human islets in hESC-derived
insulin-GFP+ cells. No significant difference was observed
between wild-type and isogenic mutant insulin-GFP+ cells (Fig-
ure S1K). The total c-peptide level of wild-type and mutant
hESC-derived insulin-GFP+ cells, as measured by ELISA, was
comparable to levels in primary human islets (Figure 1F; Table
S4). Thus, mutation of CDKAL1, KCNQ1, or KCNJ11 does not
significantly affect the generation of mature beta-like cells or
insulin production.
Mutation of CDKAL1, KCNQ1, or KCNJ11 Differentially
Impairs Insulin Secretion in Response to Multiple
Secretagogues
The major function of pancreatic beta cells is to secrete insulin/
c-peptide upon induction by secretagogues. D30-differentiated
wild-type or mutant cells were stimulated with 30 mM KCl, and
secreted human c-peptide was measured by ELISA. Wild-type
cells respond with a 4.5- ± 1.6-fold induction of c-peptide secre-
tion (Figures 2A, 2B, and S2A). CDKAL1 / cells showed a small
but insignificant decreased response, whereas KCNQ1 / and
KCNJ11 / cells were severely and significantly impaired in their
response to KCl stimulation (Figures 2A and 2B). The cells were
further queried for their response to 10 mM arginine. Again, both
wild-type and CDKAL1 / D30 cells responded well, whereas
KCNQ1 / and KCNJ11 / cells failed to respond (Figures 2C,
2D, and S2B). D30 cells were also stimulated with 20 m M forsko-
lin or 50 mM IBMX to measure cyclic AMP (cAMP)-induced insulin
secretion. Wild-type cells responded well to both, yielding 7.2- ±
1.9- and 6.2-± 1.8-fold induction of c-peptide secretion, respec-
tively (Figures 2E, 2F, and S2C). Cells carrying the three mutant
alleles were able to respond to both forskolin and IBMX stimula-
tion (Figure 2E), but compared to wild-type cells, the fold induc-
tion was significantly decreased (Figure 2F). Finally, wild-type
cells stimulated with 2 mM (low) or 20 mM (high) D-glucose
responded to high glucose with a 2.3- ± 0.8-fold induction of
c-peptide secretion, whereas all three mutant genotypes failed
to respond (Figures 2G, 2H, and S2D). Thus, loss of KCNQ1 or
KCNJ11 affects insulin secretion. Because CDKAL1 / cells
respond to KCl and arginine (Figures 2A and 2C), but not
cAMP or glucose stimulation (Figures 2E and 2G), CDKAL1
may be involved in cAMP and glucose sensing rather than
exocytosis of insulin granules.
Patch-clamp experiments were used to determine KATP chan-
nel activity in KCNJ11 / cells. To perform KATP current record-
ings, wild-type insulin-GFP+ cells were held at 0 mV to inactivate
any voltage-gated ion channels, and KATP currents were elicited
by depolarization from holding potential (HP) = 0 mV to +80 mV.
KATP channels were activated by the KATP -channel-specific acti-
vator diazoxide (Pasyk et al., 2004; Figure S2E) and inhibited
by KATP-channel-specific blocker glybenclamide. The effect of
diazoxide was reversible. After washout of diazoxide, glybencla-
mide further reduced current amplitude from 400 pA to 200 pA
(Figure S2F), suggesting that, in the absence of diazoxide, there
were basal KATP channel activities, which was likely induced by
the pipette solution. Whereas KATP currents were recorded in
wild-type insulin-GFP+ cells, diazoxide and glybenclamide did
not produce any effects in the recordings from insulin-GFP+
KCNJ11 / mutant cells (Figure S2G), suggesting the absence
of KATP channel activity.
CDKAL1–/– Insulin-GFP+ Cells Are Hypersensitive to
Glucolipotoxicity
Hyperglycemia and hyperlipidemia are two major risk factors
associated with pancreatic beta cell death in diabetic pa-
tients. Wild-type and isogenic CDKAL1 / , KCNQ1 / , and
KCNJ11 / D30 insulin-GFP + cells were cultured in the presence
of 35 mM D-glucose for 96 hr or 1 mM palmitate for 48 hr. Cells
were stained with propidium iodide (PI) to determine the cell
death rate (Figure 3A). No significant difference was detected
between wild-type and mutant insulin+ cells under control
conditions. However, the percentage of PI+ /insulin+ cells in
CDKAL1 / insulin+ cells was significantly higher compared to
wild-type insulin+ cells exposed to 35 mM D-glucose or 1 mM
palmitate, indicating that CDKAL1 / insulin+ cells are hyper-
sensitive to glucotoxicity and lipotoxicity (Figure 3B). In contrast,
neither KCNQ1 / nor KCNJ11 / insulin+ cells showed in-
creased sensitivity to glucotoxicity or lipotoxicity (Figure S3A).
Treated cells were stained with the apoptosis marker
annexin V, as well as the cell death marker 7AAD, and evaluated
by flow cytometry to measure apoptosis in insulin-GFP + cells
(Figures 3C and S3B). Consistent with the PI staining results,
the percentage of annexin V+/7AAD cells in CDKAL1 / insu-
lin-GFP+ cells was significantly higher than wild-type (Figure 3D),
KCNQ1 / , or KCNJ11 / insulin-GFP+ cells when cultured in
the presence of 35 mM D-glucose or 1 mM palmitate (Figures
S3C and S3D). We also measured the proliferation rate of wild-
type and CDKAL1 / insulin-GFP+ cells (Figure S3E), which
showed no significant difference (Figure S3F). RNA sequencing
(RNA-seq) was used to compare the gene expression profiles
in wild-type and CDKAL1 / cells cultured in the presence or
absence of palmitate. ER-stress-related genes were found
significantly upregulated in CDKAL1 / cells cultured under
palmitate conditions (Figures 3E and 3F). This suggests, consis-
tent with the literature (Brambillasca et al., 2012; Wei et al., 2011),
that loss of CDKAL1 induces elevated ER stress under exposure
to high levels of fatty acids.
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