CRISPR Gene Editing in Stem Cells

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treatment of T2DM. T5224 is able to strikingly rescue CDKAL1-
mutation-mediated pancreatic beta cell dysfunction in vivo,
which is a proof of concept for a T2DM drug candidate rescuing
a gene-specific defect in vivo.
By combining high-content chemical screening and RNA-seq,
we found the FOS/JUN pathway to be significantly upregulated
in CDKAL1 / insulin+ cells and that reducing FOS/JUNpathway
activity either chemically or genetically rescued CDKAL1 muta-
tion-induced defects. Previous studies have shown that FOS/
JUN activation is involved in cytokine and mechanical-stress-
induced beta cell death (Abdelli et al., 2007; Hughes et al.,
1990) and amylin-induced apoptosis (Zhang et al., 2002). Here,
we found that CDKAL1 / -mediated activation of the FOS/
JUN pathway through fatty acids may be a further effector of
FOS/JUN-regulated beta cell survival, providing mechanistic
insight into how CDKAL1 locus may contribute to diabetes
progression.
In summary, we established an isogenic hESC platform to
systematically evaluate the role of disease-associated loci in
the survival and function of human pancreatic beta-like cells
in vitro and in vivo. The platform can be used to study other dis-
ease-associated loci/variants with respect to beta-like cell func-
tion. It is worth noting that the glucose-responding cells derived
using the current reported protocols are not equivalent to pri-
mary human beta cells. Ca 2+ flux assays suggested that approx-
imately 30%–40% of the insulin-GFP+ cells show increased
cytosolic Ca 2+concentrations in response to glucose stimulation
(Figure S7Q), whereas robust glucose-induced signaling was
observed in more than 70% of human beta cells based on the
previous report (Rezania et al., 2014). The restricted functionality
of pancreatic beta-like cells derived using current protocols
might limit their application for evaluating subtle contributions
of genes to glucose metabolism and Ca 2+ signaling. Thus, addi-
tional work is needed to further improve the protocol to derive
mature pancreatic beta-like cells. In addition, the platform estab-
lished here can also be applied to study the role of disease-asso-
ciated loci/variants in other diabetes-related cell types, such
as hepatocytes, adipocytes, muscles, and/or intestinal neuroen-
docrine cells. Finally, the system may be used as a high-
throughput/content chemical screening platform to identify
candidate drugs correcting allele-specific defects for precision
therapy of metabolic diseases.
EXPERIMENTAL PROCEDURES
Cell Culture and Chemicals
All experiments were performed using INSGFP/W HES3 cells. hESCs were
grown on Matrigel-coated 6-well plates in mTeSR1 medium (STEMCELL
Technologies). Cells were maintained at 37 C with 5% CO2. T5224 was pur-
chased from MedChem Express (HY-12270). Human islets were provided by
IIDP (Integrated Islet Distribution Program).
Creation of Isogenic Mutant hESC Lines
To mutate the target genes, two sgRNAs targeting the first two exons of the
target gene were designed, cloned into a vector carrying a CRISPR-Cas9
gene, and validated using the surveyor assay in 293T cells. After validation,
INSGFP/W HES3 cells were dissociated using Accutase (STEMCELL Technolo-
gies) and transfected (8 3 105 cells per sample) in suspension using Human
Stem Cell Nucleofector solution (Lonza) using electroporation and following
the manufacturer’s instructions. Cells were co-transfected with the vector ex-
pressing Cas9/sgRNA at 10 nM final concentration and a vector expressing
puromycin. After replating, the transfected cells were treated with 500 ng/ml
puromycin. After 2 days of puromycin selection, hESCs were dissociated
into single cells by Accutase and replated at low density. Ten micromolar
Y-27632 was added. After approximately 10 days, individual colonies were
picked, mechanically disaggregated, and replated into two individual wells
of 96-well plates. A portion of the cells was analyzed by genomic DNA
sequencing. For biallelic frameshift mutants, we chose both homozygous mu-
tants and compound heterozygous mutants. Wild-type clonal lines from the
corresponding targeting experiments were included as wild-type controls to
account for potential nonspecific effects associated with the gene-targeting
process.
Stepwise Differentiation
Wild-type and isogenic mutant hESCs were differentiated using either of
two slightly modified protocols from what was previously reported (Rezania
et al., 2014). The details of protocol 1 and 2 are listed as Figure S1C and
described in detail in the Supplemental Experimental Procedures.
In Vivo Transplantation, GSIS, and IPGTT
Wild-type and isogenic mutant hESCs at day 30 of differentiation were resus-
pended in 40 ml DMEM+B27 and transplanted under the kidney capsule of 6-
to 8-week-old male SCID-beige mice. Two days after transplantation, the mice
were treated with 200 mg/kg STZ. To perform GSIS, mice were starved for
about 20 hr. Mouse blood was collected under fasting conditions and at
15 min after intraperitoneal injection with 3 g/kg glucose solution. The mouse
sera were analyzed using the ultrasensitive human insulin ELISA kit (ALPCO;
80-INSHUU-E01.1). To perform IPGTT analysis, the mice were fasted over-
night and treated with 2 g/kg glucose. Blood glucose level (mg/dl) in each an-
imal was measured before and every 15 min in the first hour and every 30 min in
the second hour after glucose injection. The mice transplanted with wild-type
orCDKAL1 / cells were orally treated with 300 mg/kg T5224 dissolved in pol-
yvinylpyrrolidone K 60 solution (Sigma). After 48 hr treatment, the mice were
examined for GSIS and IPGTT. The mice treated with polyvinylpyrrolidone K
60 solution (vehicle) were used as the controls. For long-term treatment, the
mice were orally treated with 300 mg/kg T5224 twice a week for 4 weeks.
GSIS and IPGTT were measured 48 hr after the last treatment.
High-Content Chemical Screening
To perform the high-content chemical screening, CDKAL1 / D30 cells were
plated on 804G-coated 384-well plates at 5,000 cells/40 ml medium/well. After
overnight incubation, cells were treated at 10m Mwith compounds froma chem-
ical collection containing the Prestwick FDA-approved drug library and drugs in
clinical trials. DMSO treatment was used as a negative control. After 48 hr incu-
bation, cells were first stainedwith 100m g/ml PI and then fixed and stained using
an insulin antibody (Dako). Plates were analyzed using a Molecular Devices
ImageXpress High-Content Analysis System. Two-dimensional analysis was
used. Compounds decreasing the cell death rate in excess of 80% and
increasing the number of insulin +cells by 2-fold were picked as primary hits.
Statistical Analysis
n = 3 independent biological replicates if not otherwise specifically indicated.
n.s. indicates non-significant difference. p values were calculated by unpaired
two-tailed Student’s t test if not otherwise specifically indicated. n = 8 mice for
in vivo experiments if not otherwise specifically indicated. p values were calcu-
lated by one-way repeated-measures ANOVA or two-way repeated-measures
ANOVA with a Bonferroni test for multiple comparisons between wild-type and
KO cells. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
seven figures, and six tables and can be found with this article online at
AUTHOR CONTRIBUTIONS
S.C., H.Z., J.G., and T.E. designed the project; H.Z. and M.G. performed most
experiments; T.Z., L.T., C.N.C., X.D., A.S.Y., and L.Y. performed other
338 Cell Stem Cell 19 , 326–340, September 1, 2016