Acta Biochim Biophys Sin, 2020, 00(00), 1–8

doi: 10.1093/abbs/gmaa047
Review

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Review

Recent advances and potential applications of

human pluripotent stem cell-derived pancreatic
β cells
Ziyu Zhou, Xiaojie Ma, and Saiyong Zhu*
MOE Laboratory of Biosystems Homeostasis and Protection, Life Sciences Institute, Zhejiang University,
Hangzhou 310058, China
∗ Correspondence address. Tel/Fax: +86-571-88981797; E-mail: saiyong@zju.edu.cn
Received 26 December 2019; Editorial Decision 23 February 2020

Abstract
Diabetes mellitus is characterized by chronic high blood glucose levels resulted from deficiency
and/or dysfunction of insulin-producing pancreatic β cells. Generation of large amounts of func-
tional pancreatic β cells is critical for the study of pancreatic biology and treatment of diabetes.
Recent advances in directed differentiation of pancreatic β-like cells from human pluripotent stem
cells (hPSCs) can provide patient-specific and disease-relevant target cells. With the improved
differentiation protocols, it is now possible to generate large amounts of functional human
pancreatic β-like cells that can response to high level of glucose both in vitro and in vivo. Combined
with precise genomic editing, biomedical engineering, high throughput profiling, bioinformatics,
and high throughput genetic and chemical screening, these hPSC-derived pancreatic β-like cells
will hold great potentials in disease modeling, drug discovery, and cell-based therapies. In this
review, we summarize the recent progress in human pancreatic β-like cells derived from hPSCs
and discuss their potential applications.

Key words: pancreatic β cells, human pluripotent stem cells, disease modeling, drug discovery, cell-based therapy

Introduction Human pluripotent stem cells (hPSCs), including human embry-

According to the International Diabetes Federation, diabetes mellitus
represents a global health epidemic and affects more than 300 mil-
lion people worldwide. As an inevitable consequence, high blood
glucose can lead to many serious life-threatening diseases, including
amputation, blindness, neuropathy, stroke, cardiovascular diseases,
and kidney diseases. While the use of insulin has become a widely
used therapeutic intervention, other promising treatment options
have also been actively investigated at academic and pre-clinic stages.
Notably, the Edmonton protocol for islet transplantation has already
shown encouraging results and will undoubtedly hold great promises
in treating or even curing diabetes, but there remain many hurdles,
including very limited supplies of healthy human islets and the issues
of life-long immunosuppression [1]. Therefore, we need to develop
novel approaches to prepare patient-specific functional pancreatic β
cells for both basic studies and clinical applications.
onic stem cells (hESCs) and human-induced pluripotent stem cells
(hiPSCs), are very promising sources to provide unlimited supplies
of functional pancreatic β-like cells. hPSCs have infinite self-renewal
capacity and can be differentiated into any specific cell types of the
human body. Very encouragingly, many efforts have already been
made to generate an unlimited supply of functional pancreatic β-like
cells from hPSCs, and in combination with an encapsulation device
or patient-specific autologous iPSCs [2]. While significant progresses
have been made with these innovative strategies, novel and cost-
effective approaches are undoubtedly required for more successful
treatments and the ultimate cures for diabetes.
In this review, we will summarize recent advances on human
pancreatic β-like cell differentiation from hPSCs and the potential
applications of these hPSC-derived pancreatic β-like cells in disease
modeling, drug discovery, and cell-based therapies.

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2 Pancreatic β cells from hPSCs

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Figure 1. Generation of hPSC-derived functional pancreatic β-like cells (A) The human pancreatic differentiation in vitro mimics human pancreas development
in vivo. (B) Timeline of four key directed human pancreatic differentiation protocols. In 2008, Kroon et al. [3] reported a five-stage protocol to obtain PPs from
hPSCs. These PP cells could mature in vivo. In 2014, Rezania et al. [10] demonstrated a seven-stage protocol to generate insulin-secreting pancreatic β-like
cells from hPSCs. Meanwhile, Pagliuca et al. [11] developed a scalable 3D differentiation protocol to generate SC-β cells from hPSCs. In 2019, Nair et al. [17]
optimized their protocols to produce more mature pancreatic β-like cells. They isolated INS-GFP-positive cells and reaggregated them into islet-sized eBCs.
Significantly, these eBCs had better glucose-stimulated insulin-secreting functions both in vitro and in vivo. Growth factors and small molecules: Act A, Activin A;
Wnt, Wnt3a; RA, retinoic acid; Cyc, KAAD-cyclopamine; Vc, vitamin C; TPB, (2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl) phenyl)-2,4 pentadienoylamino) benzolactam;
SANT, Sonic hedgehog antagonist; LDN, LDN193189; ALK5iII, ALK5 inhibitor II; T3, triiodothyronine; N-Cys, N-acetyl cysteine; CHIR, CHIR99021; PDBu, Phorbol
12,13-dibutyrate; XXI, γ-secretase inhibitor XX; TTNPB, (E)-4-(2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propen-1-yl) benzoic acid.

et al. [3] further optimized their pancreatic differentiation protocol

Generation of Functional Pancreatic β-like Cells
from hPSCs
Over the past two decades, tremendous efforts have been made
to differentiate hPSCs into insulin-secreting pancreatic β-like cells.
Briefly, stepwise differentiation protocols have been developed to
guide the directed differentiation of hPSCs into definitive endoderm
(DE), primitive gut tube, posterior foregut, and pancreatic progen-
itors (PPs). After transplantation into immunodeficient mice, these
PP cells can further mature into functional pancreatic β-like cells
in vivo [3]. Subsequently, with improved differentiation protocols,
functional pancreatic β-like cells (PB) can be derived from hPSCs
under in vitro cell culture conditions (Fig. 1).
In 2001, Assady et al. [4] first demonstrated that pancreatic β-like
cells could be randomly differentiated from hESCs using the method
of embryonic bodies (EBs), albeit with very limited efficiency. In
2005, D’Amour et al. [5] identified that SOX17 and FOXA2 double-
positive DE could be efficiently induced from hESCs by high concen-
tration of activin A and low concentration of serum. The next year,
the same group reported a five-stage protocol for the generation of
insulin-producing endocrine cells from hESCs [6]. In 2008, Kroon
and demonstrated that functional endocrine cells could be generated
in vivo after transplanting PPs. Several months after implantation,
these hESC-derived pancreatic β-like could reverse hyperglycemia
induced by streptozotocin. Besides growth factors, small molecules
also play critical roles in human pancreatic differentiation [7]. For
example, in 2009, Chen et al. [8] found that PKC activator ILV
could increase the percentage of hESC-derived PDX1-positive cells
through a large-scale chemical screening. In 2012, Rezania et al.
[9] optimized the 2D differentiation system and demonstrated that
the combination of Alk5 inhibitor with PKC activator TPB and
BMP pathway inhibitor noggin at stage 4 could increase the expres-
sion of NKX6.1. In 2014, there were several breakthroughs of
pancreatic differentiation protocols. Rezania et al. [10] developed
an improved seven-stage differentiation protocol, which can finally
produce ∼50% insulin-positive cells. At first, they added vitamin C at
stages 2 and 3 to suppress the expression of NGN3 and used thyroid
hormone (T3) at stage 5 to enhance the co-expression of NKX6.1
and insulin. Next, through a small-scale chemical screening, they
identified that R428, a tyrosine kinase receptor AXL inhibitor, could
induce MAFA expression. Finally, they demonstrated that air-liquid
Pancreatic β cells from hPSCs
interface culture from stage 5 to 7 could promote the generation of
functional pancreatic β-like cells in vitro. Although these stage-7 cells
were not identical to adult islet β cells, they could secrete insulin
and ameliorate diabetes within 40 days. Pagliuca et al. [11] reported
a scalable 3D differentiation protocol to generate large amounts
of hPSC-derived pancreatic β-like cells (SC-β cells). After testing
more than 150 combinations involving over 70 compounds, they
used a combination of 11 small molecules to obtain functional SC-β
cells. These SC-β cells contained insulin granules, responded to the
change of glucose level in vitro, and reversed hyperglycemia in vivo.
However, the authors also noted that these SC-β cells showed less
functions compared with bona fide β cells, and more improvements
were still needed. In 2015, Russ et al. [12] provided a fast and
scalable approach to produce functional human pancreatic β-like
cells. They demonstrated that the depletion of cyclopamine and
Noggin at stages 3 and 4 could increase the percentage of PDX1 and
NKX6.1 double-positive PPs. Interestingly, they found that precise
temporal activation of endocrine differentiation was critical for the
final production of functional pancreatic β-like cells. In addition,
these hPSC-derived pancreatic β-like cells could response to high
level of glucose in vitro and protect mice from diabetes in vivo
after transplantation. However, compared with adult pancreatic β
cells, these hPSC-derived pancreatic β-like cells still performed less
functional and needed further improvements.
Recently, scientists worldwide have made their efforts on opti-
mizing the pancreatic differentiation strategies from various aspects
using 3D culture system, biomimetic materials and biomedical engi-
neering [13–17]. Interestingly, by mimicking early embryonic devel-
opment, 3D culture system can promote the proliferation of PPs
and their further differentiation into pancreatic β-like cells [18–20].
In 2017, Wang et al. [13] demonstrated that a specific biomimetic
3D scaffold could help the generation of pancreatic endoderm and
endocrine cells from hPSCs. In 2018, Mamidi et al. [14] found
that the extracellular matrix could affect the fate choices of bi-
potential PPs through F-actin-YAP1-Notch mechano-signaling. In
2019, Youngblood et al. [15] used a microporous biomaterial to
guide PPs to form defined-sized clusters and promote pancreatic β-
like cell maturation. Very recently, Velazco-Cruz et al. [16] optimized
the directed differentiation protocol by resizing the pancreatic β-
like cell clusters. Significantly, Nair et al. [17] developed cell culture
conditions to closely mimic events occurring during pancreatic islet
organogenesis and β cell maturation. They isolated INS-GFP-positive
cells by FACS and then reaggregated these pancreatic β-like cells
to form islet-sized enriched β-clusters (eBCs). These eBCs could
display many physiological properties analogous to primary human
pancreatic β cells, including robust dynamic insulin secretion in
vitro, improved mitochondrial energization, and much better in vivo
functions. Moreover, incorporating pancreatic niche cells, including
endothelial and mesenchymal cells, will also be beneficial. In the
future, various tissue engineering strategies can be applied to the gen-
eration of more mature and functional pancreatic cells and tissues.
In principle, current pancreatic differentiation strategies largely
mimic pancreatic development during embryogenesis. In the past few
decades, a few model organisms, such as zebrafish, frog, and mouse,
have been very effectively used to describe the normal development
of the pancreas. These studies have identified key genes and signaling
pathways that can guide projects aimed at turning hPSCs into pan-
creatic β cells. It is necessary to understand how human pancreatic β
cells are normally made and use that information to make functional
human pancreatic β-like cells that can be used to understand and treat
diabetes. However, it is technically and ethically challenging to study
3
human early development. High-throughput sequencing approaches
have helped researchers to have a better understanding of human
pancreatic development and differentiation. In 2017, Ameri et al. [21]
identified a specific PP surface marker glycoprotein 2 (GP2) using
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a comparative gene expression analysis. The enrichment of GP2-
positive PPs could improve pancreatic β-like cell differentiation effi-
ciency. Recently, single-cell RNA-seq (scRNA-seq) has become a pro-
mising technique to identify and characterize cell types within organs
and tissues. Using scRNA-seq, Veres et al. [22] characterized different
cell types emerging during human pancreatic β-like cell differentia-
tion. After bioinformatic analysis, they found that CD49a could be
used as a specific surface marker of pancreatic β-like cells derived from
hPSCs. Later, they magnetically sorted CD49a-positive pancreatic β-
like cells with a purity up to 80%. Therefore, this work provided a
novel perspective to increase the percentage of pancreatic β-like cells.
Interestingly, Alvarez-Dominguez et al. [23] analyzed the epigenome
dynamics of human pancreatic differentiation and demonstrated
that circadian rhythms could be used to stimulate pancreatic islet
maturation. High-throughput profiling approaches will facilitate
researchers to have a better understanding of human pancreatic
differentiation and development on multiple ‘omic’ levels, including
genomic, epigenomic, transcriptomic, proteomic, and metabolomic.
The combination of CRISPR genome editing and differentiation
system provided another powerful platform to study human pan-
creatic differentiation and development. CRISPR genome editing
approaches, including CRISPR-Cas9 [24] and CRISPR-Cpf1 [25],
have many applications in human stem cell biology [24–27]. For
example, Wei et al. [26] performed a genomic CRISPR knockout
screen and identified that VDR is important for pancreatic β cell
maintenance and function. Later, they discovered that vitamin D
could switch BAF complexes to protect pancreatic β cells both in vitro
and in vivo. In another study, Li et al. [27] carried out genome-scale
CRISPR screens and identified Jun N-terminal kinase-JUN family
genes as DE barriers. Similar strategies can be applied to identify
critical barriers or enhancers of human pancreatic differentiation at
various stages and improve pancreatic β-cell functions.
In summary, many significant advances have recently been made
in pancreatic differentiation protocols. As shown in Fig. 1, the proto-
cols for the derivation of PPs are becoming more consistent, efficient,
and reproducible. Additionally, insulin-producing pancreatic β-like
cells can now be differentiated from these PP cells with various
efficiencies. However, even with the most recent protocols, these
hPSC-derived pancreatic β-like cells are still less mature than primary
islet β cells. One solution is to put more efforts on further optimizing
these in vitro culture systems to maintain the terminally differentiated
pancreatic β cells. Another approach is to promote maturation
through transplantation and taking advantage of in vivo niches.
As we have discussed earlier, better understanding of pancreatic
development of model organisms and human beings are the major
keys to generate mature and functional target cells. In the future,
the rapid advances of high-throughput profiling, high-throughput
genome-wide genetic screening, high-throughput chemical screening,
and biomedical engineering will undoubtedly provide novel infor-
mation and perspectives to human pancreas development and help
develop better pancreatic differentiation approaches.
Applications of hPSC-derived Pancreatic β-like
Cells in Disease Modeling
During the past decades, the advances of genome-wide association
studies (GWASs), hiPSC technique, directed pancreatic differentia-
4 Pancreatic β cells from hPSCs
Figure 2. Applications of hPSC-derived pancreatic β-like cells in disease modeling Unlimited number of functional pancreatic β-like cells can be generated by
directed differentiation from hPSCs. Combined with CRISPR genome editing and high-throughput profiling, these pancreatic β-like cells can be used for disease
modeling and to identify the underlying molecular mechanisms of the progression of diabetes.
Figure 3. Applications of hPSC-derived pancreatic β-like cells in drug
discovery Pancreatic β-like cells, which are derived from hiPSCs or
genome-edited hPSCs, carry many hallmarks of patient-specific genetic and
epigenetic states and are suitable for both target-focused and phenotypic
large-scale chemical screening. After further in vitro assays and animal-based
evaluations, these drug candidates will potentially provide new options for
personalized therapy.
tion protocols, and CRISPR genome editing systems have opened
new avenues for modeling and studying diabetes. Diabetes is a
multifaceted and multifactorial disease. Many genetic mutations
can cause diabetes and can be modeled and investigated using hPSCs
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combined with CRISPR genome editing [28]. In particular, there are
at least 11 described maturity onset diabetes of the young (MODY)
resulted from genetic mutations at specific genes, including MODY1
(HNF4A), MODY2 (GCK), MODY3 (HNF1A), MODY4 (PDX1),
MODY5 (HNF1B), MODY6 (NEUROD1), MODY7 (KLF11),
MODY8 (CEL), MODY9 (PAX4), MODY10 (INS), and MODY11
(BLK). Besides MODYs, neonatal diabetes is another common
form of monogenic diabetes caused by mutations in genes, such
as KCNJ11 and ABCC8. Recently, Flanagan et al. [29] identified
NKX2.2 and MNX1 as etiological genes for neonatal diabetes
and confirmed their critical roles in the development of human
pancreas. In addition, mutations in mitochondrial DNA (mtDNA)
can cause mitochondrial diabetes. The most common one linked
to mitochondrial diabetes is 3243 A>G in the MT-TL1 gene. As
carrying out mtDNA genome editing remains very difficult, patient-
specific hiPSC technologies can provide feasible ways to study this
type of diabetes.
With the advent of CRISPR-Cas system, many laboratories have
already carried out hPSC-based modeling of diabetes (Fig. 2). In
2016, Zhu et al. [30] studied eight key genes in pancreatic develop-
ment by combining CRISPR-Cas9 and directed pancreatic differenti-
ation. They found that PDX1 had haploinsufficient effect in human
pancreatic differentiation, and RFX6 could regulate the number of
PPs and endocrine differentiation. In addition, they demonstrated
that NGN3 plays different roles in humans and in mice. The loss
of NGN3 led to the absence of pancreatic β cells in mice, but a small
number of pancreatic β-like cells formed from NGN3-knockout
hPSCs. In the same year, Zeng et al. [31] combined GWAS and
directed differentiation system to study diabetes-related mutations
in CDKAL1, KCNQ1, and KCNJ11. They found that biallelic
mutations in these three genes did not influence the generation of
pancreatic β-like cells from hPSCs but affected glucose-stimulated
insulin secretion. In 2017, Shi et al. [32] reported that the deletion
of GATA6 impaired the differentiation of DEs from hPSCs. Inter-
estingly, GATA6 haploinsufficiency led to less PPs and β-like cells.
In addition, the haploinsufficiency of GATA6 was also affected by
GATA4 dosage. This study shed the new light on disease modeling
from the individual genetic defects to multigenetic modifications. In
2018, Amin et al. [33] investigated the role of GLIS3, which is asso-
ciated with T1D, T2D, and neonatal diabetes, in pancreatic differen-
Pancreatic β cells from hPSCs
Figure 4. Potential applications of hPSC-derived pancreatic β-like cells in cell-based therapy
immune rejection, safety, cost, and long-term functions. Several promising strategies, including surface modification, encapsulation, and precise genome
editing, are under active investigation. Optimistically, these efforts will eventually lead to significant advances in cell-based therapy.
tiation. They found that GLIS3 deletion impaired the differentiation
of PDX1 and NKX6.1 double-positive PPs from hPSCs. Mechanisti-
cally, they figured out that the knockout of GLIS3 led to pancreatic
β-like cell death by activating the TGF-β pathway. In another study,
Zhou et al. [34] reported that a commonly-used pesticide propargite
could induce β-like cell death. The loss of GSTT1, which increases
the risk of diabetes, made pancreatic β-like cells hypersensitive to
propargite-induced cell death. Their study demonstrated the role of
gene–environment interaction on diabetes. In 2019, Lee et al. [35]
demonstrated that FOXA2 was required in the formation of PPs.
The loss of FOXA2 would reduce the number of PPs during the
directed differentiation process and impair the recruitment of GATA6
to pancreatic enhancers. The coalescence of disease modeling and
high-throughput profiling provides novel information to understand
pancreatic development. Significantly, this phenotype was not seen
in Foxa2 conditional knockout mice. Recently, Cardenas-Diaz et
al. [36] reported that HNF1A could inhibit α cell gene expression,
regulate cellular metabolism, and maintain pancreatic endocrine cell
function. Then, they found that HNF1A could target a human-
specific long non-coding RNA, LINKA, which could regulate normal
mitochondrial respiration. Using similar approaches, other diabetes-
related mutations and more complicated subtypes can be modeled.
In the future, the combination of pancreatic differentiation sys-
tem, precise genome editing, and bioinformatics will help us con-
struct more sophisticated diabetes-related modeling and investigate
the underlying molecular mechanisms in more details.
Applications of hPSC-derived Pancreatic β-like
Cells in Drug Discovery
The pharmaceutical industry in diabetes currently faces several chal-
lenges, including the poor predictivity of efficacy and toxicity with
the preclinical biological models and the increasing costs of devel-
oping new drugs [37]. Promisingly, drug screening using hPSC-
derived pancreatic β-like cells can address some of these challenges
(Fig. 3). Patient-specific pancreatic β-like cells carry many hallmarks
of genetic and epigenetic states and are suitable for both target-
focused and phenotypic large-scale chemical screening.
To date, several research groups have used isogenic hESC-derived
pancreatic β-like cells to functionally evaluate the GWAS-identified
5
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There are many critical issues need to be addressed, including
loci and then identify candidate drugs using high-throughput chemi-
cal screening. For example, Zeng et al. [31] found that the CDKAL1
mutations could cause impaired glucose-stimulated insulin secretion
both in vitro and in vivo. These CDKAL1 knockout pancreatic β-like
cells were hypersensitive to glucotoxicity and lipotoxicity. In order
to protect CDKAL1−/− pancreatic β-like cells from glycolipid tox-
icity, they performed a high-content compound screening and then
identified a candidate drug T5224 that could rescue CDKAL1−/− -
specific defects by inhibiting the FOS/JUN pathway. In another
study from the same research group, Amin et al. [33] explored
the roles of GLIS3 gene in human pancreatic development. Using
improved differentiation protocols, they found that GLIS3−/− hESCs
showed impaired pancreatic differentiation with significant death
of pancreatic β-like cells. With a high-content chemical screening,
they found that a drug candidate, galunisertib, could effectively
inhibit the inappropriate upregulation of the TGF-β pathway and
rescue apoptosis of pancreatic β-like cells induced by GLIS3 deletion.
Therefore, the application of the directed pancreatic differentiation
system provides unlimited cell sources for drug screening with the
context of human pancreas.
Types 1 and 2 diabetes are both resulted from a deficiency
of functional pancreatic β cells. Although pancreatic β cells were
reported to proliferate in the embryo and early childhood, adult β cell
replication is barely detectable [38]. The dysfunction of pancreatic β
cells can cause type 2 diabetes, and screening chemical compounds
to protect and improve pancreatic β-cell function is in high demand.
For example, using a luciferase-based high-throughput chemical
screening strategy, Wang et al. [39] found that small molecules,
harmine and INDY, functioned as a new class of human β cell
mitogenic compounds. Mechanistically, they defined dual-specificity
tyrosine-regulated kinase-1a (DYRK1A) as the possible target for
harmine and INDY, and the nuclear factors of activated T cells
family of transcription factors as the possible mediators of human
β-cell proliferation and function [39]. As a follow-up work, Wang
et al. [40] demonstrated that inhibition of DYRK1A, SMAD, and
Trithorax pathways could synergize to induce robust replication of
hPSC-derived pancreatic β-like cells and adult human pancreatic β
cells. Likewise, screening small molecules for pancreatic β-like cell
differentiation, expansion, survival, and function will provide new
drug candidates for the treatment of diabetes.
6 Pancreatic β cells from hPSCs
It becomes more important to develop better differentiation
protocols in a sufficiently robust and reproducible manner, as to
meet the requirements of large-scale cell culture. In the next decades,
additional work, including sensitive reporter systems, high-content
analysis, data mining, and machine learning, is needed to develop
better systems for drug discovery.
Potential Applications of hPSC-derived
Pancreatic β-like Cells in Cell-based Therapy
Islet transplantation is an effective method for treating diabetes
but is severely limited by the scarceness of islet donors, the short
time duration of insulin secretion after transplantation [41], and the
adverse reactions caused by the use of immuno-suppressive drugs
[42]. Interestingly, it does not necessarily require the transplantation
of mature pancreatic β cells. Human fetal pancreatic tissues can also
develop functionally in mice [43] and human [44]. Obviously, one
major potential application of hPSC-derived pancreatic β-like cells
and tissues is for cell-based therapy (Fig. 4).
Immune rejection is a major obstacle for cell-based therapy,
and better immune protection strategies are necessary to promote
more effective and longer lasting treatments. Stem cells with specific
immunogenic modifications can allow them to escape from autoim-
mune attacks in patients with type 1 diabetes [45]. For example,
Szot et al. [46] found that hESC-derived pancreatic endoderm cells
could survive in non-diabetic mice for more than 90 days using
CTLA4Ig to inhibit T-cell activation and anti-CD154 antibody to
block the T-cell costimulatory pathway. Human leukocyte antigens
(HLAs) on the cell surface are the main cause of CD4+ T-cell-
mediated allograft rejection. In order to find a long-term solu-
tion to the immune responses, many researchers tried to generate
hypoimmunogenic hPSCs by genome editing. Lu et al. [47] disrupted
beta 2-microglobulin (β2m) and generated HLA class I-deficient
hESCs, which showed low immunogenicity. Chen et al. [48] found
that CIITA−/− hESCs could differentiate into cells with no HLA II
expression. These cells can escape the attack of receptors’ CD4+ T
cells. In addition, researchers from CRISPR Therapeutics generated
genome-edited CyT49 cells that lack β2m and express PD-L1 to
further protect cells from T-cell attack [49].
Using cell impermeable macrocysts is another promising strategy
to solve the immune rejection of xenograft in diabetic patients [50].
Encapsulated with these devices, hESC-derived PPs could further
differentiate into pancreatic β-like cells in mice [51]. Another method
of islet encapsulation is to prepare the isolated islets into alginate
microspheres. The hESC-derived pancreatic β-like cells coated with
triazolethiomorpholine dioxide alginate could regulate blood glucose
level and protect against immunosuppression for more than 170 days
in mice with normal immunity [52]. Investigators at ViaCyte used the
CyT49 cells to generate PPs [53], and then these PPs could mature
into glucose-responsive and insulin-secreting pancreatic β-like cells
in macroencapsulation devices in vivo [51]. Excitingly, PEC-Encap
clinical trial data for T1DM Phase I/II showed that the product was
safe and well tolerated, and the insulin-secreting cells could maintain
in vivo for more than 2 years [54]. Moreover, a potential advantage
of this approach is that the device can be more easily removed from
patients. Undoubtedly, these studies will set up the foundations for
further efforts to overcome immune rejection issues.
For cell-based therapy, safety is critically required, and several
innovative and promising strategies are being tested in animal mod-
els. For example, Liang et al. [55] linked cell-division gene CDK1
to a suicide gene HSV-TK to define and improve cell therapy safety.
They used hESC-derived RPE cell transplantation as an in vivo proof
of principle study and obtained very promising results. In the future,
similar approaches can also be applied for hPSC-derived pancreatic
β-like cell transplantation.
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Summary and Perspectives
Tremendous progresses have already been made to generate func-
tional pancreatic β-like cells from hPSCs. It is now possible to
create diabetes patient-specific iPSCs and their pancreatic derivatives.
In the past decade, genome-editing approaches, such as CRISPR
tools, accelerated the detailed study of pancreatic differentiation and
function. Here, we reviewed recent advances on human pancreatic β-
like cell differentiation from hPSCs and the potential applications of
these hPSC-derived pancreatic β-like cells in disease modeling, drug
discovery, and cell-based therapies.
In the future, new technologies will emerge and be critically help-
ful to achieve these goals. More precise genome-editing tools can help
to better study the underlying mechanisms of disease progression of
multiple forms of diabetes. Cost-effective high-throughput profiling
approaches can be used to better understand human pancreatic
differentiation and development on multiple ‘omic’ levels, including
genomic, epigenomic, transcriptomic, proteomic, and metabolomic.
In addition, powerful high-throughput screening will provide new
drug candidates for effectively treating diabetes. By overcoming the
issues of immune rejection, safety, cost, and long-term functions, cell-
based therapy will become a reality.
Therefore, more support and investments for the young field of
stem cell biology should be provided by governments, academics,
and industries. Very optimistically, these efforts will eventually lead
to significant advances in the field of regenerative medicine, provide
innovative therapeutics, and benefit millions of people worldwide.
Acknowledgement
We would like to thank all members of Zhu laboratory for helpful
discussions. Due to the space limitation, we apologize to those whose
findings are relevant but not cited in this review.
Funding
This work was supported by the grants from National Natural
Science Foundation of China (No. 31970818), the National Key
Research and Development Program of China (No. 2016YFC1
305300), and the Outstanding Youth Fund of Zhejiang Province
(No. R17C120002).
References
1. Shapiro AM, Ricordi C, Hering BJ, Auchincloss H, Lindblad R, Robertson
RP, Secchi A, et al. International trial of the Edmonton protocol for islet
transplantation. N Engl J Med 2006, 355: 1318–1330.
2. Bruin JE, Rezania A, Kieffer TJ. Replacing and safeguarding pancreatic β
cells for diabetes. Sci Transl Med 2015, 7: 316–323.
3. Kroon E, Martinson LA, Kadoya K, Bang AG, Kelly OG, Eliazer S,
Young H, et al. Pancreatic endoderm derived from human embryonic
stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat
Biotechnol 2008, 26: 443–452.
4. Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, Tzukerman
M. Insulin production by human embryonic stem cells. Diabetes 2001,
50: 1691–1697.
Pancreatic β cells from hPSCs
5. D’Amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, Baetge EE.
Efficient differentiation of human embryonic stem cells to definitive
endoderm. Nat Biotechnol 2005, 23: 1534–1541.
6. D’Amour KA, Bang AG, Eliazer S, Kelly OG, Agulnick AD, Smart
NG, Moorman MA, et al. Production of pancreatic hormone-expressing
endocrine cells from human embryonic stem cells. Nat Biotechnol 2006,
24: 1392–1401.
7. Ma X, Zhu S. Chemical strategies for pancreatic beta cell differentiation,
reprogramming, and regeneration. Acta Biochim Biophys Sin 2017, 49:
289–301.
8. Chen S, Borowiak M, Fox JL, Maehr R, Osafune K, Davidow L, Lam K,
et al. A small molecule that directs differentiation of human ESCs into the
pancreatic lineage. Nat Chem Biol 2009, 5: 258–265.
9. Rezania A, Bruin JE, Riedel MJ, Mojibian M, Asadi A, Xu J, Gauvin
R, et al. Maturation of human embryonic stem cell-derived pancreatic
progenitors into functional islets capable of treating pre-existing diabetes
in mice. Diabetes 2012, 61: 2016–2029.
10. Rezania A, Bruin JE, Arora P, Rubin A, Batushansky I, Asadi A, O’Dwyer
S, et al. Reversal of diabetes with insulin-producing cells derived in vitro
from human pluripotent stem cells. Nat Biotechnol 2014, 32: 1121–1133.
11. Pagliuca FW, Millman JR, Gurtler M, Segel M, Van Dervort A, Ryu JH,
Peterson QP, et al. Generation of functional human pancreatic beta cells
in vitro. Cell 2014, 159: 428–439.
12. Russ HA, Parent AV, Ringler JJ, Hennings TG, Nair GG, Shveygert M,
Guo T, et al. Controlled induction of human pancreatic progenitors pro-
duces functional beta-like cells in vitro. EMBO J 2015, 34: 1759–1772.
13. Wang W, Jin S, Ye K. Development of islet organoids from H9 human
embryonic stem cells in biomimetic 3D scaffolds. Stem Cells Dev 2017,
26: 394–404.
14. Mamidi A, Prawiro C, Seymour PA, de Lichtenberg KH, Jackson A,
Serup P, Semb H. Mechanosignalling via integrins directs fate decisions
of pancreatic progenitors. Nature 2018, 564: 114–118.
15. Youngblood RL, Sampson JP, Lebioda KR, Shea LD. Microporous scaf-
folds support assembly and differentiation of pancreatic progenitors into
beta-cell clusters. Acta Biomater 2019, 96: 111–122.
16. Velazco-Cruz L, Song J, Maxwell KG, Goedegebuure MM,
Augsornworawat P, Hogrebe NJ, Millman JR. Acquisition of dynamic
function in human stem cell-derived β cells. Stem Cell Rep 2019, 12:
351–365.
17. Nair GG, Liu JS, Russ HA, Tran S, Saxton MS, Chen R, Juang C, et al.
Recapitulating endocrine cell clustering in culture promotes maturation
of human stem-cell-derived beta cells. Nat Cell Biol 2019, 21: 263–274.
18. Toyoda T, Mae S, Tanaka H, Kondo Y, Funato M, Hosokawa Y, Sudo T,
et al. Cell aggregation optimizes the differentiation of human ESCs and
iPSCs into pancreatic bud-like progenitor cells. Stem Cell Res 2015, 14:
185–197.
19. Yabe SG, Fukuda S, Takeda F, Nashiro K, Shimoda M, Okochi H. Effi-
cient generation of functional pancreatic beta-cells from human induced
pluripotent stem cells. J Diabetes 2017, 9: 168–179.
20. Ghazizadeh Z, Kao DI, Amin S, Cook B, Rao S, Zhou T, Zhang T, et al.
ROCKII inhibition promotes the maturation of human pancreatic beta-
like cells. Nat Commun 2017, 8: 298.
21. Ameri J, Borup R, Prawiro C, Ramond C, Schachter KA, Scharfmann R,
Semb H. Efficient generation of glucose-responsive beta cells from isolated
GP2(+) human pancreatic progenitors. Cell Rep 2017, 19: 36–49.
22. Veres A, Faust AL, Bushnell HL, Engquist EN, Kenty JH, Harb G, Poh
YC, et al. Charting cellular identity during human in vitro beta-cell
differentiation. Nature 2019, 569: 368–373.
23. Alvarez-Dominguez JR, Donaghey J, Rasouli N, Kenty JHR, Helman A,
Charlton J, Straubhaar JR, et al. Circadian entrainment triggers matura-
tion of human in vitro islets. Cell Stem Cell 2019, 5909: 30466–30467.
24. Yu C, Liu Y, Ma T, Liu K, Xu S, Zhang Y, Liu H, et al. Small molecules
enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell
2015, 16: 142–147.
25. Ma X, Chen X, Jin Y, Ge W, Wang W, Kong L, Ji J, et al. Small molecules
promote CRISPR-Cpf1-mediated genome editing in human pluripotent
stem cells. Nat Commun 2018, 9: 1303.
7
26. Wei Z, Yoshihara E, He N, Hah N, Fan W, Pinto AFM, Huddy T, et al.
Vitamin D switches BAF complexes to protect beta cells. Cell 2018, 173:
1135–1149.
27. Li QV, Dixon G, Verma N, Rosen BP, Gordillo M, Luo R, Xu C,
Downloaded from https://academic.oup.com/abbs/advance-article-abstract/doi/10.1093/abbs/gmaa047/5842276 by Jamia Millia Islamia University user on 25 May 2020
et al. Genome-scale screens identify JNK-JUN signaling as a barrier for
pluripotency exit and endoderm differentiation. Nat Genet 2019, 51:
999–1010.
28. Teo AK, Wagers AJ, Kulkarni RN. New opportunities: harnessing induced
pluripotency for discovery in diabetes and metabolism. Cell Metab 2013,
18: 775–791.
29. Flanagan SE, De Franco E, Lango Allen H, Zerah M, Abdul-Rasoul MM,
Edge JA, Stewart H, et al. Analysis of transcription factors key for mouse
pancreatic development establishes NKX2-2 and MNX1 mutations as
causes of neonatal diabetes in man. Cell Metab 2014, 19: 146–154.
30. Zhu Z, Li QV, Lee K, Rosen BP, Gonzalez F, Soh CL, Huangfu D. Genome
editing of lineage determinants in human pluripotent stem cells reveals
mechanisms of pancreatic development and diabetes. Cell Stem Cell 2016,
18: 755–768.
31. Zeng H, Guo M, Zhou T, Tan L, Chong CN, Zhang T, Dong X, et al. An
isogenic human ESC platform for functional evaluation of genome-wide-
association-study-identified diabetes genes and drug discovery. Cell Stem
Cell 2016, 19: 326–340.
32. Shi ZD, Lee K, Yang D, Amin S, Verma N, Li QV, Zhu Z, et al.
Genome editing in hPSCs reveals GATA6 haploinsufficiency and a genetic
interaction with GATA4 in human pancreatic development. Cell Stem Cell
2017, 20: 675–688 e676.
33. Amin S, Cook B, Zhou T, Ghazizadeh Z, Lis R, Zhang T, Khalaj M,
et al. Discovery of a drug candidate for GLIS3-associated diabetes. Nat
Commun 2018, 9: 2681.
34. Zhou T, Kim TW, Chong CN, Tan L, Amin S, Sadat Badieyan Z,
Mukherjee S, et al. A hPSC-based platform to discover gene-environment
interactions that impact human β-cell and dopamine neuron survival. Nat
Commun 2018, 9: 4815.
35. Lee K, Cho H, Rickert RW, Li QV, Pulecio J, Leslie CS, Huangfu D.
FOXA2 is required for enhancer priming during pancreatic differentia-
tion. Cell Rep 2019, 28: 382–393 e387.
36. Cardenas-Diaz FL, Osorio-Quintero C, Diaz-Miranda MA, Kishore S,
Leavens K, Jobaliya C, Stanescu D, et al. Modeling monogenic diabetes
using human ESCs reveals developmental and metabolic deficiencies
caused by mutations in HNF1A. Cell Stem Cell 2019, 25: 273–289 e275.
37. DiMasi JA, Grabowski HG, Hansen RW. Innovation in the pharmaceu-
tical industry: new estimates of R&D costs. J Health Econ 2016, 47:
20–33.
38. Zhou Q, Melton DA. Pancreas regeneration. Nature 2018, 557: 351–358.
39. Wang P, Alvarez-Perez JC, Felsenfeld DP, Liu H, Sivendran S, Bender A,
Kumar A, et al. A high-throughput chemical screen reveals that harmine-
mediated inhibition of DYRK1A increases human pancreatic beta cell
replication. Nat Med 2015, 21: 383–388.
40. Wang P, Karakose E, Liu H, Swartz E, Ackeifi C, Zlatanic V, Wilson J,
et al. Combined inhibition of DYRK1A, SMAD, and Trithorax pathways
synergizes to induce robust replication in adult human beta cells. Cell
Metab 2019, 29: 638–652 e635.
41. Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL,
Kneteman NM, et al. Islet transplantation in seven patients with type 1
diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.
N Engl J Med 2000, 343: 230–238.
42. Lakey JR, Mirbolooki M, Shapiro AM. Current status of clinical
islet cell transplantation. In: Hornick P, Rose M eds. Transplantation
Immunology: Methods and Protocols. Totowa, NJ: Humana Press, 2006,
47–104
43. Tuch BE, Osgerby KJ, Turtle JR. Reversal of diabetes in hypergly-
caemic nude mice by human fetal pancreas. Transplant Proc 1989, 21:
2665–2666.
44. Lafferty KJ, Hao L. Fetal pancreas transplantation for treatment of IDDM
patients. Diabetes Care 1993, 16: 383–386.
45. Gjelberg HK, Hoem D, Verbeke CS, Eide J, Cooper JG, Molven A.
Hypoglycemia and decreased insulin requirement caused by malignant
8 Pancreatic β cells from hPSCs
insulinoma in a type 1 diabetic patient: when the hoof beats are from
a zebra, not a horse. Clin Case Rep 2017, 5: 761–768.
46. Szot GL, Yadav M, Lang J, Kroon E, Kerr J, Kadoya K, Brandon EP,
et al. Tolerance induction and reversal of diabetes in mice transplanted
with human embryonic stem cell-derived pancreatic endoderm. Cell Stem
Cell 2015, 16: 148–157.
47. Lu P, Chen J, He L, Ren J, Chen H, Rao L, Zhuang Q, et al. Generating
hypoimmunogenic human embryonic stem cells by the disruption of beta
2-microglobulin. Stem Cell Rev Rep 2013, 9: 806–813.
48. Chen H, Li Y, Lin X, Cui D, Cui C, Li H, Xiao L. Functional disruption of
human leukocyte antigen II in human embryonic stem cell. Biol Res 2015,
48: 59.
49. Sluch V. CRISPR-editing of hESCs allows for production of immune
evasive cells capable of differentiation to pancreatic progenitors for future
type 1 diabetes therapy. Sept 17, 2019. http://ir.crisprtx.com/static-files/
af584c8b-5264-4bdd-a409-fec52e06d365.
50. Desai T, Shea LD. Advances in islet encapsulation technologies. Nat Rev
Drug Discov 2017, 16: 338–350.
51. Agulnick AD, Ambruzs DM, Moorman MA, Bhoumik A, Cesario RM,
Payne JK, Kelly JR, et al. Insulin-producing endocrine cells differentiated
in vitro from human embryonic stem cells function in macroencapsulation
devices in vivo. Stem Cells Transl Med 2015, 4: 1214–1222.
Downloaded from https://academic.oup.com/abbs/advance-article-abstract/doi/10.1093/abbs/gmaa047/5842276 by Jamia Millia Islamia University user on 25 May 2020
52. Vegas AJ, Veiseh O, Gurtler M, Millman JR, Pagliuca FW, Bader AR,
Doloff JC, et al. Long-term glycemic control using polymer-encapsulated
human stem cell-derived beta cells in immune-competent mice. Nat Med
2016, 22: 306–311.
53. Schulz TC, Young HY, Agulnick AD, Babin MJ, Baetge EE, Bang AG,
Bhoumik A, et al. A scalable system for production of functional pancre-
atic progenitors from human embryonic stem cells. PLoS One 2012, 7:
e37004.
54. ViaCyte. Two-year data from ViaCyte’s STEP ONE clinical trial presented
at ADA 2018. Jun 23, 2018. https://viacyte.com/press-releases/two-year-
data-from-viacytes-step-one-clinical-trial-presented-at-ada-2018/.
55. Liang Q, Monetti C, Shutova MV, Neely EJ, Hacibekiroglu S, Yang H,
Kim C, et al. Linking a cell-division gene and a suicide gene to define and
improve cell therapy safety. Nature 2018, 563: 701–704.