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www.e-enm.org 191
Endocrinol Metab 2024;39:191-205
https://doi.org/10.3803/EnM.2023.1910
pISSN 2093-596X · eISSN 2093-5978
Review
Article
Scaling Insulin-Producing Cells by Multiple Strategies
Jinhyuk Choi1,*, Fritz Cayabyab1,*, Harvey Perez1, Eiji Yoshihara1,2
1The Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center, Torrance; 2David Geffen School of
Medicine at University of California Los Angeles, Los Angeles, CA, USA
In the quest to combat insulin-dependent diabetes mellitus (IDDM), allogenic pancreatic islet cell therapy sourced from deceased
donors represents a significant therapeutic advance. However, the applicability of this approach is hampered by donor scarcity and
the demand for sustained immunosuppression. Human induced pluripotent stem cells are a game-changing resource for generating
synthetic functional insulin-producing β cells. In addition, novel methodologies allow the direct expansion of pancreatic progenitors
and mature β cells, thereby circumventing prolonged differentiation. Nevertheless, achieving practical reproducibility and scalability
presents a substantial challenge for this technology. As these innovative approaches become more prominent, it is crucial to thor-
oughly evaluate existing expansion techniques with an emphasis on their optimization and scalability. This manuscript delineates
these cutting-edge advancements, offers a critical analysis of the prevailing strategies, and underscores pivotal challenges, including
cost-efficiency and logistical issues. Our insights provide a roadmap, elucidating both the promises and the imperatives in harnessing
the potential of these cellular therapies for IDDM.
Keywords: Diabetes mellitus; Islet transplantation; Stem cells; Cell expansion; Mitogens; Cryopreservation
INTRODUCTION
Diabetes is a heterogeneous metabolic disorder characterized by
chronic hyperglycemia, principally due to the loss of β cell mass
or a decline in β cell function [1-3]. Although current therapeutic
strategies mitigate symptoms and temporarily improve glycemic
control, they do not prevent or significantly forestall the progres-
sion to associated comorbidities. True remediation of diabetes
necessitates the replenishment of β cells capable of precise
blood glucose regulation. This can be accomplished primarily
through two approaches: increasing the number of endogenous
β cells or transplanting β cells from external sources. Stem cell-
based islet replacement therapy has emerged as a curative ap-
proach for insulin-dependent diabetes mellitus (IDDM) across
several preclinical animal studies [4-12] and human trials [13-
15]. In 2000, the capabilities of islet replacement therapy were
demonstrated by a hallmark islet transplantation study, wherein
all seven patients with type 1 diabetes (T1D) who underwent
transplantation with islets from cadaveric donors became insu-
lin-independent for a minimum duration of 1 year [16]. Ad-
vancements in the islet isolation protocol, along with improve-
ments in immunosuppressive regimen, known as the Edmonton
Protocol, have set a new benchmark in diabetes treatment. How-
ever, the limited availability of cadaveric donor islets in compar-
ison to the demand by T1D patients presents a substantial chal-
lenge. Over the past decades, efforts have been made to establish
Received: 27 December 2023, Revised: 20 January 2024,
Accepted: 30 January 2024
Corresponding author: Eiji Yoshihara
The Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical
Center, 1124 W Carson St, Torrance, CA 90502, USA
Tel: +1-310-781-1480, E-mail: eiji.yoshihara@lundquist.org
*These authors contributed equally to this work.
Copyright © 2024 Korean Endocrine Society
This is an Open Access article distributed under the terms of the Creative Com-
mons Attribution Non-Commercial License (https://creativecommons.org/
licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribu-
tion, and reproduction in any medium, provided the original work is properly
cited.
Choi J, et al.
Copyright © 2024 Korean Endocrine Society
192 www.e-enm.org
a reliable islet supply chain through the use of advanced islet
storage and pooling of islets from multiple donors. Another re-
newable source for the generation of β cells using human plurip-
otent stem cells (hPSCs) has been attracting attention as an alter-
native strategy to overcome the shortage of healthy donor pan-
creases [17,18]. Clinical trials typically employ hPSC-derived
pancreatic and endocrine progenitor cells for transplantation.
Viacyte’s 2014 phase 1/2 clinical trial (NCT02239354) used
pancreatic progenitor cells with a macroencapsulation device to
treat T1D, followed by a 2016 study (NCT02939118) to assess
adverse effects. Although minor adverse effects were reported,
graft longevity requires further study. Recently, a phase 1/2 clin-
ical trial (NCT04786262) by Vertex Pharmaceuticals demon-
strated that all six T1D patients receiving transplants of hPSC-
derived islets (VX-880) showed improved glycemic control
through endogenous insulin production. Moving beyond the
scope of islet transplantation from external sources, there is a
growing focus on therapeutic strategies aimed at increasing the
body’s own production of insulin-producing cells, a paradigm
shift that offers the potential for more integrated and sustainable
diabetes management. To increase the number of endogenous β
cells, one strategy is to stimulate the proliferation of existing β
cells, while another involves inducing the transdifferentiation of
non-insulin-producing cells into β cells (Fig. 1). β cell prolifera-
tion has shown promise in preclinical animal models, as well as
in select human cases, such as during pregnancy, offering poten-
tial therapeutic avenues for IDDM [19,20]. However, human β
cells exhibit a lower proliferative capacity than those in preclini-
cal animal models, achieving an average proliferation rate of
less than 1% in human adults and diminishing further with age
[21]. Current research efforts are directed toward identifying
small molecules, biologics, and pathways that can enhance the
proliferative capacity of human β cells. Compounds targeting
glucagon-like peptide 1 receptor (GLP-1R) [22] and dual-spe-
cific tyrosine-phosphorylation regulated kinase 1A (DYRK1A)
[23] are emerging as front-runners, with evidence from in vivo
Fig. 1. Sources of human insulin-secreting β-like cells in sufficient numbers for transplantation into patients with diabetes. Over a long peri-
od of time, various methods have been studied to secure sufficient β-like cells that secrete human insulin for transplantation. Various ap-
proaches have been explored, including the method involves the expansion of stem cells over multiple passages to yield a substantial num-
ber of β-like cells. Additionally, direct strategies have been employed, such as using mitogens to stimulate human β cells’ entry into the cell
cycle and the transdifferentiation of liver, gastric, intestinal, and other pancreatic cells such as α, δ, acinar, and ductal cells into β-like cells.
Finally, Significant improvements have been made in the collection of high-quality islets from donors, as well as in the processes of cryo-
preservation and cell recovery for long-term storage and transportation. hPSC, human pluripotent stem cell; LN2, liquid nitrogen.
hPSC-pancreatic cell
expansion
Passage
Mitogens
Intestinal
cell
Gastric cell
β-like cell
Ductal cell
Pooling
A B C
Donor
LN2
Acinar
cell
α cell
δ cell
Hepatocyte
Passage
β Cell proliferation
using mitogens
Non-proliferating β cell
Proliferating β cell
Transdifferentiation Islets
cryopreservation
Scalability Issues of Insulin Producing Cells
Copyright © 2024 Korean Endocrine Society www.e-enm.org 193
diabetic mouse models transplanted with human β cells, which
have shown that even a 1% to 2% proliferation increase can lead
to significant glycemic control improvements, reaching a level
of control similar to that in normal mice [24-26]. Nevertheless,
the clinical viability of these findings for enhancing adult human
β cell proliferation remains uncertain. Alternative β cell replica-
tion strategies include the transdifferentiation of liver cells
[27,28], stomach and intestinal cells [29], and other pancreatic
origin cells such as ductal cells, yet no Food and Drug Adminis-
tration-approved strategy for this purpose has been applied clini-
cally [30,31]. Despite these advances, current production meth-
ods for creating homogeneous, high-quality hPSC-derived insu-
lin-producing β-like cells (hPSC-derived insulin-producing cells
hereinafter referred to as “β-like” cells) face challenges. Large-
scale clinical application is impeded by complex differentiation
processes, batch variability, cost inefficiency, and underdevel-
oped cryostorage and delivery methods (Fig. 1). This review
discusses progressive strategies to surmount hurdles in islet
transplantation, including islet availability and the critical as-
pects of islet preservation.
SOURCES OF FUNCTIONAL HUMAN
β CELLS
While hPSC technology holds promise for addressing various
challenges in cell replacement therapies, including the genera-
tion of β cells, there are significant scalability issues that must be
addressed. The indefinite replication potential of PSCs and their
ability to differentiate into any cell type, including β cells, un-
derpins the technology’s promise. hPSCs come from two main
sources: embryonic pluripotent stem cells (ESC), and induced
pluripotent stem cells (iPSC). The same differentiation process
can be applied to both ESCs and iPSCs; however iPSCs offer an
ethical advantage over ESCs. The generation of iPSCs involves
reprogramming adult cells by targeting transcription factors like
octamer-binding transcription factor 4 (OCT4), SRY-box tran-
scription factor 2 (SOX2), Krüppel-like factor 4 (KLF4), and
cellular myelocytomatosis oncogene (c-MYC) [32,33], which
can lead to inconsistent differentiation efficiencies due to residu-
al epigenetic memory [34]. This variability can severely limit
the large-scale production of functionally homogeneous β cells.
Additionally, the differentiation stage of the β cells may influ-
ence their propensity to form teratomas, a type of tumor. Less
differentiated cells carry a higher tumorigenic risk, raising con-
cerns about the safety of cell therapies [35]. Ensuring the suc-
cess, purity, and homogeneity of differentiated β cells is critical
and remains a significant hurdle for scaling up this technology
for widespread clinical application. The differentiation of β cells
from hPSCs follows a meticulous, stepwise protocol that guides
cells through sequential lineage commitments: starting with in-
duction into definitive endoderm, followed by commitment to
foregut and pancreatic progenitor stages, further specializing
into endocrine progenitors, and culminating towards mono-hor-
monal endocrine cells, including insulin-secreting β cells, gluca-
gon-secreting α cells and somatostatin-secreting δ cells. This ap-
proach to β cell differentiation has been successfully implement-
ed in both monolayer cultures and three-dimensional (3D) cell
aggregates [36]. hPSC-derived β cells express the hallmark
markers, such as pancreatic and duodenal homeobox1 (PDX1),
NK6 homeobox 1 (NKX6-1), urocortin 3 (UCN3), MAF bZIP
transcription factor A (MAFA), insulin (INS), and neurogenic
differentiation 1 (NEUROD1), and have the functionality of
glucose-sensing and insulin-secretion at varying levels [4,5].
Despite these successes, hPSC-derived β cells still face major
issues that hinder their therapeutic application.
Identity and purity
Protocols for in vitro differentiation to generate β-like cells from
iPSCs commonly emulate the developmental progression of
pancreatic islets. Consequently, the resulting cultures typically
comprise not only β-like cells, but also α-like cells and δ-like
cells. The complex and labor-intensive nature of these protocols,
along with the dynamics of morphogen signaling gradients and
the susceptibility of different precursor cells to develop into dif-
ferent lineages, occasionally leads to the emergence of cell types
atypical to cadaveric islets. Such anomalies include enterochro-
maffin cells, which are closer to intestinal than pancreatic endo-
crine lineages, as well as polyhormonal cells that exhibit a less
developed state than monohormonal β, α, or δ cells [37-39].
Moreover, the differentiation process is not universally efficient
throughout the culture process, with some cells stalling at pro-
genitor stages, others deviating to alternative lineages, and yet
others displaying traits of both intestinal and pancreatic types.
These undesired cells can be considered contaminants that can
potentially impair the functionality of the β-like cells or alter the
differentiation trajectory of neighboring cells via direct contact
or the secretion of soluble factors. These cells can also persist
even after transplantation despite allowing the in vivo matura-
tion of derived β-like cells [39,40]. Sorting and reaggregation of
β-like cells with the removal of the undesired cell types has been
shown to enhance the functional maturation of β-like cells clus-
ters [41]. Variability in the differentiation efficiency of β-like
Choi J, et al.
Copyright © 2024 Korean Endocrine Society
194 www.e-enm.org
cells across different protocols and the disparate responsiveness
of various iPSC lines further complicate the process. Determin-
ing the optimal ratio of β-like cells to other pancreatic endocrine
cells for clinical applications remains a topic of debate. None-
theless, controlling this ratio and reducing contaminant cell pop-
ulations are critical research objectives. An emerging aspect of
β-like cell differentiation that is coming into focus is the impor-
tance of epigenetics and chromatin states. Although single cell
transcriptomic analyses have provided useful insights into the
cellular identities of cells generated by various differentiation
protocols, they fall short in explaining the emergence of non-β-
like cells and their interrelations. The prevailing theory suggests
a divergence from a common progenitor lineage into distinct
end-branches, which precludes the possibility of transdifferenti-
ation or a continuum of cellular identities. Thus, in the past few
years, strategies to improve β-like cell differentiation efficiency
have involved employing cell surface markers such as CD49a
[37], CD9 [36,42], glycoprotein 2 [43-45], CD142 [45], CD24
[46], and CD63 [47], followed by purification and/or reaggrega-
tion [41]. However, these methods might be challenging to im-
plement on a larger scale for the mass production of β-like cells.
In contrast, single cell transcriptomic analysis complemented by
single cell transposase-accessible chromatin sequencing on β-like
cells, revealed that the presence of enterochromaffin cells may
represent an intermediary transitory state of pancreatic endo-
crine cells and intestinal cells [48]. This finding suggests the
possibility of transdifferentiating enterochromaffin cells into a
β cell identity by modulating the chromatin states, timing, and
expression of key transcription factors and signals, similar to
the process of transdifferentiating stomach cells into β-like cells
[49,50]. Nonetheless, it is essential to evaluate the purity and
identity of the differentiated cells for the presence of β cell mark-
ers and the absence of stem cell and other cell type markers. Uti-
lizing single cell multiomic assays can deepen our understanding
of β cell differentiation in vitro. This approach can help analyze
the different resulting cells and may pave the way to optimize
the in vitro differentiation protocols to achieve a desirable pro-
portion of mature and functional β cells, along with other pancre-
atic endocrine cells [46].
Transplantation site and immune reaction
The selection of a transplantation site for PSC-derived β-like
cells is critical in mitigating graft rejection and immune re-
sponses [51]. To achieve functional efficacy, the loss of trans-
planted insulin-producing cells should be minimized. For this
purpose, a current focus of interest is increasing graft survival
by rapid vascularization to deliver nutrition and oxygen, as well
as protecting from harsh allogenic and autoimmune responses.
Certain transplantation sites may provide an immune-privileged
or immune-tolerant environment, potentially reducing graft re-
jection risk. Sites including the anterior chamber of the eye and
the omentum are under preclinical investigation for their effica-
cy in this regard [52,53]. Immune evasion via genetic engineer-
ing of β cells is also being tested. Clustered Regularly Inter-
spaced Short Palindromic Repeats (CRISPR)/Cas9 deletion of
the human leukocyte antigen-A/B/C (HLA-A/B/C) and class II
transactivator (CIITA) genes, and the introduction of the pro-
grammed death-ligand 1 (PD-L1), HLA-G, and CD47 genes al-
low cells to be less immunogenic [10,54-58]. The use of encap-
sulation devices is another innovative transplantation strategy.
These devices are designed to protect the cells from immune at-
tacks; closed-type devices prevent immune interactions, while
open-type devices facilitate vascularization and nutrient ex-
change [59]. These approaches are in a developmental stage and
have not yet achieved full protection of transplanted cells, pre-
senting a trade-off. Previous reviews discuss these strategies in
depth [51,60].
Maturity
Maturity in hPSC-derived β-like cells is marked by the expres-
sion of key β cell markers, including MAFA, UCN3, islet amy-
loid polypeptide (IAPP), SIX homeobox 2 (SIX2), and Wnt
family member 4 (WNT4), proper glycolysis and mitochondrial
metabolic activity, and some functional capacity for glucose
sensing and insulin secretion [7,10,18]. Although it is widely
acknowledged that these differentiated cells do not yet exhibit
definitive β cell metabolic maturity and functionality compara-
ble to human islets, they can attain in vivo maturity and reduce
hyperglycemia when transplanted in diabetic mice. Advance-
ment toward defining signals and pathways that can further ma-
ture hPSC-derived monohormonal β cells, mimicking in vivo
maturity, is a current subject of intense research. Furthermore, it
is being increasingly recognized that there is heterogeneity of
mature β cells with putatively different functions. Determining
whether current protocols are able to replicate these heteroge-
neous subtypes and how they influence β cell functionality for
transplantation and clinical applications remain to be thoroughly
investigated. The signal pathways involved in β cell maturation
have recently been reviewed in detail [18,51].
Expansion and scalability
Typically, around 3,000 islet equivalents (IEQ) per mouse and
Scalability Issues of Insulin Producing Cells
Copyright © 2024 Korean Endocrine Society www.e-enm.org 195
approximately 100,000 IEQ per kilogram of body weight in hu-
mans are required to observe a beneficial effect on glucose ho-
meostasis in diabetes (Table 1) [4,5,10-12,61-75]. Consequent-
ly, a strategic expansion of fully mature and functionally homo-
geneous β cells is necessary to standardize these therapeutic in-
terventions. The self-renewal capacity of hPSCs is expected to
provide an infinite resource for newly synthetic β cells. Howev-
er, it is known that the passage number of hPSCs influences
their differentiation function. In general, hPSCs with a higher
passage number show more variability and increased genomic
instability, and lose the function of proper differentiation [76].
In addition, different sources of human iPSCs or human embry-
onic stem cells show distinct patterns of epigenetic inheritance
[34], which can result in variability in their efficacy for generat-
ing β cells. The cell count tends to decrease during the lengthy
differentiation process, highlighting the limitations of scalability
with hPSCs. Besides relying on the self-renewal function of
hPSCs for expansion, the regulation of proliferation at the ad-
vanced stages of differentiation of hPSCs to the pancreatic lin-
eage has been explored. Distinct stages of the β cell develop-
ment have different proliferative capacities; thus, in vitro culture
of these cells is under investigation for intensive proliferation
before differentiation to increase the scalability of β cells. We
discuss these approaches in the next section.
REGULATION OF HUMAN β CELL
PROLIFERATION
The expansion of residual β cells is considered a promising
therapeutic approach for T1D and type 2 diabetes. In fact, 2% to
Table 1. Summary of Islet Quantities Required for Transplantation of Different Species
Species Types Sites Islet quantity, IEQ Reference
Donor Recipient
Mouse Mouse Primary islets Portal vein 350 [61]
Kidney capsule Approximately 1,000 [10]
Dog Dog Primary islets Kidney 3,000–5,000 [62]
Portal vein
Porcine Mouse Primary islets Portal vein 2,000 [61]
NHPs Approximately 50,000/kg (approximately 6.2×106 β cells/kg) [65]
Approximately 25,000/kg (approximately 7.9±4.8×106 β cells/kg) [66]
85,000–100,000/kg of BW [67]
Human Mouse Primary islets Portal vein Approximately 2,000 [61]
Kidney capsule Approximately 2,000 [10]
Approximately 3,000 [68]
Human Primary islets Portal vein 782,550 [69]
<10,000/kg of BW [16]
<9,000/kg of BW [71]
Human Mouse hPSC-β Kidney capsule 3×106–7×106 cells [68]
3×106–5×106 cells [5]
3×106 cells [75]
Approximately 1.25×106 cells [4]
Approximately 1.6×106 cells [72]
250–750 (diameter 100–200 μm) [11]
3.2×106–4.9×106 cells [73]
3×106 cells [74]
NHPs Omentum Approximately 17,000 [12]
Portal vein 30,000–40,000/kg of BW [75]
IEQ, islet equivalent; NHP, non-human primate; BW, body weight; hPSC-β, human pluripotent stem cell-derived β cell.
Choi J, et al.
Copyright © 2024 Korean Endocrine Society
196 www.e-enm.org
3% of human β cells are observed to divide during the infancy-
childhood β cell expansion period, a rate that gradually declines
to less than 0.5% in adulthood [77]. While mitogens that induce
β cell proliferation in rodents have been identified, the majority
of those mitogens have not been as effective in human β cells.
This discrepancy may stem from differences in cell cycle regu-
lation mechanisms between species. For example, human β cells
have high expression of cyclin-dependent kinase 6 (CDK6),
which is important for cell division in these cells, but not in ro-
dent β cells [78-80]. Additionally, rodent β cells express all three
D-cyclins, and the genetic deletion of cyclin D2 leads to β cell
hypoplasia and diabetes [81]. In contrast, human β cells express
very little or no cyclin D2 [80,82].
Inhibition of DYRK1A activity is known as a representative
pathway that induces human β cell division; therefore, DYRK1A
inhibitors such as harmine, INDY, leuketine-41, GNF4877, 5-io-
dotubericidin, TG003, AZ191, and CC-401 have been used to
promote human β cell proliferation [24,83]. DYRK1A phos-
phorylates nuclear factor of activated T-cell transcription factors
(NFATs) and prevents their translocation to the nucleus, thereby
preventing their activation. Activation of NFATs through inhibi-
tion of DYRK1A induces cell cycle regulator expression and in-
creases human β cell proliferation and mass [24]. Additionally,
DYRK1A inhibition induces human β cell proliferation through
reduced expression of the cell cycle inhibitor p27kip1 and conver-
sion of the repressive DREAM (DP, RB-like, E2F, and MuvB)
complex to the pro-proliferative MMB (MYB, MuvB, and FOXM1)
conformation [84,85]. Harmine, a prominent DYRK1A inhibitor,
can enhance adult human β cell proliferation by up to approxi-
mately 3% in vitro and in vivo [24]. In a study, streptozotocin
(STZ)-induced diabetic nonobese diabetic/severe combined im-
munodeficiency (NOD-SCID) mice that received transplanted
human islets and were treated with harmine exhibited a signifi-
cant reduction in blood glucose levels—measuring approximate-
ly 200 mg/dL within 21 days, compared to around 300 mg/dL in
the control group. Interestingly, harmine treatment not only in-
duced human β cell proliferation, but also increased the expres-
sion of important β cell transcription factors, such as PDX1,
NKX6.1, and MAFA [24]. While transforming growth factor β
(TGFβ) signaling has been shown to suppress β cell proliferation
by increasing the expression of CDK inhibitors, including P15,
P16, P21, and P57 [86,87], its suppression through inhibitors like
LY364947, ALK5, and GW788388 did not markedly increase
human β cell proliferation [25]. However, it has recently been re-
ported that leukemia inhibitory factor (LIF) signaling stimulated
the expression of cyclins and CDKs via the signal transducer and
activator of transcription 3 and CCAAT Enhancer Binding Pro-
tein Delta (CEBPD) pathways, facilitating human β cell cycle
progression [26]. Treatment with recombinant LIF (rLIF) led to
a modest 1.5% rise in human β cell proliferation in vitro [26].
rLIF treatment for 14 days improved glycemic regulation by up
to approximately 200 mg/dL (phosphate-buffered saline treat-
ment group: approximately 350 mg/dL) before a single nephrec-
tomy in STZ-induced diabetic NOD-SCID mice with human islet
transplantation. In particular, in an in vivo glucose-stimulated in-
sulin secretion test, the rLIF-treated group showed a twofold in-
crease in insulin secretion compared to controls [26]. Additional-
ly, it has been reported that γ-aminobutyric acid (GABA) signal-
ing, an inhibitory neurotransmitter, induces protein kinase B (PKB
or AKT) and cAMP-response element binding protein (CREB)
pathway activity to increase β cell proliferation, and glycogen
synthase kinase 3β (GSK3β) inhibitors such as 1-azakenpaullone,
CHIR99021, or 6-bromoindirubin-30-oxime (BIO) also induces
rat β cell survival and proliferation [88,89]. Contrary to previous
findings, it was recently reported that GABA did not restore islet
capacity and function in diet-induced obese mice, and GSK3β in-
hibitors alone were unable to increase human β cell proliferation
[83,90]. Synergistic effects—where two or more drugs, when
used in combination, produce an amplified effect—have been
harnessed in recent studies to enhance human β cell proliferation.
Research has demonstrated that combined treatment of harmine
(DYRK1A inhibitor)+LY364947 (TGFβ inhibitor) or CC-401
(DYRK1A inhibitor)+ALK5 inhibitor II (TGFβ inhibitor) can in-
crease human β cell proliferation by 4% to 8% in vitro [25,91].
Similarly, the combined treatment of harmine and GW788388
(TGFβ inhibitor) showed a synergistic effect, increasing human β
cell proliferation by 1.5% (harmine treatment group: approxi-
mately 1.2%) in NOD-SCID mice [25]. In addition, harmine also
synergized with GLP-1 agonists to induce human β cell prolifera-
tion in vitro and in vivo. The combination treatment improved
normoglycemic levels in STZ-induced diabetic NOD-SCID
gamma (NSG) mice transplanted with 500 IEQ of human islets,
and it increased human β cell proliferation approximately two-
fold over the harmine treatment group. However, a single treat-
ment with harmine failed to reduce blood glucose levels [92].
Moreover, in hPSC-β cells exhibiting a 1% cell division rate, a
triple combination of LIF+harmine+LY364947 was able to boost
cell division to approximately 5% in vitro [26]. Moreover, efforts
to increase the mass of insulin-producing cells have targeted not
only human β cell proliferation, but also the expansion of pancre-
atic progenitors [93-95]. The recent success in cultivating ex-
pandable protein C receptor positive pancreatic progenitors that
Scalability Issues of Insulin Producing Cells
Copyright © 2024 Korean Endocrine Society www.e-enm.org 197
Table 2. Human β Cell Proliferation by Mitogenic Factors
Treatment Target Molecules Experiment
types Cell types Proliferation index (vs. CON) Mechanism of
action Reference
KI67 BrdU EdU P-HH3
Single DYRK1A Harmine In vitro Human β cells 1%–3% –2% –0.3% NFAT signaling [24]
inhibitor In vitro Human β cells –3% pathway ↑ [83]
In vitro Human β cells –2% –2% –0.4% [25]
In vivo Human β cells –1%
In vitro hPSC-β cells –2.5% [26]
INDY In vitro Human β cells 1.5% –0.2% [24]
In vitro Human β cells –3% [83]
In vitro Human β cells –2% [25]
Leucettine-41 In vitro Human β cells 4% [83]
In vitro Human β cells –2% [25]
5-IT In vitro Human β cells 3% [83]
TG003 In vitro Human β cells 2%
CC-401 In vitro Human β cells 1%
DYRK1A GNF7156 In vitro Human β cells 3%–6% [97]
inhibitor+GSK3β GNF4877 In vitro Human β cells 3%–6%
inhibitor In vivo Human β cells 3%
TGFβ inhibitor SB431542 In vitro Human β cells –2.5% CDKIs (P15, [98]
In vivo Human β cells –1% P16, P21,P57) ↓
LY364947 In vitro Human β cells –1% [25]
ALK5 In vitro Human β cells –1%
GW788388 In vitro Human β cells –1%
In vivo Human β cells –1%
A83-01 In vitro Human β cells –1%
K02288 In vitro Human β cells –1%
LDN193189 In vitro Human β cells –1%
LIF Recombinant LIF In vitro Human β cells –2% STAT3 & CEBPD [26]
In vivo Human β cells –1.5% singaling
In vitro hPSC-β cells –1.5% pathway↑
GABA Recombinant GABA In vitro Human β cells –2% –0.3% PKA-CREB [89]
In vivo Human β cells –2% signaling
pathway↑
GSK3β inhibitor Tideglusib In vitro Human β cells NS [83]
CHIR99021 In vitro Human β cells NS
GLP-1 recombinant GLP-1 In vitro Human β cells NS NS [92]
Expendin-4 In vivo NS
Combination DYRK1A Harmine+SB431542 In vitro Human β cells –4% [25]
inhibitor+TGFβ Harmine+LY364947 In vitro Human β cells –7%
inhibitor Harmine+ALK5 In vitro Human β cells –7%
Harmine+GW788388 In vitro Human β cells –5%
Harmine+A83-01 In vitro Human β cells –6%
Harmine+K02288 In vitro Human β cells –5%
Harmine+LDN193189 In vitro Human β cells –4%
(Continued to the next page)
Choi J, et al.
Copyright © 2024 Korean Endocrine Society
198 www.e-enm.org
Fig. 2. Updated methodologies for β-like cell expansion at each step during stem cell-derived β cell differentiation. To date, methodologies
have been reported for cell expansion at each stage of human pancreatic stem cell (hPSC)-derived β-like cell differentiation using optimized
culture media. These methods report that cells retain their identity and capacity to differentiate through multiple passages. Exceptionally, a
methodology to expand endocrine progenitor (EP) cells has not been reported. The Hippo signaling pathway is essential for regulating pan-
creatic development, as well as β cell proliferation, differentiation and survival. Overexpression of YAP-S6A in pancreatic progenitor cells
reduces the differentiation efficiency into β-like cells, but increases the number of proliferating β-like cells [26,93,99-102]. FGF10, fibro-
blast growth factor 10; EGF, epidermal growth factor; TGFβ, transforming growth factor β; Inh, inhibitor; P, passage; Ha, harmine; LY,
LY364947; LIF, leukemia inhibitory factor; DE, definitive endoderm; FG, foregut; PP, pancreatic progenitor; TesR1, mTESR™1; BMP4,
bone morphogenetic protein 4; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; MEF, mouse embryonic fi-
broblast; HGF, hepatocyte growth factor; OE, overexpression.
Table 2. Continued
Treatment Target Molecules Experiment
types Cell types Proliferation index (vs. CON) Mechanism of
action Reference
KI67 BrdU EdU P-HH3
DYRK1A
inhibitor+GSK3β
Harmine+Tidglusib In vitro Human β cells –4% [83]
inhibitor Harmine+CHIR99021 In vitro Human β cells –4%
DYRK1A
inhibitor+TGFβ
inhibitor+LIF
Harmine+LY364947
+LIF
In vitro hPSC-β cells –5% [26]
DYRK1A Harmine+GLP-1 In vitro Human β cells –5% –3.5% –1.5% [92]
inhibitor+GLP-1 Harmine+Expendin-4 In vivo Human β cells –1%
CON, control; KI67, antigen Kiel 67; BrdU, 5-bromo-2’-deoxyuridine; EdU, 5-ethynyl-2’-deoxyuridine; P-HH3, phospho-histone H3; DYRK1A, dual-
specific tyrosine-phosphorylation regulated kinase 1A; NFAT, nuclear factor of activated T-cell transcription factor; hPSC, human pluripotent stem cell;
INDY, 1Z-(3-ethyl-5-hydroxy-2(3H)-benzothiazolylidene)-2-propanone; 5-IT, 5-iodotubericidin; GSK3β, glycogen synthase kinase 3β; GNF, Genomics
Institute of the Novartis Research Foundation; TGFβ, transforming growth factor β; CDKI, cyclin-dependent kinase inhibitors; P, protein; ALK5, activin
like kinase 5; LIF, leukemia inhibitory factor; hPSC-β, human pluripotent stem cell-derived β cell; STAT3, signal transducer and activator of transcription 3;
CEBPD, CCAAT/enhancer-binding protein delta; GABA, γ-aminobutyric acid; PKA-CREB, protein kinase A-cAMP response element-binding protein;
NS, not significant; GLP-1, glucagon-like peptide-1.
Scalability Issues of Insulin Producing Cells
Copyright © 2024 Korean Endocrine Society www.e-enm.org 199
can generate functional islet organoids in a mouse model has fur-
ther encouraged these approaches [96]. The synergistic effect of
several drugs based on the new understanding of the mechanism
of pancreatic β cell replication has significantly increased human
β cell proliferation (Table 2, Fig. 2) [24-26,83,89,92,93,97-102].
These findings hold promise for the potential clinical application
of these drug combinations in the future.
ISLET CRYOPRESERVATION
Cryopreservation of islets is a crucial component in scaling the
delivery of insulin-producing cells. This method has been ex-
tensively studied, as it offers a solution to challenges in the islet
supply chain by enabling high-quality storage and the pooling
of islets from multiple donors. However, islets are highly sus-
ceptible to cellular stress and damage during the freeze-thaw
cycle, which may lead to impaired function or cell death. Over
the past decades, various conditions have been studied for islet
cryopreservation in order to improve islet survival and function-
al recovery after thawing. In particular, major factors affecting
successful islet cryopreservation include the use of cryoprotec-
tive agents (CPAs), the management of cellular stress, and the
maintenance of the islets’ 3D structure.
Rapid freezing can cause the formation of intracellular and
extracellular ice crystals, which are detrimental to cell viability
[103]. In 1949, the discovery of glycerol’s cryoprotective prop-
erties paved the way for the use of CPAs such as dimethyl sulf-
oxide (DMSO), glycerol, ethylene glycol, and propylene, which
are introduced prior to freezing to prevent ice crystallization
[104,105]. Effective cryopreservation thus requires sufficient
time to equilibrate with CPAs within and around the cells. Fur-
thermore, it is important to set the optimal temperature and con-
centration of CPAs, as different CPAs have varying rates of dif-
fusion and levels of cytotoxicity [106].
In 1977, cryopreserved rat islets were transplanted into the
livers of diabetic rats through the portal vein [107]. The diabetic
rats transplanted with cryopreserved rat islets showed hypergly-
cemia for 6 weeks after transplantation but thereafter main-
tained normal blood glucose levels until 13 weeks. These find-
ings have spurred the development of various islet cryopreser-
vation protocols [108]. Despite success in small rodents, only
20% of pigs with transplanted cryopreserved porcine islets
achieved normal glucose regulation, highlighting the need for
improved cryopreservation methods for larger mammals, in-
cluding humans [109]. In 2001, it was shown that islet survival
improved with a protocol incorporating slow cooling at a rate of
0.25°C per minute and rapid thawing, facilitated by the addition
of 2 moles (M) DMSO to the University of Wisconsin organ
preservation solution or to a hypothermosol preservation solu-
tion [110].
Although CPAs are effective in preventing ice crystal forma-
tion, they do not alleviate the cellular stress associated with
freezing and thawing. In particular, the oxidative stress that oc-
curs during this process poses a threat to islet survival due to the
inherently low antioxidant defense of islet cells [111]. Over past
decades, research has focused on various CPA additives to re-
duce oxidative stress in islets, including taurine (an antioxidant),
metformin (an antidiabetic drug), GABA, eicosapentaenoic acid
(a polyunsaturated fatty acid), or docosahexanoic acid (a polyun-
saturated fatty acid). When combined with CPAs, these additives
have been shown to significantly lower reactive oxygen species
levels in islets, thereby enhancing their function and survival af-
ter cryopreservation [112,113] .
Human islets are aggregates of approximately 1,500 to 2,000
cells with an average diameter of 100 to 150 μm [114]. The 3D
structure of islets prevents CPAs from spreading uniformly as
the temperature of each cell within the islets changes [115]. The
differential temperature pattern formed in this way can generate
intracellular ice crystals and eventually lead to cell death. To
overcome this problem, a method was proposed to separate is-
lets into single cells, freeze them, and reassemble them into their
original spheroid form after thawing. In fact, islets reconstituted
after cryopreservation showed higher cell survival and function-
al recovery than native islets, both in vitro and in vivo [116].
In addition, improved islet survival rate and functional recov-
ery after cryopreservation have been achieved by reducing the
amount of CPAs using hollow fiber vitrification, encapsulating
islets with 1.75% alginate, or combining CPAs with ethylene
glycol and DMSO [117,118]. In particular, a recent islet cryo-
preservation protocol using cryomesh has achieved survival
rates exceeding 89% and an islet recovery rate of more than
95% in 2,500 islets after thawing. In addition, it was suggested
that clinically meaningful throughput could be achieved if a
larger-sized cryomesh and cryomesh overlapping method were
used [119]. In the near future, high-quality islet banking through
the establishment of successful islet cryopreservation methods
holds the potential to substantially reduce the geographical and
temporal barriers between donors and recipients. This advance-
ment is anticipated to markedly improve the success rates of is-
let translation by increasing the opportunity for high-dose trans-
plantation and more precise HLA matching through the pooling
of islet resources.
Choi J, et al.
Copyright © 2024 Korean Endocrine Society
200 www.e-enm.org
CONCLUSIONS
Taken together, this review highlights recent findings on novel
methodologies that provide game-changing resources for gener-
ating synthetic functional insulin-producing β cells and directly
expanding human β cells using small molecules or pooling via
islet cryopreservation. Although these developments are prom-
ising, significant hurdles remain. For instance, the production of
uniform hPSC-derived β-like cells in quantities sufficient for
clinical applications is still a challenge. Current differentiation
protocols are labor-intensive and struggle with the heterogene-
ity and nonuniformity of the resulting β cells, which are critical
issues to resolve for industrial-scale production. Despite these
challenges, the various cutting-edge methods for obtaining hu-
man β cells are paving the way toward making islet transplanta-
tion a clinically viable and more successful treatment in the
foreseeable future.
CONFLICTS OF INTEREST
No potential conflict of interest relevant to this article was re-
ported.
ACKNOWLEDGMENTS
This work was supported by grants from Beatson Foundation
(2022-006), Tobacco-Related Disease Research Program (TRDRP)
research award (T33IR6551) and National Institute of Diabetes
And Digestive And Kidney Diseases (NIDDK) of the National
Institutes of Health (NIH) under Award Number R01DK136888.
Eiji Yoshihara is supported by the Juvenile Diabetes Research
Foundation (JDRF) Career Development Award (5-CDA-2022-
1178-A-N). Jinhyuk Choi and Harvey Perez are supported by
postdoctoral fellowships from the California Institute for Re-
generative Medicine (CIRM)-training grant (EDUC4-12837).
ORCID
Jinhyuk Choi https://orcid.org/0000-0002-1490-0179
Fritz Cayabyab https://orcid.org/0000-0002-6992-5445
Harvey Perez https://orcid.org/0009-0002-6439-3897
Eiji Yoshihara https://orcid.org/0000-0001-6324-6495
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