PRINCIPLES OF TISSUE ENGINEERING 3RD EDITION - PART 2 (end) doc

I. Introduction
II. Cell Types for Pancreatic Substitutes
III. Construct Technology
IV. In Vivo Implantation
V. Concluding Remarks
VI. Acknowledgments
VII. References
Bioartifi cial Pancreas
Athanassios Sambanis
Principles of Tissue Engineering, 3
rd
Edition
ed. by Lanza, Langer, and Vacanti
Copyright © 2007, Elsevier, Inc.
All rights reserved.
I. INTRODUCTION
Diabetes is a signifi cant health problem, affecting an
estimated 20.8 million people in the United States alone,
with nearly 1.8 million affl icted with type 1 diabetes [http://
diabetes.niddk.nih.gov/dm/pubs/statistics/index.htm#7].
Type 1 diabetes results from the loss of insulin-producing
cell mass (the β-cells of pancreatic islets) due to autoim-
mune attack. Type 2 diabetes has a more complicated
disease etiology and can be the result of not producing
enough insulin and/or the body’s developing a resistance to
insulin. Although initially controlled by diet, exercise, and
oral medication, type 2 diabetes often progresses toward
insulin dependence. It is estimated that insulin-dependent
diabetics (both types 1 and 2) exceed 4 million people in the
United States. Although insulin-dependent diabetes (IDD)
is considered a chronic disease, even the most vigilant

insulin therapy cannot reproduce the precise metabolic
control present in the nondiseased state. The poor temporal
match between glucose load and insulin activity leads to a
number of complications, including increased risk of heart
disease, kidney failure, blindness, and amputation due to
peripheral nerve damage. Providing more physiological
control would alleviate many of the diabetes-related health
problems, as suggested by fi ndings from the Diabetes
Control and Complications Trial (The Diabetes Control and
Complications Trial Research Group, 1997) and its continu-
ation study (DCCT/EDIC NEJM 353(25):2643–53, 2005).
Cell-based therapies, which provide continuous regulation
of blood glucose through physiologic secretion of insulin,
have the potential to revolutionize diabetes care.
Several directions are being considered for cell-based
therapies of IDD, including implantation of immunopro-
tected allogeneic or xenogeneic islets, of continuous cell
lines, or of engineered non-β-cells. For allogeneic islet trans-
plantation, a protocol developed by physicians at the Uni-
versity of Edmonton (Shapiro et al., 2001a, 2001b, 2001c,
Bigam and Shapiro, 2004) has dramatically improved the
survivability of grafts. The protocol uses human islets from
cadaveric donors, which are implanted in the liver of care-
fully selected diabetic recipients via portal vein injection.
The success of the Edmonton protocol is attributed to two
modifi cations relative to earlier islet transplantation studies:
the use of a higher number of islets and the implementation
of a more benign, steroid-free immunosuppressive regimen.
However, two barriers prevent the widespread application
of this therapy. The fi rst is the limited availability of human

tissue, because generally more than one cadaveric donor
pancreas is needed for the treatment of a single recipient.
The second is the need for life-long immunosuppression,
which, even with the more benign protocols, results in long-
term side effects to the patients.
A tissue-engineered pancreatic substitute aims to
address these limitations by using alternative cell sources,
relaxing the cell availability limitation, and by reducing or
eliminating the immunosuppressive regimen necessary for
survival of the graft. A number of signifi cant challenges are
Chapter Forty-Two
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620 CHAPTER FORTY-TWO • BIOARTIFICIAL PANCREAS
facing the development of such a substitute, however. These
include procuring cells at clinically relevant quantities; the
immune acceptance of the cells, which is exacerbated in
type 1 diabetes by the resident autoimmunity in the patients;
and the fact that diabetes is not an immediately life-threat-
ening disease, so any other therapy will have to be more
effi cacious and/or less invasive than the current standard
treatment of daily blood glucose monitoring and insulin
injections.
In general, developing a functional living-tissue replace-
ment requires advances and integration of several types of
technology (Nerem and Sambanis, 1995). These are (1) cell
technology, which addresses the procurement of functional
cells at the levels needed for clinical applications; (2) con-
struct technology, which involves combining the cells with
biomaterials in functional three-dimensional confi gura-
tions. Construct manufacturing at the appropriate scale,

and preservation, as needed for off-the-shelf availability,
also fall under this set of technologies; (3) technologies for
in vivo integration, which address the issues of construct
immune acceptance, in vivo safety and effi cacy, and moni-
toring of construct integrity and function postimplantation.
The same three types of technology need also be developed
for a pancreatic substitute. It should be noted, however, that
the critical technologies differ, depending on the type of
cells used. With allogeneic or xenogeneic islets or beta-cells,
the major challenge is the immune acceptance of the
implant. In this case, encapsulation of the cells in permse-
lective membranes, which allow passage of low-molecular-
weight nutrients and metabolites but exclude larger
antibodies and cytotoxic cells of the host, may assist the
immune acceptance of the graft. With cell therapies based
on potentially autologous nonpancreatic cells, targeted
by gene expression vectors in vivo, or retrieved surgically,
engineered ex vivo, and returned to the host, the major
challenge is engineering insulin secretion in precise response
to physiologic stimuli. Lastly, with stem or progenitor cells,
the primary hurdle is their reproducible differentiation
into cells of the pancreatic β-phenotype. Figure 42.1 shows
schematically the two general therapeutic approaches based
on allo- or xenogeneic cells (Fig. 42.1A) or autologous cells
(Fig. 42.1B).
This chapter is therefore organized as follows. We fi rst
describe the types of cells that have been used or are of
potential use in engineering a pancreatic substitute. We
then discuss issues of construct technology, specifi cally
encapsulation methods and the relevant biomaterials, man-

ufacturing issues, and preservation of the constructs. The
challenges of in vivo integration and results from in vivo
experiments with pancreatic substitutes are presented next.
We conclude by offering a perspective on the current status
and the future challenges in developing an effi cacious, clini-
cally applicable bioartifi cial pancreas.
II. CELL TYPES FOR
PANCREATIC SUBSTITUTES
Islets
Despite several efforts, the in vitro expansion of primary
human islets has met with limited success. Adult human
islets are diffi cult to propagate in culture, and their expan-
sion leads to dedifferentiation, generally manifested as loss
of insulin secretory capacity. Although there exist reports on
the redifferentiation of expanded islets (Lechner et al., 2005;
Ouziel-Yahalom et al., 2006) and of nonislet pancreatic cells,
which are discarded after islet isolation (Todorov et al.,
2006), the phenotypic stability and the in vivo effi cacy of
these cells remain unclear. Additionally, with expanded and
A. Allo- or Xenogeneic Cells B. Autologous Cells
Implantation
Cell
retrieval
Ex vivo
manipulation
Cell storage
Cell
storage
Cell
amplification

Cell
encapsulation
Implantation
Capsule
storage
Cell
procurement
In vivo
gene therapy
A. Allo- or Xenogeneic Cells B. Autologous Cells
Implantation
Cell
retrieval
Ex vivo
manipulation
Cell storage
Cell
storage
Cell
amplification
Cell
encapsulation
Implantation
Capsule
storage
Capsule
storage
Cell
procurement
In vivo

gene therapy
FIG. 42.1. Approaches for bioartifi cial pancreas development using allo- or xenogeneic cells (A) and autologous cells (B). In (A), islets are procured from
pancreatic tissue, or cell lines are thawed from cryostorage and expanded in culture; cells are encapsulated for immunoprotection before they are implanted
to achieve a therapeutic effect; encapsulated cells may also be cryopreserved for inventory management and sterility testing. In (B), cells are retrieved surgi-
cally from the patient; manipulated ex vivo phenotypically and/or genetically in order to express β-cell characteristics, and in particular physiologically
responsive insulin secretion; the cells are implanted for a therapeutic effect either by themselves or, preferably, after incorporation in a three-dimensional
substitute; some of the cells may be cryopreserved for later use by the same individual. In in vivo gene therapy approaches, a transgene for insulin expres-
sion is directly introduced into the host and expressed by cells in nonpancreatic tissues.
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II. CELL TYPES FOR PANCREATIC SUBSTITUTES • 621
redifferentiated islets, it remains unknown whether the
insulin-secreting cells arose from the redifferentiation of
mature endocrine cells or from an indigenous stem or pro-
genitor cell population in the tissue isolate (Todorov et al.,
2006).
Animal, such as porcine, islets are amply available, and
porcine insulin is very similar to human, differing by only
one amino acid residue. However, the potential use of
porcine tissue is hampered by the unlikely but possible
transmission of porcine endogenous retroviruses (PERV) to
human hosts as well as by the strong xenograft immuno-
genicity that they elicit. Use of closed, PERV-free herds is
reasonably expected to alleviate the fi rst problem. With
regard to immunogenicity, a combination of less immuno-
genic islets, islet encapsulation in permselective barriers,
and host immunosuppression may yield long-term survival
of the implant. The use of transgenic pigs that do not express
the α-Gal (α[1,3]-galactose) epitope is one possible approach
for reducing the immunogenicity of the islets. Studies also
indicate that neonatal pig islets induce a lower T-cell reac-

tivity than adult islets (Bloch et al., 1999), even though the
α-Gal epitope is abundant in neonatal islets as well (Rayat
et al., 1998). Furthermore, it is possible that the primary
antigenic components in islet tissue are the ductal epithelial
and vascular endothelial cells, which express prominently
the α-Gal epitope; on the other hand, β-cells express the
epitope immediately after isolation but not after mainte-
nance in culture (Heald et al., 1999). It should also be noted
that the large-scale isolation of porcine islets under condi-
tions of purity and sterility that will be needed for eventual
regulatory approval pose some major technical hurdles,
which have not been addressed yet.
-Cell Lines
Recognizing the substantial diffi culties involved with
the procurement and amplifi cation of pancreatic islets,
several investigators have pursued the development of con-
tinuous cell lines, which can be amplifi ed in culture yet
retain key differentiated properties of normal β-cells. One of
the fi rst successful developments in this area was the gen-
eration of the βTC family of insulinomas, derived from
transgenic mice carrying a hybrid insulin-promoted simian
virus 40 tumor antigen gene; these cells retained their dif-
ferentiated features for about 50 passages in culture (Efrat
et al., 1988). The hypersensitive glucose responsiveness of
the initial βTC lines was reportedly corrected in subsequent
lines by ensuring expression of glucokinase and of the high-
K
m
glucose transporter Glut2, and with no or low expression
of hexokinase and of the low-K

m
transporter Glut1 (Efrat et
al., 1993; Knaack et al., 1994). A similar approach was used
to develop the mouse MIN-6 cell line that exhibits glucose-
responsive secretion of endogenous insulin (Miyazaki et al.,
1990). Subsequently, Efrat and coworkers developed the
βTC-tet cell line, in which expression of the SV40 T antigen
(Tag) oncoprotein is tightly and reversibly regulated by tet-
racycline. Thus, cells proliferate when Tag is expressed, and
shutting off Tag expression halts cell growth (Efrat et al.,
1995; Efrat, 1998). Such reversible transformation is an
elegant approach in generating a supply of β-cells via pro-
liferation of an inoculum, followed by arrest of the growth
of cells when the desirable population size is reached. When
retained in capsules, proliferating cells do not grow uncon-
trollably, since the dissolved-oxygen concentration in the
surrounding milieu can support up to a certain number of
viable, metabolically active cells in the capsule volume. This
number of viable cells is maintained through equilibration
of cell growth and death processes (Papas et al., 1999a,
1999b). Thus, growth arrest is useful primarily in preventing
the growth of cells that have escaped from broken capsules
in vivo and in reducing the cellular turnover in the capsules.
The latter reduces the number of accumulated dead cells in
the implant and thus the antigenic load to the host affected
by proteins from dead and lysed cells that pass through the
capsule material.
In a different approach, Newgard and coworkers (Clark
et al., 1997) carried out a stepwise introduction of genes
related to β-cell performance into a poorly secreting rat

insulinoma (RIN) line. In particular, RIN cells were itera-
tively engineered to stably express multiple copies of the
insulin gene, the glucose transporter Glut2, and the gluco-
kinase gene, which are deemed essential for proper expres-
sion of β-cell function. Although this is an interesting
methodology, it is doubtful that all genes necessary for
reproducing β-cell function can be identifi ed and stably
expressed in a host cell. Recently, signifi cant progress was
made toward establishing a human pancreatic β-cell line
that appears functionally equivalent to normal β-cells
(Narushima et al., 2005). This was accomplished through a
complicated procedure involving retroviral transfection of
primary β cells with the SV40 large T antigen and cDNAs of
human telomerase reverse transcriptase. This resulted in a
reversibly immortalized human β-cell clone, which secreted
insulin in response to glucose, expressed β-cell transcrip-
tional factors, prohormone convertases 1/3 and 2, which
process proinsulin to mature insulin, and restored normo-
glycemia upon implantation in diabetic immunodefi cient
mice (Narushima et al., 2005).
With regard to β-cell lines capable of proliferation under
the appropriate conditions, key issues that remain to be
addressed include (1) their long-term phenotypic stability,
in vitro and in vivo; (2) their potential tumorigenicity, if cells
escape from an encapsulation device, especially when these
cells are allografts that may evade the hosts’ immune
defenses for a longer period of time than acutely rejected
xenografts; and (3) their possible recognition by the auto-
immune rejection mechanism in type 1 diabetic hosts.
Engineered Non– Pancreatic Cells

The use of non–β pancreatic cells from the same patient,
engineered for insulin secretion, relaxes both the cell
availability and immune acceptance limitations that exist
with other types of cells. It has been shown that the A-chain/
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622 CHAPTER FORTY-TWO • BIOARTIFICIAL PANCREAS
C-peptide and B-chain/C-peptide cleavage sites on the pro-
insulin gene can be mutated so that the ubiquitous endo-
peptidase furin recognizes and completely processes
proinsulin into mature insulin absent of any intermediates
(Yanagita et al., 1992). Based on this concept, several non-
endocrine cell lines have been successfully transfected to
produce immunoreactive insulin, including hepatocytes,
myoblasts, and fi broblasts (Yanagita et al., 1993). In a differ-
ent approach, Lee and coworkers (2000) expressed a syn-
thetic single-chain insulin analog, which does not require
posttranslational processing, in hepatocytes. Although
recombinant insulin expression is relatively straightforward,
a key remaining challenge is achieving the tight regulation
of insulin secretion in response to physiologic stimuli, which
is needed for achieving normoglycemia in higher animals
and, eventually, humans.
One approach for achieving regulation of insulin secre-
tion is through regulation of biosynthesis at the gene tran-
scription level, as realized in hepatocytes by Thule et al.
(Thule et al., 2000; Thule and Liu, 2000) and Lee et al. (2000).
Besides the ability to confer transcriptional-level regulation,
hepatocytes are particularly attractive as producers of
recombinant insulin due to their high synthetic and secre-
tory capacity and their expression of glucokinase and Glut2

(Cha et al., 2000; Lannoy et al., 2002). Hepatic delivery by
viral vectors and expression of the glucose-responsive
insulin transgene in diabetic rats controlled the hypergly-
cemic state for extended periods of time (Lee et al., 2000;
Thule and Liu, 2000; Olson et al., 2003). Nevertheless, trans-
criptional regulation is sluggish, involving long time lags
between stimulation of cells with a secretagogue and
induced insulin secretion as well as between removal of the
secretagogue and down-regulation of the secretory response
(Tang and Sambanis, 2003). The latter is physiologically
more important, because it means that the cells continue to
secrete insulin after glucose has been down-regulated, thus
resulting in potentially serious hypoglycemic excursions.
Increasing the number of stimulatory glucose elements in a
promoter enhances the cellular metabolic responsiveness
in vitro (Thule et al., 2000). With regard to secretion down-
regulation, Tang and Sambanis (2003) hypothesized that the
slow kinetics of this process following removal of the tran-
scriptional activator are due to the stability of the preproin-
sulin mRNA, which continues to become translated after
transcription has been turned off. Using a modifi ed prepro-
insulin cDNA that produced an mRNA with two more copies
of the insulin gene downstream of the stop codon resulted
in preproinsulin mRNA subjected to nonsense mediated
decay and thus destabilized. This signifi cantly expedited the
kinetics of secretion down-regulation on turning off tran-
scription (Tang and Sambanis, 2003). Thus, the combina-
tion of optimal transcriptional regulation with transgene
message destabilization promises further improvements in
insulin secretion dynamics from transcriptionally regulated

hepatic cells. It should be noted, however, that despite the
time delays inherent in transcriptional regulation, hepatic
insulin gene therapy is suffi cient to sustain vascular nitric
oxide production and inhibit acute development of
diabetes-associated endothelial dysfunction in diabetic
rats (Thule et al., 2006). Hence, many aspects of the thera-
peutic potential of hepatic insulin expression remain to be
explored.
Another appealing target cell type is endocrine cells,
which possess a regulated secretory pathway and the
enzymatic machinery needed to process authentic proinsu-
lin into insulin. Early work in this area involved expression
of recombinant insulin in the anterior pituitary mouse
AtT-20 cell line (Moore et al., 1983), which can be sub -
jected to repeated episodes of induced insulin secretion
using nonmetabolic secretagogues (Sambanis et al., 1990).
Cotransfection with genes encoding the glucose transporter
Glut-2 and glucokinase resulted in glucose-responsive
insulin secretion (Hughes et al., 1992, 1993). However, limi-
tations of this approach include possible instabilities in the
cellular phenotype and the continued secretion of endoge-
nous hormones, such as adrenocorticotropic hormone
from AtT-20 cells, which are not compatible with prandial
metabolism.
In this regard, endocrine cells of the intestinal epithe-
lium, or enteroendocrine cells, are especially promising.
Enteroendocrine cells secrete their incretin products in a
tightly controlled manner that closely parallels the secretion
of insulin following oral glucose load in human subjects;
incretin hormones are fully compatible with prandial

metabolism and glucose regulation (Schirra et al., 1996;
Kieffer and Habener, 1999). As with β-cells, enteroendocrine
cells are polar, with sensing microvilli on their luminal side
and secretory granules docked at the basolateral side, adja-
cent to capillaries. Released incretin hormones include the
glucagon-like peptides (GLP-1 and GLP-2) from intestinal
L-cells and glucose-dependent insulinotropic polypeptide
(GIP) from K-cells, which potentiate insulin production
from the pancreas after a meal (Drucker, 2002). The impor-
tance of enteroendocrine cells (and, in particular, L-cells)
was fi rst put forward by Creutzfeldt (1974), whose primary
interest in these cells was for the prospect of using GLP-1 for
the treatment of type 2 diabetes. Furthermore, ground-
breaking work by Cheung et al. (2000) demonstrated that
insulin produced and secreted by genetically modifi ed
intestinal K-cells of transgenic mice prevented the animals
from becoming diabetic after injection with streptozotocin
(STZ), which specifi cally kills the β-cells of the pancreas.
This is an important proof-of-concept study, which showed
that enteroendocrine cell–produced insulin can provide
regulation of blood glucose levels. Subsequent work with a
human intestinal L-cell line demonstrated that these cells
can be effectively transduced to express recombinant human
insulin, which colocalizes in secretory vesicles with endog-
enous GLP-1 and thus is secreted with identical kinetics to
GLP-1 in response to stimuli (Tang and Sambanis, 2003,
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2004). The intestinal tract could be considered an attractive
target for gene therapy because of its large size, making it
the largest endocrine organ in the body (Wang et al., 2004);

however, enteroendocrine cell gene therapy faces serious
diffi culties due to anatomic complexity, with the entero-
endocrine cells being located at the base of invaginations of
the gut mucosa called crypts, the very harsh conditions in
the stomach and intestine, and the rapid turnover of the
intestinal epithelium.
Contrary to direct in vivo gene delivery, ex vivo
gene therapy involves retrieving the target cells surgically,
culturing them and possibly expanding them in vitro,
genetically engineering them to express the desired proper-
ties, and then returning them to the host, either as such or
in a three-dimensional tissue substitute. It is generally
thought that the ex vivo approach is advantageous, for
it allows for the thorough characterization of the genetically
engineered cells prior to implantation, possibly for the
preservation of some of the cells for later use by the
same individual and, importantly, for localization and
retrievability of the implant. However, the challenges
imposed by the ex vivo approach, including the surgical
retrieval, culturing, and in vitro genetic engineering of the
target cells are signifi cant, so such methods are currently
under development.
Differentiated Stem or Progenitor Cells
Naturally, throughout life, islets turn over slowly, and
new, small islets are continually generated from ductal pro-
genitors (Finegood et al., 1995; Bonner-Weir and Sharma,
2002). There is also considerable evidence that adult plu-
ripotent stem cells may be a possible source of new islets
(Bonner-Weir et al., 2000; Ramiya et al., 2000; Kojima et al.,
2003). However, efforts to regenerate β-cells in vitro or in

vivo by differentiation of embryonic or adult stem or pan-
creatic progenitor cells have produced mixed results. Insulin-
producing, glucose-responsive cells, as well as other pan-
creatic endocrine cells, have been generated from mouse
embryonic stem cells (Lumelsky et al., 2001). Insulin-secret-
ing cells obtained from embryonic stem cells reversed
hyperglycemia when implanted in mice rendered diabetic
by STZ injection (Soria et al., 2000). In another study, mouse
embryonic stem cells transfected to express constitutively
Pax4, a transcription factor essential for β-cell development,
differentiated into insulin-producing cells and normalized
blood glucose when implanted in STZ-diabetic mice
(Blyszczuk et al., 2003). On the other hand, other studies do
not support differentiation of embryonic stem cells into the
β-cell phenotype (Rajagopal et al., 2003). Overall, the mixed
and somewhat inconsistent results point to the consider-
able work that needs to be done before stem or progenitor
cells can be reliably differentiated into β cells at a clinically
relevant scale. Harnessing the in vivo regenerative capacity
of the pancreatic endocrine system may present a promis-
ing alternative approach.
Engineering of Cells for Enhanced
Survival In Vivo
Because islets and other insulin-secreting cells experi-
ence stressful conditions during in vitro handling and in
vivo postimplantation, several strategies have been imple-
mented to enhance islet or nonislet cell survival in pancre-
atic substitutes. Strategies generally focus on improving the
immune acceptance of the graft, enhancing its resistance to
cytokines, and reducing its susceptibility to apoptosis. Phe-

notypic manipulations include extended culturing of neo-
natal and pig islets at 37°C, which apparently reduces their
immunogenicity, possibly by down-regulating the major
histocompatibility class 1 antigens on the islet surface; islet
pretreatment with TGF-β1; and enzymatic treatment of pig
islets with α-galactosidase to reduce the a-galactosyl epitope
on islets (Prokop, 2001). However, the permanency of these
modifi cations is unknown. For instance, a-galactosyl epi-
topes reappear on islets 48 hours after treatment with α-
galactosidase. With proliferative cell lines destined for
recombinant insulin expression, selection of clones resis-
tant to cytokines appears feasible (Chen et al., 2000). Gene
chip analysis of resistant cells may then be used to identify
the genes responsible for conferring cytokine resistance.
Genetic modifi cations for improving survival in vivo
may offer prolonged expression of the desired properties
relative to phenotypic manipulations, but they also present
the possibility of modifying the islets in additional, undesir-
able ways. Notable among the various proposed approaches,
reviewed in Jun and Yoon (2005), are the expression of the
immunomodulating cytokines IL-4 or a combination of IL-
10 and TGF-β, which promoted graft survival by preventing
immune attack in mice; and the expression of the antiapop-
totic bcl-2 gene using a replication defective herpes simplex
virus, which resulted in protection of β-cells from a cytokine
mixture of interleukin-1β, TNF-α, and IFN-γ in vitro.
III. CONSTRUCT TECHNOLOGY
Construct technology focuses on associating cells with
biocompatible materials in functional three-dimensional
confi gurations. Depending on the type of cells used, the

primary function of the construct can be one or more of the
following: to immunoprotect the cells postimplantation, to
enable cell function, to localize insulin delivery in vivo, or
to provide retrievability of the implanted cells.
Encapsulated Cell Systems
Encapsulation for immunoprotection involves sur-
rounding the cells with a permselective barrier, in essence
an ultrafi ltration membrane, which allows passage of low-
molecular-weight nutrients and metabolites, including
insulin, but excludes larger antibodies and cytototoxic cells
of the host. Figure 42.2 summarizes the common types of
encapsulation devices, which include spherical microcap-
sules, tubular or planar diffusion chambers, thin sheets, and
vascular devices.
III. CONSTRUCT TECHNOLOGY • 623
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624 CHAPTER FORTY-TWO • BIOARTIFICIAL PANCREAS
Encapsulation can be pursued via one of two general
approaches. With capsules fabricated using water-based
chemistry, cells are fi rst suspended in un-cross-linked
polymer, which is then extruded as droplets into a solution
of the cross-linking agent. A typical example here is the very
commonly used alginate encapsulation. Alginate is a
complex mixture of polysaccharides obtained from sea-
weeds, which forms a viscous solution in physiologic saline.
Islets or other insulin-secreting cells are suspended in
sodium alginate, and droplets are extruded into a solution
of calcium chloride. Calcium cross-links alginate, instanta-
neously trapping the cells within the gel. The size of the
droplets, hence also of the cross-linked beads, can be con-

trolled by fl owing air parallel to the extrusion needle so that
droplets detach at a smaller size than if they were allowed
to fall by gravity; or by using an electrostatic droplet genera-
tor, in which droplets are detached from the needle by
adjusting the electrostatic potential between the needle and
the calcium chloride bath. Capsules generated this way can
have diameters from a few hundred micrometers to more
than one millimeter. Alginate by itself is relatively perme-
able; to generate the permselective barrier, beads are treated
with a polycationic solution, such as poly-l-lysine or poly-
l-ornithine. The reaction time between alginate and the
polycation determines the molecular weight cutoff of the
resulting membrane. Poly-l-lysine is highly infl ammatory
in vivo, however, so beads are coated with a fi nal layer of
alginate to improve their biocompatibility. Hence, calcium
alginate/poly-l-lysine/alginate (APA) beads are fi nally
formed. Treating the beads with a calcium chelator, such as
citrate, presumably liquefi es the inner core, forming APA
membranes. Other materials that have been used for cell
microencapsulation include agarose, photo-cross-linked
poly(ethylene glycol), and (ethyl methacrylate, methyl
methacrylate, and dimethylaminoethyl methacrylate)
copolymers (Mikos et al., 1994; Sefton and Kharlip, 1994).
Advantages of hydrogel microcapsules include a high
surface-to-volume ratio, and thus good transport proper-
ties, as well as ease of handling and implantation. Small
beads can be implanted in the peritoneal cavity of animals
simply by injection, without the need for incision. Other
common implantation sites include the subcutaneous space
and the kidney capsules. Disadvantages include the fragility

of the beads, especially if the cross-linking cation becomes
chelated by compounds present in the surrounding milieu
or released by lysed cells, and the lack of easy retrievability
once the beads have been dispersed in the peritoneal cavity
of a host. Earlier problems caused by the variable composi-
tion of alginates and the presence of endotoxins have been
resolved through the development and commercial avail-
ability of ultrapure alginates of well-defi ned molecular
weight and composition (Sambanis, 2000; Stabler et al.,
2001).
Hydrogels impose little diffusional resistance to solutes,
and indeed effective diffusivities in calcium alginate and
agarose hydrogels are in the range of 50–100% of the corre-
sponding diffusivities in water (Tziampazis and Sambanis,
1995; Lundberg and Kuchel, 1997). However, with conven-
tional microencapsulation, the volume of the hydrogel con-
tributes signifi cantly to the total volume of the implant. For
example, with a 500-µm microcapsule containing a 300-µm
islet, the polymer volume constitutes 78% of the total capsule
volume. Additionally, conventional microcapsules may not
be appropriate for hepatic portal vein implantation because,
besides their higher implant volume relative to the same
number of naked islets, they may result in higher portal vein
pressure and more incidences of blood coagulation in the
liver. To address this problem, methods have been devel-
oped for islet encapsulation in thin conformal polymeric
coats. Materials that have been used for conformal coating
include photopolymerized poly(ethylene glycol) diacrylate
(Hill et al., 1997; Cruise et al., 1999) and hydroxyethyl
methacrylate-methyl methacrylate (HEMA-MMA) (May

and Sefton, 1999; Sefton et al., 2000).
Encapsulated cell systems can also be fabricated by pre-
forming the permselective membrane in a tubular or disc-
Vascular device
Microcapsules
Membrane chambers
Titanium
housing
Immunobarrier
membranes
Silicone rubber
spacer
Tubular
Planar
Sheet
Vascular device
Microcapsules
Membrane chambers
Titanium
housing
Immunobarrier
membranes
Silicone rubber
spacer
Tubular
Planar
Sheet
FIG. 42.2. Schematics of commonly used
encapsulation devices for insulin-secreting cells.
Vascular devices and membrane chambers of

tubular, planar, or sheet architectures are gener-
ally referred to as macrocapsules, in distinction
from the much smaller microcapsules.
Ch042_P370615.indd 624Ch042_P370615.indd 624 6/1/2007 3:01:16 PM6/1/2007 3:01:16 PM
shaped confi guration, fi lling the construct with a suspension
of islets or other insulin-secreting cells in an appropriate
extracellular matrix, and then sealing the device. This
approach is particularly useful when organic solvents or
other chemicals harsh to the cells are needed for the fabrica-
tion of the membranes. Membrane chambers can be of
tubular or planar geometry (Fig. 42.2). The cells are sur-
rounded by the semipermeable membrane and can be
implanted intraperitoneally, subcutaneously, or at other
sites. Membrane materials used in fabricating these devices
include polyacrylonitrile-polyvinyl chloride (PAN-PVC)
copolymers, polypropylene, polycarbonate, cellulose nitrate,
and polyacrylonitrile-sodium methallylsulfonate (AN69)
(Lanza et al., 1992; Mikos et al., 1994; Prevost et al., 1997;
Delaunay et al., 1998; Sambanis, 2000). Typical values of
device thickness or fi ber diameter are 0.5–1 mm. Advan-
tages of membrane chambers are the relative ease of han-
dling, the fl exibility with regard to the matrix in which the
cells are embedded, and retrievability after implantation. A
major disadvantage is their inferior transport properties,
since the surface-to-volume ratio is smaller than that of
microcapsules and diffusional distances are longer.
Constructs connected to the vasculature via an arterio-
venus shunt consist of a semipermeable tube sur-
rounded by the cell compartment (Fig. 42.2). The tube
is connected to the vasculature, and transport of solutes

between the blood and the cell compartment occurs via
the pores in the tube wall. A distinct advantage of the
vascular device is the improved transport of nutrients
and metabolites, which occurs by both diffusion and con-
vection. However, the major surgery that is needed for
implantation and problems of blood coagulation at the
anastomosis sites have considerably reduced enthusiasm
for these devices.
Other Construct Systems
A common approach for improving the oxygenation of
cells in diffusion chambers is to encourage the formation of
neovasculature around the implant. This is discussed in the
following “In Vivo Implantation” section. Other innovative
approaches that have been proposed include the electro-
chemical generation of oxygen in a device adjacent to a
planar immunobarrier diffusion chamber containing the
insulin-secreting cells (Wu et al., 1999); and the coencapsu-
lation of islets with algae, where the latter produce oxygen
photosynthetically upon illumination (Bloch et al., 2006).
These were in vitro studies, however, and the ability to
translate these approaches to effective in vivo confi gura-
tions remains unknown.
In a different design, Cheng et al. (2004, 2006)
combined constitutive insulin-secreting cells with a glucose-
responsive material in a disc-shaped construct. As indicated
earlier, it is straightforward to genetically engineer non-
β-cells for constitutive insulin secretion; the challenge is
in engineering appropriate cellular responsiveness to
physiologic stimuli. In this proposed device, a concanavalin
A (con A)–glycogen material, sandwiched between two

ultrafi ltration membranes, acted as a control barrier to
insulin release from an adjacent compartment containing
the cells. Specifi cally, con A–glycogen formed a gel at a
low concentration of glucose, which was reversibly con-
verted to sol at a high glucose concentration, as glucose
displaced glycogen from the gel network. Since insulin dif-
fusivity is higher through the sol than through the gel, insu-
lin released by the cells during low-glucose periods
diffused slowly through the gel material; when switched
to high glucose, the insulin accumulated in the cell
compartment during the previous cycle was released at a
faster rate through the sol-state polymer. Overall, this
approach converted the constitutive secretion of insulin
by the cells to a glucose-responsive insulin release by the
device (Cheng et al., 2006). Again, these were in vitro studies,
and the in vivo effi cacy of this approach remains to be
evaluated.
Construct Design and In Vitro Evaluation
Design of three-dimensional encapsulated systems can
be signifi cantly enabled using mathematical models of
solute transport through the tissue and of nutrient
consumption and metabolite production by the cells.
Beyond the microvasculature surrounding the construct,
transport of solutes occurs by diffusion, unless the construct
is placed in a fl ow environment, in which case convective
transport may also occur. Due to its low solubility, transport
of oxygen to the cells is the critical issue. Models can be used
to evaluate the dimensions and the cell density within the
construct so that all cells are suffi ciently nourished and
the capsule as a whole is rapidly responsive to changes in

the surrounding glucose concentration (Tziampazis and
Sambanis, 1995). Experimental and modeling methods for
determining transport properties and reaction kinetics have
been described previously (Sambanis and Tan, 1999).
Furthermore, models can be developed to account for the
cellular reorganization that occurs in constructs with time
as a result of cell growth, death, and possibly migration
processes. Such reorganization is especially signifi cant
when proliferating insulin-secreting cell lines are encapsu-
lated in hydrogel matrices (Stabler et al., 2001; Simpson
et al., 2005; Gross et al., 2007).
Pancreatic tissue substitutes should be evaluated in
vitro prior to implantation, in terms of their ability to support
the cells within over prolonged periods of time and to exhibit
and maintain their overall secretory properties. Long-term
cultures can be performed in perfusion bioreactors under
conditions simulating aspects of the in vivo environment.
In certain studies, the bioreactors and support perfusion
circuits were made compatible with a nuclear magnetic
resonance spectrometer. This allowed measuring intracel-
lular metabolites, such as nucleotide triphosphates, as a
function of culture conditions and time, without the need
III. CONSTRUCT TECHNOLOGY • 625
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626 CHAPTER FORTY-TWO • BIOARTIFICIAL PANCREAS
to extract the encapsulated cells (Papas et al., 1999a, 1999b).
Such studies produce a comprehensive understanding of
the intrinsic tissue function in a well defi ned and controlled
environment prior to introducing the additional complexity
of host–implant interactions in in vivo experiments.

The secretory properties of tissue constructs can be
evaluated with low time resolution in simple static culture
experiments by changing the concentration of glucose in
the medium and measuring the secreted insulin. In general,
a square wave of insulin concentration is implemented,
from basal to inducing basal conditions for insulin secre-
tion. To evaluate the secretory response with a higher time
resolution, perfusion experiments need to be performed,
in which medium is fl owed around the tissue and secreted
insulin is assayed in the effl uent. Again, a square wave
of glucose concentration is generally implemented. By com-
paring the secretory dynamics of free and encapsulated
cells, one can ensure that the encapsulation material
does not introduce excessive time lags that might compro-
mise the secretory properties of the construct. Indeed,
properly designed hydrogel microcapsules introduce only
minimal secretory time lags (Tziampazis and Sambanis,
1995; Sambanis et al., 2002).
Manufacturing Considerations
Fabrication of pancreatic substitutes of consistent
quality requires the use of cells that are also of consistent
quality. Although with clonal, expandable cells this is a
rather straightforward issue, with islets isolated from human
and animal tissues there can be signifi cant variability in the
quantity and quality of the cells in the preparations. With
islets from cadaveric human donors, the quality of the iso-
lates is assessed by microscopic observation, viability stain-
ing, and possibly a static insulin secretion test. It is generally
recognized, however, that a quantitative, objective assess-
ment of islet quality would help improve the consistency of

the preparations and thus, possibly, the transplantation
outcome.
It is conceivable that encapsulated cell systems could
be fabricated at a central location from which they are dis-
tributed to clinical facilities for implantation. In this scheme,
preservation of the constructs for long-term storage, inven-
tory management, and, importantly, sterility control would
be essential. Cryopreservation appears to be a promising
method for maintaining fabricated constructs for prolonged
time periods. Although there have been signifi cant studies
on the cryopreservation of single cells and some tissues, the
problems pertaining to cryopreserving artifi cial tissues are
only beginning to be addressed. Cryopreservation of mac-
roencapsulated systems is expected to be particularly chal-
lenging and has not been reported in the literature. However,
βTC-cells encapsulated in alginate beads have been pre-
served successfully (Mukherjee et al., 2005; Song et al., 2005).
An especially promising approach involves using high
concentrations of cryoprotective agents so that water is
converted to a glassy, or vitrifi ed, state at low temperatures;
the absence of ice crystals in both the intracellular and
extracellular domains appears helpful in maintaining not
only cellular viability but also the structure and function of
the surrounding matrix (Mukherjee et al., 2005; Song et al.,
2005).
IV. IN VIVO IMPLANTATION
This section highlights results from in vivo experiments
using the different confi gurations outlined earlier. Results
with encapsulated cell systems are presented fi rst. Since
in vivo experiments with non-β-cells engineered for insulin

secretion are at present based mostly on in vivo gene therapy
approaches, these are described next. Technologies for the
in vivo monitoring of cells and constructs and the issue of
implant retrievability are then discussed.
Encapsulated Cell Systems
In vivo experiments with pancreatic substitutes are
numerous in small animals, limited in large animals, and
few in humans. Allogeneic and xenogeneic islets in hydrogel
microcapsules implanted in diabetic mice and rats have
generally restored normoglycemia for prolonged periods of
time. In the early study of O’Shea et al. (1984), islet allografts
encapsulated in APA membranes were implanted intraperi-
toneally in streptozotocin-induced diabetic rats. Of the fi ve
animals that received transplants, three remained normo-
glycemic for more than 100 days. One of these three animals
remained normoglycemic 368 days postimplantation. In the
later study of Lum et al. (1992), rat islets encapsulated in APA
membranes and implanted in streptozotocin diabetic mice
restored normoglycemia for up to 308 days, with a mean
xenograft survival time of 220 days. With all recipients, nor-
moglycemia was restored within two days postimplanta-
tion. Control animals receiving single injections of
unencapsulated islets remained normoglycemic for less
than two weeks (O’Shea et al., 1984; Lum et al., 1992). More
recently, APA-encapsulated βTC6-F7 insulinomas restored
normoglycemia in diabetic rats for up to 60 days (Mamujee
et al., 1997), and APA-encapsulated βTC-tet insulinomas in
NOD mice for at least eight weeks (Black et al., 2006). In the
latter study, it was also observed that no host cell adherence
occurred to microcapsules, and there were no signifi cant

immune responses to the implant, with cytokine levels
being similar to those of sham-operated controls. These
results are thus indicative of the potential use of an immu-
noisolated continuous β-cell line for the treatment of diabe-
tes. With the recently developed human cell line (Narushima
et al., 2005), experiments were performed with unencapsu-
lated cells transplanted into streptozotocin-induced dia-
betic severe combined immunodefi ciency mice. Control of
blood glucose levels started within two weeks postimplanta-
tion, and mice remained normoglycemic for longer than 30
weeks (Narushima et al., 2005). Besides rodents, long-term
restoration of normoglycemia with microencapsulated islets
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has been demonstrated in large animals, including sponta-
neously diabetic dogs, where normoglycemia was achieved
with canine islet allografts for up to 172 days (Soon-Shiong
et al., 1992), and monkeys, where in one animal porcine islet
xenografts normalized hyperglycemia for more than 150
days (Sun et al., 1992). More recently, one of the companies
working on islet encapsulation technology announced that
primate subjects in ongoing studies have continued to
exhibit improved glycemic regulation over a six-month
period after receiving microencapsulated porcine islet
transplants (MicroIslet Inc. Press Release, August 7, 2006).
In vivo results with vascular devices are reportedly
mixed. Implantation of devices containing allogeneic islets
as arteriovenous shunts in pancreatectomized dogs resulted
in 20–50% of the dogs becoming normoglycemic up to 10
weeks postimplantation without exogenous insulin admin-
istration. When xenogeneic bovine or porcine islets were

used, only 10% of the dogs remained normoglycemic 10
weeks postimplantation. All dogs were reported diabetic or
dead after 15 weeks (Sullivan et al., 1991). Recently, a hollow-
fi ber device composed of polyethylene-vinyl alcohol fi bers
and a poly-amino-urethane-coated, nonwoven polytetra-
fl uoroethylene fabric seeded with porcine islets provided
normalization of the blood glucose levels in totally pancre-
atectomized pigs when connected to the vasculature of the
animals (Ikeda et al., 2006). It should be noted, however, that
the overall interest in vascular devices has faded, due to the
surgical and blood coagulation challenges they pose.
Although several hypotheses exist, the precise cause of
the eventual in vivo failure of encapsulated cell systems
remains unclear. Encapsulation does not completely prevent
the immune recognition of the implant. Although direct cel-
lular recognition is prevented, antigens shed by the cells as
a result of secretion and, more importantly, lysis in the cap-
sules eventually pass through the permselective barrier and
are recognized by the antigen-presenting cells of the host.
For example, in one study, antibodies against islets in a
tubular diffusion chamber were detected in plasma two to
six weeks postimplantation, suggesting that islet antigens
crossed the membrane and stimulated antibody formation
in the host (Lanza et al., 1994). In another study, alginate-
encapsulated islets were lysed in vitro by nitric oxide pro-
duced by activated macrophages (Wiegand et al., 1993).
Passage of low-molecular-weight molecules cannot be
prevented by immunoprotective membranes imposing a
molecular weight cutoff on the order of 50 kDa. It should be
noted that in one human study involving encapsulated allo-

geneic islets, the patient had to be provided with low levels
of immunosuppression (Soon-Shiong et al., 1994). In a more
recent report, also with encapsulated allografts implanted
peritoneally, type 1 diabetic patients remained nonimmu-
nosuppressed but were unable to withdraw exogenous
insulin (Calafi ore et al., 2006).
Nonspecifi c infl ammation may also occur around the
implant and develop into a fi brous capsule, reducing the
oxygen available to the cells within. The fi brotic layer has
been found to consist of several layers of fi broblasts and
collagen with polymorphonuclear leukocytes, macrophages,
and lymphocytes. The surface roughness of the membrane
may also trigger infl ammatory responses. In one study,
membranes with smooth outside surfaces exhibited a
minimal fi brotic reaction 10 weeks postimplantation,
regardless of the type of encapsulated cells, whereas rough
surfaces elicited a fi brotic response even one week postim-
plantation (Lanza et al., 1991). Use of high-purity materials
also helps to minimize infl ammatory reactions. If a material
is intrinsically infl ammatory, such as poly-l-lysine, it can be
coated with a layer of noninfl ammatory material, such as
alginate, to minimize the host’s reaction. Such coverage
may not be suffi ciently permanent, though, resulting in the
eventual fi brosis of the implant. Indeed, several investi-
gators report improved results with plain alginate beads
without a poly-l-lysine layer, especially when allogeneic
cells are used in the capsules.
Provision of nutrients to and removal of metabolites
from encapsulated cells can be especially challenging
in vivo. Normal pancreatic islets are highly vascularized

and thus well oxygenated. There exists evidence that
unencapsulated islets injected in the portal system of the
liver become revascularized, which enhances their engraft-
ment and function. On the other hand, encapsulation
prevents revascularization, so the implanted tissue is nour-
ished by diffusion alone. Promotion of vascularization
around the immunoprotective membrane increases the
oxygenation of the implanted islets (Prokop et al., 2001).
Interestingly, transformed cells, such as the βTC3 line of
mouse insulinomas, are more tolerant of hypoxic conditions
than intact islets; such cells may thus function better
than islets in implanted capsules (Papas et al., 1996).
However, with transformed cells, too, enhanced oxygen-
ation increases the density of functional cells that can be
effectively maintained within the implant volume. Vascular-
ization is dependent on the microarchitecture of the mate-
rial, which should have pores 0.8–8.0 µm in size, allowing
permeation of host endothelial cells (Brauker et al., 1992,
1995). Vascularization is also enhanced by the delivery of
angiogenic agents, such as FGF-2 and VEGF, possibly with
controlled-release devices (Sakurai et al., 2003). Although
vascularization can be promoted around a cell-seeded
device, improved success has been reported if a cell-free
device is fi rst implanted and vascularized and the cells are
then introduced. One example of this procedure involved
placing a cylindrical stainless steel mesh in the subcutane-
ous space of rats, with the islets introduced 40 days later
(Pileggi et al., 2006). Replacement of a vascularized implant
is challenging, however, due to the bleeding that occurs. A
solution to this problem may entail the design of a device

that can be emptied and refi lled with a suspension of cells
in an extracellular matrix without disturbing the housing
and the associated vascular network.
IV. IN VIVO IMPLANTATION • 627
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628 CHAPTER FORTY-TWO • BIOARTIFICIAL PANCREAS
Gene and Cell-Based Therapies
In vivo effi cacy studies with gene therapy and non-β-
cells genetically engineered for insulin secretion are gener-
ally limited to small animals. Intraportal injection of
recombinant adenovirus expressing furin-compatible
insulin under the control of a glucose-responsive promoter
containing elements of the rat liver pyruvate kinase gene
restored near-normal glycemia in streptozotocin diabetic
rats for periods of 1–12 weeks (Thule and Liu, 2000). With
hepatic delivery of a recombinant adeno-associated virus
expressing a single-chain insulin analog under the control
of an l-type pyruvate kinase promoter, Lee and coworkers
(2000) controlled blood glucose levels in streptozotocin dia-
betic rats and NOD mice for periods longer than 20 weeks.
However, transiently low blood glucose levels observed
three to fi ve hours after glucose loading indicated a draw-
back of the transcriptional regulation of insulin expression,
which may result in hypoglycemic episodes (Lee et al., 2000).
Possible approaches toward ameliorating this problem
include optimizing the number of glucose-regulatory and
insulin-sensing elements in the promoter (Jun and Yoon,
2005) and destabilizing the preproinsulin mRNA; the latter
has been shown to expedite signifi cantly the down-regula-
tion of secretion dynamics from transcriptionally controlled

cells on removal of the secretory stimulus (Tang and Sam-
banis, 2003).
In vivo gene therapy with small animals has also shown
success when the target cells for insulin expression were
intestinal endocrine K- or gastric G-cells. Using a transgene
expressing human insulin under the control of the glucose-
dependent insulinotropic peptide (GIP) promoter, Cheung
et al. (2000) expressed insulin specifi cally in gut K-cells of
transgenic mice, which protected them from developing
diabetes following STZ-mediated destruction of the native
β-cells. Similarly, use of a tissue-specifi c promoter to express
insulin in gastric G-cells of mice resulted in insulin release
into circulation in response to meal-associated stimuli, sug-
gesting that G-cell insulin expression is benefi cial in the
amelioration of diabetes (Lu et al., 2005). Translation of
these approaches to adult animals and, eventually, humans,
requires the development of effective methods of gene
delivery to intestinal endocrine or gastric cells in vivo or the
development of effective ex vivo gene therapy approaches.
In Vivo Monitoring
Monitoring of the number and function of insulin-
secreting cells in vivo would provide valuable information
directly on the implant and possibly offer early indications
of implant failure. Additionally, in animal experiments, the
ability to monitor an implant noninvasively reduces the
number of animals that are needed in the experimental
design and helps establish a critical link between implanta-
tion and endpoint physiologic effects, the latter commonly
being blood glucose levels and animal weight.
Imaging techniques can provide unique insight into the

structure/function relationship of a construct in vitro and
in vivo. There are several imaging modalities that have been
applied to monitor tissue-engineered constructs, including
computed tomography (CT), positron emission tomogra-
phy (PET), optical techniques, and nuclear magnetic reso-
nance (NMR) imaging and spectroscopy. Among these,
NMR offers the unique advantage of providing information
on both construct integrity and function, without the need
to modify the cells genetically (e.g., through the expression
of green fl uorescent protein, used in optical methods) or
the introduction of radioactive labels (e.g., PET agents).
Furthermore, since magnetic fi elds penetrate uniformly
throughout the sample, NMR is ideally suited to monitor
constructs implanted at deep-seated locations. Its disad-
vantage is its low sensitivity. Whereas optical and radionu-
clide techniques can detect tracer quantities, NMR detects
metabolites that are available in the millimolar or, in some
cases, submillimolar range.
The ability to monitor noninvasively in vivo a pancreatic
substitute by NMR was reported recently (Stabler et al.,
2005). Agarose disc-shaped constructs containing mouse
insulinoma βTC3-cells were implanted in the peritoneal
cavity of mice. Construct integrity was visualized by MR
imaging and the metabolic activity of the cells within by
water-suppressed
1
H NMR spectroscopy (Fig. 42.3). Control
experiments established that the total choline (TCho) reso-
nance at 3.2 ppm, which is attributed to three choline
metabolites, correlated positively and linearly with the

number of viable cells within the construct, measured with
an independent assay. To obtain the TCho signal in vivo
without interference from the surrounding host tissue, such
as peritoneal fat, the central agarose disc containing the cells
had to be surrounded by cell-free agarose buffer zones. This
ensured that the MR signal arose only from the implanted
cells, even as the construct moved due to animal breathing.
A second problem that had to be resolved was that glucose
diffusing into the construct produced a resonance that inter-
fered with the TCho resonance at 3.2 ppm. For this, a unique
glucose resonance at 3.85 ppm was used to correct for the
interference at 3.2 ppm so that a corrected signal, uniquely
attributed to TCho, could be obtained. The latter correlated
positively and linearly with the number of viable cells, mea-
sured with an independent assay, on the constructs postex-
plantation (Fig. 42.3). Hence, with the appropriate implanted
construct architecture and signal processing, the number of
viable cells in an implant could be followed in the same
animal as a function of time (Stabler et al., 2005).
Labeling of cells with magnetic nanoparticles, which
can be detected by magnetic resonance, and genetically
engineering cells so that they express a fl uorescent or lumi-
nescent marker that can be optically detected are other
methods being pursued to track the location and possibly
function of implanted cells in vivo. It is expected that devel-
opment of robust monitoring methodologies will be helpful
Ch042_P370615.indd 628Ch042_P370615.indd 628 6/1/2007 3:01:17 PM6/1/2007 3:01:17 PM
not only in experimental development studies but also in
eventual clinical applications.
Retrievability

The issue of construct retrievability needs to be consid-
ered for all pertinent applications. Useful lifetimes of con-
structs are limited, so repeated implantation of cells will be
required. It is as yet unclear whether retrieval of constructs
will be necessary at the end of their useful lives. Long-term
studies on the safety challenges posed by accumulated
implants in the host need to be carried out to address this
question.
V. CONCLUDING REMARKS
Tight glycemic regulation in insulin-dependent diabet-
ics signifi cantly improves their overall health and reduces
the long-term complications of the disease. A pancreatic
substitute holds signifi cant promise at accomplishing this
in a relatively noninvasive way. However, to justify the
improved outcome, a substitute needs to be not only effi ca-
cious in terms of insulin secretion, but also immunologi-
cally acceptable. A number of approaches are being pursued
to address this obstacle and additionally develop constructs
that can be manufactured at a clinically relevant scale.
However, as the problems are being thoroughly investigated,
their solutions become more challenging. Encapsulation in
permselective barriers improves the immune acceptance of
allo- and xenografts, but it is doubtful that encapsulation
will, by itself, ensure the long-term survival and function of
the implant in nonimmunosuppressed hosts. The develop-
ment of specifi c, benign immune suppression protocols
that work in concert with encapsulation appears necessary.
Reducing the immunogenicity of the implanted cells and
modifying them so that they better withstand the encapsu-
lation and in vivo environment are appropriate strategies.

To ensure that substitutes can be fabricated at the necessary
scale, methods to expand pancreatic islets in culture, to
produce β-cells from stem cells, or to generate expandable
β-cell lines with appropriate phenotypic characteristics
need to be pursued. In alternative approaches involving
gene therapy of non-β-cells, or the ex vivo engineering of
non-β-cells retrieved surgically from the host, the major
problem is not that of cell procurement or immune accep-
tance but, rather, of ensuring precise regulation of insulin
V. CONCLUDING REMARKS • 629
Surface Coil
y = 2244x – 24
R
2
= 0.87
0
500
1000
1500
2000
2500
3000
00.20.40.60.811.2
MTT Absorbance
In Vivo TCho
Glucose-corrected
C
Surface Coil
y = 2244x – 24
R

2
= 0.87
0
500
1000
1500
2000
2500
3000
00.20.40.60.811.2
MTT Absorbance
In Vivo TCho
Glucose-corrected
A
TCho
Glucose
Lactate
B
TCho
Glucose
Lactate
FIG. 42.3. Magnetic resonance imaging and localized spectroscopy of a disc-shaped agarose construct containing βTC3 mouse insulinoma cells at an
initial density of 7 × 10
7
cells/mL agarose implanted in the peritoneal cavity of a mouse. (A)
1
H NMR image obtained with a surface coil. The inner disc,
containing the cells, is distinguishable from the surrounding cell-free buffer zone, implemented to exclude spectroscopic signal from the surrounding host
tissue. (B) Localized, water-suppressed
1

H NMR spectrum from the cells contained in the inner disc. Resonances due to total choline (TCho), glucose, and
lactate are clearly visible. The time needed to collect the spectrum was 13 min. (C) Correlation between the glucose-corrected TCho resonance at 3.2 ppm
and the viable cell number obtained postexplantation using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay. Adapted from
Stabler et al. (2005).
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630 CHAPTER FORTY-TWO • BIOARTIFICIAL PANCREAS
secretion by glucose or other physiologic stimuli. This poses
a different set of problems, which, however, are equally
challenging to those of β-cell procurement and immune
acceptance. Methods for the preservation of substitutes and
for the noninvasive monitoring of their integrity and func-
tionality in vivo are integral parts of construct development
and characterization with regard to construct manufactur-
ing and assessment of in vivo effi cacy, respectively.
As in many aspects of life, with challenges come oppor-
tunities. It is essential that multiple approaches be pursued
in parallel, because it is currently unclear which ones will
eventually develop into viable therapeutic procedures. If
more than one method evolves into a clinical application,
this would be welcome news, because it may allow fl exibility
in the personalization of therapy. For instance, in an adult
type 2 insulin-dependent diabetic, use of an encapsulated
allograft with low-level immunosuppression might consti-
tute an appropriate therapeutic modality. In a juvenile type
1 diabetic with aggressive autoimmunity, however, use of
autologous genetically engineered non-β-cells, which are
not recognized by the resident autoimmunity, may consti-
tute the therapeutic method of choice.
VI. ACKNOWLEDGMENTS
The studies in the author’s and coinvestigators’ laboratories

referenced in this chapter were supported by grants from
the Georgia Tech/Emory Center for the Engineering of Living
Tissues (GTEC), a National Science Foundation Engineering
Research Center; and by grants from the National Institutes
of Health, EmTech Bio, and the Juvenile Diabetes Research
Foundation international. The author also wishes to thank
Indra Neil Mukherjee and Heather Bara for critically review-
ing the manuscript, as well as Drs. Constantinidis and
Thulé for helpful discussions.
VII. REFERENCES
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Black, S. P., Constantinidis, I., Cui, H., Tucker-Burden, C., Weber, C. J.,
and Safl ey, S. A. (2006). Immune responses to an encapsulated alloge-
neic islet beta-cell line in diabetic NOD mice. Biochem. Biophys. Res.
Commun. 340, 236–243.
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I. Introduction
II. Engineering to Generate Insulin-Producing
Cells
III. Engineering to Improve Islet Survival
IV. Vectors for Engineering Islets and Beta-Cells
V. Conclusion
VI. References
Engineering Pancreatic Beta-Cells
Hee-Sook Jun and Ji-Won Yoon
Principles of Tissue Engineering, 3
rd
Edition
ed. by Lanza, Langer, and Vacanti
Copyright © 2007, Elsevier, Inc.
All rights reserved.
I. INTRODUCTION
The use of islet transplantation as a treatment for dia-
betes has been hampered by the limited availability of
human islets; therefore, new sources of insulin-producing
cells are needed. Expansion of beta-cells by the generation
of reversibly immortalized beta-cells and creation of insulin-
producing cells by exogenous expression of insulin in

non-beta-cells have been investigated as new sources of
beta-cells. Recently, embryonic and adult stem cells or pan-
creatic progenitor cells have been engineered to differenti-
ate into insulin-producing cells, demonstrating the possible
use of these cells for beta-cell replacement. Despite signifi -
cant progress, further studies are needed to generate truly
functional insulin-producing cells. In addition, the engi-
neering of beta-cells to protect them from immune attack
and to improve viability has been tried. Although the useful-
ness of engineered beta-cells has yet to be clinically proven,
studies utilizing different engineering strategies and careful
analysis of the resulting insulin-producing cells may offer
potential methods to cure diabetes.
Diabetes mellitus is a metabolic disease characterized
by uncontrolled hyperglycemia, which results in long-term
clinical problems, including retinopathy, neuropathy,
nephropathy, and heart disease. Diabetes affects over 150
million people worldwide and is considered an epidemic of
the 21st century. Blood glucose homeostasis is controlled by
endocrine beta-cells, located in the islets of Langerhans in
the pancreas. When the concentration of blood glucose rises
after a meal, insulin is produced and released from beta-
cells. Insulin then induces glucose uptake by cells in the
body and converts glucose to glycogen in the liver. When
blood glucose concentration becomes low, glycogen is
broken down to glucose in the liver and glucose is released
into the blood.
There are two major forms of diabetes: type 1 diabetes,
also known as insulin-dependent diabetes mellitus, and
type 2 diabetes, also known as non-insulin-dependent dia-

betes mellitus. Both types are thought to result from a reduc-
tion in the number of insulin-producing beta-cells and
defi cits in beta-cell function. In type 1 diabetes, beta-cells
are destroyed by autoimmune responses, resulting in a lack
of insulin (reviewed in Adorini et al., 2002; Yoon and Jun,
2005). In type 2 diabetes, both inadequate beta-cell function
and insulin resistance of peripheral tissues contribute to the
development of hyperglycemia, leading to eventual reduc-
tion in the number of beta-cells (reviewed in LeRoith, 2002).
Intensive exogenous insulin therapy has been used for the
treatment of type 1 diabetes, but it does not restore the tight
control of blood glucose levels or completely prevent the
development of complications. In addition, multiple daily
injections are cumbersome and sometimes cause poten-
tially life-threatening hypoglycemia. Islet transplantation
has been considered an alternative and safe method for the
treatment of diabetes (reviewed in Hatipoglu et al., 2005).
Chapter Forty-Three
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636 CHAPTER FORTY-THREE • ENGINEERING PANCREATIC BETA-CELLS
With the improvement of islet isolation techniques, the
success rate for independence from exogenous insulin is
increasing. However, the lack of suffi cient islets to meet the
demands of patients and the side effects of immunosup-
pressive drugs that are required to prevent alloimmune and
autoimmune attack against islet grafts are the major limita-
tions of islet transplantation. Therefore, various alternative
sources of insulin-producing cells are being investigated to
provide a suffi cient supply for the treatment of type 1
diabetes.

In this chapter, we discuss the use of cell engineering to
produce and expand insulin-producing beta-cells; to create
insulin-producing cells from non-beta-cells, embryonic
stem cells, and adult stem cells; and to improve islet graft
survival. Due to the publisher’s restrictions, we are unable
to cite all the references for primary data.
II. ENGINEERING TO GENERATE
INSULIN-PRODUCING CELLS
Engineering Pancreatic Beta-Cells
The pancreas is composed of endocrine and exocrine
tissues. The endocrine pancreas occupies less than 5% of
the pancreatic tissue mass and is composed of cell clusters
called the islets of Langerhans. The islets of Langerhans
contain insulin-producing beta-cells (about 80% of cells in
the islets), glucagon-producing alpha-cells, somatostatin-
producing delta-cells, and pancreatic polypeptide-produc-
ing cells. The exocrine pancreas occupies more than
95% of the pancreas and is composed of ascinar and
ductal cells, which produce digestive enzymes. The beta-cell
mass is dynamic and increases in response to environmen-
tal changes such as pancreatic injury and physiological
changes such as insulin resistance. In addition, mature
beta-cells can replicate throughout life, although at a low
level.
One approach to produce beta-cells for replacement
therapy is to expand mature beta-cells in vitro. However,
because mature beta-cells have limited proliferative capac-
ity in culture, the expression of oncogenes has been tried as
a method to establish beta-cell lines. The expression of
simian virus (SV) 40 large T antigen in beta-cells under the

control of the tet-on and tet-off regulatory system in trans-
genic mice resulted in a stable beta-cell line that could be
expanded in vitro. These cells produced less insulin in the
transformed state when T antigen was expressed, but insulin
production increased after growth was arrested by cessation
of T antigen expression, and insulin secretion was regulated
as in normal mouse islets. When these cells were trans-
planted into streptozotocin-induced diabetic mice, the mice
became normoglycemic, and normoglycemia was main-
tained for a prolonged time, without any treatment to
prevent oncogene expression (Milo-Landesman et al., 2001).
In addition to beta-cell expansion, cell engineering has
been used to improve beta-cell function. Rat insulinoma
cells that showed decreased glucose-responsive insulin
secretion were transfected with a plasmid encoding a
mutated form of GLP-1 that is resistant to the degrading
enzyme dipeptidyl-peptidase IV. These engineered cells had
increased insulin secretion in response to glucose, as com-
pared with untransfected control cells (Islam et al., 2005).
Expansion of human primary pancreatic islet cells has
also been tried. Primary adult islet cells could be stimulated
to divide when grown on an extracellular matrix in the pres-
ence of hepatocyte growth factor/scatter factor, but growth
was arrested after 10–15 cell divisions, due to cellular senes-
cence (Beattie et al., 1999). Transformation of adult human
pancreatic islets with a retroviral vector expressing SV40
large T antigen and H-ras
Val

12

oncogenes resulted in extended
life span, but eventually the cells entered a crisis phase fol-
lowed by altered morphology, lack of proliferation, and cell
death, suggesting that immortalization of human beta-cells
is more diffi cult than that of rodent beta-cells. However,
introduction of human telomerase reverse transcriptase
(hTERT) resulted in successful immortalization (Halvorsen
et al., 1999), because human cells do not express telomerase.
This immortalized cell line, βlox5, initially expressed low
levels of insulin, but insulin production subsequently fell to
undetectable levels as a result of the loss of expression of key
insulin gene transcription factors. A combination of the
introduction of a beta-cell transcription factor (Pdx-1),
treatment with exendin-4 (a glucagon-like peptide-1 [GLP-
1] homolog), and cell–cell contact was required to recover
beta-cell differentiated function and glucose-responsive
insulin production (de la Tour et al., 2001). However, Pdx-1
expression in this cell line resulted in a signifi cant decrease
in the growth rate of the cells. When streptozotocin-induced
diabetic animals were transplanted with these Pdx-1-
expressing cells, substantial levels of circulating human C-
peptide were detected and diabetes was remitted. However,
10% of the animals developed tumors, even though the
oncogenes and hTERT gene had been fl oxed by loxP sites so
that they could be deleted by expression of Cre recombi-
nase. This suggests that the Cre-expressing adenovirus and/
or Cre-mediated deletion of the oncogenes was ineffi cient
(de la Tour et al., 2001).
The limitations of previously engineered beta-cell
lines point to a need for a human beta-cell line that is func-

tionally equivalent to primary beta-cells, can be expanded
indefi nitely, and can be rendered nontumorigenic. In
another approach to establish a reversibly immortalized
human beta-cell line, human islets were transduced with a
combination of retroviral vectors expressing SV40 T antigen,
hTERT, and enhanced green fl uorescent protein to immor-
talize and mark terminally differentiated pancreatic beta-
cells. These genes were fl oxed by loxP sites to allow excision
of the immortalizing genes. Among 271 clones screened for
tumorigenicity, 253 clones were selected for further study,
and only one of these (NAKT-15) expressed insulin and the
necessary beta-cell transcription factors, such as Isl-1, Pax-
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II. ENGINEERING TO GENERATE INSULIN-PRODUCING CELLS • 637
6, Nkx6.1, Pdx-1, prohormone convertases, and secretory
granule proteins. Addition of factors that enhance insulin
expression and secretion during culture of the beta-cell line,
such as troglitazone, a peroxisome proliferator-activated
receptor-γ activator, and nicotinamide, helped to maintain
the function of beta-cells, and culture of these cells on
Matrigel matrix facilitated aggregate formation. Removal of
the immortalizing genes by Cre recombinase expression
stopped cell proliferation and increased the expression of
beta-cell-specifi c transcription factors, resulting in rever-
sion of the cells. These reverted NAKT-15 cells were func-
tionally similar to normal human islets with respect to
insulin secretion in response to glucose and nonglucose
secretagogues, although the insulin content and amount of
secreted insulin were lower than for human islets. However,
NAKT-15 cells were able to remit diabetes and clear exoge-

nous glucose when transplanted into diabetic severe com-
bined immunodefi ciency (SCID) mice. The insulin content
of these cells was higher in vivo than in vitro, suggesting that
the microenvironment may enhance cellular differentiation
(Narushima et al., 2005).
For clinical application of reversibly immortalized
human beta-cells, safety issues, particularly tumorigenicity,
should be considered. Reducing or eliminating tumorige-
nicity may be possible by using multiple selection proce-
dures. In the case of NAKT-15 cells, nontumorigenic clones
were fi rst selected by screening for tumor formation in SCID
mice. After infection of Cre-expressing adenovirus to re-
move the SV40 T antigen and hTERT, SV40T-negative cells
were selected in the presence of a neomycin analog (the
neomycin-resistance gene was positioned to be expressed
after the loxP-fl anked genes were deleted), and hTERT-neg-
ative cells were selected by purifi cation of enhanced green
fl uorescent protein-negative cells. Finally, SV40T/hTERT-
negative cells were selected by the addition of ganciclover,
because the cells had been transduced with a suicide gene,
herpes simplex thymidine kinase, which renders them
susceptible to ganciclover. These multiple selection pro-
cedures resulted in no tumor development in SCID mice
when reverted NAKT-15 cells were transplanted (Narushima
et al., 2005), although the possibility of tumorigenesis could
not be completely eliminated. Nevertheless, there are advan-
tages of reversibly immortalized human beta-cells as com-
pared with primary beta-cells. They can be easily expanded
to obtain suffi cient cells for transplantation and genetically
manipulated in vitro prior to transplantation, for example,

to confer resistance to immune attack.
Establishment of insulin-producing beta-cell lines by
reversible immortalization of primary islets is a promising
approach for replacing insulin injections, for a beta-cell line
can provide an abundant source of beta-cells for transplan-
tation. In addition, beta-cell lines can be genetically mani-
pulated to improve their function and survival. However, the
functionality of the cell lines and safety issues remain to be
further studied.
Engineering Surrogate Beta-Cells
Non-beta-cells that are genetically engineered to
produce insulin may have an advantage over intact islets or
engineered beta-cells for transplantation therapy, because
non-beta-cells should not be recognized by beta-cell-
specifi c autoimmune responses. Pancreatic beta-cells have
unique characteristics specifi c to the production of insulin,
such as specifi c peptidases, glucose-sensing systems, and
secretory granules that can release insulin promptly by
exocytosis in response to extracellular glucose levels. There-
fore, the ideal target cell to engineer for insulin production
would be non-beta-cells possessing similar characteristics.
A variety of cell types, including fi broblasts, hepatocytes,
neuroendocrine cells, and muscle cells, have been engi-
neered to produce insulin, with varying degrees of success
(reviewed in Xu et al., 2003; Yoon and Jun, 2002).
Neuroendocrine cells have received considerable atten-
tion because they have characteristics similar to those of
beta-cells and contain components of the regulated secre-
tory pathway, including prohormone convertases 2 and 3
and secretory granules. A mouse corticotrophic cell line

derived from the anterior pituitary, AtT20, expressed active
insulin after transfection with the insulin gene under the
control of a viral or metallothionein promoter but lacked
glucose responsiveness. Cotransfection of genes encoding
glucose transporter (GLUT)2 and glucokinase conferred
glucose-responsive insulin secretion in insulin-expressing
AtT20 cells. Transgenic expression of insulin in the interme-
diate lobe of the pituitary of nonobese diabetic (NOD) mice
under the control of the pro-opiomelanocortin promoter
resulted in the production of biologically active insulin.
Transplantation of this insulin-producing pituitary tissue
into diabetic NOD mice restored normoglycemia, but insulin
secretion was not properly regulated by glucose. Engineer-
ing primary rat pituitary cells to coexpress GLP-1 receptor
and human insulin resulted in GLP-1-induced insulin secre-
tion (Wu et al., 2003).
Intestinal K-cells have been explored as possible surro-
gate beta-cells. K-cells are endocrine cells located in the gut
that secrete the hormone glucose-dependent insulinotropic
polypeptide (GIP), which facilitates insulin release after a
meal. K-cells are also glucose responsive, have exocytotic
mechanisms, and contain the necessary enzymes for pro-
cessing proinsulin to insulin. A murine intestinal cell line
containing K-cells transfected with human insulin DNA
cloned under the control of the GIP promoter produced bio-
logically active insulin in response to glucose, and transgenic
mice expressing the human proinsulin gene under the GIP
promoter were protected from diabetes after treatment with
streptozotocin (Cheung et al., 2000). These results suggest
that K-cells may have great potential as surrogate beta-cells.

The strategy of engineering hepatocytes to produce
insulin has been widely studied (Nett et al., 2003). Hepato-
cytes have advantages for engineering as insulin-producing
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638 CHAPTER FORTY-THREE • ENGINEERING PANCREATIC BETA-CELLS
cells because they express components of a glucose-sensing
system somewhat similar to that in pancreatic beta-cells,
such as GLUT2 and glucokinase. In addition, there are
several hepatocyte-specifi c gene promoters that respond to
changes in glucose concentrations. The L-type pyruvate
kinase promoter (LPK) and Spot14 promoter have been
investigated as regulatory elements for glucose-responsive
insulin production in liver. Using a chimeric promoter com-
posed of three copies of the stimulatory glucose-responsive
element from the LPK promoter and an inhibitory respon-
sive element from the insulin-like growth factor–binding
protein-1 basal promoter, the expression of a modifi ed
human proinsulin gene was stimulated by glucose and
inhibited by insulin in hepatocytes. Engineering of rat hepa-
toma cells to express insulin under the control of the glucose-
6-phosphatase promoter resulted in the stimulation of
insulin production by glucose and self-limitation by insulin.
However, insulin expression by the glucose-6-phosphatase
promoter was low because of negative feedback by the pro-
duced insulin. It was recently reported that human hepa-
toma cells transduced with a furin-cleavable human
preproinsulin gene under the control of the GLUT2 pro-
moter expressed insulin in response to glucose (Burkhardt
et al., 2005).
A drawback for the regulation of insulin production

by glucose-responsive promoters in hepatocytes is slow as
compared with the rapid release by exocytosis from beta-
cells. Because a longer period of time is required for
transcriptional regulation to change the plasma levels of
insulin in response to changes in blood glucose, hypoglyce-
mia may occur. Therefore, the development of systems that
mimic insulin secretory dynamics is required. Strategies
that utilize synthetic promoters composed of multiple
copies of glucose-responsive elements for the induction of
high levels of insulin expression, insulin-sensitive elements
for feedback regulation, and methods to control of the half-
life of insulin mRNA so that it rapidly degrades may make
it possible to mimic insulin production in a glucose-
responsive fashion in non-beta-cells.
Another consideration is that most non-beta-cells do
not have the appropriate endoproteases to convert proinsu-
lin to insulin or secretory granules from which insulin can
be rapidly released in response to physiological stimuli. One
approach is the mutation of the proinsulin gene so that it
can be cleaved and converted to insulin by the protease
furin, which is expressed in a wide variety of cells. Another
approach is the development of a single-chain insulin
analog, which shows insulin activity without the require-
ment for processing. Artifi cially regulated insulin secretion
in non-beta-cells has been tried by expressing insulin as a
fusion protein containing an aggregation domain, which
accumulates in the endoplasmic reticulum and is secreted
when a drug that induces disaggregation is administered.
Although engineering somatic non-beta-cells to pro-
duce insulin is a very attractive method, no method has yet

succeeded in imitating normal beta-cells regarding the
rapid and tight regulation of glucose within a narrow physi-
ological range. Improvements, including better control of
glucose-responsive transcription of transgenic insulin
mRNA and artifi cial secretory systems, provide hope for the
potential use of insulin-producing non-beta-cells to cure
diabetes.
Engineering Stem and Progenitor Cells
An exciting advance in the last few years is the develop-
ment of cell therapy strategies using stem cells. Stem cells
are characterized by the ability to proliferate extensively and
differentiate into one or more specialized cell types. Both
embryonic and adult stem cells have been investigated as
alternative sources for the generation of insulin-producing
pancreatic islets. Although spontaneous differentiation of
beta-cells from stem cells can be observed, engineering of
stem cells for forced expression of key beta-cell or endocrine
differentiation factors should be more effi cient for driving
beta-cell differentiation.
Engineering Embryonic Stem Cells
In principle, embryonic stem (ES) cells have the poten-
tial to generate unlimited quantities of insulin-producing
cells. ES cells can be expanded indefi nitely in the undiffer-
entiated state and differentiated into functional beta-cells.
However, generation of fully differentiated beta-cells from
ES cells has been diffi cult and controversial. Beta-cell dif-
ferentiation from ES cells as determined on the basis of
immunohistochemical evidence alone has been questioned,
because insulin immunoreactivity can also result from
insulin absorption from the medium as well as from genuine

beta-cell differentiation. Therefore, these types of results
should be interpreted with caution.
Some promising results have been reported for the
differentiation of insulin-producing cells from mouse and
human ES cells (reviewed in Bonner-Weir and Weir, 2005;
Jun and Yoon, 2005; Montanya, 2004; Stoffel et al., 2004).
Pancreatic endocrine cells, including insulin-producing
cells, could be generated from mouse embryonic stem cells
by a fi ve-step protocol, including the enrichment of nestin-
positive cells from embryoid bodies, and these cells secreted
insulin in response to glucose and other insulin secreta-
gogues, such as tolbutamide and carbachol. However, these
cells could not remit hyperglycemica when transplanted
into diabetic mice. A modifi ed protocol, in which a phos-
phoinositide kinase inhibitor was added to the medium to
inhibit cell proliferation, resulted in improved insulin
content and glucose-dependent insulin release. To enrich
insulin-producing cells from mouse ES cells, a neomycin-
resistance gene regulated by the insulin promoter was
transferred to ES cells, which drove differentiation of insulin-
secreting cells, and transplantation of these cells restored
normoglycemia in streptozotocin-induced diabetic mice.
In another report, mouse ES cells were transduced with a
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II. ENGINEERING TO GENERATE INSULIN-PRODUCING CELLS • 639
plasmid containing the Nkx6.1 promoter gene, followed by
a neomycin-resistance gene to select the Nkx6.1-positive
cells, and were differentiated in the presence of exogenous
differentiating factors. The selected Nkx6.1-positive cells
coexpressed insulin and Pdx-1, and transplantation of these

cells into streptozotocin-induced diabetic mice resulted in
normoglycemia.
Exogenous expression of beta-cell transcription factors
has been used as a strategy to drive the differentiation of
insulin-producing cells from ES cells. Overexpression of
Pax4 in mouse ES cells promoted the differentiation
of nestin-positive progenitor and insulin-producing cells,
and these cells secreted insulin in response to glucose
and normalized blood glucose when transplanted into
diabetic mice (Blyszczuk et al., 2003). In the same study,
the expression of Pdx-1 did not have a signifi cant effect
on the differentiation of insulin-producing cells from ES
cells. However, another study demonstrated that the
regulated expression of Pdx-1 in a murine ES cell line
by the tet-off system enhanced the expression of insulin
and other beta-cell transcription factors (Miyazaki et al.,
2004).
It was shown that human ES cells can spontaneously
differentiate in vitro into insulin-producing beta-cells, evi-
denced by the secretion of insulin and expression of other
beta-cell markers (Assady et al., 2001). Differentiation of
insulin-expressing cells from human ES cells was promoted
when they were cultured in conditioned medium in the
presence of low glucose and fi broblast growth factor, fol-
lowed by nicotinamide (Segev et al., 2004). A recent report
suggested that human ES cells differentiated into beta-
cell-like clusters when cotransplanted with mouse dorsal
pancreas (Brolen et al., 2005). Although several in vitro
studies suggest the possibility of generating insulin-
expressing cells from human ES cells, differentiation of truly

functional beta-cells from human ES cells has not yet been
reported.
Because of their proliferative ability and capacity to dif-
ferentiate in culture, ES cells have received much attention
as a potential source of unlimited quantities of beta-cells for
transplantation therapy for diabetes. However, use of ES
cells has ethical concerns, and the mechanisms by which ES
cells differentiate to produce islets and beta-cells are not
well understood. Therefore, further studies are needed to
understand the details of the endoderm and beta-cell
differentiation process so that an effective protocol for
differentiating ES cells into insulin-producing cells can be
developed.
Engineering Adult Stem and Progenitor Cells
As with ES cells, adult stem cells have the potential to
differentiate into other cell lineages, but they do not bring
the ethical diffi culties associated with ES cells. Beta-cell
neogenesis in adults has been reported in animal models of
experimentally induced pancreatic damage, suggesting the
presence of adult stem/progenitor cells. These adult stem/
progenitor cells could be potential sources for the produc-
tion of new insulin-producing cells (reviewed in Jun and
Yoon, 2005; Montanya, 2004; Nir and Dor, 2005). Bone
marrow, mesenchymal splenocytes, neural stem cells, liver
oval stem cells, and pancreatic stem cells have been inves-
tigated for their potential to differentiate into insulin-
producing cells.
A large body of evidence suggests that the adult pancre-
atic ducts are the main site of beta-cell progenitors. Through-
out life, the islets of Langerhans turn over slowly, and new

small islets are continuously generated by differentiation of
ductal progenitors (Finegood et al., 1995). It was found that
isletlike clusters were generated in vitro from mouse pan-
creatic ducts and ductal tissue–enriched human pancreatic
islets. In addition, multipotent precursor cells clonally iden-
tifi ed from pancreatic islets and ductal populations could
differentiate into cells with beta-cell function. The expres-
sion of the Pdx-1 gene or treatment of ductal cells with Pdx-
1 protein increased the number of insulin-positive cells or
induced insulin expression. Ectopic expression of neuro-
genin 3, a critical factor for the development of the endo-
crine pancreas in humans, in pancreatic ductal cells led to
their conversion into insulin-expressing cells. In addition,
treatment of human islets containing both ductal and
ascinar cells with a combination of epidermal growth factor
and gastrin induced neogenesis of islet beta-cells from the
ducts and increased the functional beta-cell mass. In addi-
tion to ductal cells, exocrine acinar cells and other endo-
crine cells can generate beta-cells. An alpha-cell line
transfected with Pdx-1 expressed insulin when treated with
betacellulin. It was shown that treatment of rat exocrine
pancreatic cells with epidermal growth factor and leukemia
inhibitory factor could induce differentiation into insulin-
producing beta-cells (Baeyens et al., 2005). Considerable
evidence suggests that beta-cells in the pancreatic islets can
be dedifferentiated, expanded, and redifferentiated into
beta-cells by inducing the epithelial–mesenchymal transi-
tion process (Lechner et al., 2005). Nonendocrine pancre-
atic epithelial cells also have been reported to differentiate
into beta-cells (Hao et al., 2006). These results suggest that

pancreatic stem/progenitor cells are the source of new
islets.
There is also the possibility of manipulating stem/pro-
genitor cells from other organs to transform into the beta-cell
phenotype (reviewed in Montanya, 2004; Nir and Dor, 2005).
Although there are controversies regarding the differen-
tiation of bone marrow–derived stem cells into insulin-
producing cells, some successful studies have been reported.
In vitro differentiation of mouse bone marrow cells resulted
in the expression of genes related to pancreatic beta-cell
development and function. These differentiated cells released
insulin in response to glucose and reversed hyperglycemia
when transplanted into diabetic mice. In addition, ectopic
expression of key transcription factors of the endocrine
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640 CHAPTER FORTY-THREE • ENGINEERING PANCREATIC BETA-CELLS
pancreas developmental pathway, such as IPF1, HLXB9, and
FOXA2, in combination with conditioned media in human
bone marrow mesenchymal stem cells differentiated them
into insulin-expressing cells (Moriscot et al., 2005).
Because the liver and intestinal epithelium are derived
from gut endoderm, as is the pancreas, the generation of
islets from both developing and adult liver and intestinal
cells has been tried. Rat hepatic oval stem cells could dif-
ferentiate into insulin-producing isletlike cells when cul-
tured in a high-glucose environment. Fetal human liver
progenitor cells and mouse hepatocytes could differentiate
into insulin-producing cells when engineered to produce
Pdx-1, and transplantation of these cells reversed hypergly-
cemia in mice. It was reported that adult human liver cells

engineered to express Pdx-1 produced insulin and secreted
it in a glucose-regulated manner. Transplantation of these
engineered cells under the renal capsule of diabetic mice
resulted in prolonged reduction of hyperglycemia (Sapir
et al., 2005). As well, ectopic islet neogenesis in the liver
could be induced by gene therapy with a combination of
NeuroD, a transcription factor downstream of Pdx-1, and
betacellulin, which reversed diabetes in streptozotocin-
treated diabetic mice. Expression of Pdx-1 in a rat entero-
cyte cell line in combination with betacellulin treatment or
coexpression of Isl-1 resulted in the expression of insulin.
Treatment of developing as well as adult mouse intestinal
cells with GLP-1 induced insulin production, and transplan-
tation of these cells into streptozotocin-induced diabetic
mice remitted diabetes. A recent study showed that neural
progenitor cells could generate glucose-responsive, insulin-
producing cells when exposed in vitro to a series of signals
for pancreatic islet development (Hori et al., 2005). These
results suggest that the controlled differentiation of liver or
intestinal cells into insulin-producing cells may provide an
alternative source of beta-cells.
The use of adult stem/progenitor cells for generating
beta-cells for transplantation therapy appears to be promis-
ing, although most of the studies have only been done in
animal models. Further studies on the mechanisms for the
differentiation of adult stem/progenitor cells into insulin-
producing beta-cells and the characterization of the newly
generated beta-cells are required before these cells can be
considered for clinical application.
III. ENGINEERING TO IMPROVE

ISLET SURVIVAL
A hurdle to overcome for islet transplantation therapy
is the rejection and autoimmune attack against the trans-
planted beta-cells. Immunosuppressive drugs have been
used successfully, but they have many side effects. There-
fore, it is desirable to develop drug-free strategies for the
induction of tolerance to transplanted islet or beta-cells. A
variety of approaches to protect islet grafts have been
studied, such as bone marrow transplantation, treatment
with anti-T-cell agents, and inhibition of activation of
antigen-presenting cells. Another approach is engineering
islets or beta-cells to express therapeutic genes to improve
islet viability and function, such as genes for cytokines,
antiapoptotic molecules, antioxidants, immunoregulatory
molecules, and growth factors (reviewed in Giannoukakis
and Trucco, 2005; Jun and Yoon, 2005; Van Linthout and
Madeddu, 2005) (Table 43.1).
With regard to cytokines, introduction of genes for
interleukin (IL)-4 or a combination of IL-10 and transform-
ing growth factor-β improved islet graft survival by prevent-
ing immune attack in mice. In addition, islets expressing the
p40 subunit of IL-12 could maintain normoglycemia when
transplanted into diabetic NOD recipients by decreasing
interferon-γ production and increasing transforming growth
Table 43.1. Engineering islets for beta-cell survival
Strategy Molecules used
Cytokine expression Erythropoietin
Interleukin (IL)-1 receptor
antagonist
IL-4

IL-10
IL-12p40
Transforming growth factor-β
Antiapoptotic A20
molecule Bcl-2
expression Bcl-xL
Dominant-negative MyD88
Fas ligand
Flice-like inhibitory protein
IκB kinase inhibitor
Tumor necrosis factor receptor-
immunoglobulin (Ig)
Antioxidant Catalase
molecule c-Jun N-terminal kinase
expression inhibitory peptide
Glutathione peroxidase
Heme oxygenase-1
Manganese superoxide dismutase
Immunoregulatory Adenoviral E3 genes
molecule CD40-Ig
expression Cytotoxic T-lymphocyte
antigen-4-Ig
Dipeptide boronic acid
Indoleamine 2,3-dioxygenase
Growth factor Hepatocyte growth factor
expression Insulinlike growth factor-1
Vascular endothelial growth factor
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factor-β at the transplantation site. Islets engineered to
produce IL-1β receptor antagonist were also found to be

more resistant to rejection. Adenoviral-mediated gene trans-
fer of erythropoietin, a cytokine that promotes survival, in
islets resulted in protection of islets from apoptosis in
culture and destruction in vivo.
Expression of antiapoptotic molecules such as Bcl-2,
Bcl-xL, and A20, which inhibit nuclear factor-κB activation,
or an IκB kinase inhibitor was shown to protect from apop-
tosis. In addition, the expression of soluble human Fas
ligand, dominant negative MyD88, fl ice-like inhibitory
protein, or tumor necrosis factor receptor-immunoglobulin
(Ig) improved allogeneic islet graft survival. A recent study
demonstrated that silencing Fas expression with small inter-
fering RNA in mouse insulinoma cells inhibited Fas-medi-
ated beta-cell apoptosis (Burkhardt et al., 2006).
Pancreatic islets are sensitive to oxidative stress because
they produce relatively low amounts of antioxidant enzymes.
Thus, expression of antioxidant molecules such as catalase,
glutathione perioxidase, and manganese superoxide dis-
mutase in islets or insulinoma cells could protect against
oxidative stress and cytokine-induced damage. In addition,
expression of heme oxygenase in pancreatic islets protected
against IL-1β-induced islet damage. It was also found that
delivery of a c-Jun-terminal kinase inhibitory peptide into
isolated islets by the protein transduction system prevented
apoptosis (Noguchi et al., 2005).
Expression of immunoregulatory molecules that affect
T-cell activation and proliferation have been tried. Expres-
sion in islets of cytotoxic T-lymphocyte antigen-4-immuno-
globulin, which down-regulates T-cell activation, or CD40-Ig,
which blocks CD40–CD40 ligand interactions, prolonged

allogenic and xenogeneic graft survival. Transplantation
of islets overexpressing indoleamine 2,3-dioxygenase pro-
longed survival in NOD/SCID mice after adoptive transfer
of diabetogenic T-cells, probably by inhibiting T-cell prolif-
eration by the depletion of tryptophan at the transplanta-
tion site. Similarly, a proteasome inhibitor, dipeptide boronic
acid, was found to prevent islet allograft rejection by sup-
pressing the proliferation of T-cells. Expression of adenovi-
ral E3 transgenes in beta-cells was found to prevent islet
destruction by autoimmune attack through the inhibition of
major histocompatibility complex I expression.
With regard to growth factors, adenoviral-mediated
transfer of hepatocyte growth factor resulted in an improved
islet transplant outcome in animal models. As well, expres-
sion of insulinlike growth factor-1 in human islets prevented
IL-1β-induced beta-cell dysfunction and apoptosis. Insuffi -
cient revascularization of transplanted islets can deprive
them of oxygen and nutrients, contributing to graft failure.
Therefore the expression of vascular endothelial growth
factor, a key angiogenic molecule, enhanced islet revascu-
larization and improved the long-term survival of murine
islets after transplantation into the renal capsule of diabetic
mice.
Another strategy to protect islets from immune attack
is microencapsulation of islets within synthetic polymers
(Kizilel et al., 2005). Encapsulation of islets within a semi-
permeable membrane, such as alginate-poly-l-lysine-algi-
nate, blocks the passage of larger cells but allows the passage
of small molecules, thus conferring protection from auto -
i mmune attack. However, this method has limitations for

the long-term survival of islets within the microcapsules
because of the lack of biocompatibility, ischemia, and
limited protection from cytokine-induced damage. To over-
come these limitations, a bioartifi cial pancreas has been
developed, in which blood fl ows through artifi cial vessels in
close proximity to insulin-producing cells.
A variety of approaches for engineering islets or beta-
cells for improved islet graft survival and escape from
immune rejection have been successful in animal models.
However, the effi cacy of these approaches in human dia-
betic patients remains to be determined.
IV. VECTORS FOR ENGINEERING ISLETS
AND BETA-CELLS
The cells of the pancreas divide very slowly; therefore
gene transfer vehicles that can transduce quiescent cells
have been used for the delivery of transgenes, such as
nonviral plasmids and vectors based on lentivirus, adeno-
virus, helper-dependent adenovirus, adeno-associated virus
(AAV), and herpes simplex virus. In addition, protein trans-
duction using the cell penetrating peptide from HIV-1 trans-
acting protein (reviewed in Becker-Hapak et al., 2001) has
been successfully used to engineer islets (Table 43.2).
However, the choice of vector needs to be carefully made so
that the vector itself does not affect islet function or
viability.
Nonviral methods are considered safe, cost effective,
and simple to use and do not induce an immune response,
but they generally have a lower gene transfer effi cacy as
compared to viral-mediated gene transfer (reviewed in
Nishikawa and Huang, 2001). Nonviral methods for

transferring genetic material include the direct injection
of DNA, either naked or enclosed in a liposome, electro-
poration, and the gene gun method. Cationic lipid and
polymer-based plasmid delivery has been used to trans-
duce islets, and the expression of cytotoxic T-lymphocyte
antigen-4 by biolistically transfected islets improved graft
survival.
Viral vectors (reviewed in Walther and Stein, 2000) have
been widely used as a method of gene transfer to engineer
islets and beta-cell surrogates. Retroviral vectors derived
from Moloney murine leukemia virus can carry a gene effi -
ciently and integrate it in a stable manner within the host
chromosomal DNA, facilitating long-term expression of the
gene. For immortalization of human islets, retroviral vectors
expressing oncogenes or telomerase genes have been used
(Narushima et al., 2005). Although most retroviral vectors
IV. VECTORS FOR ENGINEERING ISLETS AND BETA-CELLS • 641
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642 CHAPTER FORTY-THREE • ENGINEERING PANCREATIC BETA-CELLS
only infect proliferating cells, the lentivirus genus of retro-
viruses, which includes the human immunodefi ciency virus,
has all the advantages of Moloney murine leukemia virus–
derived retroviral vectors and can infect nondividing as well
as dividing cells. Lentiviral vectors have been successfully
used to transduce islets with marker proteins (Okitsu et al.,
2003).
The adenoviral vector can harbor up to 30 Kb of foreign
DNA and can transduce nondividing cells with high effi -
ciency. In addition, a relatively high titer of virus, about 10
12

plaque-forming units/mL, can be produced. The transferred
genes are not integrated into the host genome, but remain
as nonreplicating extrachromosomal DNA within the
nucleus. Although there is no risk of alteration in cellular
genotype by insertional mutation, the duration of gene
expression may be short, and a strong cellular immune
response to the viral proteins and, in some cases, to the
transgene may be induced. Adenoviral vectors have been
widely used to transduce islets for proof-of-concept experi-
ments in vitro and in vivo. Although adenoviral vectors are
toxic because of de novo synthesized viral proteins, islet
viability and functional characteristics were not affected
when transduced in vitro. However, transduction of islets
with a high dose of recombinant adenovirus (500 MOI)
markedly reduced glucose-stimulated insulin secretion,
suggesting that an optimal dose is required to result in effi -
cient transduction without compromising islet function. In
general, adenoviral vectors result in transient transgene
expression; however, long-term (20-week) expression of the
transgene was observed in islets transduced with β-galacto-
sidase and transplanted into syngeneic diabetic mice. It was
reported that double-genetic modifi cation of the adeno-
virus fi ber with RGD polylysine motifs signifi cantly reduced
toxicity, infl ammation, and immune responses (Contreras
et al., 2003).
A new generation of adenovirus vectors has been devel-
oped that are completely devoid of all viral protein–coding
sequences and are therefore less immunogenic and less
toxic. Although these gutless viruses require the presence of

a helper virus for replication, contamination by the helper
virus can be avoided by genetically engineering a condi-
tional defect in the packaging domain of the helper virus or
fl anking the packing signal with loxP expression sites and
encoding Cre recombinase in the supporting cell line. In
Table 43.2. Vectors used for islet and beta-cell engineering
Vector Advantages Disadvantages
Nonviral plasmid vectors Easy to produce Low transduction effi ciency
Less toxic compared with viral vectors Transient expression
Retrovirus Long-term expression Only infects dividing cells
No expression of viral protein Limited insertion capacity (8 Kb)
Random integration into host chromosomal
DNA
Lentivirus Stable, long-term expression Produces low titers of virus
Infects dividing and nondividing cells Limited insertion capacity (8 Kb)
Nonimmunogenic Random integration into host chromosomal
DNA
Adenovirus Produces high titers of virus Toxic
High transduction effi ciency Host immune response to viral proteins
Infects dividing and nondividing cells Short-term expression
Gutless adenovirus Reduced toxicity and prolonged expression Helper-virus contamination
compared with adenoviral vector
Large insertion capacity
Adeno-associated virus Low immunogenicity, probably Produces low titers of virus
nonpathogenic Low transduction effi ciency
Broad host range Limited insertion capacity (4.8 Kb)
Long-term expression
Infects dividing and nondividing cells
Herpesvirus Broad host range Toxic
High insertion capacity Induces host immune response

Protein transduction No immunogenicity Short half-life
Transduces many cell types
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addition, the gutless vectors are known to have a prolonged
expression of the transgene. However, there is no report
about islet engineering using these vectors.
AAVs are nonpathogenic, replication-defective parvo-
viruses that can infect both dividing and nondividing cells.
AAVs generally have low immunogenicity; however, the
generation of neutralizing antibodies may limit readminis-
tration. This problem can be overcome by selective capsid
modifi cation of AAV to evade recognition by preexisting anti-
bodies or by direct administration of AAV to the target tissue.
The recombinant AAV vector integrates randomly into the
host chromosome or may stay in the episomal state. There
is a limitation in the size of the DNA that can be inserted (a
maximum of 4.8 Kb); however, larger inserts can be split over
two vectors and delivered simultaneously, because AAVs
tend to form concatemers, although the effi ciency of trans-
duction is often reduced (Young et al., 2006). Effi cient trans-
duction of islets was achieved using a high dose of AAVs with
an improved recombinant AAV purifi cation method, which
improved infectious titers and yield. Transduction of islets
with AAV5 is more effi cient than with AAV2, due to the low
number of receptors for AAV2 on islet cells. AAV1 was found
to be the most effi cient serotype in transducing murine islets
(Loiler et al., 2003). However, it was recently demonstrated
that intact human and murine islets could be effi ciently
transduced with a double-stranded AAV2-based vector, and
the transduced murine islets showed normal glucose respon-

siveness and viability (Rehman et al., 2005).
Herpes simplex virus type 1 (HSV-1) has also been used
as a viral vector. Based on the persistence of latent herpes
virus after infection, HSV-1 is attractive for its effi cient infec-
tivity in a wide range of target cells and its ability to infect
both dividing and nondividing cells, including islets. Trans-
fection of human islets with Bcl-2 protected beta-cells from
cytokine-induced damage. However, the transduction may
be unstable, and potential health risks of this vector remain
to be determined.
Protein transduction is an emerging technology to
deliver therapeutic proteins into cells as an alternative to
gene therapy. This method uses peptides that can penetrate
the cell membrane, such as antennapedia peptide, the HSV
VP22 protein, and human immunodefi ciency virus TAT
protein transduction domain. The therapeutic molecule
is linked to the penetrating peptide as a fusion protein,
which is then used to transduce the cell (Becker-Hapak
et al., 2001). The protein transduction method is not im-
munogenic and can transduce a variety of cell types, but it
has a short half-life. Delivery of antiapoptotic proteins
such as Bcl-xL or anti-oxidant enzymes such as copper-zinc
superoxide dismutase and heme oxygenase by protein
transduction in human and rodent islets effi ciently trans-
duced the islets and improved their viability without
affecting islet function (Embury et al., 2001; Mendoza et al.,
2005).
V. CONCLUSION
Engineering beta-cell lines and non-beta-cells, differ-
entiating embryonic and adult stem cells, and transdiffer-

entiating non-beta-cells have been studied as methods to
provide new beta-cells for cell therapy for diabetes. Expan-
sion of functional beta-cells by generation of reversibly
immortalized human beta-cell lines has been reported, but
the techniques have not been clinically proven. Generation
of insulin-producing cells from non-beta-cells is an attrac-
tive method, but it has yet to achieve tight regulation of
glucose-responsive insulin secretion. Differentiation of
insulin-producing cells from ES cells and adult stem/
progenitor cells is also a promising alternative to produce
beta-cells; however, a better understanding of the mecha-
nisms for the differentiation of beta-cells is needed to
develop a successful strategy to engineer beta-cells from
stem cells. Engineering of islets and beta-cells to improve
the survival of islet transplants has also been investigated.
Although much progress has been made, engineered beta-
cells need to be carefully analyzed for true beta-cell function
and possible tumorigenicity. It is hoped that continued
research on beta-cell engineering will offer a potential cure
for diabetes in the future.
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