Download Animal Models to Study Adult Stem Cell-derived, In Vitro

Animal Models to Study Adult Stem Cell-derived, In Vitro-generated
Islet Implantation
Ammon B. Peck, Li Yin, and Vijayakumar Ramiya
Abstract
Type 1 diabetes (T1D) is an autoimmune disease characterized by hyperglycemia following the destruction of the insulin-producing beta cells of the pancreatic islets of
Langerhans by the body’s own immune system. Although
routine insulin injections can provide diabetic patients with
their daily insulin requirements, this treatment is not always
effective in maintaining normal glucose levels. A true
“cure” is considered possible only through replacement of
the beta cell mass, by pancreas transplantation, islet implantation, or implantation of nonendocrine cells modified to
secrete insulin. With the recent success of islet implantation
to reverse T1D, this procedure has become a welcome
therapy for T1D patients. Unfortunately, this procedure is
hampered by the limited number of transplantation quality
pancreata available for the harvesting of islets. This shortage has sparked great interest in finding a replacement for
organ donation, primarily the possible use of stem cellderived islets starting with stem cells, or alternatively the
harvesting of nonhuman islets. This review focuses on
progress with growing islets in the laboratory from stem
cells and a comparison between this developing technology
and the current use of islets harvested from nonhuman
sources.
Key Words: in vitro-generated islets; islet implantation; pancreatic stem cells; type 1 diabetes; xenogeneic
transplantation
Introduction
D
iabetes mellitus refers to a group of chronic metabolic diseases characterized by hyperglycemia due to
the absence of or resistance to insulin. This state may
result from defects in insulin secretion, insulin action, or
Ammon B. Peck, Ph.D., is a Professor, and Li Yin, M.D., is an Assistant
Research Professor, in the Department of Pathology, Immunology and
Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida. Vijayakumar Ramiya, Ph.D., is an Assistant Professor in the
Department of Pediatrics, University of Florida College of Medicine,
Gainesville. All authors are affiliated with, and have a financial interest in,
Ixion Biotechnology, Inc., Alachua, Florida.
Volume 45, Number 3
2004
both (Harris 1999). Type 1 diabetes (T1D1), also known as
juvenile-onset, autoimmune, or insulin-dependent diabetes,
is a common chronic disease of children and adolescents in
both Europe and North America, and an increasing problem
elsewhere. The recent implementation of new genetic
screening programs for families and newborns to identify
“high-risk” individuals has revealed an apparent increase in
the incidence of T1D worldwide.
T1D is an especially insidious disease because clinical
symptoms are usually not recognized until after the patient’s
own immune system has destroyed more than 90% of the
total insulin-producing beta cells of the endocrine pancreas
(Eisenbarth 1986). Although routine insulin injections can
provide diabetic patients with their daily insulin requirements, blood glucose excursions are common and result in
hyperglycemic episodes. Hyperglycemia currently represents the major health problem for the diabetic patient.
When inadequately controlled, chronic hyperglycemia can
lead to microvascular complications (i.e., retinopathy and
blindness, nephropathy and renal failure, neuropathy, foot
ulcers and amputation) and/or macrovascular complications
(i.e., atherosclerotic cardiovascular, peripheral vascular, and
cerebrovascular disease). Both the Diabetes Control and
Complications Trial and the UK Prospective Diabetes Study
demonstrated a strong relation between good metabolic control and the rate/progression of complications (DCCT Research Group 1993; UKPDS Group 1998). Unfortunately, it
is difficult for most patients to achieve adequate control of
hyperglycemic excursions, and attempts to maintain euglycemia through intensive insulin treatment lead to increased
incidences of hypoglycemia. The lack of tight glycemic
control resulting in acute and chronic metabolic complications costs the healthcare system in the United States more
than one hundred billion dollars per year and highlights the
urgency for a cure.
A true “cure” for T1D relies on replacement of the beta
cell mass. Currently, beta cell replacement is accomplished
either by ectopancreas transplantation or islet implantation.
Pancreas organ transplantation is often successful in normalizing fasting and postprandial blood glucose levels,
HbA1C levels, as well as secretion of insulin and C-peptide
1
Abbreviations used in this article: AcXR, acute cellular xenograft rejection; AhXR, acute humoral xenograft rejection; HAR, hyperacute reaction;
IPC, islet progenitor cell; IPSC/IPC, islet-producing stem cells/islet progenitor cells; NOD, nonobese diabetic; PERV, procine endogenous retrovirus; T1D, type 1 diabetes.
259
in response to glucose. However, this procedure requires
long-term immunosuppression, thereby restricting it generally to patients who have end-stage renal disease and are
listed to receive a second organ transplant, or to those with
long-standing T1D who have failed insulin therapy.
Islet implantation has several notable advantages over
pancreatic organ transplantation:
1. The islets can be implanted by percutaneous catheterization of the portal vein under local anesthesia, thus
relieving the patient from undergoing general anesthesia
and invasive surgery.
2. Theoretically, islets can be genetically manipulated
in vitro to resist immune attack, either by reducing
potential islet-associated antigens or by expressing
immunomodulators.
3. The islets can be encapsulated as a way to protect them
from the immune system, but still permit insulin secretion (Drachenberg et al. 1999; Lanza and Chick 1997;
Scharp et al. 1994; Sutherland et al. 1996).
Currently, however, islet cell implantations also require immunosuppression and have been severely limited by a shortage of implantable islets from donated cadaveric pancreata.
This shortage is underscored by the recent estimation that
mortality rates for patients waiting for pancreatic grafts or
pancreatic plus kidney grafts are 42.2 and 36.2 per 1000
patients, respectively (UNOS 2002). Interestingly, the recent success using new islet implantation protocols has
popularized this intervention, a situation that no doubt has
the potential to exacerbate the shortage of implantable islets
further (Bertuzzi et al. 2003; Bretzel et al. 2001; Ricordi et
al. 2003; Ryan et al. 2001).
The possibility that stem cell-derived, in vitro-generated
islets may soon be an alternative to cadaver-derived islets
for treating diabetic patients is gaining acceptance, and has
been the topic of several recent reviews (Bonner-Weir and
Sharma 2002; Bretzel 2003; Efrat 2002; Lechner and Habener 2003; Peck et al. 2001; Peshavaria and Pang 2000;
Soria 2001). Over the past few years, several studies have
reported the production of endocrine cells, endocrine tissues
and/or islet-like clusters from embryonic stem cells, adult
stem cells isolated from the pancreas, as well as adult stem
cells from nonpancreas origins (Table 1). Although none of
these products are considered perfect yet, major advances
have been made in understanding the regulation of growth
and differentiation of beta cells and the islets of Langerhans
(reviewed in Wilson et al. 2003), thus raising hope for this
science to reach the diabetic patient in the near future.
In Vitro Generation of Islet-like Clusters
from Adult Pancreas—The Human Model
As early as the mid-1990s, Peck and coworkers presented
evidence indicating that immature, yet functional, islet-like
clusters could be grown in vitro from stem/progenitor cells
isolated from the pancreatic ducts and islets presumed to
contain epithelial stem cells from which islets of Langerhans are derived during embryogenesis (Cornelius et al.
1997; Peck and Cornelius 1995). The protocol that proved
most successful consisted of several sequential steps that
permitted stem cell/progenitor cell selection with subsequent successive growth, differentiation, and maturation of
the cultured cell populations. Today, this process has been
divided into four steps:
1. Ductal epithelial cells (in combination with islets), isolated from digested pancreas, are cultured in a growthrestrictive medium to enrich for epithelial cell
subpopulations that can form monolayers with a neuroendocrine cell-like phenotype (referred to as isletproducing stem cells, or IPSCs1).
2. Islet progenitor cells (referred to as IPCs1) are induced
to bud from the epithelial-like monolayers.
Table 1 Stem cells capable of forming endocrine-like tissues and cells
Source of stem/
progenitor cells
Differentiated tissue
References (see text)
Mouse embryo
Insulin-secreting cells, endocrine hormone-secreting
cells and islet-like clusters
Human embryo
Insulin-secreting cells and islet-like clusters
Adult mouse pancreas
Endocrine hormone-secreting cells and islet-cell
clusters
Islet-like clusters
Endocrine-hormone-producing cells, ductal cell buds
and islet-cell clusters
Islet-cell clusters
Islet-cell clusters
Kahan et al. 2003; Kim et al.
2003; Lumelsky et al. 2001;
Soria et al. 2000
Assady et al. 2001; Shiroi et
al. 2002
Cornelius et al. 1997; Ramiya
et al. 2000
Peck and Ramiya 2003
Bonner-Weir et al. 2000; Peck
et al. 2001
Zulewski et al. 2001
Yang et al. 2002
Adult pig pancreas
Adult human pancreas
Adult rat and human islets
Adult rat liver oval cells
260
ILAR Journal
3. Given a favorable growth medium that permits proliferation of IPCs and their daughter cells, islet-like cell
clusters will form that contain various endocrine cells
in various differentiation states and that are capable
of exhibiting regulated insulin responses to glucose
challenges.
4. The islet-like structures are implanted into an in vivo
environment to promote final maturation of the islet
cells.
The three in vitro stages of growth of islet-like clusters are
presented in Figure 1A.
The establishment of islet-forming cultures from human
pancreata is highly reproducible despite variations in the
health status and quality of donated pancreata. However,
one drawback to working with human cultures is the difficulty in carrying out required efficacy and toxicity studies to
determine the behavior of stem cell-derived, in vitrogenerated islets when implanted into an appropriate in vivo
environment. Thus, many aspects of using cells and tissues
grown from human stem cells must be extrapolated from
animal studies and/or studies involving implantations across
species. As a result, we turned originally to the nonobese
diabetic (NOD1) mouse model of T1D (Atkinson and Leiter
1999) to investigate a number of questions that might apply
to the use of in vitro-generated islets in treating diabetic
patients.
In Vitro Generation of Islets from Adult
Pancreatic Stem Cells—The NOD
Mouse Model
We have used the NOD mouse as a model for human T1D.
In our studies, we have shown the following: the feasibility
of obtaining IPSCs from prediabetic, postdiabetic, or normal adult mice; the ability of duplicating the human IPSC/
IPC cultures by establishing stem cell cultures for the
growth of immature functional islet-like structures; and the
potential of in vitro-generated mouse islets to reverse insulin-dependent diabetes when implanted into diabetic NOD
mice (Ramiya et al. 2000).
In Figure 1B, the temporal growth and differentiation of
epithelial monolayers derived from isolated mouse pancreatic ducts plus islets are shown, revealing the growth characteristics observed with human IPSC/IPC cultures. These
monolayers contain IPSCs capable of producing immature
islet cell-like clusters that are similar (but not identical) to
the human-derived IPSC/IPC cultures. We have noted that
the immature islets maintain their structural integrity ranging from a few days to several weeks, but they can easily be
dissociated into single cell suspensions that, if recultured,
start the process again. Thus, the clusters contain cells retaining the IPSC/IPC phenotype, including the capacity for
self-renewal, a phenomenon shown to be true for adult islets
freshly isolated from the pancreas (Zulewski et al. 2001).
Interestingly, the IPCs appear to bud from the epithelial
Volume 45, Number 3
2004
cells (IPSCs), and then proliferate into the threedimensional cell clusters. However, mouse islet cell-like
clusters appear to be more uniform and stable than human
islet cell-like clusters. The mouse IPSC/IPC cultures have
been maintained up to 3 yr through constant expansion via
repeated serial transfers. Each subculture exhibited the ability to produce increasing numbers of islet-like structures.
Based on the number of these immature islets produced
within the secondary cultures, we calculated that between
10,000 and 15,000 pancreas equivalents were produced
within the 3 yr of growth of a single cell line from five
pancreata used for initiating the primary culture.
It is critical to note that stem cell-derived, in vitro-grown
islet-like clusters exhibit temporal gene expressions and cellular organizations that mimic natural islets of Langerhans
during their organogenesis. During differentiation of cultured ductal epithelial cells to islet-like clusters, the IPSC/
IPC-derived cells temporally express various endocrine
hormones and islet cell-associated factors. A comparison of
these gene expressions for mouse and human cultures, as
detected by measuring transcripts by reverse transcriptasepolymerase chain reaction, is provided in Table 2. Included
are the genes encoding insulin I and II, insulin receptor,
hepatocyte growth factor and its receptor c-MET, glucagon,
somatostatin, glucose transporter-2, GAD-67, and insulinlike growth factors I and II. In addition, genes related to
development and differentiation are also detected, including
those encoding Reg-1, Pdx-1, Ngn-3, Isl-1, and beta2/
neuroD. Despite detectable expression of these mRNA transcripts for islet-associated genes, the cells within the islet
structures remain mostly immature. The fact that the mouse
islets generated in vitro fail to achieve full maturation before implantation into an in vivo environment is supported
by the observations that (1) many of the cells coexpressed
more than a single endocrine hormone while in culture, and
(2) insulin secretion in response to glucose challenge remained minimal. However, when nicotinamide, a reagent
known to enhance proliferation and maturation of mouse
beta cells, was added to cultures of differentiating mouse
IPSCs, the number of islets increased, the islet cells exhibited increased insulin synthesis, and the islet clusters responded with increased insulin secretion to glucose
challenges (Ramiya et al. 2000).
Functional Activity of Stem Cell-derived,
in Vitro-generated Islet Implants—A
Syngeneic and Allogeneic Mouse Model
One potential drawback of stem cell-derived, in vitro-grown
islets has been the inability to identify a culture condition
that permits full differentiation of the immature beta cells to
mature cells within the islet clusters. This last step has required exposure to an in vivo environment, suggesting that
in vitro environments do not simulate the in vivo environment and are missing important factors. The functional capacity of in vitro-grown, stem cell-derived mouse islets has
261
Figure 1 In vitro generation of islet-like clusters from (A) an adult human pancreas and (B) an adult mouse pancreas. Growth of islet-like
clusters in primary islet-producing stem cells/islet progenitor cell cultures begin with establishment and growth of epithelial-like cells from
partially digested pancreatic tissue (panels a-b). A number of islands of cells begin to produce cells that proliferate on top of the underlying
monolayers (panels c-d), which tend to aggregate into clusters of cells (panels e-f). The aggregated clusters express markers associated with
immature/mature endocrine cells, whereas the adherent monolayers express markers associated primarily with epithelial cells. When cells
within the primary cultures are dispersed with trypsin and seeded to new cultures, the entire process is repeated, thus permitting expansion
of the cultures.
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ILAR Journal
Table 2 Gene expression profiles of long-term IPSC/IPC culturesa
Human
Gene/factor
Isl-1
Reg-1
pdx-1
Hlxb9 (HB9)
ngn-3
beta2/neuroD
pax-4
pax-6
Nkx2.2
Nkx6.1
glut-2
Tyrosine hydroxylase
␤-galactosidase
Insulin growth factor-1
Insulin growth factor-2
Npy (Neuropeptide-Y)
Carbonic anhydrase-11
Cytokeratin-19
Insulin receptor
HGF
c-MET
c-Kit
CD34
INGAP
Insulin
Glucagon
Somatostatin
Pancreatic polypeptide
Amylase
Mouse
Whole
pancreas
IPSC/IPC
cultures
Whole
pancreas
IPSC/IPC
cultures
++
++
++
+
(+)b
++
++
−
(+)
−
Variable
Variable
Variable
−
+
+
+
+
+/−
+
++
Variable
++
+
+
+
+
++
++
++
++
+
+
+
+
+
++
++
++
+
++
+
+
++
++
++
(+)
(+)
+
++
++
++
++
++
++
++
(+)
(+)
−
++
++
++
+
++
Variable
(+)
−
+
++
++
Variable
+
++
++
++
++
+
++
++
+
a
IPSC/IPC, islet-producing stem cells/islet progenitor cells. Gene expressions were determined by reverse transcriptase-polymerase chain
reaction.
b
(+) = weakly positive; variable = not all lines are positive for this gene/factor.
been investigated in a set of implantation experiments
(Ramiya et al. 2000). In the first set of experiments, 300
islet-like clusters from IPSC/IPC cultures originating from
NOD mice were implanted into the spleen, the leg muscle,
or the subcapsular region of the left kidneys of individual
female diabetic NOD mice that had been maintained on
daily insulin injections for >3 wk. Within 1 wk of being
weaned from their insulin injections, mice implanted with
islets to the kidney or the spleen exhibited stable blood
glucose levels of 180 to 220 mg/dL and remained insulin
independent until euthanized for analysis of the implants.
Diabetic mice that had islets implanted into the muscle or
had not received any islet implants exhibited severe wasting
syndrome when weaned from their insulin and had to be
euthanized early.
In a second experiment, female diabetic NOD mice
maintained on insulin for 4 wk were implanted subcutaneVolume 45, Number 3
2004
ously with in vitro-grown, stem cell-derived mouse islets
with similar results, except that the blood glucose stabilized
at more normal levels (100-150 mg/dL). Islets placed subcutaneously required approximately 2 to 4 wk to achieve a
homeostatic state with the recipient. We have speculated
(Ramiya et al. 2000) that this is the time required for the
islets to become fully vascularized and establish the necessary glucose-sensing machinery for rapid and regulated insulin responses.
An interesting observation from the implant studies is
the fact that none of the syngeneic implants of in vitrogrown islets reactivated the autoimmune response in the
diabetic recipient mice, as evidenced by a lack of immune
cells in histological examinations of implant sites at time of
euthanization. In contrast, allogeneic implants of in vitrogrown islets originating from either BALB/c or C57BL/6
mice were destroyed in what appeared to be an allogeneic
263
immune response. Thus, there is clear indication that these
two immune responses are mutually distinct and that successful implantation of stem cell-derived islets will require
special attention to the allogeneic response but possibly not
to the autoimmune response. Whether this same observation
is eventually observed in human implantation will be of
great interest. Regardless, understanding why the autoimmune response is not reactivated in this system could prove
valuable for islet transplantation, in general. Furthermore,
although our ability to control the growth and differentiation
of islet stem cells potentially provides an abundant source
for beta cell reconstitution in T1D patients and insulinrequiring patients with type 2 diabetes, obtaining pancreatic tissue for autologous implants poses a number of
challenges.
Although the use of animal models, like the NOD
mouse, provides important information and invaluable insight into how stem cell-derived cells and tissues behave in
vitro and in vivo, the all-important question is whether this
knowledge can be extrapolated to human stem cell-derived
materials. The process will first require examination of human stem cell-derived, in vitro-generated islet implantations
into animal models, possibly with confirmation by appropriate cross-species implantations of stem cell-derived, in
vitro-generated islets of animal models.
Xenogeneic Implants—A Brief Overview
The transplantation of organs, tissues, or cells between individuals of different species, referred to as xenotransplantation, has been a topic of great interest and controversy due
to the conflict of a worsening shortage of available human
organs for transplantation versus perceived risks. In 2002,
nearly 6200 patients died in the United States because of the
acute organ shortage (UNOS 2002). Major issues that need
to be addressed in xenotransplantation procedures include
(1) the physiological compatibility between donor and recipient species, (2) immune attack and rejection, (3) the
potential microbiological hazards that may “jump” across
the species (zoonosis), and (4) the general attitude of society
at large toward the ethical and emotional aspects surrounding xenotransplantation.
Pancreatic islet xenotransplantation is considered to be
technically less demanding than vascularized grafts (MacKenzie et al. 2003). The absence of major physiological
incompatibilities between pig and human islets has been
established, and porcine insulin has been used clinically in
treating diabetic patients for many years. Although, from an
immunological perspective, nonhuman primates would be a
preferable source of organs for humans, almost all of these
species are either endangered or too small to provide organs
suitable for transplantation into large adult humans (Cooper
et al. 2002). Additional reasons that disfavor primates include zoonosis, their small litter size, the enormous expense
involved in breeding them in captivity, and the emotional
factor. Due to similar reasons of sentimental attachment,
264
dogs will not become a popular source of islet donors in the
clinical setting. There have been limited observations with
piscine islets (Brockmann bodies) demonstrating the feasibility of their principal islets in reversing hyperglycemia in
mice (Laue et al. 2001). In this issue, Wright and colleagues
(2004) report notable progress in piscine research. Nevertheless, fish appear to be less likely to comprise a prime
target species for islet xenotransplantation due to their phylogenetic distance from humans.
Many investigators now expect the pig to be the species
most likely to be the source of islets for xenotransplantation,
and numerous reports have already been published using
porcine islets in rodents and nonhuman primates (briefly
reviewed by Larsen and Rolin 2004, also in this issue). In
terms of physiology and size, pig pancreas and heart are
both close to that of humans with subtle differences. For
instance, pig islets activate the human blood clotting system, inducing severe damage to the islets. This process is
associated with activation of platelets, leukocytes, and the
complement system (Bennet et al. 2000b). Entrapment of
islets in large thrombi will impair neovascularization and
engraftment. In addition, there are also molecular incompatibilities, such as incompatibilities at the level of several
receptors and ligands, including activated human coagulation factors and the natural anticoagulants expressed by porcine endothelial cells (Robson et al. 2000).
One of the major hurdles of xenotransplantation with
pig organs is the immune rejection phenomenon. Human
immune responses to pig organs is a complex process that
involves hyperacute rejection (HAR1), acute humoral xenograft rejection (AhXR1), and acute cellular xenograft rejection (AcXR1). Briefly, the main components in HAR are
natural xenoreactive antibodies in combination with
complement. In the pig-to-nonhuman primate model, HAR
is generally due to the existence of naturally occurring antibodies to Gal␣1,3Gal terminal oligosaccharides (Gal).
Binding of these natural antibodies to porcine tissues and
subsequent activation of complement result in the destruction of the parenchyma, vasculature, wide-spread interstitial
hemorrhage, and thorombosis in less than 24 hr after transplantation. Several antibody depletion procedures, use of
complement activation inhibitors that include soluble
complement receptor-1, complement activation blocker-2 (a
chimeric protein from human decay-accelerating factor
CD55 and membrane cofactor protein CD46, and treatment
with cobra venom factor have been shown to delay graft
rejection (Schuurman et al. 2003).
AhXR, also known as “acute vascular rejection,” is
characterized by antibodies, complement, and polymorphonuclear granulocytes. The antibody involved in AhXR may
be anti-Gal or induced antibodies. AcXR is similar to processes seen in allograft rejection. Cellular rejection is a
relatively rare phenomenon in pig-to-human primate solid
organ transplants. More frequently, AcXR accompanies
AhXR but is less severe than AhXR (Pino-Chavez 2001).
However, because porcine islets are nonvasuclarized cellular grafts, HAR is not a great problem. Instead, rejection of
ILAR Journal
islet cells appears several days after implantation and is
assumed to be mediated mainly by cellular mechanisms.
Essentially, the whole spectrum of mononuclear cells, including T cells (CD4+ and CD8+), B cells, natural killer
cells, and macrophages, may be present in the infiltrates.
Several published studies describe the potential of islet
xenotransplantation with varying degrees of success or failure. In all of these studies, wild-type pigs were used as the
organ donors. In future studies, pigs transgenic for human
complement inhibitors (Cozzi and White 1995) or cloned
pigs lacking the Gal epitope (Phelps et al. 2003) will be
favored as donor animals and are expected to enhance engraftment success. Several studies in nonhuman primates
have already shown that vascularized organs from transgenic pigs avoid hyperacute rejection, and the same will
presumably be true for organs from Gal-knockout pigs. Expression of the Gal epitope in the islets appears to vary
depending on age, with reduced levels of expression in the
islets of adult animals compared with fetal porcine islets
(Bennet et al. 2000a).
Ever since porcine endogenous retrovirus (PERV1) was
shown to be able to infect human cells in vitro (Patience et
al. 1997), a major concern has focused on the possibility
that PERV would infect the recipients of porcine xenografts,
and that infected patients would therefore pose a public
health hazard. Interestingly, earlier studies conducted in the
1990s in which fetal porcine islets were implanted into diabetic renal transplant patients provided an opportunity to
examine porcine tissue recipients for zoonosis or crossspecies infections. Because PERV is carried in the pig germline, potentially all recipients of porcine tissues or organs
will be exposed to the virus. Using serology, polymerase
chain reaction, and reverse transcriptase assays in each islet
recipient, it was established that there was no infection with
PERV (Heneine et al. 1998). In addition, Paradis and colleagues (1999) analyzed samples collected from 160 patients who had been treated with various living pig tissues
up to 12 yr earlier. No viremia was detected in any patient
despite a persistent microchimerism (i.e., presence of donor
cells in the recipient) in 23 patients for up to 8.5 yr. Finally,
a highly controversial Mexican trial wherein pig islets were
implanted intraperitoneally inT1D patients was reported at
the XIXth International Congress of Transplantation Society (Valdes Gonzalez 2002). In this trial, 17 adolescents
were implanted with pig islets derived from specific pathogen-free pigs and isolated by Diatranz Company (New Zealand). Of the recipients, 12 remained insulin free for more
than 12 mo, and five required reduced insulin doses.
Even when all scientific issues have been satisfactorily
addressed in the future, xenotransplantation will not become
a commonplace clinical procedure unless public at large,
and patients in particular, accept this procedure as ethical
and meaningful. Given that some 100 million pigs are
slaughtered for food every year in the United States, it will
not be a serious issue for the public to accept pigs as donors
of vital organs to save the life of patients. The fact that the
US Food and Drug Administration did not agree with a
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2004
moratorium demanded by Bach and colleagues (1998)
clearly indicates the keen interest and the hope the public
has for the potential of xenotransplantation. A better understanding of PERV infection and the development of protocols to control immune responses against xenografts should
convince even skeptics to appreciate the power of xenotransplantation in the treatment of insulin-dependent diabetes. Thus, available data from xenotransplantation provide a
basis for expectations in xenogeneic stem cell implantation.
In the meantime, stem cell-based therapies remain reasonable and practical alternatives.
Future Direction
For many years, it has been speculated that a small population of islet stem/progenitor cells persisted after organogenesis within the pancreatic ducts of both healthy and
diabetic individuals (Bonner-Weir et al. 1993). Nevertheless, the ability to culture and stimulate these stem cells into
functional islets in vitro represents a major breakthrough
that has great potential for the therapeutic intervention of
T1D. Yet, results from a number of laboratories indicate
that although we are able to initiate expansion and differentiation of such stem cells, we lack some of the basic
understanding to control the process fully. Identification of
temporal gene expressions during the developmental stages
of islets may provide the information necessary for sequentially stimulating stem cells to mature to end-stage islets.
Alternatively, the immature islets may represent the more
ideal reagent for implantation because they appear to have
the potential to respond to their in vivo environment,
thereby establishing homeostasis with the recipient through
vascularization, expansion of the beta cell mass, and differentiation to insulin-secreting cells. In this regard, stem cellderived IPSC/IPC cultures and their daughter islets starting
with animal tissues clearly mimic the human cultures. Fully
understanding each stage of differentiation and identifying
the best impant material will require further investigations.
At this time, this area appears to be best studied using
animal models in which syngeneic, allogeneic, and xenogeneic implantation can be examined to answer questions pertinent to bringing this technology to the clinics and the
patients.
A second aspect of this technology is the use of autologous/allogeneic islets versus xenogeneic islets. Although
our mouse studies using syngeneic islets generated in vitro
indicate that they may be masked from the autoimmune
response, it is definitely possible for allogeneic islets to be
rejected. This potential suggests an active T cell-mediated
allogeneic response, even if there is no reactivation of the
autoimmune response. It means that potentially, the recipient of allogeneic stem cell-derived implants would still require immunosuppression or, alternatively, the implant
would have to be isolated via a form of encapsulation, no
differently than recipients of xenogeneic implants. Although the ideal situation would be to isolate the recipient’s
265
own stem cells to provide autologous implants, “one patient
at a time,” this situation is not feasible if the goal is to treat
a large number of diabetic patients. So, although most patients would probably prefer to receive autologous islet implants, the use of allogeneic islets, or possibly xenogeneic
islets, is most expedient.
In the near future, as stem cells and stem cell-derived
tissues become a legitimate source of “islets” for preclinical
trials, two different animal models are expected to precede
human trials for safety and efficacy studies. The small animal model will no doubt be the diabetic mouse model, most
likely the NOD mouse; and it appears likely that the larger
animal model will be the miniature swine model of diabetes
(Larsen and Rolin 2004). This situation could change rapidly, however, should regulatory agencies insist on studies
in nonhuman primates prior to initiating human trials. The
test article in these trials will be human stem cells or their
derivatives. Our current understanding about stem cells and
xenotransplantation will be helpful in predicting outcomes.
Because stem cells are cultured in vitro for longer periods of
time, the antigenicity may be diminished enough to permit
less threatening immunosuppression therapy, making the
procedure ethically acceptable to a larger patient population. However, the longer in vitro culturing may lead to
substantial deviations of the stem cell-derived tissue from
normal cellular phenotypes, deviations from regulated insulin secretion, abnormal migratory patterns after implantation in vivo, and/or formation of transformed cells, just to
mention a few scenarios.
Finally, one of the advantages of being able to grow
endocrine pancreas in culture from stem cells is the ease
with which the IPSC/IPC cells can be genetically modified,
possibly to create daughter cells resistant to immunological
attack. In addition, genetic modification of the isolated endocrine pancreas stem cells, whether syngeneic, allogeneic,
or xenogeneic, may allow us the opportunity to build even
healthier and stronger islets. Although our discussion in this
article is centered on adult pancreatic stem cells, we do not
discount other sources of stem cells, such as bone marrow
and oval cells (Yang et al. 2002), capable of transdifferentiating to islet-like cells. Although several major
hurdles remain before stem cell therapy is a routine procedure, the time is rapidly approaching.
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