ß-Cell Differentiation from a Human Pancreatic Cell Line in Vitro and in Vivo
Dominique Dufayet de la Tour,
Tanya Halvorsen,
Carla Demeterco,
Björn Tyrberg,
Pamela Itkin-Ansari,
Mary Loy,
Soon-Jib Yoo,
Ergeng Hao,
Stuart Bossie and
Fred Levine
University of California San Diego Cancer Center La Jolla,
California 92093-0912
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ABSTRACT
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Cell transplantation therapy for diabetes is
limited by an inadequate supply of cells exhibiting glucose-responsive
insulin secretion. To generate an unlimited supply of human ß-cells,
inducibly transformed pancreatic ß-cell lines have been created by
expression of dominant oncogenes. The cell lines grow indefinitely but
lose differentiated function. Induction of ß-cell differentiation
was achieved by stimulating the signaling pathways downstream of the
transcription factor PDX-1, cell-cell contact, and the glucagon-like
peptide (GLP-1) receptor. Synergistic activation of those
pathways resulted in differentiation into functional ß-cells
exhibiting glucose-responsive insulin secretion in vitro.
Both oncogene-expressing and oncogene-deleted cells were transplanted
into nude mice and found to exhibit glucose-responsive insulin
secretion in vivo. The ability to grow unlimited quantities
of human ß-cells is a major step toward developing a cell
transplantation therapy for diabetes.
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INTRODUCTION
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The development of expanded populations of human pancreatic
ß-cells that can be used for cell transplantation is a major goal of
diabetes research (1). A number of alternative approaches are being
pursued to achieve that goal, including using porcine tissue as a
xenograft (2), expansion of primary human ß-cells with growth factors
such as hepatocyte growth factor/scatter factor (HGF/SF) and
extracellular matrix (ECM) (3), and generation of cell lines that
exhibit glucose-responsive insulin secretion (4). Although there
has been great interest in using porcine islets, they are difficult to
manipulate in vitro, and concerns have been raised about
endogenous and exogenous xenobiotic viruses being transmitted to graft
recipients (5). With primary human ß-cells, entry into the cell cycle
can be achieved using HGF/SF plus ECM (3, 6). However, this
combination, while resulting in a 23 x
104-fold expansion in the number of cells, is
limited by cellular senescence and loss of differentiated function,
particularly pancreatic hormone expression (3, 7). Recently, the use of
pancreatic or embryonic stem cells has been proposed as a source of
cells for ß-cell replacement. Human duct cells have been shown to
differentiate in vitro. However, the extent of expansion is
limited so far (8). Murine embryonal stem cells were reported to
exhibit glucose-responsive insulin secretion when selected with an
insulin promoter driving a dominant selectable marker, but that system
remains relatively uncharacterized and has not yet been applied to
human cells (9).
The focus of our work has been on developing immortalized cell lines
from the human endocrine pancreas (10, 11, 12). The cell lines are created
by infecting primary cultures of cells from various sources, including
adult islets, fetal islets, and purified ß-cells, with a retroviral
vector expressing the potent dominant oncogenes SV40 T antigen and
H-rasval12 (12, 13). The oncogenes are flanked by
loxP DNA sequences, allowing for oncogene deletion by cre recombinase
(7). The combined effect of the oncogenes is to trigger growth factor
and ECM-independent entry into the cell cycle, as well as to prolong
the lifespan of the cells from 1015 population doublings for primary
cells to approximately 150 doubling for the oncogeneexpressing
cells (7). Further introduction of the gene encoding the hTERT
component of telomerase results in immortalization, allowing the cells
to be grown indefinitely (7).
Previously, we have shown that a cell line, TRM-6, derived from human
fetal islets, was capable of differentiating along the pancreatic
-cell lineage upon expression of the homeodomain transcription
factor PDX-1 and promotion of cell-cell contact (11). Here, we show
that ßlox5, a cell line derived from purified adult ß-cells, can be
induced to exhibit glucoseresponsive insulin secretion in
vitro and in vivo. The ability to grow an unlimited
quantity of human ß-cells has implications for cell transplantation
therapies for diabetes.
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RESULTS
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Pancreatic Hormones Are Not Expressed in ßlox5
ßlox5 is an immortalized cell line derived from a purified
population of human ß-cells by infection with retroviral vectors
expressing the SV40 T antigen, H- rasval12,
and hTERT oncogenes (12). While low levels of insulin expression could
be detected immediately after initial isolation of the cell line,
insulin subsequently became undetectable (Fig. 1A
, lane 9). Similar to other human
pancreatic cell lines that we have developed (10, 11), somatostatin
remained detectable in some samples at the limit of detection of the
RT-PCR assay (Fig. 1B
, lane 3). However, the strength of the
somatostatin signal was so weak that no meaningful quantitation or
comparison from one sample to another was possible.

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Figure 1. RT-PCR Analysis of Pancreatic Hormone Gene
Expression in ßlox5
The conditions tested were ßlox5 grown in monolayer culture, ßlox5
cells infected with a retroviral vector expressing PDX-1
(ßlox5/PDX-1), ßlox5 grown as three-dimensional aggregates, and
ßlox5 treated with exendin-4. A, Insulin; B, other pancreatic
hormones: PP, glucagon, somatostatin, and IAPP. Human adult islet cDNA
was used as positive control. In this figure as well as in Figs. 2 and 4B , the lower band on the gel represents PCR primers.
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A Subset of Insulin Transcription Factors Are Expressed in
ßlox5
To begin to address the reason for the loss of hormone expression,
the expression of some of the transcription factors that are important
in establishing and maintaining ß-cell differentiated function was
examined (Fig. 2
). Most significantly,
PDX-1, a homeodomain transcription factor that is required for ß-cell
development and function, is not expressed in ßlox5 by RT-PCR (not
shown) or electrophoretic mobility shift assay (EMSA) (Fig. 2A
, lane
2). This differs from growth-stimulated primary cells, where PDX-1
continues to be expressed (3). BETA2 (NeuroD1), a basic
helix-loop-helix (bHLH) factor important in neurogenesis as well as
pancreatic endocrine cell development and function, is not expressed in
ßlox5 (not shown and Fig. 2C
). CREB (cAMP-response element-binding
protein), which plays a critical role in cAMP responsiveness in
ß-cells, is expressed at a low level, being variably detectable at
the limit of sensitivity of the Western blot (Fig. 2D
). Pax6, which is
expressed in all endocrine cells, was expressed at high levels in
ßlox5, consistent with an origin from primary endocrine cells (Fig. 2B
, lane 5). RIPE3b, a yet uncloned factor that plays an important role
in insulin gene expression by binding to the human insulin promoter C1
element (14, 15), is present as detected by EMSA (Fig. 2E
). Overall, it
is clear that some, but not all, of the transcription factors that play
important roles in establishing and maintaining ß-cell function are
expressed in ßlox5 cells.

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Figure 2. Analysis of Transcription Factors Expressed in
ßlox5 Cells
A, EMSA for PDX-1; B, RT-PCR analysis of Pax6. cDNAs from Jurkat, a T
cell line that does not express Pax6 and from total pancreas were used
as negative and positive controls, respectively. C, RT-PCR analysis of
BETA2. Human adult islet cDNA was used as a positive control; D,
Western blot analysis of CREB. E, EMSA for RIPE3b. Nuclear extracts
from the TRM6 cell line that does not express RIPE3b was as a negative
control.
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PDX-1, Cell-Cell Contact, and Glucagon-Like Peptide 1 Receptor
(GLP-1R) Activation Work Synergistically to Activate Insulin Gene
Transcription
Previously, we showed that introduction of PDX-1 into a cell line,
TRM-6, derived from human fetal islets, resulted in an increase in
somatostatin gene expression (11). Furthermore, PDX-1 acted
synergistically with cell-cell contact to further increase the level of
somatostatin expression to 1,000-fold above that of cells not
expressing PDX-1, to a level similar to that found in normal human
islets (11). To determine whether ßlox5 could be induced to express
pancreatic hormones, PDX-1 was expressed in ßlox5 using a retroviral
vector (11). ßlox5 did not respond to PDX-1 and cell-cell contact
with an increase in hormone expression (Fig. 1
, A and B, lane 3),
despite the fact that PDX-1 expression in ßlox5 resulted in
functional protein as evidenced by its ability to bind DNA in EMSA
(Fig. 2A
, lane 3). Therefore, other factors were examined for their
ability to induce hormone expression in ßlox5 cells expressing PDX-1.
Activin has been reported to be able to induce endocrine
differentiation in cell lines and primary cells (16, 17, 18, 19). However, it
had no effect on hormone expression in ßlox5 (data not shown).
Similarly, nicotinamide and HGF, which induce endocrine differentiation
of human fetal pancreatic cells (20, 21), also had no effect.
Glucagon-like peptide-1 (GLP-1), a small peptide cleaved
from the proglucagon molecule, is an insulin secretagogue (22). A
homolog of GLP-1 derived from Gila lizards, exendin-4, has
recently been found to stimulate rodent ß-cell differentiation
in vitro and in vivo (23, 24). Therefore,
exendin-4 was tested for its ability to stimulate ß-cell
differentiation in ßlox5. By itself, exendin-4 had no effect on
hormone expression in ßlox5 (Fig. 1A
, lane 8 and not shown). However,
when ßlox5 cells expressing PDX-1 were grown in suspension culture as
three-dimensional aggregates in the presence of exendin-4, a
substantial amount of insulin mRNA was detectable (Fig. 1A
, lane 2).
The synergistic nature of the interaction between exendin-4, cell-cell
contact, and PDX-1 is evident from the fact that no insulin mRNA was
detectable with any one or two of the three inducing factors (Fig. 1A
).
Quantitative RT-PCR revealed that the level of insulin mRNA in induced
ßlox5 cells ranges from one to one-tenth that of adult islets,
depending on the quality of the islet preparation (data not shown).
Islet amyloid polypeptide (IAPP) mRNA, a hormone that is cosecreted
with insulin, was also expressed in induced ßlox5 cells (Fig. 1B
, lane 2). However, the other major pancreatic hormones, glucagon,
somatostatin, and pancreatic polypeptide (PP), were not induced
(Fig. 1B
).
Induction of ß-Cell Differentiation in ßlox5 Correlates with
Up-Regulation of BETA2/NeuroD1 and CREB Expression
The expression of transcription factors important in ß-cell
development was examined to determine whether they had been induced by
PDX-1, cell-cell contact, and exendin-4. RIPE3b binding was not altered
by PDX-1, cell-cell contact, or exendin-4 (Fig. 2E
). BETA2 was induced
only by the combination of all three inducing factors (Fig. 2C
, lane
3). In addition to acting as an insulin secretagogue (25),
GLP-1 has been reported to stimulate insulin gene promoter
activity by a protein kinase A-independent mechanism mediated by a bZIP
transcription factor related in structure to CREB (26). Therefore, it
was interesting that CREB, which plays a critical role in the cAMP
response of ß-cells, is increased in cells exposed to exendin-4,
independently of cell-cell contact and PDX-1 (Fig. 2D
). This is the
first report that activation of the GLP-1 receptor can
induce CREB expression, suggesting that GLP-1 may act at
multiple levels in promoting cAMP signaling.
Induced ßLox5 Cells Exhibit Glucose-Responsive Insulin Secretion
in Vitro
A hallmark of functional ß-cells is the ability to secrete
insulin in response to blood glucose. Primary ß-cells store large
quantities of insulin and secrete it in response to a variety of
physiological stimuli, most significantly extracellular glucose.
Induced ßlox5 cells have an insulin content between 6 and 21
pmol/µg DNA, compared with adult islet preparations, which in our
hands have insulin contents between 50 and 250 pmol/µg DNA.
Intracellular insulin is detectable by immunohistochemistry in the
induced ßlox5 cells (Fig. 3B
), whereas no cells were stained before
aggregation (Fig. 3A
). In parallel, very
few secretory granules were found in the uninduced cells (Fig. 3C
).
Those granules were pale stained, with the appearance of somatostatin
granules, consistent with the known presence of a very low level of
somatostatin mRNA in the uninduced cells (Fig. 1B
, lane 3). In
contrast, the morphology of the granules in the induced cells (Fig. 3D
)
varied from electron opaque granules with a white halo typical for
primary ß-cells, to pale staining granules that might be immature
insulin granules (Fig. 3D
, inset). In both conditions,
electron opaque structures at about the size of small mitochondria,
filled with spiral membranes, were found. These structures are easily
mistaken for large secretory granules in the overview pictures (Fig. 3
, C and D), but have a morphology consistent with remnants of
hyperproliferating endoplasmic reticulum.

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Figure 3. Insulin Immunohistochemical and Ultrastructural
Analysis of Uninduced (A and C) and Induced (B and D) ßlox5 Cells
A and B, Insulin immunohistochemistry (105x magnification); C and D,
electron micrographs (2,900x magnification, inset
22,000x).
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Consistent with the presence of morphologically mature insulin
granules, media from induced ßlox5 cells contain processed insulin
that comigrates with bovine insulin (Fig. 4A
). Interestingly, out of six duplicate
wells of induced ßlox5 cells, one did not exhibit any insulin
secretion (Fig. 4A
, lane 4) or mRNA by RT-PCR (not shown). We have
found repeatedly that occasional cultures of exendin-4-treated
ßlox5/PDX-1 aggregates fail to differentiate. The reason for this is
under investigation but seems to be due in large part to loss of PDX-1
expression over time. Previously, we have found that PDX-1 causes a
decrease in the growth rate of the human pancreatic endocrine cell line
TRM-6 (11). ßLox5 cells exhibit an even more pronounced growth arrest
in response to PDX-1 expression, leading to strong selection for loss
of PDX-1 over time in culture.

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Figure 4. Analysis of Glucose-Responsive Insulin Secretion
A, Insulin Western blot analysis of conditioned medium from induced
ßlox5 cells. Bovine insulin was used as a positive control. Fresh
tissue culture media were used as a negative control; B, RT-PCR for
glucokinase. cDNA from human total pancreas was used as a positive
control; C, Assay for insulin secreted from induced ßlox5 cells grown
in culture medium containing increasing concentrations of glucose. Two
independent cultures of induced ßlox5 cells were studied (open
boxes) and compared with a single preparation of adult human
islets (closed triangle).
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A major component of the glucose-sensing apparatus is glucokinase (27).
Induced ßlox5 cells express glucokinase mRNA, while uninduced cells
do not (Fig. 4B
). The extent to which ßlox5 cells exhibit
glucose-responsive insulin secretion was tested by culturing two
independent cultures of induced ßlox5 cells in different
concentrations of glucose and measuring insulin release in the culture
medium by ELISA (Fig. 4C
). Consistent with the lower insulin content of
the induced ßlox5 cells, the amount of insulin secreted in response
to glucose is 3- to 4-fold lower than adult islets. Induced ßlox5
cells exposed to high concentrations of glucose for prolonged periods
become depleted of insulin more rapidly than primary islets (not
shown).
Oncogene-Deleted ßlox5 Cells Exhibit Glucose-Responsive Insulin
Secretion in Vivo
To test the function of ßlox5 cells in vivo, cells
were transplanted under the kidney capsule of nude mice. To reduce the
risk of tumorigenicity after transplantation, the LTPRRTNllox
retroviral vector incorporates lox sites in the long terminal repeats
(LTRs). This allows the oncogenes to be deleted by expression of cre
recombinase. To express cre, ßlox5 cells were infected with an
adenoviral vector (Ad-cre) (28) at a multiplicity of infection of 50.
While this resulted in a large reduction in SV40 T antigen expression,
a low level of expression persisted, reflecting inefficiency of
infection and/or cre-mediated recombination (Fig. 5
). The following conditions were
studied: PDX-1 expression, oncogene deletion with Ad-cre, and exposure
of the cells to exendin-4 in vitro and/or in
vivo. Animals were monitored for tumor formation and circulating
human C-peptide levels up to a maximum of 3 months after
transplantation, when a final C-peptide determination was made (Fig. 6
). Substantial levels of circulating
human C-peptide were present only in animals grafted with cells
expressing PDX-1. Furthermore, oncogene deletion with Ad-cre and/or
exendin-4 treatment in vitro was also required for
circulating C-peptide to be present. The incidence of tumors was much
lower with oncogene-deleted cells (1 of 10 animals) compared with
oncogene-expressing cells (16 of 23 animals). The level of C-peptide
secreted by the grafted cells is comparable to the amount secreted by
primary islet cell grafts that have been shown to cure diabetes in
streptozotocin-treated nude mice (29). Interestingly, animals injected
with exendin-4 in vivo did not have any detectable
C-peptide, suggesting that continued administration is deleterious to
graft function. Significantly, C-peptide levels in fasting animals
before and after intraperitoneal glucose stimulation demonstrated that
the C-peptide secretion was glucose responsive.

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Figure 5. Western Blot of SV40LT Protein in ßlox5 before
and after Infection with Ad-cre
Whole cell extract from 293T cells was used as a positive control and
from 293 GFP as a negative control.
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Figure 6. Human C-Peptide Levels in Mice Grafted with ßlox5
Cells before and after Glucose Stimulation
Black bars represent basal C-peptide levels before
glucose stimulation. Open bars represent
glucose-stimulated Cpeptide levels. The presence (+) or absence
() of a tumor in each animal is indicated immediately beneath the
C-peptide data from that animal. Seven groups were transplanted with
ßlox5 cells: uninduced cells, Ad-Cre infected cells, ßlox5/Pdx-1
cells, Ad-Cre infected ßlox5/Pdx-1 cells, ßlox5/Pdx-1 cells exposed
to exendin-4 in vitro (induced cells), ßlox5/Pdx-1
cells exposed to exendin-4 in vivo, and ßlox5/Pdx-1
cells exposed to exendin-4 in vitro and in
vivo. Animals were scored as having a tumor if the size of the
graft was significantly greater than the 10 µl volume of cells
initially grafted.
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DISCUSSION
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Beginning with a cell line that exhibited few ß-cell
characteristics despite having been originally derived from human
ß-cells, we have successfully induced ß-cell function in
vitro and in vivo. Induction of ß-cell
differentiation in ßlox5 is complex, requiring synergistic
interactions between at least three inducing factors, PDX-1, cell-cell
contact, and activation of the GLP-1 receptor. In
addition, it is likely that those three inducing factors interact with
gene products expressed in ßlox5 to induce ß-cell differentiation,
since another cell line, TRM-6, responds to the PDX-1 and cell-cell
contact by differentiating along the
-cell lineage (11). Cell lines
such as TRM-6 (
-cell lineage) and ßlox5 (ß-cell lineage) are
powerful tools that should allow identification of the full complement
of genes that are required for endocrine cell development and function.
Furthermore, the requirement for multiple interacting inducing factors
provides an opportunity to study how different signal transduction
pathways interact with one another to control a complex differentiation
program.
A problem with our current technology is that PDX-1 expression leads to
a substantial decrease in the growth rate of the cells. Unlike TRM-6,
where we have been able to maintain stable populations of cells
expressing PDX-1 indefinitely, it has not been possible to maintain
PDX-1 expression in ßlox5. Expressing PDX-1 under inducible control
may solve this problem and such studies are underway. The level of
insulin stored and secreted by induced ßlox5 cells, while substantial
compared with rodent insulinoma cell lines, remains substantially below
that of normal human islets. Whether this level of insulin is
sufficient to cure diabetes remains to be tested. It is possible that
higher levels of insulin production could be engineered into the cells,
either directly by overexpressing the proinsulin gene or indirectly by
further manipulation of insulin gene transactivators. It is encouraging
that the shape of the glucose response curve is quite similar to that
of normal human islets. Most other insulinoma cell lines exhibit
maximal insulin secretion at a much lower glucose concentration, which
is thought to be due to an imbalance in the glucokinase/hexokinase
ratio (30).
The availability of an unlimited source of functional human
ß-cells has important implications for diabetes. One straightforward
application is in exploring aspects of ß-cell biology that would
benefit from an unlimited, homogeneous source of cells. High-throughput
screening for new diabetes drugs is one such application. Ultimately,
we hope to be able to use cells such as those presented here in a cell
transplantation therapy for diabetes. However, a number of issues must
be dealt with before an immortalized cell line can be used in humans.
One problem has to do with effects of the oncogenes on the biological
properties of the cells. Cell lines from the human endocrine pancreas
developed using SV40 T antigen plus H-rasval12
are tumorigenic (10). Although this does not prevent them from
differentiating into fully functional ß-cells in vitro,
tumorigenic cells cannot be transplanted into humans unless they are
safely encapsulated. The vector used to create the cell lines
incorporates lox sites so that the oncogenes can be deleted by
expression of the cre recombinase in the cells after expansion (12), so
it may be possible to avoid encapsulation by oncogene deletion before
transplantation. The development of improved vectors is necessary to
allow for complete oncogene deletion. Such studies are in progress.
Immune rejection of grafted cells has been a major obstacle to
successful islet transplantation, but recent advances in therapy for
allograft rejection may make this less of a concern (31, 32). A
potential advantage of using an immortalized cell line as a source of
transplantable cells is that they can be engineered to exhibit
desirable qualities, including avoidance or suppression of host immune
responses.
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MATERIALS AND METHODS
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Cells and Cell Culture
All the cells were maintained in DMEM with 5.5 mM
glucose and 10% FBS. Aggregation of cells into three-dimensional
clusters and the retroviral vector expressing PDX-1 have been described
previously (11). Infection with the PDX-1 virus was done at a
multiplicity of infection of 25. Exendin-4 (Sigma, St
Louis, MO) was used at a concentration of 10 nM in PBS.
RT-PCR
RT-PCRs for somatostatin, glucagon, IAPP, and glucokinase have
been described previously (11, 33). Quantitative insulin RT-PCR was
done by interpolation from a standard curve constructed using a plasmid
containing the human insulin cDNA (3). RT-PCR for BETA2 and Pax-6 were
performed using the following primers: BETA2
Forward-cagaaccaggacatgccc, BETA2 Reverse-atcaaaggaagggctggtg, Pax-6
Forward-gccttttattgtgagagtgg, Pax-6 Reverse-ctcacacatccgttggacac. PCR
amplification conditions were 5 min at 94 C followed by 40 cycles of 94
C for 45 sec, 60 C for 45 sec, 72 C for 45 sec. All cDNA preparations
were checked for the quality and concentration of cDNA by RT-PCR for
the housekeeping gene porphobilinogen deaminase (11).
Protein Expression
EMSA for Pdx-1 and RIPE3b were performed using probes derived
from the human insulin promoter A5 and C1 elements, respectively (11, 14). Western blot for CREB was performed using a polyclonal antibody (a
kind gift from Dr. Marc Montminy) at a dilution of 1:200 on 10 µg of
nuclear extracts. Western blot for insulin was done using a monoclonal
antibody (Insulin 2D11-H5, Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) at a dilution of 1:500 on 40 µl conditioned media.
Anti-SV40 T antigen antibody (PharMingen, La Jolla, CA)
was used at a concentration of 1 µg/ml. Induced ßlox5 cells were
cultured in DMEM containing a single concentration of added glucose for
1 h. Medium was harvested and assayed for insulin by RIA
(Diagnostic Products, Los Angeles, CA) or
enzyme-linked immunosorbent assay (ELISA) (Ultrasensitive human insulin
ELISA; Mercodia, Uppsala, Sweden).
Immunohistochemistry
ßlox5 aggregates or single cells were fixed in 4%
paraformaldehyde for 3 h, embedded in 2% agarose, fixed for
another 21 h, processed for paraffin embedding, and sectioned in 5
µm slices. Specimens were stained for insulin with an antihuman
insulin antibody (Santa Cruz Biotechnology, Inc.) and a
peroxidase/diaminobenzidine detection system providing a brown color
precipitate (EnVision+, DAKO Corp., Glostrup,
Denmark).
Ultrastructural Analysis
ßlox5 aggregates or single cells were washed in 0.1
M cacodylate buffer and fixed in 2% glutaraldehyde in
cacodylate buffer for 1 h. Specimens were then rinsed in
cacodylate buffer and dH2O, postfixed in 1%
OsO4 in dH2O for 1 h,
rinsed in dH2O and 70% ethanol, stained en
bloc with 2% uranyl acetate in 70% ethanol, dehydrated in a
graded series of ethanol, embedded in Epon 812 (34), sectioned in 80-nm
slices, and contrasted with 2% lead citrate and 2% uranyl acetate.
The specimens were randomly photographed in a transmission electron
microscope (Hitachi-600; Hitachi Scientific Instruments, Inc., Tokyo, Japan) at an acceleration voltage of
75 kV.
Transplantation Studies
Human cell transplants were performed by inserting 10 µl of
packed aggregated cells under the kidney capsule of 7-week-old
NIH Swiss homozygous athymic nude mice as previously described
(35).
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ACKNOWLEDGMENTS
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We thank the Juvenile Diabetes Foundation Islet Isolation
Program and particularly the Islet Transplant Centers at the University
of Minnesota and University of Miami for providing human islets for
these studies.
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FOOTNOTES
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Address requests for reprints to: Dr. Fred Levine, Center for Molecular Genetics, University of California San Diego School of Medicine, La Jolla, California 92093-0634. E-mail: flevine{at}ucsd.edu
This work was supported by grants to Fred Levine from the NIDDK
(DK-55065 and DK-55283), the Juvenile Diabetes Foundation (197035), and
the Stern Foundation. Bjorn Tyrberg was supported by the Swedish
Academy of Pharmaceutical Sciences Fellowship. Soon-Jib Yoo was
supported by a research scholarship from the Catholic University of
Korea.
Received for publication September 19, 2000.
Revision received October 26, 2000.
Accepted for publication November 21, 2000.
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