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Transplantation of the fetal rat pancreasQuantitative morphological analysis of islet tissue growth.

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Transplantation of the Fetal Rat Pancreas: Quantitative
Morphological Analysis of Islet Tissue Growth
ORION D. HEGRE, ROBERT J. LEONARD, JAMES D. RUSIN AND
ARNOLD LAZAROW 1
D e p a r t m e n t of A n a t o m y , U n i v e r s i t y of M i n n e s o t a ,
M i n n e a p o l i s , M i n n e s o t a 55455
ABSTRACT
When pancreases from fetal rats were transplanted beneath the
kidney capsule of isologous normal adult recipients, continued growth and differentiation of the endocrine portion of the pancreas occurred. While limited
amounts of acinar tissue were identifiable in the early transplant period ( 7 days),
such cells were absent in long term transplants ( 1 4 and 21 days). In contrast,
while few definitive islet beta cells were present a t the time of transplantation,
following 21 days at the kidney site large circumscribed islets comprised of
heavily granulated beta cells in association with duct epithelial cells predominated. Mitotic figures were seen in both these cell populations. Total islet mass
had increased over 20-fold during the transplantation period. Similar results were
observed if fetal pancreases were grown in organ culture for ten days prior to
transplantation. Continued islet and duct cell growth, as evidenced by mitotic
figures and an increase in absolute islet cell mass was obtained in such cultured
explants when transplanted to either isogenic or allogenic recipients.
These observations support the hypothesis that fetal pancreas may be the best
source of donor material for transplantation to diabetic recipients, in part, due
to the continued growth and differentiation of the islet tissue during the transplantation period.
Growth as well as survival of a wide
variety of transplanted organ slices has
been reported. Various transplantation
sites have been utilized (anterior eye chamber, kidney capsule, testis, subcutaneous
pocket and cheek pouch) and organs examined have included adrenal (Dameron, '50;
Martinovitch, '55), thymus (Dameron, '50;
Metcalf et al., '65), skin cerebrum, stomach, kidney (Browning, '49), ovary, testis,
thyroid (Dameron, 'SO), and pancreas
(Browning and Resnick, '51; Coupland,
'60; House et al., '61; Gonet and Renold,
'65; Sak et al., '66; Macchi et al., '68;
Hegre et al., '70, '72a; Hultquist, '72;
Brown et al., '74). Among investigators
studying the effects of donor age on graft
survival and growth, increased success
with younger aged tissue has been consistently reported for both translanted pancreas, (Browning and Resnick, '51; House
et al., '61; Macchi et al., '68) as well as
other organ slices (Greene, '43; Browning,
'49; Martinovitch, '55). While neonatal
AXAT. REC., 185: 209-222.
pancreatic tissue was in general superior
to adult pancreas, in those studies where
fetal tissue comparisons were made
(Browning and Resnick, '51; House et al.,
'61) fetal pancreas was found to exhibit
the best islet survival with continued differentiation and growth of islet reported
during the transplantation period.
Estimates of pancreatic growth have
been based on gross morphological measurements or on qualitative microscopic
examination of the grafted tissue. The observation of mitotic figures within grafted
islet beta cells (Sak et al., '66) and increased insulin content of the transplanted
pancreatic fragments (Gonet and Renold,
'65; Hegre et al., '72a) have further supported the hypothesis that growth of islet
tissue occurs at the transplant site. However, because of the qualitative nature of
the morphological observations, accompanReceived Oct. 17, '75. Accepted Dec. 17, '75.
1 Deceased, June 25, 1975.
209
210
0. HEGRE, R. LEONARD, J. RUSIN AND A. LAZAROW
ied by the fact that most investigators reported loss of the acinar component of the
pancreatic graft (Browning and Resnick,
'51; Coupland, '60; House et al., '61; Gonet
and Renold, '65; Macchi et al., '68; Brown
et al., '74) and the presence (or development) of large amounts of adipose tissue
within the graft (Browning and Resnick,
'51; Coupland, '60; House et al., '61; Macchi et al., '68; Brown et al., '74) it is possible that the reported increases in islet tissue represent relative changes as opposed
to absolute increases in mass.
To examine the extent, if any, of islet
growth during the transplantation period,
we morphologically quantitated the fetal
pancreatic islet mass prior to and following transplantation under a number of
experimental conditions. In the present
experiments, transplants of fetal pancreas
and organ cultured fetal pancreas were
placed at two sites; beneath the kidney
capsule and into the anterior chamber of
the eye. Both sites have been successfully
utilized previously for pancreatic transplantation (Coupland, '60; Hegre et al.,
'70; Hegre et al., '72a; Hultquist, '72;
Brown et al., '74) and each permitted the
easy recovery of the transplanted tissue
for subsequent quantitative morphological
analysis. The results reported in this communication establish that continued differentiation and growth of fetal pancreatic
islet tissue occurs during the transplantation period resulting in absolute increases
in islet tissue mass of up to 20-fold.
MATERIALS AND METHODS
Animals
Rats of the inbred Fischer strain (FSH,
ARS/Sprague-Dawley Technical Center)
were used in the isotransplantation studies. Allotransplants were made between
fetuses of the outbred Sprague-Dawley
strain (SD, Thorpe Industries) and their
maternal hosts. The age of the fetuses was
calculated from witnessed matings within
our colonies.
Fetuses were removed by sterile hysterectomy on day 17.5 or day 19.5 postcoitum. Bisection of pancreases from 17.5
day fetuses (FSH) resulted i n an appropriate sized slice (0.5 mg) for either explantation or transplantation. Pancreases
from 19.5 day fetuses ( S D ) were dissected
into somewhat larger (1.0 m g ) explants.
Organ culture
Explants were grown in our organ culture system for four or ten days on a
millipore filter grid supported by a platform of stainless steel at the gas-liquid
media interface. The medium consisted
of equal parts of chicken (GIBCO) or rooster serum and freshly prepared 12-day
chick embryo extract (Hegre et al., '70).
The atmosphere in the culture chamber
(Falcon 3010) was 95% 0,-5% CO, and
was renewed each day. Glucose concentration of the medium averaged 165 m g /
100 ml. Explants were transferred to fresh
medium at 48-hour intervals. Following
organ culture, explants were removed from
their millipore grids and either fixed for
microscopic study or prepared for transplantation.
Transplantation
Two to three segments of fetal pancreas
were placed at the transplant site of an
adult host. Details of the procedures have
been reported (Hegre, '70). Briefly, transplants were made at the kidney site
through a small opening made in the renal
capsule with a scalpel blade. The transplant was inserted under the capsule onto
the cortex with microdissection forceps.
For transplantation to the eye site, the
bulb was delivered through a slit in a
piece of rubber dam and the anterior
chamber was opened by incising the cornea just above the corneal-scleral junction.
The tissue was inserted with microdissecting forceps so that it rested on the iris
opposite the incision.
Organ cultures of fetal pancreas were
transplanted in an identical manner except
that they were removed from their supportive grid and washed in Tyrode's solution
prior to transplantation.
At various times ( 4 through 21 days)
following transplantation, the host was
sacrificed and the grafts removed, fixed in
Bouin's solution and prepared for histological study. In some cases, with the
recipient anesthetized, transplants were
examined with a dissecting microscope
prior to fixation.
FETAL PANCREATIC TRANSPLANTATION
Histological preparatioii and quantitation
of tissue
Tissues were fixed in Bouin's solution,
embedded in paraffin, and serially sectioned at 4 pm. Sections were stained with
aldehyde fuchsin (Gomori, '50) and Ponceau de xylidine. Tissues containing well
circumscribed islets (with centrally located
aldehyde fuchsin positive beta cells and
periphically located aldehyde fuchsin negative cells) offered no problem in quantification. However, in many of the tissues
considerable cellular reorganization had
taken place during the experimental period. In these cases, only cells with aldehyde fuchsin positive granules were classified as islet tissue. Tissue components
were quantitated by the linear scan method
the details of which have been described
(Lazarow and Carpenter, '62; Carpenter
and Lazarow, '62; Hegre et al., '72b). The
scanning instrument consists of a microscope equipped with a mechanically driven
stage and a reference point in the eyepiece.
The stage can be advanced horizontally by
turning a n y of five micrometers, each of
which records the distance travelled over
a different tissue component. The stage is
then advanced vertically by a measured
increment using a nonrecording micrometer, a second parallel scan is made across
the section, and the components are similarly recorded. Successive parallel scans
were made throughout the entire section.
To ensure representative sampling, sections were scanned at a constant interval
until the entire block had been processed.
Using this method of systematic scanning
we have previously shown that the recorded distances ( m m of scan) are directly
proportional to tissue mass or weight
(Hegre et al., '72b).
RESULTS
Eighteen-day fetal pancreas isotransplanted directly to the renal
subcapsular site
Transplants were well vascularized and
appeared translucent macroscopically.
Grafts examined two and three weeks posttransplantation frequently contained large
areas of adipose tissue. (fig. 1 ) Upon
removal, the transplant frequently re-
211
mained adherent to the renal capsule. The
underlying kidney cortex appeared undamaged by the transplant. Twenty-four of
these pancreatic isoimplants were prepared
for microscopic examination. No evidence
of tissue rejection was observed. Although
acinar cells were readily observable after
only seven days of transplantation (fig. 2 ) ,
their number decreased and only occasional acinar cells were seen after 21 days
of transplantation. Ducts, in contrast, were
the predominant feature of the implants.
Mitotic figures were irequently observed
in this cell population (fig. 3 ) . Associated
with the duct elements were well-circumscribed regions of aldehyde fuchsin positive islets (figs. 4, 5). The islets appeared
to be comprised almost exclusively of heavily granulated beta cells (fig. 4 ) . The total
mass of the implanted tissue had increased
by about 6-fold two to three weeks following isotransplantation. Quantitative analysis of these implants revealed an islet mass
of 6.8 2 1.1 units compared to 0.3 t 0.1
islet mass units prior to transplantation
(table I), representing a 23-fold increase
in islet tissue mass during the transplant
period (p = < 0.001).
Organ cultures of 18-day fetal pancreas
isotransplanted to the renal subcapsular
or anterior eye chamber site
Following ten days of organ culture, explants consisted primarily of duct and islet
tissue with few acinar cells (fig. 6 ) . Such
cultures were placed beneath the renal
capsule or into the anterior eye chamber
of an isologous host. Following isotransplantation cultures appeared similar to
fresh grafts; highly vascularized and
translucent. However no areas of adipose
tissue were observed, The transplants at
the renal site, although adherent to the
kidney cortex, had not embedded themselves within it. At the eye site, the transplants were inseparable from the underlying iris. Five of these organ cultured
isografts w-ere removed from each site and
prepared for microscopic examination. Acinar cells were absent from the transplant
while duct cells, in association with islet
areas, were the predominant feature following transplantation (fig. 7). Duct cells
in mitosis were frequently observed. Fol-
212
0. HEGRE, R. LEONARD, J. RUSIN AND A. LAZAROW
TABLE 1
Islet mass (mm scan)
Type of transplant
(donor/recipient)
Isotransplant
(FSH/FSH >
Isotransplant '
(FSH/FSH)
Allotransplant
( SD/SD )
1
Fetal
donor
age
Transplantation
site
in
culture
18
Kidney
0
D?ys
Pretransplant
0.3+-0.1
(6)
1.120.2
(2)
2.8 C 0.4
\
18
Kidney
20
Eye
10
4
Posttransplant
7-10-day
14-2 1-day
-
6 . 8 2 1.1
2.3 t 0.8
(2)
4.9 t- 0.6
3.6 2'0.3
(7)
,
(6)
(3)
-
(8)
Mean f S.E.M.
(N)
lowing twenty-one days at the renal site,
aldehyde fuchsin positive beta cells were
the predominant feature of the transplants
(fig. 8 ) . Many of the beta cells were columnar shaped and arranged around a central lumen (figs. 8, 9 ) . Polarization of the
cells was evident with the aldehyde fuchsin
positive granules concentrated away from
the luminal surface on the side of the cell
approximating the stroma (fig. 9 ) . After
culture but prior to transplantation quantitative analysis of the cultures revealed an
islet mass of 1.1 f 0.2 islet mass units
(table 1 ) . Following two to three weeks
of isotransplantation, although the total
mass of the cultured grafts had not increased, grafts at the kidney contained
3.6 = 0.3 islet mass units, representing a
nearly 3.5-fold increase in islet mass during the transplant period.
Organ cultures of 20-day fetal pancreas
allotransplanted to the anterior
chamber of the eye
Following four days in organ culture,
explants of 20-day fetal pancreases were
composed primarily of duct and islet cells
with some acinar cells persisting. Mitoses
were seen in the duct cell population (fig.
10). Cultures were subsequently placed
in the anterior eye chamber of adult maternal hosts. Four days following allotransplantation transplants were highly vascularized and were inseparable from the
underlying iris. No areas of adipose tissue
were observed in the transplants. Microscopic examination of 28 grafts after four
days of transplantation revealed few if
any acinar cells while ducts with associated
islet tissue predominated in the tissue (fig.
11). Mitotic figures were observed within
islet beta and duct cells (figs. 11, 12). By
ten days posttransplantation, many grafts
showed regions of lymphocytic infiltration
with associated degenerating islets; however, aldehyde fuchsin positive islets were
identifiable in 95% of the 44 transplants
examined (fig. 13). Although the total
mass of cultured transplants remained
unchanged following allotransplantation,
quantitative analysis of the transplants revealed an increase in islet mass. After ten
days of allotransplantation, 4.9 2 0.6 islet
mass units were measured in the tissue
compared to 2.8 i 0.4 islet mass units
following culture but prior to transplantation (table 1). This represented a nearly
60% increase in islet mass during the
transplantation period ( p = 0.015).
DISCUSSION
Quantitative morphological analysis of
isotransplants of fetal rat pancreas has
established that a selective differentiation
and growth of islet tissue occurs during the
transplantation period; in a manner similar to that previously observed in vitro
(Murrell, '66; Vecchio, '67; Hegre et al.,
'72b). We have previously suggested that
growth of dispersed islet tissue occurs
following intraperitoneal isotransplantation
(Leonard et al., '73; Hegre et al.,'75). The
presence of mitotic figures in beta cells of
grafts recovered from reversal recipients
supported this hypothesis (Leonard et al.,
'73). Further, the extended latent period,
frequently observed between the time of
transplantation and the time of reversal of
the diabetic state (Leonard et al., '73;
Hegre et al., '75) suggested that although
the amount of viable islet tissue initially
transplanted may not be sufficient to
reverse the diabetic state, adequate islet
mass may be provided by subsequent
TRANSPLANTATION
growth of the transplanted islet tissue
eventually to secrete enough insulin to
control the diabetes.
In the present study, following two to
three weeks of isotransplantation at the
renal subcapsular site of a normal recipient, islet and duct cells predominated while
only occasional acinar cells were found in
the fetal pancreatic grafts. The total islet
mass of the transplant, quantitated by
linear scanning, had increased by over
20-fold after the isotransplantation period
establishing an absolute increase i n islet
cell mass during transplantation. The fact
that the growth potential of fetal islet
tissue continues to be expressed during the
transplantation period, may explain why a
comparatively small amount of donor fetal
pancreas is required for reversal of streptozotocin (Brown et al., '74) and alloxan
induced diabetes (Leonard et al., '75)
when contrasted to the amount required
when adult or late neonatal donor islet tissue is used (Leonard et al., '73; Steffes et
al., '75; Weber et al.,'75). The islet tissue
growth observed i n these studies reflects
primarily changes within the beta cell (insulin producing) population of the transplants. Other cell types known to be present in the islets (including alpha [A,] and
delta [A,]), not identifiable by the routine
aldehyde fuchsin staining method, can be
localized by immunocytochemical techniques (Erlandsen et al., '75; Hegre et al.,
'76). Studies utilizing specific immunocytochemical localization of a number of islet
hormones are being carried out to further
characterize the types and amounts of
various islet cells present in these transplants. I n addition, studies are in progress
to establish the extent of fetal islet growth
during longer transplant periods in the
recipients and to compare the growth of
islet tissue from various aged fetal and
neonatal pancreases at a number of different transplantation sites.
We have previously reported that organ
cultured fetal rat pancreas, which has undergone selective islet differentiation and
growth in organ culture (Hegre et al.,
'72b), remains physiologically responsive
following allotransplantation as evidenced
by beta cell degranulation in response to
hyperglycemia (Hegre et al., '70) and by
amelioration of the diabetic state in allox-
213
anized recipients (Hegre et al., '72a).
Changes in total insulin content of the
explants during the transplantation period
further suggested that the mass of islet
tissue in these organ cultures had increased
during the transplantation period (Hegre
et al., '7%).
In the present study, quantitative morphometric analysis of these organ cultured
transplants has confirmed that a selective
islet growth continues during transplant ation. Under optimized conditions of allotransplantation (fetal donor, maternal host
anterior eye site) islet and duct tissue in
the transplanted organ cultures survived
for ten days in a normal host while acinar
cells did not develop. By ten days posttransplantation, the total mass of islet tissue in the transplant had increased by
6 0 % . Similarly, when isotransplants of
organ cultured fetal pancreas were made,
acinar cells disappeared from the grafts
while islet and duct cells predominated.
At the renal subcapsular site a &fold increase in islet mass was observed following 21 days of isotransplantation. The
successful combination of organ culture
with subsequent transplantation indicates
that such an in vitro period may be useful
in the storage of donor tissue. I n addition,
the ability to culture the donor tissue prior
to transplantation provides a n opportunity
for future attempts at donor tissue modification directed toward improving transplantability .
As with the isotransplants of uncultured
fetal pancreas, many duct cells were still
present in the grafts of organ cultures.
However, the islet tissue in the cultured
transplants appeared less well organized.
Few circumscribed islets were present (cf.
figs. 4 and 8). Rather, most of the aldehyde fuchsin positive beta cells were arranged in cords of cuboidal or columnar
shaped cells and many of these cellular
cords were in duct-like configurations surrounding a central lumen. A similar histological appearance has been described
for certain fetal transplants at the intratesticular site (Gonet and Renold, '65).
The histological organization of these beta
cells (fig. 9) suggests that they may have
differentiated from ductal precursors during the transplant period. A similar observation has been made for adult pancreatic
214
0. HEGRE, R. LEONARD, J. RUSIN AND A. LAZAHOW
slices of duct-ligated pancreas transplanted
to the anterior eye site (Hultquist, '72).
Evidence for the differentiation of islet
cells from ductal precursors has been reported during embryological development
(Hard, '44; Bencosme, '54; Like and Orci,
'72), during differentiation in vitro (Pictet et al., '71; Brown et al., '71; Hegre et al.,
'72b) and during regeneration of islet tissue in the adult pancreas (Zweens and
Bouman, '67; Boquist, '68). The probability that pancreatic ductal cells are an important source of new islet tissue and that
this process of differentiation continues
during the transplantation period offers a n
advantage to the use of either dispersed
whole pancreas or whole pancreatic fragments as opposed to isolated islets as
sources of donor material for transplantation to diabetic recipients.
We have previously demonstrated that
the successful reversal of the alloxan diabetic state with dispersed pancreas is indirectly related to the age of the starting
donor pancreases ( a greater success rate
is achieved with younger pancreatic tissue)
and that the younger the donor pancreatic
material is, the less of it is required to reverse the same degree of diabetes (Leonard
et al., '75). The present data provide quantitative morphological evidence that the
islet tissue components of the fetal pancreas will continue to grow and differentiate at transplantation sites in adult
recipients. These observations support the
hypothesis that fetal pancreas may be the
best source of donor material for transplantation to diabetic recipients, in part,
due to the continued growth and differentiation of the islet tissue during the transplantation period.
ACKNOWLEDGMENTS
This research has been supported by
U.S.P.H.S. Grants AM 0215, HD 412, a
Research and Development Award of the
American Diabetes Association, the Kroc
Foundation and a grant from the Graduate
School.
The authors wish to thank Sue Marshall,
Nancy Latvala, Mary Miller and Janet
Agrell for their valuable technical assistance.
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PLATE 1
E X P L A N A T I O N O F FIGURES
Figs. 1-5 Isotransplants of 18-day fetal pancreas from the renal subcapsular site of adult recipient. Fixed in Bouin’s and stained with aldehyde
fuchsin ( A F ) and Ponceau d e xylidine.
Following 21 days of isotransplantation. Adipose tissue observed
macroscopically at the time of autopsy c a n be verified histologically
( A ) . A small islet in the center of the field ( I ) is near two duct elements ( D ) . x 620.
Following seven days of isotransplantation. The accumulation of
specific cell secretion granules h a s taken place during the transplant
period. Thus, in the transplant, acinar cells (arrows) a n d islets ( I )
are easily recognizable in association with duct elements. x 810.
Following 21 days of isotransplantation. Duct cells are t h e predomin a n t feature of the transplant. Numerous mitotic figures within the
wall of the duct elements were observed (arrows). An islet i s visible
appearing to bud from the duct. x 810.
Following seven days of isotransplantation. A large well circumscribed islet comprised almost exclusively of AF+ beta cells is found
next to a duct. x 620.
Following 21 days of isotransplantation. Islets tended to remain well
circumscribed following transplantation (cf., figs. 8 and 9 ) ; however,
the peripherally located regions of the AF- cells characteristic of
neonatal and adult rat islets were not observed in the long term
transplants. X 620.
216
FETAL PANCREATIC TRANSPLANTATION
0. Hegre, R . Leonard, J. R u s h and A. Laaarow
PLATE 1
21 7
PLATE 2
EXPLANATION OF FIGURES
Figs. 6-9 Isotransplants of 18-day fetal pancreas grown in organ culture for ten
days and subsequently transplanted to the renal subcapsular site of adult recipients.
Fixed i n Bouin’s and stained with aldehyde fuchsin (AF) and Ponceau de xylidine.
Organ cultured fetal pancreas prior to isotransplantation. The normal histoarchitecture of the pancreas is vastly altered during development i n vitro. Few acinar
cells are identifiable. Irregular masses of AF+ cells are spread throughout the
tissue intimately associated with duct elements. Mitotic figures within both duct
(arrow) and islet cell populations were observed. x 810.
Following seven days of isotransplantation. N o acinar cells are observed i n the
grafts. In contrast, duct and islet cells predominate. Islet beta cells are found as
isolated cells i n the duct wall (arrow) or as groups of cells budding from the
ducts. x 620.
Following 21 days of’ isotransplantation. Islet tissue had continued to grow and
differentiate during the transplant period. AF+ islet beta cells appear to be the
predominant cell type. Separation nf duct elements from islets is difficult since
most cells forming the wall of the duct elements contain a varying degree of
aldehyde fuchsin positive granulation. x 620.
Following 21 days of isotransplantation. Many aldehyde fuchsin positive beta
cells are now tall cuboidal or columnar shaped and form part of a duct wall.
These are polarized with the aldchyde fuchsin negative portion of the cell bordering o n the luminal space, while that part of the cell containing the aldehyde
fuchsin positive beta cell granulation is bordering on a capillary ( c ) . The impression is that most of the duct cells havc differentiated into insulin storing beta
cells; however, this is not proven. x 1,000.
218
FETAL PANCREATIC TRANSPLANTATION
0. Hegre, K. Leonard, J. Rusm and A . Lazarow
PLATE 2
219
PLATE 3
EXPLANATION OF FIGURES
Figs. 10-13 Allotransplants of 20-day fetal pancreas grown i n organ culture for
four days and subsequently transplanted to the anterior eye chamber of maternal recipients. Fixed i n Bouin’s and stained with aldehyde fuchsin (AF) and Ponceau de
xylidine.
10
Organ cultured fetal pancreas prior to allotransplantation. A selective growth and
differentiation of islet tissue occurred during the i n vitro period. Heavily granulated islets developed in close association with ducts. Mitotic figures (arrow) were
€requently observed. Further development of acinar cells was not fostered under
these culture conditions. x 1000.
11
Following four days allotransplantation. The acinar tissue remaining following
organ culture had disappeared from the transplanted tissue; however, duct and
islet cells continued to survive. Mitotic figures were seen within A F + islet beta
cells (arrows) and AF+ cells were frequently seen forming part of the ductal
wall. x810.
12
Following four days allotransplantation. Duct cells were the predominant cell
type and mitotic figures were frequently seen i n this cell population (arrow).
X 1000.
13
220
Following ten days of allotransplantation. Although signs of rejection were present
i n most transplants, regions of duct and islet cells were still present. Most of the
islet cells appeared to be AF+, although the irregular contours of these islet areas
made distinction between AF - duct cells and AF - islets cells nearly impossible.
x 620.
FETAL PANCREATIC TRANSPLANTATION
0. Hegre, K. Leonard, J. Rusin and A. Lazarow
PLATE 3
22 1
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growth, isle, analysis, rat, pancreasquantitative, tissue, morphological, fetal, transplantation
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