Nestin expression in pancreatic exocrine cell lineages

Nestin expression in pancreatic exocrine cell lineages

Alexandra Delacoura, Virginie Nepotea, Andreas Trumppb, Pedro Luis Herreraa,*

aDepartment of Morphology, room 5040, University of Geneva Medical School, 1 rue Michel-Servet, CH-1211 Geneva 4, SwitzerlandbSwiss Institute for Cancer Research (ISREC), Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland

Received 19 September 2003; received in revised form 20 November 2003; accepted 21 November 2003

Abstract

Expression of nestin has been suggested to be a characteristic of pancreatic islet stem cells. To determine whether nestin is indeed

expressed in such putative cells during embryonic development, or in the adult pancreas after injury, we performed a cell lineage analysis

using two independent lines of transgenic mice encoding Cre recombinase under the control of rat nestin cis-regulatory sequences, each

crossed with loxP-bearing R26R mice. F1 animals produced the reporter molecule b-galactosidase only upon Cre-mediated recombination,

thus solely in cells using (or having used) the transgenic nestin promoter. In early pancreatic primordia, b-galactosidase was observed in

mesenchymal and epithelial cells. At later developmental stages or in adults, vast clusters of acinar cells and few ductal cells were labeled, in

addition to fibroblasts and vascular cells, but no endocrine cells were tagged by b-galactosidase. This correlated with the transient expression,

observed with an anti-nestin antibody, of endogenous nestin in about 5% of epithelial cells during development (whether in cord-forming

arrangements or in nascent acini), and in vascular and mesenchymal structures. After partial pancreatectomy, there was a transient increase of

the number of anti-nestin-labeled endothelial cells, but again, no endocrine cells bore b-galactosidase. Together, these findings show that

nestin is expressed in the pancreatic exocrine cell lineage, and suggest that consistent nestin expression is not a major feature of islet

endocrine progenitor cells.

q 2003 Elsevier Ireland Ltd. All rights reserved.

Keywords: Pancreas; Islet; Development; Transgenic; Mouse; Cre; Cell tracing; Lineage; Nestin; Insulin; Progenitor; Stem cell

1. Introduction

The isolation of islet or pancreas stem cells requires the

identification of molecular markers characteristic of such

lineage precursors. During murine embryogenesis, the

pancreas begins to develop at embryonic day E9.5 from

evaginations of the endoderm at the level of the anterior

intestinal portal (AIP), forming two primordia (dorsal and

ventral) capped by mesenchymal cells (Pictet et al., 1972;

Wells and Melton, 1999), which are subsequently brought

together to form a single gland. Proliferation of the

pancreatic epithelial endoderm results in the formation of

a tubular complex from which clusters of endocrine and

exocrine cells progressively differentiate (Herrera et al.,

1991; Slack, 1995). The four main pancreatic endocrine cell

types (b, a, d and PP cells, which produce insulin, glucagon,

somatostatin and pancreatic polypeptide, respectively) arise

from epithelial progenitor cells that can be identified for

their expression of transcription factor Ngn3 (Gu et al.,

2002; Herrera et al., 2002). In adult mice, pancreatic stem

cells seem to reside in the ducts and within the islets

(Bonner-Weir and Sharma, 2002); nevertheless, it is not

known whether they are Ngn3 þ , and no marker for such

putative precursor/stem cell has been identified so far. Since

pancreatic and intestinal endocrine cells share many

markers with neuronal cells, namely: peptidic hormones,

neuropeptide-processing enzymes, glucose transporters,

transcription factors (Ngn3, Pax-4, Pax-6, Isl1, NeuroD/

Beta2, Nkx 2.2 and Nkx 6.1) (Ahlgren et al., 1997; Alpert

et al., 1988; Gradwohl et al., 2000; Naya et al., 1997; Sander

et al., 2000; Sosa-Pineda et al., 1997; Sussel et al., 1998;

Turque et al., 1994), among others, it was thought that the

Class IV intermediate filament nestin, a specific marker of

neuronal stem cells (Cattaneo and McKay, 1990; Johansson

et al., 1999; Lendahl et al., 1990), which is widely expressed

during development but is very rare in adult organs, could

be a marker of islet progenitor cells as well (Hunziker and

Stein, 2000). Nestin has been detected in many proliferating

regions of the central nervous system (CNS) and in

0925-4773/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.mod.2003.11.004

Mechanisms of Development 121 (2004) 3–14

www.elsevier.com/locate/modo

* Corresponding author. Tel.: þ41-22-379-5225; fax: þ41-22-379-5260.

E-mail address: [email protected] (P.L. Herrera).

http://www.elsevier.com/locate/modo

developing mesenchyme and blood vessels throughout the

embryo (Dahlstrand et al., 1995; Frojdman et al., 1997;

Kachinsky et al., 1994, 1995; Mokry and Nemecek, 1998a,b,

1999; Yang et al., 2000), where it could be involved in

epithelium–mesenchymal interactions (About et al., 2000)

and in cell migration (Doyle et al., 2001). Whether nestin is

a marker of a multipotent pancreatic stem cell population is

debated (Blyszczuk et al., 2003; Duvillie et al., 2003; Klein

et al., 2003; Lardon et al., 2002; Lumelsky et al., 2001;

Selander and Edlund, 2002; Zulewski et al., 2001). Nestin

has been reported in cells located within adult pancreatic

islets (Hunziker and Stein, 2000); when isolated, these

nestin-positive cells can be grown ex vivo into different

pancreatic cell types (Zulewski et al., 2001). Others reported

that ES cells selected for their expression of nestin can

differentiate in vitro into the four islet endocrine cell types

(Lumelsky et al., 2001). Taken together, these results have

suggested that nestin-positive cells represent a multipotent

pancreatic stem cell population, which could be used in

future cell replacement therapies to cure diabetes. However,

other experimental evidences have toned down the hope of

generating new b-cells from such hypothetical nestin-

positive precursors. Nestin has been shown by immuno-

histochemistry to be expressed in mesenchymal cells during

pancreatic development, but not in epithelial cells of the

developing mouse pancreas (Klein et al., 2003; Lardon et al.,

2002; Selander and Edlund, 2002), where the islet cells

form, whereas in the pancreas of adult mice nestin was only

found in stellate and endothelial cells (Lardon et al., 2002).

Other reports failed to reproduce the results obtained with

nestin-enriched ES cells (Rajagopal et al., 2003).

In this study we sought to determine whether islet

endocrine cells derive from nestin-expressing progenitors.

We thus have systematically analyzed in vivo, during

embryogenesis and in adulthood, the progeny of cells that

transcribe the nestin gene by marking them genetically

using the Cre/loxP approach (Gannon et al., 2000; Gu et al.,

2002, 2003; Herrera, 2000; Herrera et al., 1998, 2002;

Kawaguchi et al., 2002). Briefly, in doubly transgenic mice

(nestin-Cre/R26R), the cells transiently (or permanently)

expressing nestin also produce Cre and are genetically

tagged, at the Rosa26 locus (R26R mice, Soriano, 1999), so

that their progeny stably expresses the b-galactosidase

reporter. We found that nestin is expressed not only in

mesenchymal tissues but also in epithelial cells of the

exocrine lineage, and that islet endocrine cell progenitors do

not express this marker.

2. Results

1. Nestin is expressed in mesenchymal and epithelial

cells of the developing pancreas (Fig. 1). In order to

determine whether nestin is expressed during pancreas

development we used an anti-mouse nestin monoclonal

antibody. At the earliest stage analyzed, E10.5,

mesenchymal stained cells were abundant around the

pancreatic primordia (not shown). At E12.5, stained cells

were frequent in the mesenchyme surrounding the branch-

ing primordium, as described (Klein et al., 2003; Selander

and Edlund, 2002) (Fig. 1B,C); however, contrary to

previous reports (Hunziker and Stein, 2000; Klein et al.,

2003; Lardon et al., 2002; Selander and Edlund, 2002; Piper

et al., 2002), nestin-positive cells forming clusters within

the growing epithelial cords were also found: 5.7% of the

epithelial cells were nestin-positive (Figs. 1B,C,4G). At

E15.5 and E17.5, anti-nestin antibody clearly labeled

mesenchyme and vessels (Fig. 1E,F,H) as well as some

acini (Fig. 1E,F,H), but no staining was ever seen in nascent

islets (Fig. 1I). The proportion of nestin-expressing

epithelial cells decreased as differentiation progressed

(3.9% at E15.5, 0.2% at E17.5), so that after birth, nestin-

expressing epithelial cells were absent (Fig. 4G).

2. The expression pattern of the two nestin-Cre

transgenes accurately reflects that of endogenous nestin

gene (Figs. 2,3). Nestin-expressing cells and their progeny

were traced in vivo using the Cre/loxP system, as in

previous pancreatic cell lineage analyses (Gannon et al.,

2000; Gu et al., 2002; Herrera, 2000; Herrera et al., 1998,

2002; Kawaguchi et al., 2002) (Fig. 2). We used two

different transgenic strains of mice, encoding Cre under the

control of rat nestin cis-regulatory sequences, which were

generated independently in different laboratories (Tronche

et al., 1999; Trumpp et al., 1999). Both yielded the same

expression pattern, indicating that the phenotype was likely

not affected by the integration site and the number of copies

of the transgenes. Throughout development, doubly trans-

genic embryos displayed a fully penetrant expression of the

reporter b-galactosidase gene in the central nervous system

and in other organs such as the eyes, vessels and hair

follicles (Fig. 3A,B,D,E), where nestin expression has been

demonstrated by immunostaining (Kachinsky et al., 1995;

Li et al., 2003; Mokry and Nemecek, 1998b; Yang et al.,

2000). In early pancreatic primordia (E10.5–12.5), b-

galactosidase was found in cells that were stained with anti-

nestin antibody, indicating that the activity of nestin-Cre

transgenes is accurate (Fig. 3F,G). Nestin-expressing cells

were slightly more abundant than X-Gal þ cells (5.7 vs.

4.8% in the epithelia, respectively—see below); since there

is a delay of several hours between Cre expression and

activation of the recombined reporter transgene (Davidson

et al., 2003), an early population of pancreatic nestin þ

precursor cells might not be X-Gal-labeled in the buds of

E10.5–12.5 transgenic embryos. Later and post-natally, the

proportion of b-galactosidase þ cells increased (see

below), concomitantly with a decrease to almost total

dismiss of anti-nestin-labeling, since b-galactosidase

expression, once triggered, is irreversible (Fig. 2).

3. The reporter b-galactosidase transgene is activated in

mesenchymal and epithelial cells of early pancreatic

primordia, but not in endocrine cells (Figs. 4,5). We started

the systematic analysis of X-Gal staining at E12.5. At this

A. Delacour et al. / Mechanisms of Development 121 (2004) 3–144

stage, the blue labeling was observed in mesenchymal and

vascular cells, as well as in 4.8% of epithelial cells

(Fig. 4A,G), which correlates with the expression of endo-

genous nestin, as shown above. From E15.5 to adulthood, the

proportion of stained epithelial/acinar cells, which were

distributed within the entire gland, increased from 6.5 to

almost 20% (Figs. 4B–G,5D,6C). Frequently, within stained

acini, not all cells bore b-galactosidase (Fig. 4E). Excretory

ducts, mesenchymal and vascular cells were also stained

(Fig. 4). Within the islets, no staining was found (Fig. 4F). By

combining X-Gal staining and immunohistochemistry, we

performed a precise identification of traced cells. Islet

endocrine cells were not labeled (Fig. 5A–C). X-Gal þ cells

were stained with anti-amylase (acinar cells, Fig. 5D), anti-

cytokeratin (ducts), anti-vimentin (fibroblasts), and anti-

myosin or anti-pecam (blood vessels) antibodies (not

shown). That hormone-containing endocrine cells were

never X-Gal þ indicates that they had not been tagged by

Cre expression, and suggests that neither they nor their

progenitors express significant amounts of nestin.

4. Pancreas remodeling after partial pancreatectomy is

characterized by a transient over-expression of endothelial

nestin (Fig. 6). In order to determine whether expression of

nestin is a feature of the cells that generate new endocrine

cells during islet neogenesis in the adult pancreas, we

performed a partial pancreatectomy of about 60% of the

gland (Bonner-Weir et al., 1983) in four wild type mice and

in four nestin-Cre/R26R animals (two of each strain), and

the pancreatic remnants (which correspond to the duodenal

pancreas) were quantitatively analyzed either 1, 3 or 13 days

after the operation, or 1 month later. The nestin-Cre/R26R

transgenics were only analyzed 13 or 30 days post-surgery,

when the process of regeneration has been completed. In the

adult pancreas, occasional nestin-expressing endothelial

cells were observed (Fig. 6A). After partial pancreatectomy,

the number of cells stained with anti-nestin antibody in the

remaining pancreas increased dramatically starting 3 days

post-removal, both in islets and in the exocrine parenchyma

(Fig. 6A,B). Thirty days after pancreatectomy the anti-

nestin staining pattern was indistinguishable from that of

Fig. 1. Nestin is expressed in mesenchymal and epithelial cells of the developing pancreas. Nestin-positive cells were detected by immunohistochemistry at

different stages of mouse pancreas development with an anti-mouse nestin antibody. Negative controls (A,D,G) were sections incubated without the primary

anti-nestin antibody, and showed no background staining. At E12.5 (A–C), nestin (brown staining) was found in some epithelial cells, as well as in

mesenchymal and vascular cells. At E15.5 (D–F), nestin was detected in some epithelial and acinar cells of the developing pancreas. Nestin-positive cells in

mesenchyme and vessels were less conspicuous than at earlier stages (F). At E17.5 (G–I), nestin immunostaining was observed in some acinar cells (H). Very

few nestin-containing mesenchymal or vascular cells were detected at this stage, whereas no stained cells were seen in the growing islets (I). Dashed lines

indicate epithelial cords in B and C. Scale bars, 20 mm.

A. Delacour et al. / Mechanisms of Development 121 (2004) 3–14 5

non-operated pancreata (Fig. 6A), showing the transitory

character of the increase in nestin expression. The nestin-

expressing cells, whether within the islets or amongst the

acini, were revealed to be endothelial with anti-pecam

staining (Fig. 6B). In nestin-Cre/R26R mice, the proportion

of cells having expressed the nestin-Cre transgenes (i.e.

having b-galactosidase) after partial pancreatectomy was

similar to that in non-operated controls (Fig. 6C), and no

islet endocrine cell was stained using anti-b-galactosidase

antibody or the substrate X-Gal, indicating that cells having

expressed the nestin gene contribute to adult pancreas

remodeling to a similar extent as during embryonic

development.

3. Discussion

Tracking the progeny of a particular cell in transgenic

mice expressing Cre recombinase is an informative

approach with broad potential applications. The direct

pancreatic cell tracing experiments reported herein suggest

two main conclusions: first, about one-fifth of adult exocrine

cells descend from nestin-expressing progenitors, and

second, nestin is probably not a general marker of the

endocrine lineages. Furthermore, after partial pancreatect-

omy, nestin is transiently expressed in endothelial cells that

are probably involved in tissue remodeling.

Tracking the fate of cells in vivo: limitations of

transgenesis. In this study we used two different, previously

generated, nestin-Cre transgenic strains (Tronche et al.,

1999; Trumpp et al., 1999). The pattern of b-galactosidase

activity was the same in both cases, which strengthens the

conclusions supported by our observations. Nevertheless,

since the nestin regulatory elements used are incomplete,

Cre expression may not accurately recapitulate the entire

pattern of endogenous nestin expression in the embryo; in

the early developing pancreas, however, all X-Gal þ cells

were stained with anti-nestin antibody.

Fig. 2. Tracking the fate of cells in transgenic mice. General strategy used to irreversibly label cells using the nestin regulatory elements. In doubly transgenic

mice, if a progenitor cell expresses nestin, whether transiently or not, then it should also express the transgenic Cre, which would induce the irreversible

excision of the floxed STOP sequence at the R26R locus, thus allowing the expression of b-galactosidase in that particular cell, and in its progeny, and therefore

the tracing of its lineage.


Fig. 3. The nestin cis-regulatory sequences-driven transgenes express Cre accurately. (A–C) X-Gal-staining in whole mount embryos. Cre activity corresponds

to nestin expression in early E10.5 nestin-Cre/R26R embryos, in which the encephalon and neural tube were deeply stained in the presence of X-Gal, as

described (Zimmerman et al., 1994) (A). At E15.5, the retina, hair follicles (double arrow) and blood vessels (arrow) were clearly stained (B), as well as many

cells in the pancreas (C). (D,E) Sagittal (D) and transverse (E) sections of the neural tube (developing spinal cord, E10.5) at the level indicated with a box in

panel A. b-Galactosidase activity (D) and anti-nestin antibody (E) marked the ependymal layer (e), which is very thick at this early stage. (F,G) Doubly

transgenic E12.5 pancreatic buds were double-stained using X-Gal (pale blue) and anti-nestin antibody (brown). In these sections, endogenous nestin and

b-galactosidase were detected in the same cells. An arteriole (F) and mesenchymal cells (G) are shown. Scale bars, 0.5 mm (A–C) and 20 mm (D–G). a,

amnion; c, central canal (neural lumen); d, duodenum; da, dorsal aorta; dp, dorsal pancreas; e, ependymal layer; m, muscle; vp, ventral pancreas.


The main limitation of classical transgenesis by pro-

nuclear microinjection of DNA into zygotes is variegation,

i.e. reliability of transgene expression, which is site- and

copy number-dependent, even within the same litter.

Transgene silencing (loss of expressivity) may result from

a number of alterations, whether genetic (intra-locus

recombination, when there is a high number of tandem

copies) or epigenetic (hypermethylation). For this reason,

Fig. 4. b-Galactosidase is expressed in exocrine and interstitial cells of the growing pancreas. (A) At E12.5, b-galactosidase expression, revealed by

histochemistry (X-Gal staining) on pancreatic sections from nestin-Cre/R26R mice, was detected in mesenchymal and endothelial cells, and in some epithelial

cells (arrows). (B) At E15.5, b-galactosidase activity was detected in some acini (arrow), ductal cells (d), mesenchymal cells, and in endothelial cells. Reporter

gene expression in E17.5 fetal (C,D) and P12 post-natal (E,F) pancreata was seen a fraction of acini (C,E,F), vessels (D), ducts and interstitial cells (E,F). In

labeled acini, not all acinar cells were stained (E). In islets, no stained endocrine cells were found (F). (G) Graph representing the proportions of nestin-

expressing cells and of X-Gal þ cells during pancreas development and in the adult. Scale bars, 20 mm. d, duct; e, epithelial cord; i, islet; v, blood vessel.


this technique requires the production of large numbers of

founder mice; in most cases, these allow for the selection of

good strains in which there is reproducible transgene

expression, without aberrant activity.

More recently, new approaches, such as the use of very

large stretches of DNA in bacterial artificial chromosomes

(BACs) comprising entire enhancer/promoter sequences, or

the generation of knock-in mice, may help overcome the

problems of accuracy in transgene expression. In both cases,

homologous recombination is used to replace or modify, by

introducing the transgenic DNA, the transcribed region of

the gene whose regulatory sequences are to be used in the

transgenic animal. Even though the generation of knock-in

nestin-Cre mice would probably be the best solution to

address the Cre expression concerns, these techniques have

their own problems, as for classical transgenes, since genes

may have unknown internal regulatory elements that are

replaced, or split, or altered somehow by the inserted

foreign DNA. In other situations, which may not apply to

nestin, the generation of knock-in mice may not be the best

solution, such as for genes located on the X chromosome, or

parentally imprinted, or expressed monoallelically, or even

for those genes whose normal activity is only obtained when

both alleles are expressed.

Nestin is expressed in pancreatic epithelial cells. The

expression pattern of nestin in the pancreas is controversial.

The detection of nestin transcripts in early pancreatic bud

cells (E12.5 total RNA) was recently reported in gene

expression profiling analyses (Chiang and Melton, 2003).

The presence of nestin has been described, by immuno-

histochemistry, in islet cells that do not contain hormones

(Hunziker and Stein, 2000; Zulewski et al., 2001) or in some

ductal cells (Zulewski et al., 2001) or only in mesenchymal

cells surrounding the developing gland (Selander and

Edlund, 2002). Other studies on adult pancreata show that

pancreatic nestin is found in capillaries (Klein et al., 2003)

and also in pericytes after duct ligation, or during

development (Lardon et al., 2002). Our observations using

anti-mouse nestin antibody confirm that nestin is much more

abundant in the developing pancreas than in the adult, and is

mainly expressed in mesenchymal and vascular cells (Klein

et al., 2003; Lardon et al., 2002; Selander and Edlund,

2002). However, we show, together with the accompanying

paper by Esni and co-workers, that nestin is also expressed

in early epithelial cords and, later in development, in some

acini as well as in ductal cells. That nestin is expressed in

certain epithelial cells during development has been

demonstrated in other organs such as the mesonephros,

eye and hair follicles (Frojdman et al., 1997; Mokry and

Nemecek, 1998b). The difference with previous reports on

pancreatic primordia probably reflects the different sensi-

tivity of the antibodies or the immunohistochemical

techniques used.

We have reported that some 20% acinar (and ductal)

cells are labeled in the adult nestin-Cre/R26R mice.

Whereas Esni and colleagues (this issue) have obtained

very similar results with pancreatic primordia grown in

vitro, this observation is discordant with a recently

published report (Treutelaar et al., 2003) also using the

nestin-Cre mice generated by one of us (Trumpp et al.,

1999, which is one of the two strains used in this study). The

reason for this discrepancy is not clear at this time.

Between E15.5 and adult life, i.e. in a period where

nestin is not expressed, the proportion of epithelial cells that

express b-galactosidase increases from 6.5 to 20%. This

observation suggests that, for some reason, the progeny of

Fig. 5. Characterization of the progeny of nestin-expressing progenitor cells. E15.5 pancreata from nestin-Cre/R26R embryos were doubly stained with X-Gal

and with antibodies against islet hormones, as well as endothelial, epithelial or mesenchymal markers (see text). b-Galactosidase did not co-localize with

insulin or glucagon (A), somatostatin (B) or PP (C). The majority of the b-galactosidase þ cells were also stained with anti-amylase (acinar cells, D). Arrows,

arrowheads and dashed line indicate the same cells in every pair of consecutive panels. Scale bars, 20 mm.


Fig. 6. Nestin expression is transiently upregulated after partial pancreatectomy. (A) Sections of pancreata from adult pancreatectomized (PX) non-transgenic

or double transgenic animals were incubated with anti-nestin antibody either 1 (PX-d1), 3 (PX-d3), 13 (PX-d13) or 30 days (PX-d30) after ablation. In control

mice and 1 day after PX (PX-d1), nestin-positive cells were rare. Three and 13 days post-surgery, nestin-expressing cells were, on the contrary, very abundant,

but 1 month after the operation (PX-d30) the nestin-expression pattern was as in non-operated animals. Scale bars, 20 mm. Dashed lines delimitate islets. (B)

Confocal images of pancreatic sections from Px-d13 animals stained with anti-nestin and anti-pecam antibodies revealed that nestin-expressing cells were

endothelial, whether within the islets (B) or among the acini (not shown). Scale bar, 20 mm. (C) Estimation of the area occupied by b-galactosidase-stained

cells in nestin-Cre/R26R mice, whether non-operated (control, n ¼ 2) or pancreatectomized (PX-d30, n ¼ 2). The distributions were not significantly different

ðP , 0:01Þ: An example of b-galactosidase staining pattern in nestin-Cre/R26R PX-d13 is shown on the right of the histogram (Scale bars, 20 mm).


nestin þ cells has a proliferative advantage as compared

with that of epithelial cells not expressing nestin. It is

noteworthy that, in the majority of X-Gal þ acini, only a

fraction of acinar cells were stained, suggesting that in vivo,

acini, as islets (Deltour et al., 1991), have a polyclonal

origin (Percival and Slack, 1999); alternatively, a nestin-

negative early acinar progenitor may give rise to nestin þ

and nestin 2 progeny in some acini. The biological

significance of these acinar and ductal cell populations

derived from nestin-expressing progenitors is not clear at

this time, and they do not appear to contribute to adult

regeneration differently than non-stained acini. Indeed,

whether these tagged exocrine cells represent a versatile

population of cells that, by transdifferentiation, could

directly contribute to the rise of new endocrine cells during

regeneration after injury seems unlikely, since our results

using a model of partial pancreatic ablation suggest the

contrary.

Whether the patchiness of the acinar b-galactosidase

staining is due to a lack of fully expressivity of the Cre

transgenes is unlikely, the proportions of nestin- and

b-galactosidase-expressing epithelial cells are very similar

in early pancreatic primordia, where b-galactosidase and

nestin colocalize. Therefore, the acinar cells bearing

b-galactosidase post-natally probably derive from early

epithelial cells, which expressed nestin transiently (as we

have shown with an anti-nestin antibody), further illustra-

ting an heterogeneity of the exocrine pancreas that is not

associated with a distinct arrangement or distribution of the

X-Gal-stained acini (for instance, ‘peri-‘ vs. ‘teleinsular’,

Adelson and Miller, 1989).

The expression of a neuronal progenitor marker such as

nestin represents a convergence between neurons and

acinar cells; in this perspective, the reported expression of

acinar cell markers such as Mist1 and p48 in various

neuronal cell types (Hewes et al., 2003; Obata et al., 2001)

is noteworthy.

Our results also indicate that endocrine progenitor cells

do not appear to express nestin. Esni and co-workers

provide a similar conclusion in an elegant accompanying in

vitro study, and Treutelaar and colleagues also report that

nestin is not expressed in islet endocrine cells (Treutelaar

et al., 2003); furthermore, studies using primary human

embryonic pancreatic cells in vitro suggest similar

conclusions (Humphrey et al., 2003). If true, this fact

would represent an important divergence between nerve

cells and islet endocrine cells. However, several consider-

ations must be addressed. It may be that endocrine cells

arise from a fraction of nestin þ cells in which nestin

expression results from cis-regulatory elements not present

in the transgenes used by us and Treutelaar et al.

Furthermore, Cre expression may be insufficient to tag

cells in which nestin is transcribed at very low levels or for

a short period of time, or both. Nevertheless, taken

together, our results suggest that there is no major

contribution of cells expressing nestin to the formation of

the endocrine cells of the islets of Langerhans.

What may be the role of nestin-expressing cells?

Previous in vitro studies suggest that nestin-expressing

cells are precursors to endocrine cells: cultures of nestin-

containing islet-derived cells (Zulewski et al., 2001), or ES

cells selected for the expression of nestin (Lumelsky et al.,

2001), generated islet-like structures in vitro; since no direct

cell tracing analyses were performed in those experiments,

nestin þ cells might act indirectly (i.e. non-cell autono-

mously) in such differentiation protocols. Whether different

mechanisms (involving different cell types and different

gene expression pathways) are implicated in the production

of new b-cells in different situations, such as during

development, or in the adult in pathological conditions, or

in experimental protocols in vitro from ES cells (Blyszczuk

et al., 2003; Lumelsky et al., 2001) or from other

undifferentiated cells (Zulewski et al., 2001), needs to be

further explored.

Anti-nestin staining confirmed that nestin-expressing

cells are rare in the adult pancreas. After partial

pancreatectomy, however, this is transitorily different, for

there is a massive increase in the numbers of nestin-

expressing endothelial cells. Others reported similar results

after duct ligation, another experimental model of pan-

creatic injury that triggers regeneration: nestin was shown

in new capillaries and in peri-acinar stellate cells,

indicating that nestin is an angiogenic marker (Lardon

et al., 2002). In nestin-Cre/R26R transgenics, the tempor-

ary rise of pancreatic endothelial nestin-expressing cells

after the partial ablation of the gland was not followed by a

subsequent increase of the proportion of cells stained with

either X-Gal or anti-b-galactosidase antibody. This indi-

cates that the transient nestin-expressing endothelium does

not represent a specific pool of islet progenitor cells, but

rather a population of cells that die without leaving any

labeled progeny.

Stellate cells, which are more abundant in the regenerat-

ing pancreas and liver (Kalinichenko et al., 2003; Zimmer-

mann et al., 2002), are a source of NGF and neurotrophins

(Cassiman et al., 2001), factors that have previously been

implicated in the regulation of islet neogenesis (Scharf-

mann, 1997). Nestin expression is a feature of de-

differentiation: it is re-expressed during repair processes in

a variety of organs such as liver (stellate cells, Friedman,

1999; Messing, 1999; Niki et al., 1999), teeth (odontoblasts,

About et al., 2000), muscle (myoblasts, Vaittinen et al.,

2001), or the CNS (astrocytes and endothelia, Duggal et al.,

1997; Ha et al., 2002). Expression of nestin may therefore

be viewed as a trait of cells involved mainly indirectly in the

genesis of new cells during regeneration, and possibly

during development, not only in the pancreas but also in

other organs. This could be via stellate or endothelial cells,

or both, and should be of relevance in devising new

protocols for cell replacement therapies in a variety of

diseases.


4. Materials and methods

4.1. Transgenic mice

Two different nestin-Cre mice were generated indepen-

dently in two different laboratories (Tronche et al., 1999;

Trumpp et al., 1999). The transgenics produced by Klein

(Tronche et al., 1999) were purchased from The Jackson

Laboratories (strain: B6.Cg (SJL)-TgN(NesCre)1Kln; stock

#003771). Both strains bear a Cre recombinase gene under

the control of the rat nestin 50Flanking (5.8-kb) and 2nd

intron (1.8-kb) sequences, as described (Zimmerman et al.,

1994). The ROSA26R ubiquitous reporter mice (R26R)

(Soriano, 1999) were obtained from The Jackson Labora-

tories (strain: B6.129S4-Gtrosa26tm1Sor; stock #003474).

Doubly transgenic animals were genotyped by PCR and/or

directly with X-Gal or anti-b-galactosidase staining. The

oligonucleotides used as primers yielded two fragments of

300 and 500 bp for Cre recombinase and Lac Z genes,

respectively; their sequences were: 50-ATGCTTC-

TGTCCGTTTGCCG-30/50-CCTGTTTTGCACGTTCAC-

CG-30 for Cre, and 50-ACGGCAAACGACTGTCCTGG-30/

50-CGTGACTGGGAAAACCCTGG-30 for Lac Z, using a

standard 30-cycle PCR profile.

4.2. X-Gal staining

Total embryos or pancreatic buds at different develop-

mental stages, and adult pancreata, were harvested in cold

phosphate buffer (pH 7.4) and fixed for 1 h at 4 8C in 4%

PFA. Tissues were then incubated overnight at 37 8C in

X-Gal substrate (5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6,

2 mM MgCl2, 0.01% Na deoxycholate, 0.02% NP40,

1 mg/ml X-Gal in phosphate buffer, pH 7.4). Subsequently,

stained samples were embedded in paraffin, sectioned, and

counterstained with hemalun, for high-resolution analyses

of b-galactosidase activity.

4.3. Immunohistochemistry

Embryos at different developmental stages, and adult

pancreata, were harvested in cold PBS and fixed in 4% PFA

overnight at 4 8C. Then, the tissues were embedded in

paraffin and 5 mm-thick tissue sections were mounted on

adhesive-coated slides.

For detection of nestin and b-galactosidase, the slides

were dewaxed, rehydrated and soaked in 10 mM citrate

buffer (pH 6) in a microwave oven at 700 W for 10 min, and

then an additional 20 min at room temperature (‘antigen

retrieval’ procedure). When diaminobenzidine (DAB) was

used to reveal the immunostaining, sections were pre-

treated with 3% H2O2 (in PBS) for 10 min, in order to

quench endogenous peroxydases. Sections were sub-

sequently incubated with blocking reagent (5% donkey

serum, 0.1% Tween, free biotin—Vector Laboratories—in

PBS) for 30 min and then overnight at 4 8C with either

mouse anti-rat nestin (Chemicon, clone 401, dilution 1/100)

or rabbit anti-b-galactosidase (Abcam, dilution 1/250)

antibody in blocking reagent (free biotin was replaced by

free streptavidin), and then incubated for 1 h with

biotinylated anti-mouse or anti-rabbit (Jackson Immuno-

research, dilution 1/200) antibody, respectively. Immuno-

staining was revealed by either immunofluorescence with

streptavidin-Alexa 488 (for nestin) or 568 (for b-galacto-

sidase) (Molecular Probes, dilution 1/100, during 1 h at

room temperature), or with streptavidin-hrp (Jackson

Immunoresearch, dilution 1/300, 1-h)/DAB.

The surface occupied by epithelial cells stained with

X-Gal or anti-nestin antibody was measured on pancreatic

sections from two embryos per developmental stage

(i.e. E12.5, E15.5 and E17.5) by outline/integration using

the NIH’s Image J software (version 1.25).

All other antibodies were used on paraffin sections of

pancreata previously stained with X-Gal. For PanK and

myosin, antigen retrieval was required as well. In these

protocols, non-specific binding sites were blocked by

treating the sections for 30 min with 3% BSA, 0.1%

Tween in PBS. Primary antibodies were then incubated

for 1 h in blocking reagent. The antibodies and their dilution

used in the present analysis were as follows: guinea pig anti-

porcine insulin (Dako, dilution 1/400), mouse anti-porcine

glucagon (Sigma, dilution 1/1000), rabbit anti-human

somatostatin (Sigma, dilution 1/200), rabbit anti-human

PP (Bachem, 1/200), mouse anti-human PanK (Sigma,

dilution 1/100), goat anti-human vimentin (Sigma, dilution

1/20), rabbit anti-PECAM (kind gift of Dr Aurrand-Lyons,

University of Geneva Medical School, dilution 1/500) and

mouse anti-myosin (kind gift of Dr Piallat-Bochaton,

University of Geneva Medical School, at 1/20). Then,

sections were washed and incubated with specific secondary

antibodies coupled to Cy3 (Jackson Immunoresearch).

The specificity of the different immunostainings was

confirmed by controls in which primary antibodies were

omitted. Sections were examined and photographed with a

Nikon epifluorescence microscope (Eclipse TE200)

equipped with a Nikon CoolPix 990 digital camera, and

when indicated, the specimens were analyzed using a Zeiss

LSM510 confocal microscope.

4.4. Partial pancreatectomy

Partial (60%) pancreatectomy (PX) was performed in

wild type C57/B6xCBA/J mice (2 month-old, Iffa Credo)

for the analysis of endogenous nestin expression and on two

males of each strain of nestin-Cre/R26R mice. Briefly, the

pancreas was removed by gentle abrasion with cotton

applicators (Q-tips), leaving intact the major blood vessels

supplying other organs, as described in rats (Bonner-Weir

et al., 1983). The residual pancreas was easily defined as the

tissue within 1–2 mm of the common pancreatic duct that

extends to the first part of the duodenum. Littermates of the

same sex were not operated, and used as controls.


One, 3, 13 (for wild type mice) and 30 days (for the

nestin-Cre/R26R) after pancreatectomy, pancreas of con-

trols and pancreatectomized mice were collected and fixed

overnight in 4% PFA, at 4 8C. Then, the tissues were

embedded in paraffin and sections of 5 mm of thickness

were performed and mounted on coated slides for the

analysis of nestin and b-galactosidase expression, as

described above.

The surface occupied by cells having b-galactosidase

was measured using sections of pancreas from PX and

sham-operated nestin-Cre/R26R mice that were stained

using X-Gal or anti-b-galactosidase antibody. Four sections

per pancreas were randomly chosen and photographed with

the digital camera. Nestin- and b-galactosidase-stained

areas were quantified using the NIH’s Image J software. The

distributions were compared using a non-parametric Mann–

Whitney test.

Acknowledgements

We are grateful to Drs P. Vassalli, J.-D. Vassalli,

I. Rodriguez, K.A. Johansson, Y. Dor and S. Leach for

carefully reading the manuscript. We thank Mr Nicolas

Steiner, Mr Olivier Fazio, and Mr Angelo Gradia for their

skillful technical assistance. P.L.H. is supported by grants of

the Juvenile Diabetes Research Foundation, the Swiss

National Science Foundation and the NIH/NIDDK. A.T. is

supported by the Swiss National Science Foundation, the

Swiss Cancer League and the Leenards Foundation. A.T. is

a member of the EMBO Young Investigator Programme.

This study was part of the Geneva Programme for Metabolic

Disorders, and of the Frontiers in Genetics National Center

for Competence in Research.

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Nestin expression in pancreatic exocrine cell lineages

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