In vitro differentiation into insulin-producing β-cells of

Digestive and Liver Disease 45 (2013) 669–676
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Digestive and Liver Disease
journal homepage: www.elsevier.com/locate/dld
Liver, Pancreas and Biliary Tract
In vitro differentiation into insulin-producing ␤-cells of stem cells isolated from
human amniotic fluid and dental pulp
Gianluca Carnevale a,∗,1 , Massimo Riccio a,1 , Alessandra Pisciotta a , Francesca Beretti a , Tullia Maraldi a ,
Manuela Zavatti a , Gian Maria Cavallini a , Giovanni Battista La Sala b , Adriano Ferrari c , Anto De Pol a
a
Department of Surgery, Medicine, Dentistry and Morphological Sciences with Interest in Transplant, Oncology and Regenerative Medicine, University of Modena and Reggio Emilia,
Modena, Italy
b
Department of Medical and Surgical Sciences, IRCCS Arcispedale Santa Maria Nuova, University of Modena and Reggio Emilia, Reggio Emilia, Italy
c
Department of Biomedical, Metabolic and Neuroscience, Children Rehabilitation Special Unit, IRCCS Arcispedale Santa Maria Nuova, University of Modena and Reggio Emilia, Reggio
Emilia, Italy
a r t i c l e
i n f o
Article history:
Received 23 November 2012
Accepted 6 February 2013
Available online 3 May 2013
Keywords:
␤-Cells
Diabetes
hAFSCs
hDPSCs
Insulin
a b s t r a c t
Aim: To investigate the ability of human amniotic fluid stem cells and human dental pulp stem cells to
differentiate into insulin-producing cells.
Methods: Human amniotic fluid stem cells and human dental pulp stem cells were induced to differentiate
into pancreatic ␤-cells by a multistep protocol. Islet-like structures were assessed in differentiated human
amniotic fluid stem cells and human dental pulp stem cells after 21 days of culture by dithizone staining.
Pancreatic and duodenal homebox-1, insulin and Glut-2 expression were detected by immunofluorescence and confocal microscopy. Insulin secreted from differentiated cells was tested with SELDI-TOF MS
and by enzyme-linked immunosorbent assay.
Results: Human amniotic fluid stem cells and human dental pulp stem cells, after 7 days of differentiation started to form islet-like structures that became evident after 14 days of induction. SELDI-TOF
MS analysis, revealed the presence of insulin in the media of differentiated cells at day 14, further confirmed by enzyme-linked immunosorbent assay after 7, 14 and 21 days. Both stem cell types expressed,
after differentiation, pancreatic and duodenal homebox-1, insulin and Glut-2 and were positively stained
by dithizone. Either the cytosol to nucleus translocation of pancreatic and duodenal homebox-1, either
the expression of insulin, are regulated by glucose concentration changes. Day 21 islet-like structures
derived from both human amniotic fluid stem cells and human dental pulp stem cell release insulin in a
glucose-dependent manner.
Conclusion: The present study demonstrates the ability of human amniotic fluid stem cells and human
dental pulp stem cell to differentiate into insulin-producing cells, offering a non-pancreatic, low-invasive
source of cells for islet regeneration.
© 2013 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1. Introduction
Diabetes is a chronic disease characterized by hyperglycemia
resulting from defects in insulin secretion or insulin action on
cells [1]. Although exogenous use of insulin remains the most
successful therapy, it fails to prevent hyperglycemia, resulting in
acute episodes of hypoglycemic and/or chronic complications like
nephropathy, retinopathy, neuropathy, impotence and cardiovascular diseases [2,3]. For these reasons, over the past two decades
∗ Corresponding author at: Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia, Via Del Pozzo 71,
41125 Modena, Italy. Tel.: +39 059 2054828; fax: +39 059 2054859.
E-mail address: [email protected] (G. Carnevale).
1
Contributed equally.
the allogeneic transplantation of pancreatic islet cells has become
a subject of intense interest and activity as a potential care for diabetes [4]. Unfortunately, islets transplantation faces a great number
of side effects. Particularly, the difficulty in obtaining sufficiently
large numbers of purified islets due to shortage donor organs and to
the loss during the purification process [5], limits their therapeutic
usage thus prompting a search for alternative sources of islet cells.
The use of stem cells, a potential renewable source of pancreatic ␤cells, is being currently investigated as an alternative therapeutic
option for the treatment of diabetes mellitus. Recently, attention
has been aroused on embryonic [6], hematopoietic [7], pancreatic islet [8], human biliary tree [9] and mesenchymal stem cells
[10] isolated from foetal and adult tissues, cultured and differentiated in vitro. Concerning mesenchymal stem cells, several reports
have focused great interest on human bone marrow-derived stem
cells (hBMSCs), dental pulp stem cells (hDPSCs) and amniotic fluid
1590-8658/$36.00 © 2013 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dld.2013.02.007
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G. Carnevale et al. / Digestive and Liver Disease 45 (2013) 669–676
stem cells (hAFSCs). Under appropriate experimental conditions
and chemical stimuli, hBMSCs [11] and hDPSCs [12] can differentiate into insulin-producing cells. On the contrary, Trovato et al. [13]
demonstrated that under experimental conditions, used in their
study, cultured hAFSCs failed to differentiate into ␤-cells. Only Li
et al. [14] demonstrated that hAFSCs can be differentiated into functional insulin-producing cells by knocking down neural restrictive
silencing factor. Despite their differentiation potential, the invasive
procedures required to isolate hBMSCs as well as the scarce percentage of these cells in bone marrow can limit their use. On the
contrary, hDPSCs show a large pool of donors and are easily accessible. Regarding hAFSCs, they can be obtained following human
amniocentesis backup and, as described by De Coppi et al. [15], are
pluripotent stem cells capable of giving rise to multiple lineages
including representatives of all three embryonic germ layers.
For these reasons the aim of the study is to develop a simple
method as well as to improve the differentiation of hDPSCs and
hAFSCs in pancreatic islet cells producing insulin and to compare
their ability along the differentiation pathways. Particularly, we
demonstrated that hDPSCs and hAFSCs, under appropriate stimuli,
express genes related to pancreatic ␤-cell development and function, such as pancreatic and duodenal homebox-1 (PDX-1) and
insulin. Therefore, hDPSCs and hAFSCs are able to differentiate in
insulin-producing cells. The two different stem cell lines could represent a promising tool for the treatment of diabetes mellitus.
2. Materials and methods
2.1. Cell culture isolation and seeding
Human AFSCs were isolated and cultured as previously
described [15,16]. Supernumerary amniocentesis samples were
provided by the Laboratorio di Genetica, Ospedale Santa Maria
Nuova (Reggio Emilia, Italy). Back-up human amniocentesis cultures were harvested by trypsinization and subjected to c-Kit
immunoselection by MACS® technology (Miltenyi Biotec).
Human DPSCs were isolated from third molar of adult subjects
(18 and 35 years of age) and were subjected to immunoselection
by magnetic cell sorting to obtain the CD34+ /c-Kit+ /STRO-1+ population as previously described by Riccio et al. [17].
All samples were collected with informed consent of the
patients according to Italian law and guideline of ethical committee
of Modena and Reggio Emilia University.
(Euroclone), supplemented with 5% FBS, 5 ␮M trans-retinoic acid
(RA) and 0.5 mM ␤-mercaptoethanol (BME)] for two days. On
the third day, the pre-induction medium was replaced with the
induction medium [H-DMEM (Euroclone), serum-free, plus 10 mM
nicotinamide (NA), 5 ␮M RA, 0.5 mM BME]. On the fifth day the
medium was replaced with maintaining-1 medium (H-DMEM supplemented with 5% FBS, 10 mM NA, 10 ␮M zinc sulphate and
10 ␮M selenium). The maintaining-1 medium was replaced at day
7 with maintaining-2 medium (L-DMEM supplemented with 5%
FBS, 10 mM NA, 10 ␮M zinc sulphate and 10 ␮M selenium). The
maintaining-1 and the maintaining-2 media were alternated every
2 days until day 21. A time table of treatments is shown in Fig. 1.
Cells cultured in medium without inducers were used as controls.
All chemicals were purchased from Sigma–Aldrich unless otherwise indicated.
2.4. Medium protein profile by SELDI-TOF MS
Proteomic analyses to evaluate the presence of insulin in differentiating and control medium were performed using SELDI-TOF
MS (Bio-Rad). Particularly after 14 days of differentiation, the proteomic profiles, between maintaining-1 medium of hAFSCs, hDPSCs
and the medium from control cells were compared to the proteomic
profiles obtained from maintaining-1 medium supplemented with
21 pg of recombinant human insulin.
Chip preparation and sample application were performed
according to the manufacturer’s instructions using the Biomek
3000 Laboratory Automation Workstation (Beckman Coulter,
Fullerton, CA, USA). Briefly, 3 ␮l of each medium was loaded on the
spot of gold ProteinChip arrays and incubated for 15 min at room
temperature.
After air drying, 1 ␮l of matrix (saturated sinapinic acid solution
in 50% acetonitrile in water containing 1% trifluoroacetic acid) was
applied to each spot twice. Spot adsorbed protein patterns were
analyzed on a ProteinChip Reader (Series 4000, Bio-Rad Laboratories, Hercules, CA, USA) and spectra detection was performed with
ProteinChip Data Manager software (Bio-Rad Laboratories Inc., Hercules, CA, USA).
The obtained spectra were baseline subtracted, normalized for
total ion current (TIC) in the range of mass-to-charge ratios (m/z)
between 4000 and 10,000 Da and finally mass aligned.
2.2. In vitro multilineage differentiation
2.5. Immunofluorescence and confocal microscopy
To induce osteogenic, adipogenic, myogenic and neurogenic differentiation, hAFSCs and hDPSCs were cultured in the appropriate
induction media as previously described [18].
In order to verify adipogenic differentiation cells were stained
with oil red O solution and counterstained with Harris haematoxylin for 1 min.
Myogenic differentiation was verified by double immunofluorescence staining, using an anti-human nuclei antibody (Ab)
(anti-hNu; Millipore, Billerica, MA, USA) and an anti-Myosin
Ab (Sigma–Aldrich), in order to verify the formation of hybrid
myotubes between hDPSCs or hAFSCs and murine C2C12 cells. The
expression of ␤3-Tubulin was analyzed by immunofluorescence
staining as marker of neurogenic differentiation.
Undifferentiated or differentiated hAFSCs and hDPSCs were
fixed for 20 min in 4% ice-cold paraformaldehyde in phosphatebuffered saline (PBS), permeabilized with 0.1% Triton X-100 in PBS
for 5 min and then processed as previously described [19]. The following primary Abs were used: rabbit anti-c-Kit, mouse anti-CD34,
mouse IgM anti-STRO-1, rabbit anti-Glut-2, Santa Cruz Biotechnology; goat anti-PDX-1, rabbit anti-insulin, rabbit anti-␤3-Tubulin,
AbCam; rabbit anti-myosin, Sigma–Aldrich; mouse anti-human
nuclei, Millipore; diluted 1:100. Secondary Abs (goat anti-mouse
Alexa405, goat anti-rabbit Alexa488, bovine anti-goat Alexa488
and goat anti-rabbit Alexa546, Molecular Probe; goat anti-mouse
IgM Cy3, Jackson) were diluted 1:200. Nuclei were stained by
1 ␮g/mL 4 ,6-diamidino-2-phenylindole (DAPI) in PBS. Negative
controls consisted of samples not incubated with the primary Ab.
The multi-labelling immunofluorescence experiments were carried out avoiding cross-reactions between primary and secondary
Abs.
Confocal imaging was performed by a Nikon A1 confocal laser
scanning microscope as previously described [20]. The confocal
serial sections were processed with ImageJ software to obtain
2.3. In vitro ˇ-cells differentiation of hAFSCs and hDPSCs
Differentiation of hAFSCs and hDPSCs was carried out in three
stages. Briefly, cells were seeded at the density of 10,000 cell/cm2
and cultured until reaching 80% of confluence. On day 1 culture medium was replaced with Pre-induction medium [L-DMEM
G. Carnevale et al. / Digestive and Liver Disease 45 (2013) 669–676
671
Fig. 1. Schematic representation of the multistep differentiation protocol used to generate islet-like structure from hAFSCs and hDPSCs. RA: trans-retinoic acid; BME:
␤-mercaptoethanol; NA: nicotinamide.
three-dimensional projections and image rendering was performed by Adobe Photoshop Software.
test (GraphPad Prism Software version 5 Inc., San Diego, CA, USA).
In any case significance was set at p < 0.05.
2.6. Determination of insulin release and dithizone staining
3. Results
Insulin concentrations were measured in the culture media of
hAFSCs and hDPSCs at 0, 7, 14 and 21 days of differentiation. Moreover, after 21 days of differentiation the total release of insulin was
detected following the stimulation with 25.0 mM glucose.
Briefly, cells were gently washed with PBS once and then were
incubated with L-DMEM and H-DMEM containing 5.5 or 25.0 mM
glucose respectively, at 37 ◦ C for 3 h. After 3 h of incubation, supernatants from wells were harvested and the levels of insulin from
cells exposed to low or high glucose were measured. Media from
undifferentiated cells were used as control.
The quantitative measurement of human insulin concentration was determined using the Insulin Human enzyme-linked
immunosorbent assay (ELISA) kit (AbCam Cambridge, UK) according to the manufacturer’s instructions. Islet-like structures were
assessed in hAFSCs and hDPSCs after 21 days of culture following
Dithizone staining [21].
3.1. In vitro multilineage differentiation
2.7. Western blotting
Whole cell lysates were obtained from undifferentiated and
differentiated hAFSCs and hDPSCs at 7, 14 and 21 days of differentiation, and were processed as described by Pisciotta et al. [18].
40 ␮g of total proteins from each sample were separated by 12%
SDS-PAGE and then transferred to PVDF membranes.
Blots were incubated with primary Abs (goat anti-PDX-1,
AbCam; rabbit anti-Glut-2, rabbit anti-PARP, Santa Cruz) diluted
1:1000, revealed by HRP-conjugated secondary Abs (anti-goat;
anti-rabbit; Pierce Antibodies, Thermo Scientific) diluted 1:300. All
membranes were revealed by using Enhanced Chemio Luminescence (Amersham). Anti-actin Ab was used as control of protein
loading in timing experiments. Densitometry was performed on
three independent experiments by NIS software (Nikon). An equal
area was selected inside each band and the mean of grey level (in a
0–256 scale) was calculated. Data were then normalized to values
of background and of control actin band.
In order to verify the phenotype of isolated hAFSCs and hDPSCs
surface immune-labelling was performed by using c-Kit, CD34, and
STRO-1 specific Abs. Isolated hAFSCs showed a clear expression of
c-Kit confirming that the correct population was obtained. In the
same manner hDPSCs showed positivity to c-Kit, CD34, and STRO-1
Abs, by co-expressing these antigens on their surface (Fig. 2A).
Multi-differentiation experiments were also performed for both
the cell types to confirm multipotent properties of isolated stem
cells.
After 3 weeks of culture with osteogenic medium, both hAFSCs
and hDPSCs showed nodular structures and the presence of a dense
extracellular matrix. Particularly, extracellular matrix showed a
positivity to the Alizarin Red staining (Fig. 2B).
The adipogenic potential of hAFSCs and hDPSCs was assessed
by treating cells with adipogenic medium for 21 days. Particularly,
lipid vacuoles were noticeable under light microscopy as early as
10 days after adipogenic induction and visualized by staining with
Oil Red O after 21 days (Fig. 2B).
The ability of hAFSCs and hDPSCs to differentiate towards myogenic lineage was verified by co-culturing the two human stem cell
types with C2C12 mouse myoblasts. After 14 days of co-culture
myotubes formation was observed in both co-cultures. Particularly, myotubes appear multi-nucleated indicating that cell fusion
occurs. Double staining with anti-hNu and anti-myosin Abs indicates that mature hybrid myotubes were formed between mouse
C2C12 and hAFSCs or hDPSCs. Myotubes not labelled by anti-hNu
Ab and therefore formed only by C2C12 cells were also present
(Fig. 2B).
After 10 days of addition of neurogenic medium, most of hAFSCs
and hDPSCs showed a typical neuronal appearance. Furthermore,
cells with neuron-like morphology showed a positive staining
against the ␤3-Tubulin neuronal marker (Fig. 2B).
3.2. ˇ-Cell differentiation
2.8. Data analysis
Values are reported as the mean ± SD obtained by groups of
4–5 samples each. Differences between two experimental samples
were analyzed by paired, Student’s t test. Three or more experimental samples were analyzed by ANOVA followed by Dunnett’s
hAFSCs and hDPSCs were cultured in differentiation medium
for 21 days according to the multistep protocol described in Fig. 1.
The induced cells were examined daily for morphological changes
under light microscope. Particularly, undifferentiated hAFSCs and
hDPSCs showed a typical fibroblast-like morphology. Conversely,
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G. Carnevale et al. / Digestive and Liver Disease 45 (2013) 669–676
the peaks with mass-to-charge ratio (m/z) 5800 Da that matched
with the detection peak obtained in the medium supplemented
with insulin (Fig. 3B). Moreover, as shown in Fig. 3B, the spectra analysis of undifferentiated hAFSCs and hDPSCs media did not
show any peaks with m/z 5800 Da. This result suggests that after 14
days of culture in differentiation media the two stem cell types are
already committed towards the pancreatic ␤-cells and are able to
produce and release insulin.
3.3. Insulin release assay and immunocytochemistry assay
Fig. 2. Surface phenotype and multi-differentiation ability of hAFSCs and hDPSCs. A:
Immunofluorescence staining of hAFSCs by anti-c-Kit Ab (green) and triple immunofluorescence of hDPSCs by anti-CD34 (blue), anti-c-Kit (green) and anti-Stro-1 (red)
Abs. B: In the first line is shown Alizarin staining of hAFSCs and hDPSCs osteogenic
differentiation. The second line shows oil red/haematoxylin staining of hAFSCs and
hDPSCs differentiated for two weeks towards adipogenic lineage. The third line
represents immunofluorescence images of hAFSCs/C2C12 and hDPSCs/C2C12 coculture stained by DAPI, anti-hNu (green) and anti-myosin (red) Abs. Images, in
fourth line represent anti-␤3-tubulin immunofluorescence labelling on hAFSCs and
hDPSCs differentiated towards neurogenic lineage. Bar 10 ␮m.
after 7 days of differentiation the cells started to form islet-like
structures that became evident after 14 days of induction. The
number and size of these islet-like structures markedly increased
and their boundary became clearer on 21st day of differentiation
(Fig. 3A). In order to evaluate whether morphological changes were
coupled with the release of insulin in the culture medium, the
proteomic profiles of media from undifferentiating and differentiating hAFSCs and hDPSCs were compared. Spectrum obtained from
medium supplemented with recombinant human insulin was used
as positive control. Particularly, after 14 days of differentiation the
spectra obtained from hAFSCs and hDPSCs culture media showed
To further clarify the function of differentiated hAFSCs and hDPSCs, insulin concentrations in the culture media were quantitatively
measured by using the Human Insulin ELISA kit after 0, 7, 14 and
21 days of induction.
As shown in Fig. 4A, 7 days after differentiation the insulin
secretion from hAFSCs was significantly higher (p < 0.05) when
compared to the respective control (time 0). Furthermore, the
insulin release statistically increased after 14 and 21 days of differentiation (30.7 ± 5.5 and 42.0 ± 4.1 ␮UI/ml) p < 0.01 and p < 0.001
respectively (Fig. 4A).
Likewise, after 7 days of induction the insulin amount in hDPSCs
medium was significantly higher in comparison to the undifferentiated hDPSCs (time 0; p < 0.05; Fig. 4A). Moreover, after 14 and
21 days of differentiation the insulin concentration in the medium
increased in significant manner (27.6 ± 4.7 and 42.8 ± 0.9 ␮UI/ml)
p < 0.01 and p < 0.001 respectively (Fig. 4A).
Furthermore, at day 14 the expression of PDX-1, insulin and
Glut-2 in undifferentiated and differentiated hAFSCs and hDPSCs
was evaluated by immunofluorescence assay. The results showed
that differentiated hAFSCs and hDPSCs after 14 days of culture
were intensely labelled by anti-PDX-1, anti-insulin and anti-Glut-2
Abs (Fig. 4B). Furthermore, undifferentiated hDPSCs express PDX-1
showing a weak staining for anti-PDX-1 and anti-Glut-2 Abs, and
are not labelled by anti-insulin Ab. The same results were found in
undifferentiated hAFSCs (data not shown).
To confirm immunofluorescence data, the presence of PDX-1
and Glut-2 was evaluated by Western blotting (WB) in hAFSCs and
hDPSCs at three different times of differentiation (7, 14 and 21
days). Densitometric analysis was carried out on the WB bands to
obtain a semi-quantitative analysis. In differentiating culture, both
cell types expressed PDX-1 and Glut-2 with the immunoreactive
bands corresponding to 31 kDa and 62 kDa respectively (Fig. 4C).
Particularly, densitometric analysis revealed that in undifferentiated cells, the protein bands corresponding to PDX-1 and Glut-2
were weakly expressed, while in differentiating hAFSCs and hDPSCs the expression of PDX-1 and Glut-2 was significantly increased
(p < 0.001).
3.4. Dithizone staining
After 21 days of induction islet-like structures were stained
by DTZ. Particularly, differentiated hAFSCs and hDPSCs cultures
showed the typical “crimson red” staining. Undifferentiated cells
were not stained by DTZ (Fig. 4D).
3.5. Effect of glucose on PDX-1 translocation and insulin secretion
In order to investigate the intracellular localization of PDX-1 and
the expression of insulin in response to the changes of glucose concentration, islet-like structures formed by hAFSCs and hDPSCs, at
day 21, were incubated in media at two different glucose concentrations: 5.5 mM and 25.0 mM. Immunofluorescence assay revealed
that in hAFSCs and hDPSCs maintained in 5.5 mM glucose PDX-1
was mainly localized in the cytoplasm of the cells forming the islets
G. Carnevale et al. / Digestive and Liver Disease 45 (2013) 669–676
673
Fig. 3. Morphological features and SELDI analysis of hAFSCs and hDPSCs ␤-cell differentiation. A: Phase contrast microscopy of the two cell types at different time of
differentiation. Bar 100 ␮m. B: Proteomic profile of media from differentiating cultures as indicated. Value indicated on the top of the peaks indicates the m/z ratio of the
detected protein.
(Fig. 5A,C; G,I). Under the same condition cells were very weakly
stained by anti-insulin Ab (Fig. 5B,C; H,I).
After 3 h of incubation in high glucose concentration (25.0 mM),
PDX-1 translocated from cytosol to nucleus. As a matter of fact,
as showed in Fig. 5D and J the nuclei of both hAFSCs and hDPSCs
were strongly stained by anti-PDX-1 Ab. Under these conditions the
intracellular expression of insulin increased (Fig. 5E, F; K,L).
Moreover, in order to confirm whether the amount of insulin
secreted in the medium increased in response to glucose stimulation, ELISA test was performed on media from hAFSCs and hDPSCs
maintained in the two glucose concentrations (5.5 mM or 25.0 mM).
Particularly, as shown in Fig. 5M and N, the insulin release values in
both hAFSCs and hDPSCs were low at 5.5 mM glucose. Insulin values increased in a significant manner (46.2 ± 4.1 ␮UI/ml in hAFSCs
and 47.6 ± 11.3 ␮UI/ml in hDPSCs) when cells were maintained in
25.0 mM glucose for 3 h. On the other hand, insulin concentration
in media from undifferentiated hAFSCs and hDPSCs was very low
under both glucose concentrations.
In order to verify whether the differentiation protocol used in
this study influenced cell viability, Poly(ADP-ribose) polymerases
(PARP) expression was evaluated in islet-like structures formed
by both cell types. Fig. 5O shows WB analysis of PARP in hAFSCs
and hDPSCs maintained in 5.5 mM or 25.0 mM glucose. Densitometric analysis revealed that the native form of PARP was clearly
expressed while the cleaved form (cPARP), involved in apoptotic
process, was undetectable. Conversely, positive control (etoposide
treated HL60) clearly showed the band corresponding to cPARP,
therefore demonstrating that cell death did not occur in hAFSCs
and hDPSCs samples.
4. Discussion
The aim of this study is to explore the potential of hAFSCs
and hDPSCs as a source of insulin-producing cells. Particularly
we developed a simple multistep method, through a non-viral
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Fig. 4. Characterization of hAFSCs and hDPSCs ␤-cell differentiation. A: Insulin release in the medium from 0 to 21 days differentiating cultures, detected by a specific ELISA
assay (ANOVA followed by Dunnett’s test: *p < 0.05, **p < 0.01, ***p < 0.001, vs 0 days). B: Immunofluorescence staining of 14 days differentiating hAFSCs and hDPSCs by DAPI
(blue), anti-PDX-1 (green), anti-Insulin (red) and anti-Glut-2 (green) Abs. Undifferentiated hDPSCs used as control show a scarcely detectable signal from anti-Insulin Ab.
The same result was obtained for hAFSCs (not shown). Bar: 10 ␮m. C: Western blot analysis of PDX-1 and Glut-2 expression in ␤-cell differentiating hAFSCs and hDPSCs.
Actin bands were presented as control of the protein loading. Densitometric analysis is reported in the bottom (ANOVA followed by Dunnett’s test: ***p < 0.001 diff hAFSCs vs
undiff hAFSCs; §§§ p < 0.001 diff hDPSCs vs undiff hDPSCs). D: Phase contrast images of dithizone staining (crimson red) of islet-like structures formed by hAFSCs and hDPSCs
as indicated.
genomic reprogramming approach, to direct the differentiation of
these two stem cell types as a source of glucose-regulated insulinproducing cells. In the first step we used a medium that included
trans RA and BME. As demonstrated by Micallef et al., trans-RA
promotes expression of the endogenous PDX-1 gene and therefore
provides a reliable marker of cells within the pancreatic endoderm
differentiation pathway [22]. In the second step the low glucose
medium was replaced with the medium at high glucose concentration and supplemented with NA, zinc and selenium. It is well
known that high-glucose condition is a crucial factor for stem cells
differentiation into insulin-producing cells [23]. Zinc is an essential
micronutrient required for insulin synthesis, secretion, signalling
and packaging [24]. An integral part of insulin forms crystals for
2-Zn-insulin hexamer, as well as free ionized zinc in the extragranular space that acts as a reservoir for granular zinc [24–26].
Moreover, selenium stimulates pancreatic ␤-cell gene expression
and enhances islet function [27]. In the second step NA was also
used. NA is a PARP inhibitor that can induce islet formation from
pancreatic progenitor cells, trans-differentiation and maturation
of liver stem cells into insulin-producing cells [28,29]. NA is able
to induce the differentiation and increase ␤-cell mass in cultured
human foetal pancreatic cells [28], as well as to protect cells from
prolonged exposure to large amounts of glucose [29].
Our results are in accordance to these findings. Particularly,
after 7 days of differentiation hAFSCs and hDPSCs morphology
changed rapidly by forming islet-like structures after 14 and 21
days of induction. Moreover, after just one week of differentiation
the expression of PDX-1 and Glut-2 in hAFSCs and hDPSCs as well
as the secretion of insulin in the media was observed. As a matter of
fact, the transcription factor PDX-1 is the first molecular marker that
temporally correlates with the pancreatic commitment and plays
a critical role in pancreas development, ␤-cells differentiation and
maintenance of ␤-cells function [5,30]. The expression of PDX-1
as well as the expression of Glut-2 was maintained and temporally
increased indicating that both cell types were committed to ␤-cells
differentiation and improved glucose receptor expression. Besides,
also insulin concentrations increased throughout the time of differentiation, although both cell types were alternatively cultured
at high glucose and low glucose concentrations. No cellular death
was observed according to the possible mechanisms that explain
the protective effects of NA. Therefore, PARP inhibitors prevent ␤cell death, induce and maintain insulin secretion [31]. The main
role of pancreatic ␤-cells is the secretion of insulin in response to
glucose and although we did not transplant the insulin-producing
cells into experimental diabetic animals, we then studied whether
or not insulin-producing hAFSCs and hDPSCs were able to respond
to the change of glucose concentration in vitro, by simulating the
physiological stimulus. Particularly, our results suggest that both
differentiated hAFSCs and hDPSCs are able to secrete insulin in
response to a glucose change thus maintaining their function. These
data are supported by the nuclear translocation of PDX-1 after high
glucose stimulation. Particularly, under low glucose culture, PDX-1
G. Carnevale et al. / Digestive and Liver Disease 45 (2013) 669–676
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Fig. 5. Effect of glucose on PDX-1 translocation and insulin secretion. A–L: Immuno-staining by DAPI (blue), anti-PDX-1 (green) and anti-Insulin (red) Abs of islet-like structures
formed by hAFSCs (A–F) and hDPSCs (G–L), exposed to 5.5 mM (A–C; G–I) and 25.0 mM (D–F; J–L) glucose. M–N: Insulin release in the medium from glucose exposed islet-like
structures formed by hAFSCs (M) and hDPSCs (N), detected by a specific ELISA assay (paired t-test: *p < 0.05, 25.0 mM glucose vs 5.5 mM glucose). O: Western blot analysis of
PARP in hAFSCs and hDPSCs cultured in 5.5 mM and 25.0 mM glucose. HL60, treated with etoposide, were loaded as positive control of the presence of cleaved PARP (cPARP).
Actin bands were presented as control of the protein loading. Densitometric analysis is reported in the right side: no significant differences were observed in c-PARP levels
comparing glucose treated cells vs controls (ANOVA followed by Dunnett’s test).
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was mainly localized into the cytoplasm in both hAFSCs and hDPSCs, while in response to high glucose stimulation translocated to
the nucleus. Previous studies indicated that nuclear translocation
of PDX-1 occurs as a consequence of glucose concentration changes
[32,33]. In the nucleus PDX-1 binds to the insulin gene [34,35] thus
leading to insulin gene transcription [36]. Moreover, nuclear PDX-1
expression prevents deleterious effect of glucose and consequently
preserves survival and function of ␤-cells [33]. These observations
represent a very important consideration for the future islets transplantation programme by using hAFSCs and hDPSCs. Even though
other stem cell types, such as adipose derived stem cells and biliary tree stem cells [9,37], appear appropriate for this purpose,
hAFSCs and hDPSCs present the advantage to be easily collected
from routine medical practice and to be rapidly expanded in culture. Moreover the possibility to culture hDPSCs in human serum
containing media, maintaining an higher proliferative potential,
represents a further advantage for the application to human cell
therapy [18].
In conclusion, the present study confirms the potential and the
ability of the two stem cell types, isolated from human amniotic fluid and dental pulp, to differentiate into insulin-producing
cells. Furthermore, no substantial differences have been observed
between the two stem cell types along the differentiation pathways, thus offering another non-pancreatic, low-invasive source of
cells for islet regeneration and a possible new therapeutic target
for the treatment of diabetes mellitus.
Conflict of interest statement
No conflicts of interest exist.
Acknowledgements
Authors are grateful to Dr. Emanuela Monari for SELDI assistance. This work was supported by grants from “Progetto Strategico
per lo sviluppo nella sede di Reggio Emilia della Facoltà di Medicina e Chirurgia” Prot: 2010 0007725, Arcispedale S. Maria Nuova
di Reggio Emilia and MIUR FIRB Accordi di Programma 2010 Prot:
RBAP10Z7FS.
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