Original Paper
|
|
|
file on Synergy OPEN |
Acta Biochim Biophys
Sin 2008, 40: 830-839
doi:10.1111/j.1745-7270.2008.00459.x
Human amnion epithelial cells
can be induced to differentiate into functional insulin-producing cells
Yanan Hou1,2,
Qin Huang1,2, Tianjin Liu1,3,
and Lihe Guo1,3*
1 Institute of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
Shanghai 200031, China
2 Graduate School of the Chinese Academy of
Sciences, Beijing 100049, China
3 Cellstar Biotechnologies Company, Shanghai
201210, China
Received: April 30,
2008
Accepted: May 28,
2008
This work was
supported by a grant from the Science and Technology Department of Shanghai
Research Fund (No. 05DZ19329)
*Corresponding
author: Tel/Fax, 86-21-54921392; E-mail, [email protected]
Pancreatic
islet transplantation has demonstrated that long-term insulin independence may
be achieved in patients suffering from diabetes mellitus type 1. However,
limited availability of islet tissue means that new sources of
insulin-producing cells that are responsive to glucose are required. Here, we
show that human amnion epithelial cells (HAEC) can be induced to differentiate
into functional insulin-producing cells in vitro. After induction of
differentiation, HAEC expressed multiple pancreatic b-cell genes, including
insulin, pancreas duodenum homeobox-1, paired box gene 6, NK2 transcription
factor-related locus 2, Islet 1, glucokinase, and glucose transporter-2, and
released C-peptide in a glucose-regulated manner in response to other
extracellular stimulations. The transplantation of induced HAEC into streptozotocin-induced
diabetic C57 mice reversed hyperglycemia, restored body weight, and
maintained euglycemia for 30 d. These findings indicated that HAEC may be a new
source for cell replacement therapy in type 1 diabetes.
Keywords amnion epithelial cell; diabetes; differentiation;
transplantation
Type 1 diabetes is characterized by the autoimmune destruction of
pancreatic b-cells. The resulting lack of insulin production leads to
hyperglycemia and serious long-term complications. Clinical trials have proven
that allogeneic islet transplantation is an alternative treatment for patients
with type 1 diabetes [1]. However, the shortage of islet donors limits the
large-scale use of this therapy. Recent studies have demonstrated that
embryonic stem cells and adult stem/progenitor cells isolated from the pancreas
[2–6],
liver [7,8], salivary gland [9], adipose [10], or nerve system are capable of
differentiation into insulin-producing cells [11]. Stem cells hold great
promise for supplying sufficient donor cells for transplantation.Amniotic epithelial cells are generated from amnioblasts on the
eighth day after fertilization and constitute the inner layer of the amnion
[12]. Recent studies have shown that human amniotic epithelial cells (HAEC) are
endowed with stem cell characteristics and have the potential to differentiate
into cells of all three germ layers, including endodermal pancreatic cells
[13]. It has also been shown that after induction of differentiation, the
transplantation of HAEC into streptozotocin (STZ)-induced diabetic mice can
normalize blood glucose [14]. However, it is unclear whether HAEC could be
induced to differentiate into functional insulin-producing cells and whether,
with regard to its immune privilege property [15], HAEC could normalize blood
glucose levels after transplantation into diabetic C57 mice. In the present study, we showed that, after induction of
differentiation, human amniotic epithelial cells exhibit b-cell-like
characteristics, including the expression of genes related to b-cell
development and function as well as C-peptide production and release response
to glucose and other extracellular stimulations. Transplantation of induced
HAEC into STZ-induced diabetic C57/B1 mice resulted in perfect control of blood
glucose for 30 d and allowed body weight to be restored.
Materials and Methods
Isolation of HAEC
The study and use of human amnion were approved by the Patients and
Ethics Committee of the Shanghai Institute of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
(Shanghai, China). Human amnion membranes were mechanically peeled from the
chorion of a placenta obtained from healthy mothers undergoing cesarean
sections. The membrane was extensively scraped out to remove the underlying
tissues (i.e. the spongy and fibroblast layers) to obtain a pure epithelial
layer with an intact basement membrane. The tissue was minced and incubated at
37 ?C with 0.25% trypsin (Difco Laboratories, Detroit, USA) containing 0.53 mM
EDTA for 30 min. Afterwards, the digested tissue was passed through a 200 mm filter and
then a 74 mm filter to remove larger fibrous tissue remnants. The amniotic epithelial
cells were collected by centrifugation and suspended in medium. Standard
culture medium is Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% fetal bovine serum (PAA, Pasching, Austria), 2 mM L-glutamine, 1%
non-essential amino acid, 55 mM 2-mercaptoethanol, 1 mM sodium pyruvate, and 1%
antibiotic-antimycotic (all from Gibco, Grand Island, USA). Incubation was
carried out in a 5% CO2 atmosphere at 37 ?C.
Induction of differentiation
To induce differentiation into insulin-producing cells, HAEC (1107) were seeded in a 10-cm culture dish and cultured in serum-free
DMEM containing 25 mM D-glucose, N2 supplement (Gibco), 1%
antibiotic-antimycotic, and 10 mM nicotinamide (Sigma, St. Louis, USA) for 1–3 weeks. Medium
was replaced twice a week. Cell culture supernatants were collected at 0, 7,
14, and 21 d, centrifuged briefly to remove cell debris, and then tested for
C-peptide using human C-peptide radioimmunoassay (RIA) kit (China Diagnostics
Medical Corporation, Beijing, China).
Reverse
transcription-polymerase chain reaction (RT-PCR)
Total RNA was prepared from HAEC at different times of induction (0,
7, 14 and 21 d) using TRIzol reagent (Invitrogen, Carlsbad, USA). Prior to RT,
RNA samples were digested with DNase I (1 U/mg RNA; Fermentas, Glen
Burnie, USA) for 30 min at 37 ?C to eliminate genomic DNA contamination. Total
RNA (2 mg) underwent reverse transcription-polymerase chain reaction using
Moloney murine leukemia virus reverse transcriptase (Promega, Madison, USA) in
a 25 ml volume containing 0.5 mg oligo dT, 400 mM deoxynucleotide
triphosphate, and buffers supplied by the manufacturer. As described in Table
1, 1 ml cDNA was subjected to PCR amplification using human primer pairs.
C-peptide release
After induction for 7 d, cells (2106 each
well) were seeded in 6-well plates and grown in DMEM containing 25 mM glucose,
10 mM nicotinamide and 10% fetal bovine serum for 2 d. The cells were then
washed twice in DMEM containing 0.5 mM glucose and cultured in this medium for
1 h. The medium was then replaced by 2 ml DMEM containing 0.5, 5, or 25 mM
glucose, respectively, for each well. As a control, the non-induced HAEC (at
day 0) were washed twice in DMEM containing 0.5 mM glucose and cultured in DMEM
containing 25 mM glucose. The culture supernatants were collected after 12 h of
incubation, centrifuged briefly to remove cell debris, and then tested for
C-peptide using human C-peptide RIA kit. For KCl and tolbutamide stimulation experiments,
the cells were washed twice in DMEM containing 2 mM glucose and cultured in
this medium for 1 h. The medium was then replaced by 2 ml DMEM containing 20 mM
glucose and KCl (0, 10, 20, 30, and 40 mM, respectively, for each well) or
tolbutamide (0, 50 and 100 mM, respectively, for each well; Sigma) for 1 h. The culture
supernatants were collected after 12 h of incubation, centrifuged to remove
debris, and then tested for C-peptide. The cells were harvested for
quantitation of the cell number. After induction for 7 d, cells (2106 each
well) were seeded in 6-well plates and grown in DMEM containing 25 mM glucose,
10 mM nicotinamide and 10% fetal bovine serum for 2 d. The cells were then
washed twice in DMEM containing 0.5 mM glucose and cultured in this medium for
1 h. The medium was then replaced by 2 ml DMEM containing 0.5, 5, or 25 mM
glucose, respectively, for each well. As a control, the non-induced HAEC (at
day 0) were washed twice in DMEM containing 0.5 mM glucose and cultured in DMEM
containing 25 mM glucose. The culture supernatants were collected after 12 h of
incubation, centrifuged briefly to remove cell debris, and then tested for
C-peptide using human C-peptide RIA kit. For KCl and tolbutamide stimulation experiments,
the cells were washed twice in DMEM containing 2 mM glucose and cultured in
this medium for 1 h. The medium was then replaced by 2 ml DMEM containing 20 mM
glucose and KCl (0, 10, 20, 30, and 40 mM, respectively, for each well) or
tolbutamide (0, 50 and 100 mM, respectively, for each well; Sigma) for 1 h. The culture
supernatants were collected after 12 h of incubation, centrifuged to remove
debris, and then tested for C-peptide. The cells were harvested for
quantitation of the cell number.
Immunofluorescence
Following isolation from amniotic membrane, HAEC were seeded on
glass cover slides in 6-well culture plates. After induction of
differentiation, induced and non-induced HAEC were rinsed in phosphate-buffered
saline (PBS) after 14 d of induction, fixed with 4% polyformaldehyde in PBS at
room temperature for 15 min, and permeabilized with 0.1% Triton X-100 in 0.1%
sodium citrate at room temperature for 15 min. Preparations were blocked by 5%
goat serum in PBS at room temperature for 30 min, and then incubated overnight
at 4 ?C with primary guinea pig anti-insulin polyclonal antibody (1:200; Dako,
Carpinteria, USA) and rabbit anti-Pdx-1 polyclonal antibody (1:200; Chemicon,
Temecula, USA). For insulin staining, the cells were incubated in Texas
Red-labeled anti-guinea pig secondary antibody (1:500; Santa Cruz
Biotechnology Inc., Santa Cruz, USA) at room temperature for 1 h. For Pdx-1
staining, the cells were incubated in fluorescein-isothiocyanate-labeled
goat-anti-rabbit secondary antibody (1:500; Jackson ImmunoResearch
Laboratories, West Grove, USA) at room temperature for 1 h. Nuclei were stained
with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Goat serum was used
as a negative control.Kidneys were removed from HAEC-transplanted mice and sham-operated
mice at 30 d after transplantation and then fixed with 4% polyformaldehyde in
PBS, rinsed with PBS, and cryoprotected overnight in phosphate buffer
containing 30% sucrose. Tissues were embedded in optimal cutting temperature
compound (Tissue-Tek, Sakura-Finetek, Torrance, USA), frozen, sectioned at 6 mm and collected
on gelatin pre-coated slides. Immunostaining was preformed. After
immunofluorescence analysis, the sections were stained with hematoxylin,
dehydrated, and mounted for light microscopic examination. Three mice from each
group were analyzed.
Transfection of HAEC with
green fluorescence protein (GFP)
Lentiviral vectors (pWPT-GFP, pMDlg/pRRE, pMD2.G and pRSV-REV) were
kindly provided by Dr. Didier Trono (University of Geneva, Geneva, Switzerland)
[16]. The pseudotyped viral particles were produced by four-plasmid transient
cotransfection into 293T cells with the calcium phosphate transfection system,
harvested, and concentrated by ultracentrifugation (72,000 g, 120 min,
4 ?C). Concentrated supernatants were titrated with serial dilutions of vector
stocks on 1?105 HeLa
cells, followed by fluorescence-activated cytometric analysis (FACSCalibur flow
cytometer; BD Biosciences, San Jose, USA). Primary cultured HAEC were
transduced by 24 h exposure (three rounds of infection) to
lentivirus-containing supernatant at 30 multiplicity of infection in the
presence of polybrene (8 mg/ml), followed by induction of differentiation. After induction
for 7 d, the cells were trypsinized for transplantation.
Animal and cell
transplantation
All animal experiments were performed in compliance with the
standard institutional guidelines for animal care (Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences). Six- to eight-week-old male
C57/B1 mice were made hyperglycemic by two intraperitoneal injections of STZ
(Sigma) of first 180 mg/kg, and then 60 mg/kg of body weight at 3 d interval.
Animals with blood glucose level16.8 mM for 1 week were either transplanted
with induced HAEC (2–3?106 each) or
injected with saline (sham operation) into the left subrenal capsule. Blood
glucose levels were monitored twice a week in samples obtained from the tail
vein of mice using the GlucoTrend glucose detector (Roche Diagnostics,
Indianapolis, USA). The body weight of mice was also monitored twice a week.
For intraperitoneal glucose tolerance test (IPGTT), at d 30 after
transplantation, normal non-diabetic mice (n=5), diabetic mice with
normalized glucose levels following the HAEC transplantation (n=5),
and diabetic mice (n=5) were fasted for 8 h and then given an
intraperitoneal injection of glucose (1 g/kg of body weight). Blood glucose was
monitored at 0, 15, 30, 60, 90, and 120 min after injection. Serum samples
were collected at 30 d after transplantation from HAEC-transplanted mice with
euglycemia (n=5), sham-operated mice (n=5) and normal control
mice (n=5) for human C-peptide levels analysis. The mice were fasted for
8 h and given an intraperitoneal injection of glucose (1 g/kg of body weight)
before blood samples were collected from the orbital plexus. The
ultrasensitive human C-peptide ELISA kit (Mercodia, Uppsala, Sweden) was used
according to the manufacturer’s instruction.
Statistical analysis
Data were expressed as mean±SD. Statistical differences between
groups were assessed by Student’s t-test. P<0.05 was considered statistically significant.
Results
Gene expression of HAEC after
induction of differentiation
To determine whether the HAEC had undergone pancreatic
differentiation, gene expression profiles for pancreatic b-cells
differentiation markers and hormones were assessed using RT-PCR. Insulin and
other pancreatic b-cell related genes, such as pancreas duodenum homeobox-1 (Pdx-1),
paired box gene 6 (Pax-6), NK2 transcription factor-related locus 2 (Nkx-2.2),
Islet 1 (Isl-1), glucokinase (GCK), and glucose transporter-2 (Glut-2),
were expressed at 7, 14 and 21 d of induction. The expression of GCK and
Glut-2 indicated that the HAEC might have glucose-sensing ability after
induction of differentiation. As shown in Fig. 1(A), the expression of
Octamer-4 (Oct-4), a pluripotent cell marker, was detected in
non-induced HAEC, but not in induced HAEC at 7 d of induction and thereafter.
Immunostaining for insulin and Pdx-1 were also observed at 14 d of induction,
but not in non-induced HAEC [Fig. 1(B)]. These results indicated that
HAEC could be induced to differentiate into b-cell-like cells in vitro.
C-peptide release
Because of the controversy surrounding insulin uptake into cells
from media supplements [17,18], we measured C-peptide in the cell culture
supernatants to further investigate the function of insulin synthesis in the
induced HAEC. C-peptide is the byproduct of de novo insulin synthesis,
co-secreted from the pancreas with and in equimolar amounts (i.e., an equal
number of each molecule) to insulin [19]. Therefore, the presence of C-peptide
in HAEC supernatants at 7 (111.43±0.7 pM per 106 cells),
14 (110.83±0.86 pM per 106 cells), 21 d (108.6±2.48 pM per
106 cells) of induction indicated that proinsulin is synthesized and
processed in these cells. In addition, there was no detectable amount of
C-peptide in HAEC supernatants at day 0 of induction [Fig. 2(A)].To determine whether HAEC were responsive to different
concentrations of glucose, KCl and tolbutamide after induction of
differentiation, the release of C-peptide into the culture medium was measured
using human C-peptide RIA kit. The results showed that the induced HAEC could
secret C-peptide in a glucose-regulated manner. No detectable C-peptide release
was observed in the non-induced HAEC culture medium, even in the presence of
glucose stimulation [Fig. 2(B)]. Direct depolarization of the induced
HAEC by adding sequential concentrations of KCl consistently resulted in
notable increases in secreted C-peptide in a concentration-dependent manner
during 1 h incubations [Fig. 2(C)]. We inferred the presence of
functional sensitive potassium (KATP) channels in the cells from
increases in C-peptide release over basal levels upon the addition of
tolbutamide, an inhibitor of KATP-channels [Fig. 2(D)]
[20]. These results indicated that HAEC could be induced to differentiate into
functional insulin-producing cells, exhibiting secretory characteristics
similar to that of pancreatic b-cell.
Reversal of hyperglycemia in
STZ-induced diabetic mice
To determine whether the induced HAEC possessed the capacity to
correct hyperglycemia in diabetic mice, C57/B1 mice were either induced to
become diabetic with STZ before transplantation with induced HAEC (2–3?106 per mouse) or treated with sham operation. As
shown in Fig. 3(A), glucose levels in the HAEC-implanted mice decreased
and were below 13.9 mM within 30 d following transplantation. In contrast,
blood glucose levels in the diabetic control mice and in mice receiving sham
operation remained elevated (P<0.01). The body weights of the HAEC-implanted mice were restored gradually, whereas the mice in the other two groups persistently lost weight [Fig. 3(B)]. These results suggested
that induced HAEC are functional in vivo and capable of reversing hyperglycemia
in diabetic mice. To further evaluate the function of the implanted HAEC, we performed
an IPGTT on induced HAEC-implanted mice (n=5), sham-operated mice (n=3)
and non-diabetic control mice (n=3) after 30 d of normalized glucose
levels following the transplantation. As illustrated in Fig. 3(C), blood
glucose levels in normal control mice rose rapidly, with peak values obtained
at 15 min, followed by a return to the normal range between 60 and 120 min.
Blood glucose levels in the HAEC-implanted mice were generally higher, but
likewise displayed a peak at 15 min and returned to the normal range at 120
min. Blood glucose levels in the sham-operated mice kept higher than 20 mM
during the test. These results indicated that the implanted HAEC were indeed
responsive to a glucose challenge in vivo. To confirm the presence and function of HAEC in vivo, HAEC
were infected with lentivirus coding GFP before induction of differentiation.
The C-peptide-secreting characteristics and hyperglycemia-reversing ability of
induced HAEC were not changed by the lentivirus infection (data not shown).
The mice were killed, and the transplanted cells in the subrenal capsule were
examined for insulin expression by anti-insulin antibody. Transplanted HAEC
were positive for both insulin and GFP expression at 30 d after transplantation
[Fig. 4(A)]. As in the sham-operated controls, no diffuse interstitial
expansion, tubular atrophy and massive leukocytic infiltration were observed
at the site of transplantation in the HAEC-transplanted mouse kidneys [Fig.
4(B)]. To confirm glucose-responsive insulin secretion in HAEC-transplanted
mice, human C-peptide levels in the serum of the mice after glucose injection
were measured using the ELISA kit. After injection, a significant increase of
human C-peptide in HAEC-transplanted mice serum samples was detected [Fig.
4(C)]. Discussion
Although recent studies have demonstrated the feasibility of
generating insulin-producing cells from stem/progenitor cells of various cellular
sources or genetically engineered somatic cell lines [2–10,21,22], some obstacles,
such as immune rejection and autoimmunity against newly formed
insulin-producing cells derived from pancreatic stem cells, still remain.
Despite their promising potential, it may also prove difficult to obtain enough
autologous adult stem cells from these organs.
To overcome these limitations, we explored the possibility of using
HAEC as sources for differentiation into insulin-producing cells under specific
in vitro culture conditions. HAEC are a monolayer of epithelial cells
endowed with unique secretory properties that line the human amniochorion
[23]. HAEC develop from the epiblast by 8 d after fertilization and before
gastrulation, suggesting that they might maintain the plasticity
of pregastrulation embryo cells. Because of the lack of major histocompatibility
complex class II antigens and mild expression of major histocompatibility
complex class I antigens [15], HAEC have been used in researching the treatment
of neural degenerative diseases [24]. Previous studies have proven that HAEC
can be induced to differentiate into endodermal pancreatic cells and can
normalize blood glucose levels after transplantation into STZ-induced diabetic
nude mice [13,14]. In the present study, we further detected the production and
release of C-peptide of differentiated HAEC induced by nicotinamide. The
secretion process can be regulated. By transplanting HAEC into diabetic C57
mice, the cells can normalize the blood glucose of the mice and maintain
euglycemia for 30 d.Previous studies have shown that nicotinamide can induce
differentiation and maturation of human fetal pancreatic islet cells [25], and
that duct tissue from human tissue treated with nicotinamide can be directed
to differentiate into glucose responsive islet tissue in vitro [5]. The
nicotinamide-induced differentiation activated a number of genes related to
pancreatic b-cell development and function in HAEC, such as insulin, Pdx-1,
Pax-6, Nkx-2.2, Isl-1, and the expression of GCK
and glut-2, two important components in “glucose-sensing
apparatus” of pancreatic -cells [26], indicating that these cells might
have glucose-sensing ability. Unlike previous reports [13,14], we have not
detected glucagon expression during the period of induction, and the expression
of Pdx-1 could not be detected before induction of differentiation.
Additionally, the expression of Nkx 6.1 and Pax-4 were not detected during
differentiation (data not shown). Because there was no obvious change in the
expression of the detected transcription factors Pdx-1, Pax-6, Nkx-2.2
and Isl-1 at different times during induction, we speculated that the
nicotinamide-induced differentiation process did not recapitulate a normal
pancreatic development pathway in vivo. As demonstrated by immunofluorescence
analysis, the expression of insulin and Pdx-1 after 14 d of induction further
proved that HAEC can exhibit b-cell-like characteristics after induction of differentiation with
nicotinamide for a longer period in vitro. Although the proportions of
immunopositive cells are small, the possibility that the cells further
differentiated into b-cell-like cells in vivo after transplantation can not be
ruled out [27,28].The secretion of human C-peptide in HAEC cell culture detected by
RIA kit indicated that insulin de novo synthesis and processing may be
present in induced HAEC. Although the secretion of human C-peptide did not
exhibit a time-dependent manner after induction of differentiation, the cells immunopositive
for insulin and Pdx-1 could not be detected until 14 d of induction in vitro.
These results indicated that cell differentiation proceeded during the
induction process.Glucose-stimulated insulin secretion from pancreatic b-cells is
regulated by a series of electrogenic events leading to exocytosis of
insulin-containing granules [29]. The induced HAEC exhibited glucose
responsiveness in C-peptide release [Fig. 2(B)], indicating that the
induced HAEC may possess the major function of b-cell, namely, insulin
release in response to changes in extracellular glucose concentration. The
induced HAEC were also responsive to KCl [Fig. 2(C)], suggesting the
presence of insulin-containing secretory granules. Insulin secretion from
pancreatic b-cells is acutely regulated by a complex interplay of metabolic and
electrogenic events. The electrogenic mechanism regulating insulin secretion
from b-cells is commonly referred to as the KATP
channel-dependent pathway [30]. Depolarization of b-cell by closing the KATP channel results in the opening of voltage-dependent Ca2+ channels, and influx of Ca2+ is the main trigger for
insulin secretion [31]. Our results showed that the C-peptide release of
induced HAEC was responded to tolbutamide, a KATP
channel inhibitor, suggesting the presence of functional KATP channels. The C-peptide secreting characteristics of induced HAEC
indicated that these cells could function similarly to normal pancreatic b-cells in
vitro.Induced HAEC have the ability to replace b-cell function in vivo.
After implantation of induced HAEC, blood glucose levels of diabetic C57 mice
decreased and remained normal within 30 d, while body weights continued to
increase. One problem inherent in our study is that normoglycemia reached
after transplantation might be due to regeneration of b-cell mass. However, there
are reasons that argue against this possibility. None of the sham-operated
diabetic mice, which remained hyperglycemic throughout the study, survived
more than 8 weeks after surgery, and untreated diabetic mice all remained
hyperglycemic and died within 10 weeks. HAEC-implanted mice reached
normoglycemic within 1 week after implantation, even though it was reported
that pancreas regeneration in mice takes approximately 6–8 weeks in the
case of 60% of pancreatectomized animals [32]. About 60% of normoglycemic
HAEC-implanted mice developed hyperglycemia within 7–8 weeks after the
transplantation, indicating that this glucose decrease process is reversible;
reversibility likely depends on the half-life of the implanted cell cluster,
suggesting the absence of contribution of b-cell mass regeneration to
glycemia normalization. As tested by IPGTT, the blood glucose levels of transplanted
diabetic mice exhibited a similar kinetics to that of normal control mice,
representing the recovery of insulin secretion ability of the HAEC-transplanted
mice. The immunofluorescence analysis of kidney sections showed that induced
GFP-labeled HAEC survived and presented insulin-positive staining 30 d after
transplantation. It was demonstrated that mild mononuclear cell infiltration
existed in kidneys receiving surgical operations. However, no intensive
immunorejection was observed in HAEC-transplanted kidneys, indicating that the
immune response was caused by surgery and that the transplanted cells did not
cause severe immunorejection in vivo. Although the mouse serum samples
caused high background signal in the ELISA assay, a statistically significant
increase of human C-peptide in HAEC-transplanted mouse serum could be
detected. Taken together, these results suggest that these induced cells have
similar function to b-cells in vivo and could be used as a b-cell
replacement.In summary, our findings present evidence that HAEC could be induced
to differentiate into functional insulin-producing cells, which may provide a
source of b-cells for the treatment of type 1 diabetes by transplantation.
Acknowledgements
We thank Dr. Xuejun Zhang and Dr. Bao Zhang for their technical
assistance and helpful discussion. This study received technical support from
Cellstar Biotechnologies Company (Shanghai, China).
References
1 Shapiro AM, Lakey JR, Ryan EA, Korbutt GS,
Toth E, Warnock GL, Kneteman NM et al. Islet transplantation in seven patients
with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive
regimen. N Engl J Med 2000, 343: 230–238
2 Soria B, Roche E, Berna G, Leon-Quinto T,
Reig JA, Martin F. Insulin-secreting cells derived from embryonic stem cells
normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 2000, 49:
157–162
3 Assady S, Maor G, Amit M, Itskovitz-Eldor J,
Skorecki KL, Tzukerman M. Insulin production by human embryonic stem cells.
Diabetes 2001, 50: 1691–1697
4 Lumelsky N, Blondel O, Laeng P, Velasco I,
Ravin R, McKay R. Differentiation of embryonic stem cells to insulin-secreting
structures similar to pancreatic islets. Science 2001, 292: 1389–1394
5 Bonner-Weir S, Taneja M, Weir GC,
Tatarkiewicz K, Song KH, Sharma A, ONeil JJ. In vitro cultivation of
human islets from expanded ductal tissue. Proc Natl Acad Sci U S A 2000, 97:
7999–8004
6 Gao R, Ustinov J, Pulkkinen MA, Lundin K,
Korsgren O, Otonkoski T. Characterization of endocrine progenitor cells and
critical factors for their differentiation in human adult pancreatic cell
culture. Diabetes 2003, 52: 2007–2015
7 Yang L, Li S, Hatch H, Ahrens K, Cornelius
JG, Petersen BE, Peck AB. In vitro trans-differentiation of adult
hepatic stem cells into pancreatic endocrine hormone-producing cells. Proc Natl
Acad Sci U S A 2002, 99: 8078–8083
8 Zalzman M, Anker-Kitai L, Efrat S.
Differentiation of human liver-derived, insulin-producing cells toward the b-cell phenotype.
Diabetes 2005, 54: 2568–2575
9 Okumura K, Nakamura K, Hisatomi Y, Nagano K,
Tanaka Y, Terada K, Sugiyama T, et al. Salivary gland progenitor cells
induced by duct ligation differentiate into hepatic and pancreatic lineages.
Hepatology 2003, 38: 104–113
10 Timper K, Seboek D, Eberhardt M, Linscheid P, Christ-Crain
M, Keller U, M?ller B et al. Human adipose tissue-derived mesenchymal
stem cells differentiate into insulin, somatostatin, and glucagon expressing
cells. Biochem Biophys Res Commun 2006, 341: 1135–1140
11 Hori Y, Gu X, Xie X, Kim SK. Differentiation
of insulin-producing cells from human neural progenitor cells. PLoS Med 2005,
2: e103
12 Sandler TW. In: Coryell P eds. First Week of
Development: Ovulation to Implantation Langmans Medical Embryology. 7th edn.
Baltimore, MD: William and Wilkins 1995
13 Miki T, Lehmann T, Cai H, Stolz DB, Strom SC.
Stem cell characteristics of amniotic epithelial cells. Stem Cells 2005, 23:
1549–1559
14 Wei JP, Zhang TS, Kawa S, Aizawa T, Ota M,
Akaike T, Kato K et al. Human amnion-isolated cells normalize blood glucose
in streptozotocin-induced diabetic mice. Cell Transplant 2003, 12: 545–552
15 Adinolfi M, Akle CA, McColl I, Fensom AH,
Tansley L, Connolly P, Hsi BL et al. Expression of HLA antigens, b 2-microglobulin
and enzymes by human amniotic epithelial cells. Nature 1982, 295: 325–327
16 Zufferey R, Dull T, Mandel RJ, Bukovsky A,
Quiroz D, Naldini L, Trono D. Self-inactivating lentivirus vector for safe and
efficient in vivo gene delivery. J Virol 1998, 72: 9873–9880
17 Hansson M, Tonning A, Frandsen U, Petri A,
Rajagopal J, Englund MC, Heller RS et al. Artifactual insulin release
from differentiated embryonic stem cells. Diabetes 2004, 53: 2603–2609
18 Paek HJ, Morgan JR, Lysaght MJ. Sequestration
and synthesis: the source of insulin in cell clusters differentiated from
murine embryonic stem cells. Stem Cells 2005, 23: 862–867
19 Sherry NA, Tsai EB, Herold KC. Natural history
of b-cell function in type 1 diabetes. Diabetes 2005, 54 Suppl 2: S32–S39
20 Sturgess NC, Ashford ML, Cook DL, Hales CN.
The sulphonylurea receptor may be an ATP-sensitive potassium channel. Lancet
1985, 2: 474–475
21 Qin XY, Shen KT, Song LJ, Zhang X, Han ZG.
Regulated production of mature insulin in rat hepatoma cells: insulin
production is up-regulated by dexamethasone and down-regulated by insulin.
Acta Biochim Biophys Sin 2006, 38: 89–94
22 Zhang Y, Yao L, Shen K, Xu M, Zhou P, Yang W,
Liu X et al. Genetically engineered K cells provide sufficient insulin
to correct hyperglycemia in a nude murine model. Acta Biochim Biophys Sin 2008,
40: 149–157
23 Matthews RN, Faulk WP, Bennett JP. A review of
the role of amniotic membranes in surgical practice. Obstet Gynecol Annu 1982,
11: 31–58
24 Kakishita K, Nakao N, Sakuragawa N, Itakura T.
Implantation of human amniotic epithelial cells prevents the degeneration of
nigral dopamine neurons in rats with 6-hydroxydopamine lesions. Brain Res 2003,
980: 48–56
25 Otonkoski T, Beattie GM, Mally MI, Ricordi C,
Hayek A. Nicotinamide is a potent inducer of endocrine differentiation in
cultured human fetal pancreatic cells. J Clin Invest 1993, 92: 1459–1466
26 Newgard CB, McGarry JD. Metabolic coupling
factors in pancreatic beta-cell signal transduction. Annu Rev Biochem 1995,
64: 689–719
27 Davani B, Ikonomou L, Raaka BM, Geras-Raaka E,
Morton RA, Marcus-Samuels B, Gershengorn MC. Human islet-derived precursor
cells are mesenchymal stromal cells that differentiate and mature to
hormone-expressing cells in vivo. Stem Cells 2007, 25: 3215–3222
28 Tang DQ, Cao LZ, Burkhardt BR, Xia CQ,
Litherland SA, Atkinson MA, Yang LJ. In vivo and in vitro
characterization of insulin-producing cells obtained from murine bone marrow.
Diabetes 2004, 53: 1721–1732
29 Easom RA. Beta-granule transport and
exocytosis. Semin Cell Dev Biol 2000, 11: 253–266
30 Straub SG, Sharp GW. Glucose-stimulated
signaling pathways in biphasic insulin secretion. Diabetes Metab Res Rev 2002,
18: 451–463
31 MacDonald PE, Wheeler MB. Voltage-dependent
K(+) channels in pancreatic beta cells: role, regulation and potential as
therapeutic targets. Diabetologia 2003, 46: 1046–1062
32 Martin F, Andreu E, Rovira JM, Pertusa JA,
Raurell M, Ripoll C, Sanchez-Andr?s JV et al. Mechanisms of glucose
hypersensitivity in beta-cells from normoglycemic, partially pancreatectomized
mice. Diabetes 1999, 48: 1954–1961