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Histological study of the role of stem cells on experimentally induced diabetes mellitus

El-Nashar, Eman M.a,b; Metwaly, Hala G.a,b; Ibrahem, Sali O.a,b; Salam, Sherifa Abdela,b; El-Gendy, Enas M.a,b

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The Egyptian Journal of Histology: December 2011 - Volume 34 - Issue 4 - p 849-858
doi: 10.1097/01.EHX.0000407701.18895.e2
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Abstract

Introduction

Diabetes is a threat to global health. According to the WHO, at least 171 million people worldwide (2.8% of the population) suffer from diabetes. Its incidence is increasing rapidly, and it is estimated that by the year 2030, this number will almost double [1]. Diabetes mellitus (DM) is a group of metabolic disorders sharing the common underlying feature of hyperglycemia [2].

Type 1 DM is characterized by the loss of insulin-producing β cells of the islets of Langerhans in the pancreas [3,4]. Alloxan selectively destroys the cells of the pancreas that secrete insulin [5].

Islet transplantation has been performed experimentally. This measure is not yet practical in regular clinical practice partly because of the limited number of β-cell donors and allogeneic rejection [6].

The use of stem cells for cell replacement strategies [mesenchymal stem cells (MSCs) from the bone marrow stroma] has recently experienced a high level of attention because they can be easily isolated and acquired in virtually unlimited numbers. Also, MSCs are capable of committing to multiple lineages [7,8].

Stem cells serve as an internal repair system; when a stem cell divides, each resulting cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, such as a muscle cell, a red blood cell, or a brain cell [9]. Insulin-producing cells (IPCs) have been derived from mouse embryonic stem cells (ESCs) [10] and human ESCs [11]. However, the use of ESCs in a clinical setting is limited.

Adult stem cells possess a strong regenerative capability to replenish the senile or sick cells [12]. One of the most extensively studied populations of multipotent adult stem cells has been MSCs from the bone marrow [13].

Several recent studies in rodents have indicated that the adult pancreas contains some types of endocrine progenitor cells that can differentiate into β cells [14]. Several studies seem to support the conclusion that the endocrine precursor cells are present not only in the duct but also within the islets themselves [15].

Recent reports have also described putative stem cells in the liver, spleen, central nervous system, and bone marrow, which can differentiate into IPCs [16].

The present study was designed to test the therapeutic effect and the role of the regeneration capacity of MSCs from the bone marrow in diabetic dogs.

Materials and methods

Nine adult male dogs were obtained from the animal house of the Faculty of Veterinary Medicine, Cairo University. The dogs were acclimatized to laboratory conditions and fed a basal diet formed of ordinary bread, fresh milk, and different vegetables, with a liberal supply of water; they were divided into two major groups. Group I including one dog considered as the −ve control. Group II (eight dogs) diabetic models received a single intravenous injection of alloxan (50 mg/kg) to induce diabetes, and were further subdivided into three subgroups: subgroup IIA (two dogs) served as the +ve control, subgroup IIB (two dogs) received undifferentiated MSCs at a dose of 5 × 106 intrahepatic percutaneously, and subgroup IIC (four dogs) also received the same dose of differentiated MSCs (IPCs). Dogs of group II were given a single intravenous injection of alloxan monohydrate (Loba Chemie, Loba chemie: Jehangir Villa, 107, Wode House Road, Colaba, Mumbai 400 005. India) 50 mg/kg to induce diabetes [17].

Isolation and preparation of mesenchymal stem cells

Bone marrow extracts contain heterogeneous cell populations that represent a small fraction of the total mononucleated cells within the bone marrow [8]. MSCs were obtained under complete aseptic conditions [18].

Bone marrow was collected in 50 ml falcon tubes containing 200 U/ml heparin. The sample was diluted with RPM (Gibco BRL, Grand Island, NY 14072, USA) I, and then Ficall-paque was added to it (1 : 3 ratios); on centrifugation, the upper layer aspirated, leaving the mononuclear cell layer undisturbed. The mononuclear cell layer was carefully transferred at a new 50 ml conical tube, the tube was filled with RPMI to 10 cm, and then centrifuged; the precipitation was completed with RPMI to 1 cm. The cell pellet was resuspended in 4 cm of solution, with 20% fetal calf serum up to 1 cm, antimycotic 100 mm, and antibiotic 100 mm (equal volume), to a final volume of up to 5 cm. The cells were incubated in a humidified 5% CO2 and 95% air atmosphere at 37°C for 3 weeks.

Separation of mesenchymal stem cells from the flask

The colonies of MSCs adherent to the flasks’ borders were left for 10 days [19]; then, the cells were harvested after being treated with 25% trypsin-EDTA (Biochrom AG, Berlin, Germany), and rinsed with RPMI twice for 3 min. The conical tube was filled with a suspension of culture media with trypsin and washed with RPMI centrifuged. The supernatant was removed completely and the precipitate was taken to assess cell viability.

Assessment of cell viability by microscopy

Cell viability was detected by adding equal volumes of the cell suspension and the trypan blue stain, and counting with a hemocytometer.

Flow cytometry

One million cells per 5 ml conical tube were incubated according to the manufacturer’s instructions at room temperature for 10 min with monoclonal antibodies labeled with phycoerythrin against one of CD34 or CD44. Flow cytometry was used as an additional means of characterization of the MSC preparations.

Transfection procedure

One day before transfection, 0.5–2 × 105 cells were plated in 500 ml of growth medium without antibiotic per well so that they will be 90–95% confluent at the time of transfection, and then DNA lipofectamine 2000 complexes were prepared. Cells were incubated at 37°C in a CO2 incubator for 24–48 h until they were ready to be assayed for transgenic expression. Transfected cells reflect a fluorescent green light.

In-vitro differentiation of mesenchymal stem cells into functional insulin-producing cells

MSCs with 80% confluence were induced to differentiate into IPCs. The cells were cultured (37°C, 5% CO2) in basic medium for 2 weeks in the presence of high-glucose media (23 mmol/l), and then cultured for an additional 5–7 days in the presence of 20 nmol/l exendin-4 Sigma, (Pharmaceutical Industries, Mubarak Industrial City, Quesna, menofya, Egypt) (modified from the technique of Tang et al. [20]).

Functional assessment of differentiated cells

Antigenic detection of insulin in the cytoplasm of cultured cells was detected by immunohistochemistry, and the result was observed microscopically. Glucose levels were monitored by tapped leg vein blood under fasting condition every day after transplantation for 7 days.

Transplantation of differentiated mesenchymal stem (insulin-producing cells) cells to alloxan-induced diabetic dogs

Under general anesthesia, the dogs received a transplant of 5 × 106 differentiated mesenchymal stem cells (D-MSCs or undifferentiated MSCs) intrahepatic percutaneously into the liver parenchyma using a Spinocan spinal needle (22 G, 3.5 in. long; Ghatwary Medical, Alexandria, Egypt) under real-time ultrasonic guidance (Acuson XP10; Pyramid management LLC, Los Alamitos, USA).

Histological study

Each dog was sacrificed with an overdose of sodium pentobarbital at 7 days after implantation. The liver and the tail part of pancreas were removed and fixed in 10% neutral-buffered formalin for 72 h at room temperature. Tissue specimens were embedded in paraffin and stained with H&E [21].

Immunohistochemistry for the detection of insulin-secreting cells

Sections from the liver and the pancreas were immunostained for the detection of insulin-secreting cells, according to Shi et al. [22].

Quantitative morphometric measurements

The area % of positive insulin immunoreactivity in the pancreas and the liver were estimated using the ‘Olympus BX40: image analyzer computer system, Hopkinton, MA 01748, USA)’. Measurements were performed within 10 non-overlapping fields for each animal at × 400 magnification. Morphometric measurements were carried out in the Histology Department, Faculty of Medicine, Cairo University.

Statistical analysis

The data obtained from the image analyzer were analyzed using the statistical software 'statistical for windows'; the parameters were tested using the f test, and the results were considered significant when P < 0.05.

Results

Assessment of cell viability

The viability of MSCs was confirmed by trypan blue exclusion. The live cells were not stained, and the dead cells were stained blue (Fig. 1).

F1-22
Figure 1:
A photomicrograph of mesenchymal stem cells suspended in culture. L, live cells (nonstained); D, dead cells (stained). Trypan blue, × 1000.

Phenotype characteristics of expanded undifferentiated bone marrow-derived mesenchymal stem cells

Flow cytometric analysis of the immunophenotype of the MSCs showed that these cells were negative for CD34 and expressed high levels of CD44. These results indicated that relatively purified bone marrow-derived mesenchymal stem cells (BM-MSCs) were isolated (Fig. 2).

F2-22
Figure 2:
Flow cytometric analysis of the mesenchymal stem cells (MSCs), showing that MSCs are negative for CD34. They expressed high levels of CD44.

Functional assessment of differentiated cells

Antigenic detection of insulin in the cytoplasm of D-MSCs (IPCs) was performed by immunohistochemistry in vitro (Fig. 3).

F3-22
Figure 3:
A photomicrograph of the immunohistochemical staining of differentiated mesenchymal stem cells (insulin-producing cells) suspended in culture in vitro, showing positive immunostaining of the cells (dark brown) for insulin. Peroxidase antiperoxidase immunohistochemistry, × 400.

Fluorescent microscopic examination

Fluorescence microscopy images demonstrated the green fluorescence of MSCs and differentiated cells labeled with enhanced green fluorescent protein in vivo 1 week after implantation into the liver parenchyma in group IIB (Fig. 4) and group IIC (Fig. 5).

F4-22
Figure 4:
Fluorescence microscopy image of the liver of a dog from group IIB demonstrating the green fluorescence of undifferentiated mesenchymal cells labeled with enhanced green fluorescent protein in vivo 1 week after implantation (arrows indicate green fluorescent cells). Immunofluorescent technique, × 400.
F5-22
Figure 5:
A fluorescence microscopy image of the liver of a dog from group IIC demonstrating the green fluorescence of differentiated mesenchymal stem cells labeled with enhanced green fluorescent protein in vivo 1 week after implantation. The arrows indicate green fluorescent cells. Immunofluorescent technique, × 400.

Histological result (H&E)

Group I (ve control)

The pancreas showed normal islets of Langerhans, formed of clumps of cells separated by blood capillaries with no apparent capsule. The islets were surrounded with normal exocrine tissue formed of serous acini, the lining acinar cells of which showed basal basophilia, apical acidophilia, and round basal nuclei (Fig. 6).

F6-22
Figure 6:
A photomicrograph of a section from the pancreas of a dog from group I (−ve control) showing normal islets of Langerhans (arrow) and normal exocrine tissue (arrow heads). H&E, × 400.

Group IIA (+ve control)

This group showed a decreased size of the islets of Langerhans (atrophied islets), loss of uniform cellular distribution, fewer cells, vacuolated cytoplasm, pyknotic nuclei, and perivascular inflammatory cells. Dilated blood vessels were observed. Pancreatic acini were maintained (Fig. 7). Some islets showed the deposition of a hyaline material (Fig. 8).

F7-22
Figure 7:
A photomicrograph of a section from the pancreas of a dog from group IIA (+ve control) showing a few islet cells, loss of uniform cellular distribution, vacuolated cells (v) and pyknotic nuclei (N) congested dilated blood vessel (BV), and perivascular polymorph nuclear cells (arrows). H&E, × 400.
F8-22
Figure 8:
A photomicrograph of a section from the pancreas of a dog from group IIA (+ve control) showing a few islet cells, loss of uniform cellular distribution, large vesicular nuclei (N), and deposition of a hyaline material in the islet (arrows). H&E, × 400.

Group IIB (received intrahepatic undifferentiated mesenchymal stem cell)

The pancreas showed less organized pancreatic islets. The cells were hypertrophied with large vesicular nuclei (Fig. 9).

F9-22
Figure 9:
A photomicrograph of a section from the pancreas of a dog from group IIB, which received intrahepatic mesenchymal stem cells, showing less organized pancreatic islets (arrows) and hypertrophied islet cells with large vesicular nuclei. H&E, × 400.

Group IIC (insulin-producing cells transplants)

The pancreas showed a histological structure similar to that of group IIB; some islets were atrophic, whereas others showed hypertrophic cells with large vesicular nuclei (Fig. 10).

F10-22
Figure 10:
A photomicrograph of a section of the pancreas of a dog from group IIC, which received intrahepatic insulin-producing cells, showing pancreatic islets (arrow) with loss of uniform cellular distribution and hypertrophied islet cells with large vesicular nuclei. H&E, × 400.

Immunohistochemical results for the detection of β cells

Group I (ve control)

Positive insulin expression in β cells of the pancreas had dark-brown reaction in the cytoplasm, whereas the nuclei were not stained (Figs 11 and 12).

F11-22
Figure 11:
A photomicrograph of a section from the pancreas of a dog from group I (−ve control). The islets stained positive for insulin (dark brown) (arrows). Immunohistochemistry: peroxidase antiperoxidase, × 100.
F12-22
Figure 12:
A photomicrograph of a section from the pancreas of a dog from group I showing pancreatic islets (arrow) with cytoplasmic staining of β cells of the islet (dark brown). Immunohistochemistry: peroxidase antiperoxidase, × 400.

The liver showed negative immunostaining for insulin (Fig. 13).

F13-22
Figure 13:
A photomicrograph of a section from the liver of a dog from group I (−ve control) showing negative immunostaining of the liver. Immunohistochemistry: peroxidase antiperoxidase, × 100.

Group IIA (+ve control)

There were fewer insulin immunopositive cells in each pancreatic islet compared with the −ve control (Fig. 14).

F14-22
Figure 14:
A photomicrograph of a section from the pancreas of a dog from group IIA after destruction of islet β cells by alloxan, showing a few insulinpositive cells in the pancreatic islets (arrow). Immunohistochemistry: peroxidase antiperoxidase, × 400.

Group IIB

There were more insulin immunopositive cells in the pancreatic islets in this group compared with that of the positive control group (Fig. 15) and insulin immunopositive cells in the liver (Fig. 16).

F15-22
Figure 15:
A photomicrograph of a section from the pancreas of a dog from group IIB, which received intrahepatic undifferentiated mesenchymal stem cells, showing immunopositive cells (arrows). Immunohistochemistry: peroxidase antiperoxidase, × 400.
F16-22
Figure 16:
A photomicrograph of a section from the liver of a dog from group IIB, showing immunopositive cells (arrows) 1 week after intrahepatic injection of undifferentiated mesenchymal stem cells. Immunohistochemistry: peroxidase antiperoxidase, × 400.

Group IIC (insulin-producing cells transplants)

Insulin immunopositive cells in the pancreatic islets increased compared with that of the positive control group (Fig. 17). The liver showed a positive immunostaining of IPC transplants (Fig. 18).

F17-22
Figure 17:
A photomicrograph of a section from the pancreas of a dog from group IIC, which received intrahepatic differentiated mesenchymal stem cells, showing positive reaction of pancreatic islet (arrow). Immunohistochemistry: peroxidase antiperoxidase, × 400.
F18-22
Figure 18:
A photomicrograph of a section from the liver of a dog from group IIC, showing insulin-producing cell transplants located in the liver, expressing insulin (arrows) 1 week after intrahepatic injection of differentiated cells. Immunohistochemistry: peroxidase antiperoxidase, × 400.

Morphometric results

The area % of positive insulin immunoreactive cells in the liver and the pancreas increased significantly (P < 0.05) in groups IIC and IIB as compared with group IIA (Table 1 and Histogram 1).

T1-22
Table 1:
Comparison between the mean and SD area % of insulin immunopositive cells in different group
F19-22
Histogram 1. Comparison between the mean and SD of the area % of insulin immunopositive cells of different groups: 1=group IIC, 2=group IIB, 3=group IIA, and 4=group I control.

Biochemical results

The fasting blood glucose level measurements showed an improvement in the group that received D-MSCs (IPCs), as the mean was 400 mg/dl before intrahepatic transplantation of cells, and then reached 302 mg/dl on day 4 and 240 mg/dl on day 6 after transplantation. However, the group that received undifferentiated MSCs showed no improvement in the blood glucose level, as the mean was 410 mg/dl before intrahepatic transplantation of cells, and then reached 390 mg/dl on day 4 and 370 mg/dl on day 6 after transplantation of cells.

Discussion

DM results when there is a progressive failure of functional β cells. Pancreatic islet transplantation has sparked new interest in the treatment of DM. Unfortunately, islet transplantation has historically been hampered by immune rejection and the scarcity of donor islets [23,24]. Also, the immunosuppressive drug regimen necessary to protect islets from a recurrent autoimmune response and allorejection may, with time, irreversibly damage kidney function [25].

An alternative for islet transplantation would involve the use of a renewable source of stem cells capable of self-renewal and differentiation, and capable of insulin production [20].

BM-MSCs reside in the bone marrow, and can differentiate into lineages of mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, adipocytes, chondrocytes, osteocytes [13,10,11,14–27], and also into endodermal and epidermal cells, such as vascular endothelial cells, neurocytes, lung cells, and hepatocytes [28–30].

MSCs have high multiplication potency; their cell-doubling time is 48–72 h, and cells could be expanded in culture for more than 60 doublings [31]; MSC donors (autologous transplantation) would not cause any rejection [32]. Several in-vitro studies have shown that bone marrow-derived stem cells could be reprogrammed to become functionally IPCs under certain culture conditions [20,33,34].

Recent studies have demonstrated the feasibility of generating IPCs obtained from progenitor cells of various cellular sources, including the pancreas [35,36], the liver [37] and intestinal epithelium [38], adipose tissue-derived stem cells [39] and pluripotent ESCs of mouse [20,40] and human [41] origins.

In the present study, BM-MSCs were isolated, cultured, and characterized. Also, functional IPCs were generated from the BM-MSCs by an in-vitro differentiation procedure, and the presence of insulin production was confirmed by immunohistochemical study.

BM-MSCs were induced to differentiate into IPCs by glucose-rich medium supplemented with exendin-4 (GLP-1 agonist). Glucose is a growth factor for β cells; it induces in-vitro differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells and increases the insulin content in cell lines derived from ESCs [40–43].

Exendin-4 could stimulate both β-cell replication and neogenesis from ductal progenitor cells, and inhibit the apoptosis of β cells [44,45].

In this study, the immunophenotype of BM-MSCs is negative for CD34 and positive for CD44, indicating that the bone marrow-derived stem cells capable of generating IPCs might be BM-MSCs [46,47].

In the present study, MSCs were marked with green fluorescence protein, which can be visualized easily by fluorescence microscopy. Green fluorescence protein transfection was used to detect the fate of the injected cells.

Alloxan induces diabetes through selective destruction of the pancreatic islet β cells (IPCs) [5].

The dogs were fasted for 12 h before and after the injection of alloxan; the unfed animals are more susceptible to alloxan-induced diabetes [48,49].

In the present study, diabetic dogs that received the differentiated IPCs showed an improvement in the fasting blood glucose level as it was 400 mg/dl before intrahepatic transplantation of differentiated cells and reached 240 mg/dl on day 6 after transplantation [20,50]. We conclude that glucose levels in the differentiated MSC-implanted mice decreased and normalized within 1 week after transplantation [51]; we detected proinsulin-positive and insulin-positive cells in the liver, adipose tissue, spleen, bone marrow, and thymus in diabetic mice.

In the present study, H&E staining of the tail of the pancreas of diabetic dogs showed a reduction in the size and number of islets, loss of uniform cellular organization, necrotic changes in pancreatic islets, nuclear changes and karyolysis. Some islet cells were hypertrophied, with large vesicular nuclei (compensatory hypertrophy) with the deposition of a hyaline material and dilated blood vessels.

In the current study, immunohistochemical staining of the pancreas of diabetic dogs that received intrahepatic undifferentiated or differentiated MSCs showed a significant increase in the area % of insulin immunoreactive cells compared with the positive control group, which showed a loss of β cells and the insulin immunoreactivity was scattered, if any. A significant increase in the area % of positive immunostaining for insulin was also detected in the liver of the groups that received undifferentiated or D-MSCs.

Conclusion

BM-MSCs are easily accessible, easily isolated, expanded in culture, and could differentiate into IPCs. Intrahepatic transplantation of autologous differentiated MSCs (IPCs) improved fasting blood glucose levels in diabetic dogs.

T2-22
Table:
No title available.

Acknowledgements

Conflicts of interest

There is no conflict of interest to declare.

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Keywords:

alloxan; diabetes; immunostaining; stem cells transplantation

© 2011 The Egyptian Journal of Histology