Function of Rat Diabetic Islets Improved By Coculturing with Pancreatic Mesenchymal Stromal Cells

Research Article

J Stem Cell Res Transplant. 2015;2(1): 1016.

Function of Rat Diabetic Islets Improved By Coculturing with Pancreatic Mesenchymal Stromal Cells

Maryam Mohammadi Khajehdehi1, Durdi Qujeq2*, Habibollah Peirovi3, Narges Karbalaie4, Seyed Javad Mowla5

1Department of Genetics, Science & Research Branch, Islamic Azad University, Tehran, Iran

2Department of Biochemistry and Biophysics, Faculty of Medicine, Babol University of Medical Sciences, Babol, Iran

3Nanomedicine & Tissue Engineering Research Center, Taleghani Hospital, Tehran, Iran

4Department of Physiology, Faculty of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

5Molecular Genetics Department, School of Biological Sciences, Tarbiat Modares University, Tehran, Iran

*Corresponding author: Dr Durdi Qujeq, Department of Biochemistry and Biophysics, Faculty of Medicine, Babol University of Medical Sciences, Babol, Iran

Received: November 26, 2014; Accepted: February 19, 2015; Published: February 24, 2015

Abstract

Developing strategies to prevent beta cells mass reduction and/or increasing their in vivo and in vitro extension are promising choices for cell therapy of type 1 and type 2 diabetes. MSCs (Mesenchymal stem cells)are multipotent stromal cells with the ability to self-renew and differentiate into various cell types of its host tissues. Recently, the effects of transplanted rBMSCs(rat Bone Marrow Mesenchymal Stem Cells) on survival and function of isolated islets have been reported. In this research, rP-MSCs (rat pancreatic Mesenchymal Stem Cells) were isolated and characterized before being co-cultured with injured islets of diabetic rat models in vitro. The effects of rP-MSCs on insulin secretion of islets were examined after 5 days. Furthermore, the extracted cells were labeled by DiI dye and their migration and incorporation within the injured pancreatic islets were visualized after 24 hours. Altogether, our data revealed that after co-culturing of the injured islets with rP-MSCs, the insulin secretion level of the islets were significantly elevated (p value<0.001). This finding implies that rPMSCs have a potential ability in repair of diabetic islets by either releasing some growth and immuno-modulatory factors, or by direct incorporation within the damaged islets.

Keywords: Type 1 diabetes; Pancreatic Stromal Cells; Injured islets; Coculture

Introduction

Diabetes Mellitus is the most common endocrine disorder, with more than 200 million people suffering from the disease worldwide. More importantly, it is estimated that the number of cases will reach to 300 million by 2025 [1-3]. The disease is a chronic metabolic syndrome, and is diagnosed by an elevated sugar level in blood which is referred to hyperglycemia. Type 1 diabetes is usually caused by an autoimmune disturb of pancreatic islet's beta cells which is consequently followed by a shortage or lack of insulin in blood. For this reason, the person will need a permanent supply of external insulin to survive [4]. While the external insulin sources can improve the blood sugar level, managing its routine application, and hence permanent blood's sugar control, is very complicated. Therefore, transplantation of Pancreas tissue or insulin producing cells has been an attractive potential therapy to cure the disease permanently [5].

The main aim of the cell-based therapy that has emerged as a strategic treatment for many human diseases is to replace, repair and/ or enhance the biological function of damaged tissues in an organ. The main biological materials for this purpose are "stem cells" which is obtained either from embryonic or adult tissue-specific stem cells [6]. According to some recent studies, the insulin secreting cells can be generated in vitro from mouse bone marrow stromal cells [7] and mature pancreatic cells including mouse and human pancreatic duct cells [8,9]. In this regard, the epithelial cells of pancreatic duct contain progenitor cells which are involved in pancreas growth and renewal [10]. Furthermore, there are other cell types within the pancreas that are able to proliferate and differentiate to beta cells [11,12]. These cells are recognized by the presence of some cell surface markers that are related to a specific linage of stem cells.

The application of pancreas transplantation, as a therapeutic strategy for diabetic patients, is limited because of a shortage in organ donors [13,14]. In case of islet transplantation to diabetic patients, a good supply of insulin producer cells is required [15]. At the same time, maintaining the survival of islets can be another challenge [16], as the injuries made during islet isolation may lead to a functional drop of islets and transplantation failure [17]. Therefore, improvement of the methods to preserve the function and survival of islets are very important in transplantation success.

Some researchers have tried to overcome the aforementioned problem by co-culturing islets with different cell types. The first study in this subject was performed by [18], when they co-cultured pancreatic islets of new born rats with mouse fibroblasts and observed an increase in survival and function of the cultured islets. Similar efforts have been made by using pancreatic duct cells [19] or bone marrow cells [6,20,21] to improve the islets function.

In this study, we have investigated a new strategy to improve the function of isolated injured islets obtained from rat diabetic models, by co-culturing them with pancreatic mesenchymal stromal cells.

Materials and Methods

Isolation and culture of rP-MSCs

Male Wistar rats (Rattus norvegicus; weight 90-110grams; 3-4 weeks old) were obtained from Pastor Institute in Tehran, and were stored under standard conditions of constant humidity (55-65%) and temperature (22-24°C), 12 hours dark/light cycle, with unrestricted access to food and water. Animal housing and surgical procedures were carried out in accordance with the Animal Care and Use Committee regulations of Tarbiat Modares University (TMU) to reduce animal suffering and the number of used animals.

The animals were anaesthetized with 60mg/kg pentobarbital (Sigma, Germany); their pancreases were removed under sterile conditions, minced into small pieces and washed with cold PBS. The enzymatic digestion was performed by collagenase P (Roche, Germany) enzyme at 1mg/mL in cold PBS, with an incubation time of 13 minutes. Digestion was then stopped with addition of cold PBS. After washing and discarding the supernatant, DMEM (Gibco- Invitrogen, USA) media supplemented with 20% fetal bovine serum (Biowest, USA) was added to the pellet, resuspended and cultured in a 6-well plate.

RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was isolated from passage 1 of rP-MSCs cell cultures, using Trizol reagent (Invitrogen, USA) and as instructed by the manufacturer. The RNA of PSCs (Pancreatic Stem Cells)and islets (as positive controls) were reverse-transcribed using the primescript™ RT reagent kit (TaKaRa, Japan). The obtained cDNA of PSCs and islets was then added to PCR mix consisted of 10X PCR buffer, 4μl dNTPs, 1μl mix primer, 4μl MgCl2 and 0.25μl TaKaRa Ex Taq™.The PCR products were then analyzed by 2% agarose gel electrophoresis. The sequences of the designed primers for selected genes are listed in Table 1.

Induction of diabetes with Alloxan

Diabetes was induced by intravenous injection of a freshly prepared solution of alloxan (Sigma-Aldrich, USA; 40mg/kg body weight) to rats. The blood sugar level was measured in 48 hours, and animals with glucose ranging from 300-380 mg/dl, showing clear signs of polyuria, polydipsia and polyphagia, were considered as diabetic and were analyzed 48 hours after alloxan treatment.

Isolation of islets from normal and diabetic rats

The rats were starved for 12 hours before surgery, and their pancreatic ducts were clamped. The exposed pancreas was subjected to collagenase P solution (0.5 mg/ml in cold PBS) and incubated in 37°C for 17 minutes. Next, the endocrine parts were isolated from the other parts by PBS washing and suction for several times. The islets were then handpicked under a stereo microscope.

Staining of islets with DTZ

The diabetic and normal islets were stained by DTZ (dithizone), (Millipore,USA). For islets staining, we added 10μl of 100X DTZ stains solution to 1ml culture medium. After 3-5 minutes, the cells were examined under an invert microscope.

Co-culturing of rP-MSCs with normal & diabetic islets

For co-culture experiments, pancreatic stromal cells were employed as feeder cells. Isolated islets (diabetic and normal) were categorized in two groups: half of the strainers (BD Falcon, Mexico) containing islets from each group were cultured with rP-MSCs and the rest of the islets were cultured alone. In order to study the effects of rP-MSCs on islets' function, co-cultures of days 1, 3 and 5 were chosen randomly. At the end of each day, strainers containing islets were incubated first in L-DMEM for 1 hour and then in H-DMEM for another 1 hour. Finally the culture media were collected and the amount of released insulin was measured by rat insulin ELISA kit (Mercodia, Denmark), according to the manufacturer instruction.

Labeling of rP-MSCs by DiI vital dye and monitoring their migration into injured islets

rP-MSCs were labeled with 10μg /ml DiI dye (invitrogen, USA), as instructed with the manufacture. Then, they were examined under a florescent microscope, before and 24 hours after being co-cultured with injured islets.

Statistical analysis

Each experimental group included 72 islets. Co-culture experiments were performed in 8 different subgroups, and 9 times. Data are reported as mean ± SD by Prism 5 software (GraphPad Prism version 5.0.0.288 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com). Then they were analyzed by two-way ANOVA and paired t-test with the same software. Differences between the experimental and control groups were regarded as statistically significant when p<0.05.

Results

Isolation and Characterization of Rat Pancreatic Mesenchymal Stromal Cells (rP-MSCs)

Isolated cells from enzymatically digested minced pancreases were cultured and their morphology were examined routinely. On first day of culture, fibroblast-like mesenchymal cells adhered to cultured plates (Figure 1A).The cells were then propagated to a confluency of 70% by 72 hours (Figure 1B). The rate of growth and morphology of the cells were not changed during consequent passages. In 8th day of culture, the confluent cells started to generate cell colonies which were morphologically similar to pancreatic islets (Figure 1C). Gradually, more cellular clusters were found in cultures, and step by step maturation took place (Figure 1D).