Type 1 diabetes is characterised by absolute insulin deficiency caused by T-cell-mediated destruction of pancreatic
Transcription factors control biological processes such as differentiation, proliferation, and apoptosis. They bind to the specific sequence within the region of the promoter or enhancer and activate specialized genes’ expression. It has been reported that a number of transcription factors were involved in pancreas
Recently, MSCs were chosen as target cells for transplantation, because of their ability to differentiate into multiple cell types [
There are some findings demonstrating the feasibility of inducing a functional alteration in cultured MSCs by expression of a single master pancreatic regulator gene [
The genes of mouse transcription factors PDX-1, NeuroD1, and MafA (gene ID: 18609, 18012, 378435) were obtained by total gene synthesis and gene sequencing to validate that the synthesis was correct. The encoding sequences of PDX-1, NeuroD1, and MafA were amplified and ligated with an internal ribosome entry site sequence-green fluorescent protein (IRES-GFP) by PCR, then cloned into a shuttle vector pDONR221 by BP clonase II enzyme mix (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The corrcet recombinant plasmids were then cloned into the pAD/CMV/V5-DEST adenoviral vectors (Invitrogen, Carlsbad, CA, USA) by LR clonase II enzyme mix (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Electrophoretic analysis and DNA sequencing were performed to identify the recombinant vectors.
After the cells were counted, the packaging cell line 293A in logarithmic growth phase was incubated in a 6-well culture plate at 37°C, 5% CO2 the day before transfection. The sequences of the recombinant adenovirus vectors pAd-Mouse PDX-1-IRES-GFP, pAd-Mouse NeuroD1-IRES-GFP and pAd-Mouse MafA-IRES-GFP were confirmed by gene sequencing and linearized with Pac I and then transfected into the adenovirus packaging cell line 293A using Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA). 48 hours after transfection, cells were detached and transferred to a petri dish. Fresh nutrient medium was added every two or three days. The supernants were collected from 293A cells when most of the cells showed significant cytopathic effect (CPE). Primary adenoviruses were harvested after 3 times of freeze-thawing of the supernants. The primary adenoviruses were used to infect 293A cells in 10 cm petri dishes to make adenoviruses concentrated. Finally the concentrated adenoviruses were stored at −80°C. The control adenovirus expressing green fluorescent protein (Ad-GFP) was prepared as the above-mentioned method. The titer of the adenovirus was determined by an immune method, as follows: the HEK-293 cells that had been infected with adenovirus in different concentrations were reacted with rabbit antiadenovirus polyclonal antibody (1 : 1000) for 1 hour and were then incubated for additional 1 hour with horseradish peroxidase labeled anti-rabbit antibody. After 3,3′-diaminobenzidine (DAB) staining, the titer of the adenovirus was calculated in terms of the number of brown particles formed in different dilutions.
Bone marrow mMSCs from mice were enriched and expanded in vitro by using the whole bone marrow adherence method according to the previous protocol published from our laboratory [
Cells were collected from a highly proliferative mMSC culture at passage 3 and plated into 96 well culture plates at the same density. On the next day cells were infected with freshly harvested Ad-GFP at different multiplicity of infection (MOI) from 5 to 5000 in medium containing 2% FBS and incubated for 24 h. Infection efficiency was determined by fluorescence microscope after a further three days, and toxicity was determined by Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). 10 uL of CCK-8 solution was added into every well according to the instructions on the manufacturers’ kit, followed by incubation at 37°C with 5% humidified CO2 for 2 hours. The absorbance of the infected cells was measured by a microplate reader. The optimal MOI were identified from infection efficiency and toxicity. Ad-Mouse PDX-1-IRES-GFP, Ad-Mouse NeuroD1-IRES-GFP and Ad-Mouse MafA-IRES-GFP were prepared, and then mMSCs were infected with viruses containing the three factors at an optimal MOI. Each of the adenovirus has the same contribution to the optimal MOI, single gene delivery and double infection were also performed. The following day the cells were switched to differentiation medium supplemented with EGF. The infection was repeated in the following days. The cells were infected with Ad-GFP as a control.
Total cellular RNA was isolated using the MicroElute Total RNA Kit (OMEGA BIO-TEK, GA, USA) according to the manufacturer’s instructions. After quantification by spectrophotometry, 2
After being cultured for 21 days, infected cells were seeded on glass slides in a 12-well culture plates and fixed in 4% paraformaldehyde in PBS. Permeabilizing and blocking was performed in 10% fetal calf serum, 3% bovine serum albumin, and 0.2% triton X-100 in PBS. Then the cells were incubated with primary antibody (rabbit anti-mouse insulin polyclonal antibody 1 : 50; Santa Cruz Biotechnology, CA, USA.) overnight at 4°C. For insulin staining, the cells were further incubated for 2 h at room temperature in the dark, with secondary antibody (Cy3 anti-rabbit 1 : 50; Proteintech Group, Chicago, IL USA). After Hoechst staining for additional 15 min, the slides were washed and examined under the microscope. Images were captured using an Olympus phase contrast fluorescent microscope (Olympus Corporation, Tokyo, Japan).
The infected cells or noninfected cells were preincubated with Krebs-Ringer buffer (KRB) for 1 h, followed by incubation for an additional 1 h in KRB containing 10.0 mM glucose. The buffer was collected and frozen at −80°C until assay. Insulin enzyme-linked immunosorbent assay (ELISA; Cusabio Biotech Co., Wuhan, Hubei, China) was used for the quantitative determination of insulin levels in the collected buffer according to the manufacturer’s protocol. All values were determined against a standard curve prepared with mouse insulin.
To set up models of mice with diabetes, adult C57BL/6J mice were injected intraperitoneally with Streptozotocin (STZ; Sigma Chemical Company, St. Louis Missouri, USA) at a dose of 160 mg/kg. Hyperglycemia had been made by the administration of this dose of STZ within 7 days. Blood glucose reached levels >16.7 mmol/L and kept hyperglycemia for 2 weeks at least. Cells for transplantation were prepared at the same time. Transcription factors PDX-1, NeuroD1, and MafA were delivered into mMSCs 3 days before transplantation. Mice were anesthetized with intraperitoneal injection of sodium pentobarbital at 50 mg/kg, followed by the abdominal incision. About 1-2 × 106 infected cells or non-infected cells suspended in 0.2 ml PBS were transplanted into the liver parenchyma of mice with diabetes. For the glucose tolerance test, mice were injected intraperitoneally with 2.0 g of glucose per kg body weight after overnight fast. Blood glucose levels were monitored at the indicated time points (0–120 min) in samples obtained from the tail vein of mice by using One-Touch II portable blood glucose monitor (Lifescan Inc., Milpitas, CA, USA). The mice were sacrificed two weeks after transplantation. To witness the survival of the IPCs and detect the insulin secretion of triple infected cells in the liver tissues, the livers were removed and fixed in 10% formalin. Forty-eight hours later they were cut into serially sections and analyzed by immunohistochemistry. Negative controls were also set up. mMSCs infected with Ad-GFP or mMSCs without any infection were transplanted into the livers as a control. Terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling (TUNEL) assay was also performed using the In Situ Cell Death Detection Kit, Fluorescence (Roche Applied Science, Mannheim, Germany) to determine whether the injected cells were apoptotic. The livers were taken out immediately for making frozen tissue sections. Frozen tissue sections were rinsed with PBS and treated with 1% Triton X-100 in PBS for 2 min on ice. Slides were rinsed in PBS and incubated for 60 min at 37°C with 50
The sequences of the resultant recombinant adenoviruses, which encode PDX-1, NeuroD1, MafA, and GFP, were confirmed by gene sequencing and restriction endonuclease digestions with Pac I. Pac-I-digested adenoviral vectors were transfected into the 293A cell line to produce a crude adenoviral stock and the adenovirus was amplified by infecting 293 A cells. Various kinds of cells can be infected with this adenovirus.
The unattached cells from the bone marrow samples were removed through medium changes, and the adherent mMSCs were cultured for propagation. After subsequent passaging, most of the adherent cells exhibited fairly uniformly appearance. Immunophenotypes of the cells at passage 3 were assayed by flow cytometry analysis. The majority of the cells expressed high levels of CD29, CD44, and CD105. Meanwhile, the markers CD14, CD34, and CD45 displayed extremely low expression. mMSCs were incubated in osteogenic and adipogenic differentiation medium to identify the multipotency character, and it showed osteogenic differentiation and adipogenic differentiation after 21 days (Figure
mMSC were induced into osteoblasts and adipocytes in vitro under different differentiation medium. After incubation for 21 days, the differentiated cells were stained with alkaline phosphatase (a) or oil red (b) for their multipotent characteristic.
The susceptibility of different types of cells to adenovirus is variable and differs significantly among the cell types. To achieve the optimal infection with adenovirus, we chose the optimal MOI to raise the infection efficiency and also to have the least amount of cytotoxicity to the mMSCs simultaneously. The results indicated that the infection efficiency improved constantly with the increasing MOI. When cells were infected with freshly adenovirus at MOI of 100, the infection efficiency was over 80%. When cells were infected with fresh adenovirus at an MOI of more than 2,400, the infection rate was almost 100%. On the other hand, the cell survival became significantly inhibited with increasing MOI values beyond 600. The survival curves showed that the number of living infected cells decreased markedly when the MOI value was greater than 600 (Figure
The toxic effect of adenovirus on mMSCs at different multiplicity of infection was determined by CCK-8. 10
To determine whether the endogenous insulin gene started transcription, gene expression profiles of exogenous transcription factors and insulin gene were evaluated by RT-PCR. As illustrated in Figure
Adenovirus-mediated expression of PDX-1, NeuroD1, and MafA together induced expression of the insulin gene in infected mMSCs. mMSCs were infected with diverse single recombinant adenoviruses, both of the two adenoviruses, a combination of the three adenoviruses, or Ad-GFP. Total RNA from mMSCs was isolated 3 d after infection, and RT-PCR analysis was performed to examine expression of the specified genes. Cultured mMSCs without infection served as the negative control.
Quantitation of the amount of insulin1 and insulin2 that were produced by the infected mMSCs. The amount of insulin1 (a) and insulin2 (b) mRNA expression in mMSCs with triple infection was significantly larger compared with any other group.
After infection, to determine the biosynthesis of insulin and assay the insulin expression at the protein level, the differentiated mMSCs were documented by immunofluorescence analyses. All the cells on the slides were incubated with anti-mouse insulin and then red fluorescence was clearly visualized in the nucleus and cytoplasm after the triple infection (Figure
Expression of insulin protein in the infected cells. After culturing them for 21 days, all the infected cells were incubated with anti-mouse insulin antibody. Nuclei were stained blue with Hoechst dye (a). Most of the transgenic cells were stained positively for insulin (b). In contrast, the mMSCs infected with Ad-GFP or null were negative for insulin.
To further determine whether the function of insulin secretion of the differentiated mMSCs, the amount of insulin released by the cells in vitro at indicated concentrations of glucose was measured using a mouse insulin ELISA kit. As illustrated in Figure
Insulin secretion of the infected cells which were transferred into PDX-1, NeuroD1, MafA, or GFP at different differentiation stages in vitro. The cells were incubated in KRB containing the indicated concentration of glucose. The buffer was then collected for assay of insulin release in each experimental group. One asterisk, *
Take a step further to determine whether the induced IPCs give full scope to normal physiological functions of
Immunohistochemistry assay for insulin of the survival infected mMSCs in the liver tissues of mice with diabetes. (a) Positive control, anti-mouse insulin staining of mouse pancreatic specimen showing an intense expression of insulin (enlargement ×100). (b) Anti-mouse insulin staining of pancreatic specimen of mice with STZ-induced diabetes showing a markedly decreased expression of insulin (enlargement ×100). (c) Infected mMSCs were injected into the livers of mice with diabetes three days after infection. The positive staining of mouse insulin expression can be clearly observed in the liver. (d) An enlargement of induced IPCs in the liver.
TUNEL assay was performed to see whether the injected cells were apoptotic. (a) Frozen tissue sections of the livers were stained with hoechst. (b) The immunofluorescent stainings of TUNEL were negative in the transplanted cells.
Glucose responses to glucose tolerance test of mice with diabetes after transplantation. The infected cells which expressed combination of PDX-1, NeuroD1, and MafA were transplanted into the livers of mice with STZ-induced diabetes, glucose tolerance test was performed at 7 (a) and 14 days (b) following transplantation, compared with a normal control and diabetes models without any treatment. Data are presented as mean ± SD.
In recent years, cell transplantation has become a research hotspot concerning surgical methods for the treatment of diabetes. In order to obtain surrogate
The
Previous research has suggested that stable expression of PDX-1 in adult human mesodermal tissues activated expression of all four islet hormones including insulin and reversed hyperglycemia in vivo, but more factors that stimulate cells further toward differentiated normal
In the experiments of gene detection, genetic transformation of PDX-1 activated the expression of endogeneous NeuroD1 and endogeneous PDX-1 could be activated by exogenous NeuroD1 or MafA. The experimental results indicated that adjustment or interaction may really exist between each transcription factor. However, PDX-1 and MafA, together with endogeneous NeuroD1 were unable to exert as strong an influence on the expression of the insulin gene as delivery of a combination of the three transcription factors. We assume that fine synergism could not be achieved due to the low expression level of induced factors.
Intracellular GFP of the mMSCs was subsequently initiated to expression at 3 days after gene delivery, close together with the factors. However, one week, later, the intensity of the fluorescence decreased with the degradation of partial mitochondrial DNA. Therefore, induced efficiency was significantly inhibited without a repetition of infection. Cell transplantation in liver parenchyma was done to further verify the function of induced IPCs in vivo. Both intraperitoneal injection and high carbohydrate feeding are the methods recommended by researchers for glucose tolerance test. Comparatively, intraperitoneal injection goes in a more accurate way for mice and is also simple to perform. The results of an IPGTT demonstrated the ability of these implanted cells to dispose of a glucose load, and the glucose tolerance was close to normal mice. However, it should be noted that impaired glucose tolerance was found after another 7 days. It may be the case that the implanted induced IPCs failed to proliferate. Strategies that make stable expression of the factors in mMSCs may possibly help to evaluate the long term effect of the treatment.
Bone marrow mesenchymal stem cell has been known for their multiplex differentiation potential and relative ease to obtain. They were able to be modified to develop epigenetic changes, which were controlled by a series of several distinct related genes, and then differentiated into functional
In conclusion, our findings demonstrated that genetic manipulation producing infection by a combination of PDX-1 NeuroD1, and MafA and their subsequent expression significantly promoted insulin-producing function of mMSCs. Although substantial work has been done, the effective approach related to generation of surrogate
This work was supported by the National Natural Science Foundation of China (no. 81070654).