Isolation, purification and identification of human pancreatic progenitor cells
The present study was approved by the Clinical Research Ethics Committee of China-Japan Friendship Hospital and conducted according to the principles of the Declaration of Helsinki. Five human fetal pancreases at the 10th to 12th gestational week were obtained from abortion patients in China-Japan Friendship Hospital, in which one was a spontaneous abortion due to low progesterone level and the other four were intended abortions according to the mothers’ choice. All the tissues were obtained following medical ethics and all with patient informed consent.
Pancreas tissues at the 10th to 12th gestational week were confirmed to be abundant with islet-like structures which were CD133 positive but insulin negative by immunohistochemistry staining. The pancreatic tissues were digested with XI collagenase (Sigma, Shanghai, China), and the islet-like structures extracted were suspended in (D)MEM/F12 (Sigma) in a 35-mm cell culture dish. After slowly hand-shaking the dish, the islet-like structures would move to the middle of the dish and were picked up using a pipette under a stereomicroscope (Nikon, Beijing, China). The islet-like structures were resuspended and cultured in a 37°C, 5% CO2 incubator in (D)MEM/F12 medium containing 5% fetal calf serum for stem cell, 40 μg/L leukemia inhibitor factor (LIF), 10 μg/L basic fibroblast growth factor (bFGF), 10 μg/L epidermal growth factor (EGF), 105 U/L penicillin and 100 mg/L streptomycin  Adherent cells that grew from the islet-like structures after 24 hours were trypsinized for passage with 0.1% trypsin/0.1% ethylenediaminetetraacetic acid (EDTA) solution at confluence. The propagated cells were saved for further study. The control human islets were isolated from a section of pancreas after pancreatectomy from a patient with a pancreatic tumor, as previously described .
RT-PCR was employed to detect the following markers for proliferated stem cells: Oct4, ATP-binding cassette superfamily G member 2 (ABCG2), stem cell factor (SCF), CD133, carbonic anhydrase II (CAII), cytokeratin 19 (CK19), PDX-1 and neurogenin 3. The expression of PDX-1 and Neurogenin 3 (Ngn3) was also confirmed by immunofluorescence staining using goat anti-human PDX-1 antibody (Abcam, Cambridge, MA, USA) and rabbit anti-human Ngn3 antibody (Abcam). After two, five and ten passages, cells were collected to measure the expression levels of smooth muscle actin (SMA), vimentin, stem cell markers (Oct4, PDX-1 and CA II) and mature cell markers (insulin and glucagon) by real-time PCR.
Induced differentiation of human pancreatic progenitor cells
Human fetal pancreatic progenitor cells were induced in M199 medium containing 15% fetal bovine serum (FBS), 10 mmol/L nicotinamide, 30 ng/ml all-trans retinoic acid and 42 ng/ml glucagon-like peptide-1 (gift of Shanghai Huayi Bio-Lab Co. Ltd) for four weeks. The medium was replaced every three days and 50 ng/ml activin A was added to the medium in the last week. The flowchart of the differentiation protocol is as follow (Nico, nicotinamide; RA, all-trans retinoic acid):
Formation of islet-like structures
After four weeks of directed differentiation, the cells were harvested and aggregated in suspension culture under special conditions. In brief, the cells were resuspended in M199 containing 20% FBS, 1.5 mmol/L Ca2+, 1 mmol/L ATP, 2 mmol/L glutamine, 2 μg/ml laminin, 5 μg/ml Type IV collagen and 3 μg/ml fibronectin. The mixture was transferred to a nontreated flask or siliconized centrifugal tubes and placed into a 37°C, 5% CO2 incubator. Gentle agitation was applied every eight hours. After a 24-hour incubation, small cell aggregations formed and were harvested by centrifugation at 1,200 rpm for eight minutes. The morphology of islet-like structures was examined under a stereomicroscope.
The islets differentiated from progenitor cells were selected manually, embedded in optimal cutting temperature (OCT) compound and prepared into frozen sections. The expressions of insulin and glucagon were examined and compared with adult islets as control. The frozen sections were then fixed for one minute with alcohol (95%), washed three times with PBS solution and blocked for 30 minutes at room temperature with 0.1% BSA/PBS. Then anti-insulin and anti-glucagon antibodies (Sigma) were incubated overnight, respectively, with the cells at 4°C. After the sections were rinsed three times with PBS solution, Alex488-labeled secondary antibody (Invitrogen Inc, Grand Island, NK, USA) was added and incubated with the cells for another 30 minutes. A fluorescent microscope was used to observe the results.
Transmission electron microscopy
Adult islets and differentiated cells were collected by centrifugation and washed with PBS, then fixed with 2.5% glutaraldehyde for 30 minutes at room temperature. Cells were washed, post-fixed, dehydrated and embedded with Epon 812 (Electron Microscopy Sciences, Beijing, China). The sections were examined under a JEM-1010 electron microscope (JEOL Inc, Tokyo, Japan). Five fields were examined for each sample.
Total RNA was extracted from cells 0, 3 and 4 weeks post differentiation and from islet-like structures 4 weeks post differentiation using a RNEasy Mini kit (Qiagen Inc, Hilden, Germany). A total of 1 μg RNA was used for reverse transcription using omniscript reverse transcriptase (Qiagen). Real-time PCR was performed on an Applied Biosystems instrument (ABI 7500 system), using SYBR Green PCR Master Mix (ABI Inc, Foster city, CA, USA) and 40 reaction cycles. Markers for pancreatic stem cells, endocrine, exocrine and mesenchymal cells were measured at different time points of expansion and differentiation At the end of each reaction, melting-curve analysis was performed to confirm the absence of primer dimers. The expression levels of genes of interest were normalized to the expression level of GAPDH. Data were analyzed using the 2- Δ Ct method. The pancreatic stem cell markers (CAII, PDX-1, Neurogenin 3; endocrine markers: insulin, glucagon, GLUT1, GLUT2 and VDCC), as well as exocrine markers (HES-1 and amylase) were evaluated during differentiation. The mesenchymal markers SMA and vimentin were evaluated during pancreatic stem cell expansion. The gene-specific primer sequences are shown in additional file (see Additional file 1).
Insulin release assay by ELISA
After pre-incubation with Krebs-Ringer buffer at 37°C for 90 minutes, the progenitor cell-derived islets (100 IEQs/ml) were incubated with Krebs-Ringer buffer containing 2.5 mM glucose or 25 mM glucose at 37°C for different durations. Conditioned medium was then collected, centrifuged and assayed for insulin content by an ELISA kit (Linco Inc, St Charles, MO, USA).
Diabetic animal models
The animal study was approved by the Animal Ethics Committee of China-Japan Friendship Hospital. Twelve male nude BALB/c mice, four- to six-weeks-old, were purchased from Vital River Laboratories (quality certificate: SCXK (Beijing) 20060009)–. All animals were housed in the specific pathogen free (SPF) facility of the Institute of Clinical Research at our hospital. The diabetic model was induced in the mice by an intraperitoneal injection of streptozocin (200 mg/kg body weight; Sigma, St. Louis, MO, USA) freshly dissolved in citrate buffer (pH 4.5). Blood samples were taken from the tail vein of the animals under non-fasting conditions (8:00 A.M.; standard laboratory diet ad libitum overnight) and examined for blood sugar level using a glucometer (Onetouch®Ultra Easy™, Johnson & Johnson, Shanghai, China). Only animals exhibiting blood glucose concentrations >20 mM in three consecutive measurements were employed in the study. They were assigned randomly into a control group and a progenitor cell-derived islet group (n = 6 each).
Transplantation of differentiated islet-like structures
To evaluate the function of progenitor cell-derived islet-like structures, they were transplanted under the capsule of the left kidney of diabetic mice. Blood glucose was measured regularly after transplantation. When euglycemia was maintained for one week, the left kidney with transplants was excised surgically and examined by immunohistochemistry for human C-peptide and human mitochondrion antigen as described below. Blood glucose was monitored regularly after surgery.
In order to verify further the functionality of human pancreatic progenitor cell-derived beta cells in vivo, the animals were sacrificed at the end of experiment. Left murine kidneys were embedded in paraffin. Then murine kidneys were immunostained for human C-peptide (mouse anti-human C-peptide, Chemicon, Temecula, CA, USA) and human mitochondrion antigen (mouse anti-human mitochondrion, Millipore, Billerica, MA, USA) to confirm the survival and functionality of transplanted human islet-like structures.
The analysis was conducted with SPSS11.0 software and the data were expressed as mean ± SD. The t test was uesed for comparison between two groups and one way analysis of variance (ANOVA) for comparisons among multiple groups. P <0.05 and P <0.01 denoted statistical significance.