| Objective:Study on a three-step approach for the direct differentiation of rat liver epithelial stem-like WB-F344cells (WB cells) into functional insulin-producing cells (IPCs) using small molecules. To observe whether the induced pancreatic precursor cells can further induce to IPCs into DM rats and evaluate function of IPCs in vivo and vitro. These findings lay the foundation for further study to differentiate the liver stem cells to pancreatic β cells.Methods:1. Induction in vitro1.1Induction protocolThree-step-induction1) Step1:WB cells were dedifferentiated to WB-1cells(1) WB cells were treated with5μM5-AZA for48hours and then treated with100nM TSA for24hours.(2) A basal medium containing knockout-serum DMEM and the main components:1mM β-mercaptoethanol,1%non-essential amino acids,1%B27supplement,2mM L-glutamine,2%N2supplement,20ng/ml fibroblast growth factor,20ng/ml epidermal growth factor,100U/ml penicillin, and100mg/ml STZ.2) Step2:WB-lcells were induced to pancreatic precursor WB-A cells(1) WB-1cells were treated with1×ITS,2mM RA for7days.(2) Induction medium was low glucose (1g/L) and the main components.3) Step3:WB-A cells were induced to IPCs(1)10mM Nicotinamide for7days.(2) Differentiation medium:Basical medium without bFGF and EGF.1.2Test index1) The changes of cell shape in every stage were observed by phase contrast microscope.2) The genes of cells in every stage were identified by RT-PCR, immunofl- uorescence.3) Glucose-stimulated insulin release assay in vitro to evaluate functin of IPCs4) Structure analysis of IPCs by TEM.2. Induction in vivo2.1Induction protocolPancreatic precursor WB-A cells were induced to IPCs by transplanting into DM rats in vivo.1) Observe the changes of glucose and weight after1×106/rat WB-A cells were transplanted into the left renal subcapsular space of DM rats.2) WB-A cell-implanted rats divided two groups at day36. One group underwent nephrectomy. Another group did not undergo nephrectomy.3) IPGTT and glucose-stimulated insulin release assay at day60after Tx.4) HE and immunofluorescence were observed at day60after Tx.2.2Test index1) Dynamic observation of the changes of glucose after Tx.2) The change of fasting serum rat insulin levels after and before Tx.3) Function evaluation:IPGTT and glucose-stimulated insulin release assay.4) Gene expression by RT-PCR.5) The tissue of WB-A cell Tx was analysis by HE and immunofluorescence staining.Results:1Induction in vitro1.1Cell morphological changes1) During the stage1:Parental WB cells were initially small and polygonal, after stage1, the rate of WB cells in shape proliferation decreased with metamorphosis into spindle-shaped cells as WB-1cells.2) During the stage2:The cells continued to differentiate to form smaller cells with a higher nucleus/cytoplasm ratio than WB cells as WB-A cells.3) During the stage3:The cells differentiated to a decreased nucleus/cytoplasm ratio with an increased cytoplasm but did not form aggregates as IPCs.1.2RT-PCR1) In stage1:Expression of the hepatocyte dedifferentiation marker C/EBPβ gene was drastically downregulated, while liver genes of AFP and ALB were undetectable after stage1to show dedifferentiation of WB cells.2) In stage2:The WB-A cells expressed multiple genes characteristic of the key pancreatic early-stage developmental genes including Pdx1, Ngn3, NKX2.2and the endocrine genes insulin I in WB-A cells. Non-β-cell endocrine markers, such as somatostatin, glucagon, PP and exocrine marker amylase were not detected.3) In stage3:The islet cell-specific marker genes Pdxl, NeuroD, and insulin I were also expressed, whereas the Pax4, Pax6and MafA genes, the β cell functional GK, Kir6.2and the endocrine insulin II gene were activated.1.3ImmunofluorescenceOver5%of all WB cells within a population definitively differentiated into Pdxl-expressingWB-A cells. Insulin was positive in near10%Pdx1expressing cells.1.4Glucose-stimulated insulin release assay in vitroWB-A cells were not capable of glucose-responsive insulin release. IPCs were capable of glucose-responsive insulin release.1.5TEMInduced IPCs showed scattered cytoplasmic globular structures containing insulin secretory granules that were similar to those seen in β cells.2.Induction in vivo2.1Changes of blood glucose1) Rats receiving the transplant began to have normalized blood glucose levels within15days.2) Ex of the implanted WB-A cells, around day36, persistent hyperglycemia returned, while animals that retained the transplanted cells maintained fairly normal glucose levels.2.2Changes of fasting serum rat insulin levelsFasting serum rat insulin levels were also increased to2fold in WB-A Tx rats at day30after Tx compared to those at day3before Tx.2.3Function evaluationRats receiving WB-A cells had a similar pattern to normal control rats without cell transplantation by IPGTT and glucose-stimulated insulin release assay.2.4RT-PCRAfter60days in vivo, the transplanted cells, IPCs expressed the PCI, PC2, Pax4, Pax6, MafA, insulin Ⅱ, GK and Kir6.2genes, which were not expressed in WB-A cells, and which were upregulated compared with IPCs in vitro. Ngn3was downregulated more than in IPCs in vitro and WB-A cells. Expression of amylase, PP, somatostatin, and glucagon was not detected.2.5HE and immunofluorescenceAfter60days in vivo the tissue of explanted WB-A cells formed glandular structure-like β-cells using HE. Immunofluorescence staining for insulin protein showed that some of the implanted cells expressed insulin.Conclusion:Some small molecules are capable of inducing liver stem cells WB cells into IPCs. An essential step in this protocol was the generation of pancreatic precursor cell that express Pdxl based on induction by a combination of5-AZA, TSA, RA, ITS. Induced pancreatic precursor can further induce into IPCs in a high-glucose microenvironment in vivo with the ability to ameliorate hyperglycemia. Induced IPCs are functional and highly selective β-like cells. These findings lay the foundation for further study to differentiate the liver stem cells into pancreatic β cells and provide potential cell source and new protocol for DM therapy. |
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