The In Vitro Potential Of Proliferation And Differentiation Of Nestin-Positive Cells From The Brain And Pancreas In Nestin-EGFP Transgenic Mouse

Background:Recently the research of stem cell has become a hot topic in biomedical field and interested so many researchers because it provided a new idea for curing the complicated diseases in medicine. The research of some adult stem cells was studied deeply such as haematopoietic stem cells and neural stem cells. However some just began, sucn as pancreatic stem cells, skin stem cells and hepatic stem cells. All in all, the research of stem cells is on the first stage because there are so many unresolved problems: stem cells devoid of specific markers to isolate and purify; the faulty culture systems in vitro; unclear molecular mechanism of proliferation and differentiation in stem cells; immature methods to induce stem cells into differentiated cells and so on. Moreover, there are correlation with stem cells research and ethical question of embryo and the clone of human being. Objective:In this study, we generated the transgenic mouse by the promoter/enhancer of second intron of nestin driving EGFP. The nestin-positive cells from the brain and pancreas of transgenic mice were isolated by flow cytometry. Then we studied whether the nestin-positive cells from brain had the ability to proliferate and differentiate as neural stem cells and whether the nestin-positive cells could represent the neural stem cells. We further studied whether the nestin-positve cells were the pancreatic stem cells. It will provide a simple and efficient method to isolate/purify the neural stem cells and establish the methods of isolation, culture and identificationof pancreatic stem cells. It has significance in study of the developmental process andmechanisms and curing the disease of neural systems and diabetes.Methods:1. To make the transgenic mice: We purified the gene fraction nestin-hsp68-EGFP and injected it into pronucleus of mouse zygotes to generate the transgenic mice.2. To identify the expression pattern of nestin in the transgenic mice: The positive transgenic mice were identified and selected by PCR and the fluorescent microscopes. The tissueses of brains and pancreases from embryonic and adult mice were made into frozen slices to examine the expression pattern of nestin by immunofluorecence.3. The research on the potential of proliferation and differentiation of the nestin-positive cells in the brains of the embryonic transgenic mice in vitro: The forebrains from Embryonic Day 14.5 positive transgenic mice were removed and dissociated into single cells by 0.125% trypsin. The nestin-positive cells were sorted by FACS on the fluorescent intensity of EGFP and were cultured in serum-free medium as neurospheres. The expression of nestin in neurospheres was examined by RT-PCR and the fluorescent microscope. The proliferation potential of the nestin-positive cells was detected by Brdu labeling analysis and the clonal forming rate analysis. The differentiation potential of the nestin-positive cells was identified by immunocytochemistry in serum medium. The neurospheres were able to reserved by freezing.4. The research on the potential of proliferation and differentiation of nestin-positive cells in the pancreases of the embryonic transgenic mice in vitro: The pancreases from Embryonic Day 14.5 positive transgenic mice were removed and dissociated into single cells by 2mg/ml collagenase IV. The cells were sorted by the fluorescent intensity of the EGFP and PE-CD24a. The sorted cells were cultured in RPMI1640 medium with EGF and bFGF to observe the ability to proliferate. After induced differentiation by factors HGF, Activin-A, exendin-4 and nicotinamide, the cells were examined by RT-PCR and immunocytochemistry.Results:1. The transgenic mice were obtained: We obtained three independent lines ofnestin-EGFP transgenic mice (5#, 25#, 42#). The three lines of transgenic mice were fertile and showed no apparent abnormality in development and behavior. EGFP transgenes were stably transmitted to subsequent generations. The data presented here were obtained from experiments using heterozygous mice from a single strain (5#).2. The identification of the expression pattern of nestin in the transgenic mice: The data showed that the expression of nestin matched with that of EGFP both in the brain and pancreas in positive transgenic mice. The nestin and EGFP coexpressed in cerebral cortex and ventricular zone in embryonic brain but only in ventricular zone in adult brain. The nestin and EGFP coexpressed in exocrine acinar cells, but not in vascular endothelial cells, ductal epithelium cells, P -cells in embryonic pancreas. However, nestin and EGFP were coexpressed in some channels in adult pancreas. The EGFP coexpressed with Vffl factors related antigen, which was the marker of the vascular endothelial cells, but only weakly coexpressed with CK19 which was the marker of ductal epithelium cells. This suggested that the nestin expressed mainly in vascular endothelial cells in adult pancreas.3. The research on the potential of proliferation and differentiation of the nestin-positive cells in the brains of the embryonic transgenic mice in vitro: The percentage of nestin-positive cells in El4.5 forebrain was 70-80%. We sorted the 15-20% of the brightest fluorescent intensity of EGFP cells into serum-free medium to culture. The forming neurospheres were found the brightest fluorescent intensity of EGFP and nestin expressed in mRNA level by RT-PCR. It suggested that the nestin-positve cells had the characteristic of neural stem cells. The Brdu-positive cells were found in the neurospheres. The cells from neurospheres in primary culture and secondary culture had the ability to clone, but the more the time of passage, the fewer clone can be formed. These data showed nestin-positive cells from brains had the ability to proliferate. After induced differentiation, the nestin-positive cells could be differentiated into NF-positive cells, GFAP-positive cells and GC-positive cells. So these data showed the nestin-positive cells from the brain of transgenic mouse had the ability of neural stem cells to proliferate and differentiate.4. The research on the potential of proliferation and differentiation of nestin-positive cells in the pancreases of the embryonic transgenic mice in vitro:The percentage of nestin-positive cells from E16.5 pancreas of positive transgenic mice was 19.26 ± 7.60%. So the same quantity of nestin-positive cells and nestin-negative cells were sorted into RPMI 1640 medium with EGF and bFGF. The nestin-negative cells proliferated well in this condition, but the nestin-positive cells could not proliferate. It suggested that the nestin-negative cells had the potential to proliferate. To further characterize the nestin-negative population, it was necessary to select other cell surface markers combing with EGFP to sort this population. CD24 was a cell surface glycoprotein that was used as marker to sort the haematopoietic stem cells and neural stem cells. So the PE-mCD24a was selected with EGFP to sort the whole embryonic pancreatic cells into four subpopulations as follow: CD24high/+EGFpneg 636? ± io.63%,CD24high/+EGFPpos 14.24 ± 8.81%, CD24iow/-EGFpneg 17.55 + 8-96o/O5 CD24low/-EGFPpos 4.93 + 3.60%. The sorted cells were cultured in RPMI 1640 medium with EGF and bFGF. Only the CD24low/"EGFPneg population could live and proliferate for a long-term period of five months in the medium and the immature islet-like cell clusters (ICCs) appeared in the long-term culture in this condition. The immature ICCs were induced by the combined factors for a period of 10 days. It showed that more insulin-positive cells existed in ICCs, glucagons-positive cells located close to the periphery of ICCs, somatostatin-positiye cells and pdxl-positive cells also appeared in ICCs by immunocytochemistry. It was found that after differentiation the cells expressed the endocrine markers insulin 1, glucagons, somatostatin by RT-PCR, however, these endocrine markers cannot be found in uninduced cells. This data demonstrated that the CD24low/'EGFPneg cells indeed enriched the pancreatic endocrine progenitors that had the capacity to self-renew and generate endocrine cells. Conclusion:1. The expression pattern of nestin and EGFP matched in the brains and pancreases of the nestin-EGFP transgenic mice. So the nestin-positive cells were sorted on the fluorescent intensity of the EGFP by fluorescent-activate-cell-sorting (FACS). It provided an available method to isolate/purify stem cells combing transgenic...