Design Of Glucagon Like Peptide-1 Analogues And Murine Pluripotent Stem Cells That Escape Differentiation Inside Teratomas Maintain Pluripotency

Type 2 diabetes is a major threat to human health.The disease process is accompanied by insulin resistance and βcell dysfunction Glucagon like peptide-1(GLP-1)is a peptide that can increase insulin secretion only when glucose concentration is higher than normal level.In addition,GLP-1 can reduce βcell apoptosis,promote its regeneration So,GLP-1 provides a new choice for the treatment of type 2 diabetes.However,the half-time of GLP-1 is only 2-5 minutes in vivo.Because of its extreme short half-time,GLP-1 in clinical treatment is greatly limited.In response to this situation,the current research mainly focus on prolonging biological activity of GLP-1.In this project,we considered the characteristics of GLP-1 and GIP to design the new peptide.The amino acid sequence of glucagon like peptide-1(GLP-1)analog provided in this study is as follows:Xaal-Ser-Glu-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaal 6-Xaal 7-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30.The second amino acids of GLP-1 were replaced by Ala,and other sites amino acids were modified according to the above formula.We expect to obtain new peptide that have both biological functions of GLP-1 and GIP.In vivo experiments showed that SGP-13 has ability to reduce blood glucose,and the dose needed is lower than that of wild type GLP-1.SGP-13 is promising for the development of drugs for type 2 diabetes mellitus.Pluripotent stem cells(PSCs)offer immense potential as a source for regenerative therapies.The teratoma assay is widely used in the fielk of stem cells and regenerative medicine,but the cell composition of teratoma is still elusive.We utilized PSCs expressing enhanced green fluorescent protein(EGFP)under the control of the Pou5f1 promoter to study the persistence of potential pluripotent cells during teratoma formation in vivo.OCT4-MES(mouse embryonic stem cells)were isolated from the blastocysts of 3.5-day OCT4-EGFP mice(transgenic mice express EGFP cDNA under the control of the Pou5f1 promoter)embryos,and TG iPS 1-7(induced pluripotent stem cells)were generated from mouse embryonic fibroblasts(MEFs)from 13.5-day OCT4-EGFP mice embryos by infecting them with a virus carrying OCT4,SOX2,KLF4 and c-MYC.These pluripotent cells were characterized according to their morphology and expression of pluripotency markers.Their differentiation ability was studied with in vivo teratoma formation assays.Further differences between pluripotent cells were examined by real-time quantitative PCR(qPCR).The results showed that several OCT4-expressing PSCs escaped differentiation inside of teratomas,and these escaped cells(MES-FT,GFP-positive cells separated from OCT4-MES-derived teratomas;and iPS-FT,GFP-positive cells obtained from teratomas formed by TG iPS 1-7)retained their pluripotency.Interestingly,a small number of GFP-positive cells in teratomas formed by MES-FT and iPS-FT(MES-ST,GFP-positive cells isolated from MES-FT-derived teratomas;iPS-ST,GFP-positive cells obtained from teratomas formed by iPS-FT)were still pkuipotent,as shown by alkaline phosphatase(AP)staining,immunofluorescent staining and PCR.MES-FT,iPS-FT,MES-ST and iPS-ST cells also expressed several markers associated with germ cell formation,such as Dazl,Stella and Stra8.In summary,a small number of PSCs escaped differentiation inside of teratomas,and these cells maintained pluripotercy and partially developed towards germ cells.Both escaped PSCs and germ cells present a risk of tumor formation Therefore,medical workers must be careful in preventing tumor formation when stem cells are used to treat specific diseases.