Characteristics Of Human Amniotic Mesenchymal Stem Cells And Their Therapeutic Efficiency On Type 2 Diabetic Mice

Background and purposes:Diabetes including type 1(deficiency of pancreatic beta cells)and type 2(insulin resistance,T2D)diabetes,is a severe disease which ranks as third after cardiovascular diseases and cancers in the world.At present,the treatments of T2 D mainly include oral hypoglycemic drugs and exogenous insulin injection.If the hypoglycemia could not be efficiently controlled in patient,many complications such as progressive β-cell damage,renal failure,heart disease and fundus lesions would be finally developed.Mesenchymal stem cells(MSCs)are a kind of pluripotent stem cells with the potential of self-renewal and multi-lineage differentiation,which have the characteristics of immune regulation,inhibition of inflammation and so on.In recent years,the application of MSCs in the treatment of type 2 diabetes and its complications has become a research hotspot in the field of diabetes treatment.Compared with MSCs from other sources(such as umbilical cord,bone marrow,cord blood,etc.),MSCs derived from human amniotic membrane have the advantages of abundant source,easy separation,strong proliferation ability,non-tumorigenicity,low immunogenicity and high histocompatibility.This study aims to characterize hAMSCs first and then the therapeutic efficiency and mechanisms of the cells in treatment of T2 D will be explored in mouse diabetic models,to provide a new method for treatment of T2 D clinically.Methods:1.Isolation,culture and identification of hAMSCs: 1)Amniotic membrane was isolated from placenta and then digested by trypsin and collagenase to obtain single cell suspension of hAMSCs.2)The isolated hAMSCs was inoculated into 10 cm culture dish for routine culture.3)Expressions of mesenchymal stem cells markers,hematopoietic stem cells markers,embryonic stem cells markers and immunogenic molecular markers were detected by RT-PCR,flow cytometry and immunofluorescence.4)Then we induced in vitro to explore the multi-lineage differentiation potential of hAMSCs by adipogenic and osteogenic differentiation.2.Detection of tumorigenicity of hAMSCs: 1)hAMSCs was inoculated in doublelayer soft Agar to observe its colony formation ability and verify its tumorigenicity in vitro.2)hAMSCs was inoculated into NOD/SCID mice subcutaneously to detect its tumorigenicity in vivo.3.The therapeutic effects of hAMSCs transplantation on hyperglycemia in T2 D mice: 1)T2D mouse model was established by high fat feeding combined with STZ injection.2)The hAMSCs(experimental group)or PBS(control group)were injected into T2 D mice via tail vein,then the blood glucose levels including random blood glucose and fasting blood glucose were measured,and the glucose tolerance,insulin tolerance and serum C-peptide content were also detected in the mice.3)The alternations of islet beta cells and pancreatic morphology were detected by HE staining and immunofluorescence staining.4.The mechanism of hAMSCs in ameliorating hyperglycemia in T2 D mice: 1)The histological alternations of liver in mice were detected by HE staining.2)The effect of hAMSCs on glycogen synthesis in liver tissue of T2 D mice was detected by PAS staining.3)The effects of hAMSCs on glycolysis and gluconeogenesis in the liver of T2 D mice were detected by Q-PCR,and the signal pathway related to the improvement of insulin resistance in the liver of T2 D mice was detected by western blot.Results:1.hAMSCs were successfully isolated and cultured from human amniotic membrane.RT-PCR,flow cytometry and immunofluorescence assays showed that hAMSCs were positive for embryonic stem cell markers Nanog,OCT4,SSEA-4,mesenchymal stem cell markers CD29,CD73,CD90,CD105 and negative for hematopoietic stem cell markers CD34,CD45,CD133.Although the major histocompatibility protein HLA-ABC was expressed in hAMSCs,its co-stimulatory molecules such as CD80,CD86,CD40 were not detectable.In addition,there was no expression for the major histocompatibility protein HLA-DR in the cells.Under adipogenic and osteogenic differentiation conditions,the hAMSCs were able to differentiate into adipocytes and osteocytes,respectively.2.The results of double layer soft Agar assay and tumorigenesis assay demonstrated that hAMSCs had no tumorigenicity both in vitro and in vivo.3.Injection of hAMSCs into T2 D mice via tail vein could significantly decrease the level of random and fasting blood glucose,and ameliorate the glucose tolerance and insulin tolerance in mice.In addition,the islet morphology in T2 D mice was partially reconstructed after the injection of hAMSCs;4.The injection of hAMSCs could attenuate the damage of liver tissues,and increase glycogen synthesis in T2 D mice.The expressions of gluconeogenesis related genes PGC-1α and G6 Pase and inflammation-related genes IL-1β and TNF-α were decreased.In contrast,the expressions of Glycolysis-related gene L-PK,PFK were increased.Western blot revealed that hAMSCs significantly increased the expressions of insulin sensitivity related proteins p-IRS-1 and GLUT4,and the expressions of IGF-1R,p-IGF-1R,PI3 K,AKT and p-AKT were also up-regulated.Conclusion:The hAMSCs expresses mesenchymal stem cells markers and embryonic stem cell markers with many important advantages such as high differentiation potential,low immunogenicity and no tumorigenicity.Transplantation of hAMSCs can significantly alleviate hyperglycemia,ameliorate islet injury induced by STZ and longterm high blood glucose,attenuate the inflammation in liver tissue,inhibit hepatic gluconeogenesis,promote liver glycolysis and improve liver insulin sensitivity in T2 D mice.Finally,the improvement of hyperglycemia symptoms in T2 D mice may be related with the activation of the PI3K/AKT/INS signaling pathway.