Therapeutic Efficiency Of Human Amniotic Mesenchymal Stem Cells On Type Ⅰ Diabetic Mice And Its Mechanism

Background and objectives:With the change of lifestyle and the acceleration of population aging,the incidence of diabetes has been increasing rapidly,and diabetes has become the third major disease that poses a threat to human health beyond cardiovascular diseases and cancer.China is the largest country with diabetes patients in the world.Diabetes is mainly divided into type 1 diabetes(T1D)and type 2 diabetes(T2D)clinically.T1 D is an autoimmunemediated disease,in which the permanent destruction of pancreatic β cells was caused by autoimmune,resulting in hyperglycemia due to insufficient insulin secretion.At present,the treatment of T1 D mainly relies on oral hypoglycemic drugs and injection of exogenous insulin,but the occurrence of complications such as diabetic foot and diabetic nephropathy cannot be avoided.Mesenchymal stem cells(MSCs)are derived from the mesoderm mesenchyme of tissues and have the potential for self-proliferation and multi-differentiation.In recent years,stem cell therapy has become a hot spot in diabetes research.Numerous studies have shown that MSCs from different sources(such as bone marrow,adipose,umbilical cord etc.)have therapeutic effects on T1 D.Compared with MSCs derived from other sources,human amniotic mesenchymal stem cells(hAMSCs)have the advantages of abundant sources,easy acquisition,no tumorigenicity,low immunogenicity and high histocompatibility.However,there are few reports about the application of hAMSCs in the treatment of T1 D.Therefore,this study aims to explore the therapeutic potential and underlying mechanisms of hAMSCs on T1 D mice,providing an experimental evidence for subsequent clinical applications.Methods:1.Isolation,culture and identification of hAESCs and hAMSCs: 1)Isolation of hAESCs and hAMSCs: The amniotic membrane was isolated from the placental tissue which was retrieved from hospital and was digested by trypsin to obtain human amniotic epithelial stem cell(hAESCs),and the remaining tissue was digested by collagenase to obtain hAMSCs;2)Culture of the stem cells: hAESCs and hAMSCs were inoculated into 10 cm petri dishes containing corresponding media,respectively;3)Identification of the stem cells: The expressions of hAESCs and hAMSCs marker genes,such as embryonic stem cells(ESCs),MSCs,hematopoietic stem cells and immune-related marker molecules,were detected by flow cytometry,RT-PCR and immunofluorescence;4)Differentiation potential experiment: The multidirectional differentiation potential of hAMSCs was determined by the osteogenic and adipogenic differentiation experiments.Flow cytometry and adipogenic and osteogenic differentiation experiments were also used to detect the characteristics of GFP-labeled hAMSCs(GFP-hAMSCs);5)In vivo and in vitro tumor formation experiments: In vivo and in vitro tumorigenicity of hAESCs and hAMSCs were measured by NODSCID mice transplantation and soft agar colony formation assay.2.Related experiments of hAMSCs transplantation in T1 D mice: 1)Generation of T1 D mouse model: The T1 D mouse model was established by intraperitoneal injection of streptozocin(STZ)for 5 days consecutively;2)Transplantation of stem cells in vivo: hAESCs and hAMSCs or PBS(control group)were injected into T1 D mice via tail vein on the 3rd,5th,and 7th day of STZ injection,respectively.After 3 days of cells injection,the random blood glucose of mice in each group were tested,and the fasting blood glucose and glucose tolerance assays were carried out after 21 days;3)HE staining and immunofluorescence detection: HE staining and immunofluorescence were used to detect the morphology of the islets and the β cells in the pancreas of each group;4)GFP-hAMSCs tracer: GFP-labeled hAMSCs were injected into T1 D mice,and the distribution of GFP-hAMSCs in mouse tissues was detected by whole-body fluorescent imaging and immunofluorescence;5)Immunofluorescence detection: Cell proliferation(Brdu+),angiogenesis(CD31+)and islet cells apoptosis(TUNEL+)in the pancreas of each group were detected by immunofluorescence.3.3D culture of hAMSCs and damaged islets in vitro: 1)Generation of injured islet model in vitro: Sufficient and intact islets were extracted from healthy mice in vitro and divided into 3 groups equally.The islets were treated with 5 mmol/L STZ for 30 min to establish injured islet model;2)Generation of islet organoids: 3D culture of injured islets and hAMSCs to form an organoid structure in vitro.The experiments were divided into normal group,injured islets group and hAMSCs 3D coculture group;3)Morphological observation of islets: The growth of the islets in each group was observed,the size of the islet spheroids in each group was measured by Image J,and the insulin secretion function of islets was detected by Elisa.Results:1.hAESCs and hAMSCs have the characteristics of stem cells: 1)hAESCs:RT-PCR,immunofluorescence and flow cytometry detection analyses showed that hAESCs expressed ESCs marker genes OCT4,Nanog,SSEA-4,epithelial stem cell marker genes E-cadherin and CK7,and MSCs marker genes CD29,CD73,CD105,but did not express MSCs marker genes CD90,hematopoietic stem cell marker genes CD34,CD45,histocompatibility class II antigen HLA-DR,and low expressed major histocompatibility class I antigen HLA-ABC;2)hAMSCs: RT-PCR,flow cytometry and immunofluorescence detection analyses showed that hAMSCs expressed ESCs marker genes Nanog,OCT4,SSEA-4 and MSCs marker genes CD29,CD73,CD90,CD105,and did not express hematopoietic stem cell marker genes CD34,CD45,CD133.hAMSCs did not express major histocompatibility class II antigen HLA-DR,low expressed major histocompatibility class I antigen HLA-ABC,and did not express costimulatory molecules CD80,CD86,CD40;3)hAMSCs could successfully differentiate into adipocytes and bone cells,with multi-directional differentiation potential;4)The tumorigenicity experiments showed that hAESCs and hAMSCs were not tumorigenic in vivo and in vitro.5)Flow cytometry,adipogenic and osteogenic differentiation experiments showed that the introduction of GFP did not change the properties of hAMSCs.2.hAMSCs could significantly improve the blood glucose of T1 D mice by promoting the proliferation of islet progenitor cells and inhibiting the apoptosis of islet β cells,while hAESCs had no significant effect on improving blood glucose in T1 D mice: 1)After hAESCs and hAMSCs were transplanted into T1 D mice via tail vein,random blood glucose,fasting blood glucose and glucose tolerance tests showed that hAMSCs could improve the blood glucose of T1 D mice and increase the size of islets and the number of β cells significantly,while hAESCs could not improve the hyperglycemia of T1 D mice significantly;2)Whole-body fluorescent imaging and immunofluorescence detection showed that GFP-hAMSCs were enriched in pancreatic tissues and distributed around the islets;3)Immunofluorescence and TUNEL assay showed that hAMSCs transplantation could significantly promote the proliferation of pancreatic cells and islet progenitor cells,and inhibit the apoptosis of β cells.3.In vitro 3D culture,hAMSCs were be able to protect islet cells from STZinduced damage: 1)An islet injury model could be successfully established in islets treated with STZ(5 mmol/L)for 30 min in vitro;2)3D culture of the damaged islets and hAMSCs could significantly inhibit β cell apoptosis and improve β cell function.Conclusion:Both hAESCs and hAMSCs have the characteristics of low immunogenicity,high histocompatibility,multi-directional differentiation potential and no tumorigenicity in vivo and in vitro.hAESCs transplantation had no obvious therapeutic effect on T1 D mice,while hAMSCs could specifically migrate around the damaged islets,and improve the hyperglycemia of T1 D mice by promoting the proliferation of islet progenitor cells and inhibiting the apoptosis of islet β cells.In addition,3D culture of hAMSCs and damaged islets could form a structure similar to islets,which could inhibit STZ-induced β cell apoptosis and improve β cell function.