Effect Of IGF-1 Gene Transfection On The Biological Activity Of Urinary Stem Cells And Its Potential Significance In The Treatment Of Erectile Dysfunction

Background:The prevalence of diabetes is on the rise worldwide at present.Diabetes mellitus erectile dysfunction（DM ED）is one of the most significant complications that seriously affect the quality of life of male patients.The pathogenesis of DM ED is complex,involving cavernous endothelial and smooth muscle dysfunction,cavernous neuropathy,and endocrine abnormalities.Therefore,conventional diabetes or ED treatment is less effective in patients with DM ED than in non-diabetic ED patients,however,with more side effects.Thus,how to effectively treat DM ED has become an urgent problem in medical science.With the development of regenerative medicine,stem cell transplantation therapy is becoming more and more familiar.Mesenchymal stem cells（MSCs）are widely used in the field of cell transplantation therapy because of their multidirectional differentiation and paracrine secretion.We previously performed Bone marrow-derived mesenchymal stem cells（BM-MSCs）transplantation to treat ED and muscle derived stem cells（MDSCs）transplantation in the treatment of stress urinary incontinence animal models can achieve good efficacy.However,the acquisition of both BMSCs and MDSCs requires invasive procedures,and this disadvantage limits their future clinical application.Urine derived stem cells（USCs）are a new type of adult stem cells,which have many advantages,such as wide source,easy access,low cost,strong proliferation,and differentiation ability,etc.,and are a new choice for transplantation in the treatment of DM ED.However,there are some problems with stem cell transplantation therapy,such as the short survival time of stem cells after injection.There are several ways to increase the activity of stem cells,of which genetic modification is the most widely used.In our previous studies,we found Insulin like growth factor-1（IGF-1）improved erectile function in aged rats via restoration the integrity of smooth muscle of corpus cavernosum and modulation of NO-c GMP pathways.IGF-1 is a cell growth promoter that promotes the ability of cells to proliferate.In this study,IGF-1 gene was used to modify USCs,so that the IGF-1 protein secreted by USCs could play a synergistic therapeutic role while enhancing the activity of USCs.To explore the feasibility of IGF-1 gene modified USCs for the treatment of DM ED in vitro.Objective:To isolate and culture the primary USCs from urine of volunteers,and identify its cell surface markers,their multi-differentiation potential and examine their paracrine cytokine profiles in vitro.transfect IGF-1 gene into USCS cells using a lentiviral vector system,detect the stability of IGF-1 protein secreted by USCs after transfection and the change of biological function of USCs,evaluate the safety and effectiveness of lentivirus transfection system.Method:Nine sterile urine samples were collected from 3 healthy adult male volunteers,and primary USCs were isolated and cultured.USCS（P3）cell surface markers were identified by flow cytometry.The multi-differentiation potential of USCs was identified by osteogenic and adipogenic induction differentiation.In vitro paracrine cytokine profiles of USCs were detected by ELISA.USCs were infected with a lentiviral vector carrying the IGF-1 gene and IGF-1 gene expression was tested by Western-blot,RT-q PCR and ELISA.CCK-8 and Transwell assay were used to evaluate the cell proliferation and migration in each group.Results:Primary USCs were successfully isolated and cultured.Phenotypic identification showed that USCs were negative for the HSCs markers CD31,CD45,and strongly positive for the MSCs markers D44,CD73,CD90 and CD146.USCs can be induced to differentiate into bone and fat tissue.ELISA assay showed that USCs could secret various kinds of angiogenic growth factors（VEGF and FGF）,neurotrophic factors（IGF and PDGF）and colony stimulating factor CSF.After the transduction of lentivirus vector into USCs,the continuous expression of green fluorescence was observed under fluorescence microscope.USCs^IGF-1 can continuously and steadily secrete large amounts of IGF-1 protein.CCK-8 and Transwell experiments showed that IGF-1 could significantly promote the proliferation and migration of USCs.Conclusion:Urine derived stem cells expressed surface markers of mesenchymal stem cells（CD44,CD73,CD90,CD146）,but did not express surface markers of hematopoietic stem cells（CD31,CD45）;USCs have multidirectional differentiation potential;USCs can secrete a variety of growth promoting factors in vitro.Lentiviral vector mediated gene transfection is safe and effective for urine derived stem cells.After the transfection of IGF-1 gene,the urine derived stem cells could secrete a large amount of IGF-1 protein stably and continuously,and the proliferation and migration ability of the USCs were improved.