The Role And Mechanism Of Exosomes Secreted By IPSCs-MSCs In Repairing Of Ischemic Tissue Damage

Background and Objective:Ischemic tissue injury is a common pathological process in ischemic diseases, heart ischemia, brain ischemia and peripheral vascular ischemia all cause tissues or brgans damaging due to ischemia and hypoxia. Recently, more and more researches focus on transplantation of stem cells in repairing ischemic damage. Mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs) have showed more powerful function in alleviating ischemic damage, promoting angiogenesis and functional recovery when compared with adult MSCs, which exhibited a good application prospect in the field of stem cell therapy. However, there were still some risks for stem cell transplantation in clinical treatment, such as block vessels and tumor formation and so on. Exosomes are endosomal origin small membrane vesicles with a size of 40 to 100nm in diameter, and wrapped with proteins and miRNAs. The latest study have shown that transplantation of stem cell-derived exosomes directly played a similar role of transplantation of stem cells. Therefore, it was worth to further research if exosomes secreted by iPSCs-MSCs (iMSC-Exo) have powerful function in repairing ischemic tissue. Now in our experiment, we established hind-limb ischemia model and cerebral ischemia model to simulate human common ischemic diseases in peripheral and central nervous system, then we research if iMSC-Exo achieve its repairing function by promoting angiogenesis, further more, we investigated the function and possible mechanism in promoting angiogenesis in vitro. Through the study, we want to provide a safe and effective method in curing ischemic diseases.Methods:1. iPSCs differentiated into iMSCs and iMSC-Exo collection1.1 iPSCs differentiated into iMSCs:Human iPSCs were differentiated into iMSCs using a modified one-step method; Flow cytometry was used to identify cell markers in iMSCs; Osteogenesis, adipogenesis and chondrogenesis were used to identify iMSCs multipotent differentiation potency.1.2 iMSC-Exo collection and identification:iMSCs were cultured in MSC serum-free medium for 48h; Ultracentrifugation was used to collect iMSC-Exo; Transmission Electron Microscopy (TEM), Tunable Resistive Pulse Sensing (TRPS) technique and Western Blot were used to identify iMSCs-Exo.2. In vivo observe iMSC-Exo repair ischemic injury and promote angiogenesis2.1 iMSC-Exo repair ischemic hind-limb muscle and promote angiogenesis2.1.1 Established hind-limb ischemia (HLI) model in mice, multi-point injection of iMSC-Exo in quadriceps.2.1.2 Comparison of infarction extent and muscle cell morphology in ischemic limb:statistical limb infarction extent in each group at day 21; H&E itaining was used to detect morphology change of ischemic quadriceps at day 21.2.1.3 Laser Dopplerwas used to detect blood recovery in ischemic limb at day 1,7,14,21 post-surgery.2.1.4 CD31 and CD34 staining was used to detect ischemic muscle microvessel density in each group at day 14 and 21 post-surgery.1.2 iMSC-Exo repair ischemic brain tissue and promote angiogenesis2.2.1 Established middle cerebral artery occlusion (MCAO) model in rat, transplantation of iMSC-Exo by tail vein.2.2.2 mNSS was used to test nerve function before surgery, and day 1,3,7, 14,21,28 post-surgery. TTC staining was used to test brain ischemic volume at day ,28 post-surgery.2.2.3 The location of iMSC-Exo was detected in brain and cells at 30min, 1h, 5h post-surgery.2.2.4 CD31 and CD34 staining was used to detect microvessel density in schemic brain at day 14,28 post-surgery.3. In Vitro analyze the mechanism of iMSC-Exo in promoting angiogenesis3.1 Primary human umbilical vein endothelial cells (HUVECs) isolation and identification.3.2 Immunofluorescence double staining was used to observe the efficiency of HUVECs swallow iMSC-Exo.3.3 Real Time Cellular Analysis (RTCA) and scratched assay were used to detect HUVECs migration; CCK8 assay was used to detect HUVECs proliferation; Matrigel tube formation assay was used to detect HUVECs tube formation.3.4 qRT-PCR was used to detect angiogenesis related genes expression level in HUVECs. ELISA was used to detect the level of angiogenesis related proteins secreted by HUVECs.3.5 miScript miRNA PCR Array Human Serum& Plasma 384HC was used to detect the type and content of miRNAs in iMSC-Exo.Results:1. Successfully acquaired iMSCs and collected iMSC-Exo1.1 Using a modified one-step induction protocol, we successfully get iMSCs; iMSCs was positive for CD29, CD44, CD73, CD90, CD105, and CD146, while negative for CD34, CD45, CD133, and HLA-DR; iMSCs had osteogenic, adipogenesis, chondrogenesis differentiation ability.1.2 TEM revealed that iMSCs-Exo exhibited a cup-or round-shaped morphology with a size of 40～100nm; TRPS showed the median of iMSC-Exo diameter was 82nm; Western blot confirmed that iMSC-Exo express CD63, CD81 and CD9.2. iMSC-Exo repaired ischemic tissue and promoted angiogenesis2.1 iMSC-Exo repaired ischemic limb and promoted angiogenesis:Compared to control group, iMSC-Exo group had less amputation and necrosis. H&E staining showed that there was remarkably less muscle degeneration in iMSCs-Exo group. Laser Doppler showed that blood perfusion recovered gradually in iMSC-Exo group,. CD31 and CD34 staining showed that ischemic muscle in iMSC-Exo group have nore CD31 and CD34 positive cells than control group.2.2. iMSC-Exo repaired ischemic brain and promoted angiogenesis:mNSS results showed that iMSC-Exo group had a better neural function recovery after stroke; TTC staining showed that brain ischemic volume in iMSC-Exo group was much smaller; iMSCs-Exo can locate into ischemic brain. CD31 and CD34 staining showed that ischemic brain tissue in iMSC-Exo group have more CD31 and CD34 positive cells than control group.3. iMSC-Exo can promote angiogenesis in vitroRTCA and scratched wound assay results showed iMSC-Exo can promote HUVECs migration; CCK8 showed iMSC-Exo can promote HUVECs proliferation; Vlatrigel tube formation assay results showed iMSC-Exo can promote HUVECs tube formation. qRT-PCR results showed iMSC-Exo can promote HUVECs express higher level angiogenesis related genes and proteins compared to control group. miScript miRNA PCR Array showed that there were 32 angiogenesis related miRNAs in iMSC-Exo,7 pro-angiogenesis miRNAs including miR210、miR126、 miR103、miR424、miR378、let-7α、let-7e are highly expressed in iMSC-Exo.Conclution:1. Transplantation of iMSC-Exo can alleviate ischemic limb injury and promote ischemic limb function recovery; Transplantation of iMSC-Exo can alleviate ischemic brain injury and promote ischemic brain function recovery.2. Transplantation of iMSC-Exo can promote angiogenesis in ischemic limb nuscle; Transplantation of iMSC-Exo can promote angiogenesis in ischemic brain.3. iMSC-Exo interventions in vitro can promote HUVECs migration, proliferation and tube formation, and improve angiogenesis related genes expression and proteins secretion in HUVECs.4. iMSC-Exo riched of various angiogenesis related miRNAs, which may participated in the angiogenesis of iMSC-Exo.The results in the study indicated that transplantation of iMSC-Exo mainly through promote angiogenesis and improve blood perfusion in ischemic tissue, which benefited to reduce ischemic tissue injury and promote function recovery. The results indicated that transplantation of iMSC-Exo can alternate stem cell as a new strategy in repairing ischemic tissue injury.