The Reparative Mechanisms Of Bone Marrow And Cardiac Mesenchymal Stem Cells For Treating Tissue Damage

Mesenchymal stem cells(MSCs) are non-hematopoietic stem cells originally isolated from bone marrow, which participate in the hematopoietic microenvironment of bone marrow and have obvious effect on supporting proliferation and differentiation of hematopoietic stem cells. MSCs have the potential of self-renewal and multi-directional differentiation into osteoblasts, fat cells and cartilage cells in vitro. MSCs have traditionally been considered that it could differentiate into particular cells to replace the damage tissues and organs. MSCs can participate in the reparative process of large section bone defects or cartilage damages by replacing damaged cells directly. Subsequent researchers found that MSCs have the function of nutritional support and immune regulation and paly a more important role in the repair of tissue damages and treatment of disease. For example, MSCs could inhibit cell apoptosis induced by ischemia, suppress scar formation, and stimulate angiogenesis and activate endogenous stem cells through trophic mediators in a paracrine fashion. In the aspect of immunomodulation, MSCs could inhibit proliferation of T cells stimulated by motigen, increase the proportion of regulatory T cells(Tregs) and decrease the ratio of Th1/Th2, which results in Th17+ cells decline; MSCs could inhibit the proliferation and activation of B cells and its antibody secretion and affect the chemotaxis function of B cells. MSCs could inhibit proliferation, cytotoxic effects and the secretion of cytokines of NK cells. MSCs could also affect the antigen presented function of antigen presenting cells(APC), and inhibit T cell activation by down regulation of MHC molecules and costimulatory molecules(CD40, CD86, CD80) on the surface of APC and inhibition of DC cells mature. The main molecules involved in the immunomodulation of MSCs including TGF?, PGE-2, TSG6, NO and IDO. However, inhibition of the expression of these molecules does not completely reverse immune regulating function of MSCs, which indicate that some other unknown regulatory factors and pathways are involved. Therefore, it is of great significance to systematic describe the mechanisms of MSCs involved in wound repair and promote its clinical application by looking for the new regulatory factors and pathways.In our previous RNA-seq results, we found that the expression of insulin-like growth factor bingding protein 7(IGFBP7) was highest in the subpopulation of MSCs that have strongest immunomodulatory effects. It indicated that IGFBP7 may participate in the immune modulation of mouse MSCs. Nontheless, the underlying mechanisms are worth exploring in the future.Many studies in recent years have shown that except for the bone marrow, MSCs also existed in the fat, umbilical cord, kidney and heart, and showed similar ability to repair damage tissues. In our studies using Nestin-GFP transgenic mice, we found that Nestin was widely distributed in the bone marrow, pancreas, testis, heart, kidney and retina of newborn mice except for central and peripheral nervous system. GFP+ cells that isolated form testis and kidney have self-renewal and mesenchymal differentiation ability. These results proved that Nestin is a specific marker of MSCs in adult tissues and provided an important basis in obtaining the subpopulation of MSCs that is suitable for damage repair. Further, Nestin+ cells from heart of Nestin-GFP transgenic mice were also isolated and found that they had the characteristics of MSCs, and showed a better therapeutic effect in repairing the damage of myocardial tissue than bone marrow MSCs. Therefore, whether MSCs have better therapeutic effect on their target tissues and the underlying mechanisms of repairing tissue damages by MSCs remain to be further elucidated.This study consists of 2 sections:Section 1: The mechanism of insulin-like growth factor binding protein(IGFBP7) involved in the immunoregulation of mouse MSCs.Section 2: The mechanism of cardiac-derived Nestin+MSCs to treat acute myocardial infarction in mice.Section 1: The mechanism of insulin-like growth factor binding protein(IGFBP7) involved in the immunoregulation of mouse MSCs1. Objective At present, there are many immunoregulatory molecules that participate in the immunoregulatory of MSCs, including NO, IDO, PGE2, IL-10, TSG6, Galectin1 and TGF?. Besides these molecules, other factors involved in the immune regulation of MSCs are the key scientific questions worth exploring. Existing research shows that MSCs, derived from patients who have IFN- gamma receptor mutation, will still have immune regulating function, and it prompts that IGFBPs may participate in immune regulation, but the underlying mechanism is still unknown. In our previous RNA-seq results, the expression of insulin-like growth factor bingding protein 7(IGFBP7) was highest in the subpopulation of MSCs that had strongest immunomodulatory effects. It indicated that IGFBP7 may participate in the immune modulation of mouse MSCs. Therefore, this study systematically reveals the mechanism of IGFBP7 involved in the immune regulation of MSCs.2. Method 2.1 The gene expression of members of IGFBPs family(IGFBP1~7) in mouse bone marrow MSCs was analyzed by Q-PCR. 2.2 We successfully silenced the gene of IGFBP7 in MSCs using RNA interference. And the efficiency of interference of IGFBP7 was analyzed by Q-PCR and Western blot. 2.3 The proliferation, surface markers and multi-differentiation potential(osteogeic, adipogenic and chondrogenic) between control and interference group of MSCs were analyzed. 2.4 In in vitro experiments, we used co-culture system to explore whether IGFBP7produced by MSCs has direct suppression on proliferation and cytokine secretion of T cells and its subpopulations. The change of fluorescence intensity of CFSE analyzing by flow cytometry reflects proliferation of T cells and the percentage of TNF-? and IFN-? were analyzed by intracellular cytokine staining using flow cytometry. 2.5 The apoptosis of T cells was analyzed by Annexin V and PI staining and the percentage of CD4+CD25+Foxp3+ Tregs was analyzed by flow cytometry. 2.6 Cell cycle analysis of T cells by flow cytometry and Western blot were used to clarify the underlying mechanisms of the immunosuppression of IGFBP7, respectively. 2.7 In in vivo experiments, mouse experimental colitis was induced by trinitro-benzene-sulfonicacid(TNBS), and MSCs or saline were transplanted to treat mice through intraperitoneal injection. Mouse weight, symptom and death of each group were recorded in the seven-day modeling process. The general appearance and length of colon were observed and analyzed. The degree of inflammation infiltration of the intestinal tissue was reflected by H&E staining; the changes of immune cell types were reflected by immunofluorescent staining. To investigate if MSCs secreted IGFBP7 have a suppression role on the proliferation and cytokine secretion of T cells in MLNs, MLNs were obtained from colitis mouse 2 days after TNBS induction and analyzed by flow cytometry directly or after 3 days co-culture with MSCs.3. Result 3.1 Q-PCR revealed that IGFBP7 was the most abundant factor of the IGFBPs family in mouse bone marrow MSCs. 3.2 The interference of IGFBP7 did not change the expression of surface markers(like Sca-1, CD106 and CD44), the proliferation and multi-differentiation potential of MSCs. 3.3 In in vitro experiments, IGFBP7 secreted by mouse MSCs could inhibit proliferation and pro-inflammation cytokines(TNF-? and IFN-?) secretion of T cells, but not influence the apoptosis of T cells and the percentage of Tregs.3.4 In in vitro experiments, IGFBP7 secreted by mouse MSCs could block cell cycle process of T cells in G0/G1 phase through inhibiting PI3K/AKT and MAPK/ERK signaling pathway; IGFBP7 suppressed pro-inflammation cytokines secretion through inhibiting NF-?B signaling pathway. 3.5 In in vivo experiments, MSCs-produced IGFBP7 could mitigate the symptom of experimental colitis, including effective suppression of losing weight, improving the quality of the survival rate and survival of mice, as well as inhibiting shortened and hyperemia and edema of colon, at the same time reducing the degree of inflammation infiltration in colon, which finally effectively inhibit inflammation response of intestine. 3.6 Mouse MSCs inhibit the proliferation and TNF-? or IFN-? secretion of T cells in MLNs via IGFBP7, but did not affect the percentage of Tregs in MLNs.4. Conclusion 4.1 This study first clarify that IGFBP7 could participate in the immunoregulation of mouse MSCs and is characterized by inhibiting T cell proliferation and the secretion of proinflammatory factor. 4.2 The results showed that IGFBP7 could block cell cycle process of T cells in G0/G1 phase through inhibiting PI3K/AKT and MAPK/ERK signaling pathway and then inhibit proliferation of T cells. IGFBP7 suppressed pro-inflammation cytokines secretion of T cells through inhibiting NF-?B signaling pathway. 4.3 In in vivo experiments, the knock down of IGFBP7 in MSCs significantly inhibited the therapeutic effect of MSCs for treating experimental colisis in mice, leding to a decline in body weight, low survival rate, short of colon tissue and inflammatory cell infiltration in the colon. These results further confirmed that IGFBP7 play an important role in the immunomodulation of MSCs.Section 2: The mechanism of cardiac-derived Nestin+MSCs to treat acute myocardial infarction in mice1. Objective Many studies in recent years have shown that except for the bone marrow, MSCs also existed in the fat, umbilical cord, kidney and heart, and showed similar ability to repair damage tissues. In our previous study, we proved that Nestin+ testicular and kidney mesenchymal stem cells have the reparative function on their target tissue damages. Further, Nestin+ cells from heart of Nestin-GFP transgenic mice were also isolated and found that they have the characteristics of MSCs, and showed a better therapeutic effect in repairing the damage of myocardial tissue than bone marrow Nestin+MSCs. However, the underlying mechanisms of repairing tissue damages are still unknown and need to be further elucidated.2. Method 2.1 We isolated cardiac Nestin-GFP+ cells from 7-days old Nestin-GFP transgenic mouse using flow cytometry, and cultured these cells in vitro with serum-free medium containing a variety of growth factors, including b FGF, EGF, N2, B27 and CT-1. 2.2 Single Nestin+ cell was put into one well of 96-well plate, and its self-renewal ability was observed at different incubation time(0, 3, 7 and 10 days). 2.3 The osteogenic, adipogenic and chondrogenic differentiation abilities of Nestin+ cells were investigated. 2.4 The expressions of MSCs-related surface markers on Nestin+ cells were analyzed by flow cytometry, including Sca-1,c-ki,CD44,CD106,CD90,CD45 and CD11 b. 2.5 Establish the AMI model in mice through permannent ligation of left ventricular anterior descending coronary artery. And 25?l saline(Saline group)?25?l saline including 3×105 bone marrow derived Nestin+MSCs(Nes+MSCs group)or cardiac derived Nestin+c MSCs(Nes+c MSCs group)were tranplanted into infarct area throughintramyocardial injection. 2.6 The indicators of cardiac function in mice were monitored by echocardiography at 1 week and 3 weeks after AMI, including LVEF, LVFS, LVEDV and LVESV. The degree of myocardial fibrosis of heart was reflected by masson-trichrome staining at 3 weeks after AMI. 2.7 The changes of immune response were reflected using H&E staining;Macrophages were reported to play the key role in the repair process of AMI, and the changes of macrophages in vitro and in vivo experiments were reflected using immunofluorescent staining,flow cytometer analysis,Q-PCR and ELISA. 2.8 Remove macrophages in mice using anionic clodronate liposomes and reuse the above methods to detect the cardiac function and immune response of AMI model mice influenced by macrophages depletion. 2.9 Different kinds of Nestin+ c MSCs-produced paracrine factors that involved in damage repair were analyzed by Q-PCR. 2.10 Knock down the expression of periostin in Nestin+ c MSCs using RNA interference and then the changes of therapeutic effect and the macrophages under the action of Nestin+ c MSCs were investigated using the above testing methods in vitro and in vivo.3. Result 3.1 Cardiac Nestin+ cells had strong self-renewal ability,expressed some mesenchymal stem cells related surface markers and were capable of differentiating into mesodermal cell lineages, such as osteocytes, adipocytes and chondrocytes. Thus we defined the group of cells that inside the heart as Nestin+ cardiac mesenchymal stem cells(Nestin+c MSCs). 3.2 3×105 Nestin+MSCs(Nes+MSCs group)and Nestin+c MSCs(Nes+c MSCs group)were tranplanted into infarct area of AMI model through intramyocardial injection. Echocardiography revealed that Nestin+c MSCs and Nestin+MSCs treatment could significantly increased LVEF and LVFS as well as obviously decreased LVEVD andLVESV when compared to Saline group; Nestin+c MSCs and Nestin+MSCs were still able to significantly reduce the range of myocardial fibrosis and finally promoted the repair of the heart. And we found that Nestin+c MSCs had a better therapeutic effect on promoting the recovery of cardiac function than Nestin+MSCs. 3.3 Nestin+c MSC could effectively reduce the inflammatory response in the myocardial infarction areas after AMI and we further found that it is due to partly inhibiting macrophages into the infarction region and effectively increase the percentage of M2 macrophages, which has an anti-inflammation role. 3.4 In vitro co-culture experiments also indicated that Nestin+c MSCs could inhibit actived macrophages polarize to M1 type(pro-inflammatory) and promote them differentiate to M2 macrophages(anti-inflammatory). As well as, Nestin+c MSCs inhibited the secretion of TNF-? and IFN-? by M1 macrophages and increased IL-10 production by M2 macrophages. 3.5 The therapeutic effects of Nestin+c MSCs on AMI were significantly weakened after macrophages removal in vivo using anionic clodronate liposomes. It indicated that macrophages play a key role in the treatment of AMI using Nestin+c MSCs.3.6 The periostin molecule had the highest expression in all the paracrine factors of Nestin+c MSCs as screened by Q-PCR. 3.7 When periostin was knocked down in Nestin+c MSCs, the percentage of M2 macrophages can not be effectively increased, which therefore blocked the suppression of inflammation reaction in infarct area and eventually fail to promote repair of heart tissue and the recovery of cardiac function. 3.8 Periostin secreted by Nestin+c MSCs not only increase the percentage of M2 macrophages in infarct area, but also promote M2 macrophages to secrete IL-10, which play a vitral role in immunosuppression after AMI.4. Conclusion 4.1 Cardiac-derived Nestin+ cells are a group of cells with mesenchymal stem cell properties; therefore, we defined them as Nestin+ cardiac mesenchymal stem cells(Nestin+c MSCs). 4.2 Compared to bone marrow Nestin+MSCs, Nestin+c MSCs could be more effective to relieve the symptoms of myocardial infarction, promote myocardial repair and the recovery of cardiac function. It was shown that Nestin+c MSCs significantly increase LVEF and LVFS as well as obviously decrease LVEDV and LVESV, and reduce the area of myocardial fibrosis. 4.3 Nestin+c MSCs play the role on wound repair by inhibiting pro-inflammatory M1 macrophages and increasing the percentage of anti-inflammatory M2 macrophages in infarct area. 4.4 Periostin secreted by Nestin+c MSCs promoted the percentage of M2 macrophages and its IL-10 secretion, thereby promote the repair of heart tissue and help to improve cardiac function.