The Research On Mechanisms Underlying Exogenous MSC Transplantation-mediated Durable Functional Recovery Of Recipient MSCs

Background Recently, MSCs have been widely applied to therapy of multiple human diseases. Previous studies indicated that, in animal disease models and clinical trials, MSCT efficiently relieve the disease phenotype of Gv HD, rheumatoid arthritis, inflammatory bowel disease, myocardial infarction, liver fibrosis, and multiple sclerosis. Our previous studies also found that, MSCT efficiently rescues SLE, promotes angiogenesis during wound healing, induces multiple myeloma cell apoptosis and prevents cutaneous hypertrophic scar and vocal fold scar formation.Given the efficient therapeutic effects generated by MSCT, the researchers are continuously exploring the mechanisms underlying the therapeutic effects. Earlier studiesindicated that the transplanted cells could survive in recipient, and differentiate into multiple adult cells for tissue regeneration. Later, more and more experimental evidences indicate that in most cases, donor MSCs would not survive in the recipient in long enough time for differentiation. Conversely, more and more experimental evidences show that, MSCs could regulate immune cell functions, promote angiogenesis and facilitate tissue regeneration through secretion of soluble cytokines including PGE2, TSG-6, VEGF and IL-10. Meanwhile, it is demonstrated that MSCs can interact with immune cells and induce immune tolerance. Although some of the mechanisms underlying MSCT have been explored, many phenomena observed during MSCT application have not been explained. Especially, it is very interesting that in most cases of MSCT, MSC infusion is just performed once. However, the therapeutic effects generated by the one-time MSCT could last for a long term. These evidences indicate that MSCT generate durable therapeutic effects through regulation of the recipient cell functions, and MSCT generate a “therapeutic memory”. The mechanisms underlying these phenomena have not been explored yet.Epigenetic modification is relatively stable regulation of gene expression without changes of DNA sequences, mainly including DNA methylation, histone modification and micro RNA. Numerous studies demonstrate that epigenetic modification has important roles in disease development and in maintenance of pathological status of some diseases. Meanwhile, multiple therapeutic approaches aimed at reversing epigenetic aberrations in disease conditions cansustain therapeutic effects.In summery, we hypothesize that, during MSCT, donor MSCs generate durable therapeutic effects through regulating the epigenetic status of the recipient cells. In our previous study, we found that, similar to SLE patient, SLE mice(MRL/lpr mice with Fas gene mutation) have impaired osteogenic differentiation potential of BMMSCs and significant osteoporotic disease phenotype. Meanwhile, we found, a single MSCT durably and efficiently rescues the differentiation potential of BMMSCs derived from MRL/lp rmice and rescues their osteoporotic disease phenotype. In this study, we use the MRL/lpr mice model to further explore the molecular regulation mechanisms underlying thedurable therapeutic effects generated by MSCT.Aim In MRL/lpr mice model, we will determine the MSCT-generated durable therapeutic effects on osteoporotic disease phenotype and osteogenic differentiation capacities of the recipient BMMSCs. Then, we will explore the role of epigenetic modifications, especially DNA methylation, in the durable therapeutic effects. Finally, we will dissect the mechanisms underlying donor MSCs regulate the epigenetic status of the recipient BMMSCs. This study provides new intervention strategy for rescuing abnormal epigenetic status, and further provide theory basis.Methods 1. The short-term and long-term of MSCT-generated therapeutic effects. Donor MSCs were systemically infused into MRL/lpr mice model. The short-term and long-term therapeutic effects on osteoporotic disease phenotype were determined by micro-CT and calcein green labeling experiments. The osteogenic differentiation capacities of MRL/lpr BMMSCs were evaluated by Alizarin red staining, Western blot and subcutaneousbone formation in nude mice.2. The regulation of recipient BMMSC epigenetic status by MSCT. DNA methylation array was used to analyze global methylation pattern changes of MRL/lpr BMMSCs before and after MSCT. Then, the inhibiting effects of 5-Azacytidine on MSCT were evaluated.3. The regulation of DNA methylation levels of Notch signaling genes by Dnmt1. Western blot was used to detect Dnmt1 expression in MRL/lpr BMMSCs before and after MSCT. Then, si RNA knockdown approach and Alizarin red staining were used to determine the role of Dnmt1 in osteogenic differentiation of BMMSCs. The si RNA knockdown and overexpression of Dnmt1 and DAPT were further used to evaluate whether Dnmt1 regulated osteogenic differentiation through Notch signaling.4. The regulation of Dnmt1 by mi R-29 b. The mi RNA array was performed for detection of the mi RNA levels, which were recovered after MSCT. Real-time PCR was performed to confirm the level of mi R-29 b. After treatment of mi R-29 b inhibitor in vivo, the effects ofmi R-29 b inhibitor on Dnmt1 expression, Notch signaling activation, recipient BMMSC differentiation capacities and osteoporotic disease phenotype were evaluated. Then, after treatment of mi R-29 b mimics in vitro, the effects of mi R-29 b mimics on Dnmt1 expression, Notch signaling activation and BMMSC differentiation capacities were evaluated.5. The regulation of mi R-29 b level in the recipient BMMSCs by donor MSC-derived exosomes. Transwell was used to establish a co-culture system for co-culture of normal BMMSCs and MRL/lpr BMMSCs. After co-culture, MRL/lpr BMMSC functions were detected. Rab27 a si RNA was used to inhibit exosome secretion and evaluate the role of exosome secretion in MSCT. After systemic infusion of exosomes, the levels of Dnmt1 and Notch genes, and BMMSC functions and osteoporotic disease phenotype of MRL/lpr mice were determined.6. The recipient BMMSC secretion of mi R-29 b regulated by reused donor-derived Fas. The si RNA knockdown and overexpression of Fas were performed to investigate the role of Fas in mi R-29 b secretion. After co-cultured with exosomes derived from normal BMMSCs and MRL/lpr BMMSCs respectively, the functions of MRL/lpr BMMSCs were determined. The Fas-EGFP fusion protein was constructed for Fas protein tracing. Exosomes containing Fas-EGFP fusion protein were co-cultured with MRL/lpr BMMSCs and infused into MRL/lp rmice respectively. Then the Fas-EGFP was detected by immunofluorescent staining in BMMSCs in vitro and in vivo.Results 1. MSCT generates durable therapeutic effects. Compared with BMMSCs derived form normal control mice(C3H/He J mice), BMMSCs derived from MRL/lpr mice had lower osteogenic differentiation capacities in vitro and in vivo. Meanwhile, compared with normal control mice, MRL/lpr mice had significant osteoporotic disease phenotype. Four weeks after MSCT, the impaired BMMSCs functions and osteoporotic disease phenotype of MRL/lpr mice were rescued. More importantly, twelve weeks after MSCT, the therapeutic effects on BMMSC functions and the osteoporotic disease phenotype were maintained.2. MSCT recovered the DNA methylation status of MRL/lpr BMMSCs. BMMSCs derived from MRL/lpr mice had abnormal global DNA methylation pattern and lower DNA methylation level, which were rescued after MSCT. The demethylating agent 5-Azacytidine treatment blocked MSCT-mediated therapeutic effects on osteogenic differentiation capacities of MRL/lpr BMMSCs and the osteoporotic disease phenotype of MRL/lprmice. 3. MSCT recovers the DNA methylation levels of Notch signaling genes of the recipient BMMSCs. The MRL/lpr BMMSCs had lower DNA methylation levels and higher expression levels of Notch signaling genes, which were rescued after MSCT. In in vitro and in vivo studies, DAPT treatments significantly rescued the osteogenic differentiation capacities of MRL/lpr BMMSCs and rescued the osteoporotic disease phenotype of MRL/lpr mice. 4. Dnmt1 regulates DNA methylation levels of Notch signaling genes. MRL/lpr BMMSCs had lower Dnmt1 expression. In C3H/He J BMMSCs, knockdown of Dnmt1 decreased the DNA methylation level of Notch1, active Notch signaling and inhibit osteogenic differentiation of BMMSCs. In MRL/lpr BMMSCs, Dnmt1 overexpression rescued the DNA methylation level of Notch1, Notch signaling activation level and osteogenic differentiation capacities. 5. Mi R-29 b downregulates Dnmt1 expression. MRL/lpr BMMSCs had higher expression of mi R-29 b, which was recovered by MSCT. In in vivo study, inhibition of mi R-29 b significantly rescued Notch, Dnmt1 expression level and osteogenic differentiation of MRL/lpr BMMSCs, and rescued the osteoporotic disease phenotype of MRL/lpr mice. In C3H/He J BMMSCs, overexpression of mi R-29 b decreased Dnmt1 expression and DNA methylation level of Notch1, activated Notch signaling and inhibited osteogenic differentiation capacities of BMMSCs. 6. Exosomes secreted by donor MSCs downregulate mi R-29 b level in the recipient BMMSCs. Co-culture with C3H/He J BMMSCs rescued mi R-29 b, Dnmt1 and Notch1 levels and osteogenic differentiation capacities of MRL/lpr BMMSCs, which could be partially blocked through inhibition of exosome secretion by MRL/lpr BMMSCs.Meanwhile, inhibition of exosome secretion by donor MSCs significantly blocked MSCT-generated rescuing effects on mi R-29 b, Dnmt1 and Notch1 levels and osteogenic differentiation capacities of MRL/lpr BMMSCs. Direct infusion of exosomes significantly rescued mi R-29 b, Dnmt1 and Notch1 levels and osteogenic differentiation capacities of MRL/lpr BMMSCs, and osteoporotic disease phenotype of MRL/lpr mice. 7. The recipient BMMSCs reuse donor exosome-derived Fas for mi R-29 b secretion. In C3H/He J BMMSCs, knockdown of Fas increased intracellular mi R-29 b level and decrease mi R-29 b secretion. Meanwhile, in MRL/lpr BMMSCs, Fas overexpression decreased intracellular mi R-29 b and promoted mi R-29 b secretion. However, neither Fas knockdown nor Fas overexpression affected original transcription level of mi R-29 b. Exosomes derived from C3H/He J BMMSCs could rescue the intracellular and secreted mi R-29 b levels, whereas exosomes derived from MRL/lpr BMMSCs did not have such functions. Exosomes containing Fas-EGFP fusion protein were co-cultured with MRL/lpr BMMSCs and infused into MRL/lpr mice respectively. Then, Fas-EGFP fusion protein could be detected in MRL/lpr BMMSCs in in vitro and in vivo studies.ConclusionThis study demonstrates that, in MRL/lpr mice model,elevated intracellular levels of mi R-29 b, caused by Fas-deficient-mediated failure of releasing, downregulate expression of Dnmt1 in MRL/lpr BMMSCs.This results in hypomethylation of Notch signaling gene promoters and activation of Notch signaling, in turn leading toreduced osteogenic differentiation of MRL/lpr BMMSCs and osteoporotic disease phenotype of MRL/lpr mice. During MSCT, exosomes secreted due to MSCT reduce intracellular levels of mi R-29 b in recipient MRL/lpr BMMSCs, and that exosome-derived Fas is reused by these BMMSCs. Finally, the reduced intercellular mi R-29 b levels result in recovery of Dnmt1-mediated hypomethylation of Notch signaling gene promotersand thereby improve MRL/lpr BMMSC function, and significantly rescued the osteoporotic disease phenotype of MRL/lpr mice.In this study, by demonstrating the Fas/mi R-29b/Dnmt1/Notch epigenetic cascade, we are the first to reveal the mechanisms underlying the durable therapeutic effectsgenerated by MSCT, indicating that stem cell therapy can durably regulate the functions of the recipient cells through epigenetic modifications. Thus, this study provides new therapeutic method for reversing the abnormal epigenetic status during disease development. Meanwhile, we are also the first to find that, the components of donor cells can be reused by recipient cells for rescuing their own functions, indicating that the interactions between donor cells and recipient cells play an important role in MSCT. Therefore, this study provides further theory basis for clinical applications of stem cell therapy.