Immunomodulatory properties and senescence-associated secretory phenotypes in human mesenchymal stem cell aging

Abstract

Because human mesenchymal stem cells (hMSC) have profound immunomodulatory effects, many attempts have been made to use hMSCs in preclinical and clinical trials. For hMSCs to be used in therapy, a large population of hMSCs must be generated by in vitro expansion. However, the immunomodulatory changes following the in vitro expansion of hMSCs have not been elucidated. In the first and second part of this study, I evaluated the effect of senescence on the immunomodulatory ability of hMSCs. Late-passage hMSCs showed impaired suppressive effect on mitogen-induced mononuclear cell proliferation. Interestingly, late-passage hMSCs had a significantly compromised protective effect against mouse experimental colitis, which was confirmed by gross and histologic examination. Among the anti-inflammatory cytokines, the production of prostaglandin E2 (PGE2) and the expression of its primary enzyme, cyclooxygenase-2 (COX-2), were profoundly increased by pre-stimulation with interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), and this response was significantly decreased with consecutive passages. I demonstrated that the impaired phosphorylation activity of p38 MAP kinase (p38 MAPK) in late-passage hMSCs led to a compromised immunomodulatory ability through the regulation of COX-2. These results indicate that the immunomodulatory ability of hMSCs gradually declines with consecutive passages via a p38-mediated alteration of COX-2 and PGE2 levels.<br/> Low oxygen environment have been reported to inhibit senescence and maintain undifferentiated states in somatic stem cells. In the second part of study, I evaluated the effects of hypoxia on cellular senescence and the immunomodulatory abilities of hMSCs. Hypoxic-cultured human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) showed enhanced proliferation and had increased immunosuppressive effects on mitogen-induced mononuclear cell proliferation. I found that BMI1, a member of the polycomb repressive complex protein group, showed increased expression in hypoxic-cultured hUCB-MSCs, and the further knock down of BMI1 in hypoxic cells induced decreased proliferative and immunomodulatory abilities in hUCB-MSCs, along with COX-2/PGE2 down-regulation. Furthermore, the expression of phosphorylated p38 MAP kinase increased in response to the over-expression of BMI1 in normoxic conditions, suggesting that BMI1 regulates the immunomodulatory properties of hUCB-MSCs via p38 MAP kinase-mediated COX-2 expression. More <br/> importantly, I identified BMI1 as a direct repressor of MAP kinase phosphatase-1 (MKP-1)/DUSP1, which suppresses p38 MAP kinase activity. The results demonstrate that hypoxia induced BMI1 plays a key role in the regulation of the immunomodulatory properties of hMSCs.<br/> Defects in the nuclear lamina occur during physiological aging and as a result of premature aging disorders. Aging is also accompanied by an increase in transcription of genes encoding cytokines and chemokines, a phenomenon known as the senescence-associated secretory phenotype (SASP). Progerin and prelamin A trigger premature senescence and loss of function of hMSCs, but little is known about how defects in nuclear lamin A regulate SASP. In the third part of study, I show that both progerin overexpression and ZMPSTE24 depletion induce paracrine senescence, especially through expression of monocyte chemoattractant protein-1 (MCP-1), in hMSCs. Importantly, I identified that GATA binding protein 4 (GATA4) is a mediator regulating MCP-1 expression in response to prelamin A or progerin in hMSCs. Co-immunoprecipitation revealed that GATA4 expression is maintained due to impaired p62-mediated degradation in progerin-expressing hMSCs. Furthermore, depletion of GATA4 abrogated SASP-dependent senescence through suppression of NF-ĸB and MCP-1 in hMSCs with progerin or prelamin A. Thus, my findings indicate that abnormal lamin A proteins trigger paracrine senescence through a GATA4-dependent pathway in hMSCs. This molecular link between defective lamin A and GATA4 can provide insights into physiological aging and pathological aging disorders.<br/> In conclusion, cellular senescence can alter immunomodulatory abilities and paracrine senescence phenotypes in hMSCs. The molecular mechanisms demonstrated in this study can provide insights into physiological aging and pathological aging disorders. These findings also suggest a strategy to enhance the functionality of hMSCs for use in therapeutic applications.<br/>