Regulation of stemness and aging in human mesenchymal stem cells

Abstract

Adult stem cells in mammalian organs play pivotal roles in the maintenance and repair of these organs throughout the adult life of the organism, and the main function of adult stem cells is to maintain the proper homeostasis of a tissue or organ through precisely regulated molecular signaling. Thus, first part of this study mainly focused on the identification of the specific marker to maintain the stemness and enhance multipotent of human adult stem cells. I demonstrated that the expression of CD49f regulated the ability of human mesenchymal stem cells (hMSCs) to form spheres and was associated with an activation of the PI3K/AKT signaling pathway. Furthermore, the forced expression of CD49f modulated the proliferation and differentiation potentials of hMSCs through prolonged activation of PI3K/AKT and suppressed the level of p53. The pluripotency factors OCT4 and SOX2 were recruited to the putative promoter region of CD49f, indicating that OCT4 and SOX2 play positive roles in the expression of CD49f. Indeed, CD49f expression was up-regulated in human embryonic stem cells (hESCs) compared with hMSCs. The elevated level of CD49f expression was significantly decreased upon embryoid body formation in hESCs. In hESCs, the knockdown of CD49f down-regulated PI3K/AKT signaling and up-regulated the level of p53, inducing differentiation into three germ layers. These data suggest that the cell-surface protein CD49f has novel and dynamic roles in regulating the differentiation potential of hMSCs and maintaining pluripotency.

Similar to normal somatic cells, adult stem cells experience a lifelong exposure to stressors, which leads to an age-associated decline in their number and function. The second and third part of this study showed the molecular regulatory mechanism of HMGA2 and ZMPSTE24, which are an important regulator of hMSCs aging. The human high-mobility group protein A2 (HMGA2) protein is an architectural transcription factor that transforms chromatin structure by binding to DNA. Recently, it has been reported that HMGA2 is highly expressed in fetal neural stem cells and has the capacity to promote stemness. I evaluated the direct effects of HMGA2 on the cellular aging and proliferation of hMSCs and investigated potential regulatory mechanisms responsible for the corresponding functions. The overexpression of HMGA2 enhanced proliferation and reduced or even reversed the in vitro aging process of hMSCs. This effect was accompanied by the increased expression of cyclin E and CDC25A and the significantly decreased expression of cyclin-dependent kinase inhibitors. Furthermore, HMGA2 inhibition compromised cell proliferation and adipogenic differentiation in early-stage hMSCs. From the molecular/cellular functional analysis of microarray data, I found that HMGA2 overexpression induced a PI3K/Akt/mTOR/p70S6k cascade, which in turn suppressed the expression of p16INK4A and p21CIP1/WAF1 in hMSCs.

ZMPSTE24 is involved in the post-translational maturation step of lamin A. Defects in ZMPSTE24 are associated with the accumulation of prelamin A in the nuclear envelope and lead to premature senescence. hMSCs down-regulated ZMPSTE24 and accumulated prelamin A during replicative or HDAC inhibitor-mediated senescence. The miR-141, which is over-expressed during the senescence process, was able to decrease the ZMPSTE24 expression levels and led to an up-regulation of prelamin A and a DNA damage marker in hMSCs. The transfection of anti-miR-141 prevented the reduction of ZMPSTE24 in VPA/SB-treated cells, resulting in the suppression of the induction of abnormal nuclear morphology. In addition, epigenetic histone markers of the chromatin configuration on the miR-141 promoter region were transcriptionally activated during senescence.

According to these results, following conclusions have been drawn: i) CD49f, a cell-surface molecule, plays important roles in MSC sphere formation and in the determination of the differentiation potential through direct regulation of OCT4 and SOX2. ii) HMGA2 regulates in vitro aging by repressing p16INK4A, p21CIP1/WAF1 expression through the PI3K/AKT/mTOR/p70S6K signaling pathway. iii) Histone deacetylase regulates ZMPSTE24 and induces prelamin A accumulation through miR-141 up-regulation. Taken together, these results provide novel insights into the mechanism by which CD49f, HMGA2 and ZMPSTE24 regulate the stemness and aging of human mesenchymal stem cells.