Kyung-Rok Yu

CD49f Enhances Multipotency and Maintains Stemness Through the Direct Regulation of OCT4 and SOX2

CD49f (integrin subunit α6) regulates signaling pathways in a variety of cellular activities. However, the role of CD49f in regulating the differentiation and pluripotency of stem cells has not been fully investigated. Therefore, in this study, human mesenchymal stem cells (hMSCs) were induced to form spheres under nonadherent culture conditions, and we found that the CD49f‐positive population was enriched in MSC spheres compared with MSCs in a monolayer. The expression of CD49f regulated the ability of hMSCs to form spheres and was associated with an activation of the phosphatidylinositol 3‐kinase (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. We showed that 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 upregulated 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 downregulated PI3K/AKT signaling and upregulated the level of p53, inducing differentiation into three germ layers. Taken together, our data suggest that the cell‐surface protein CD49f has novel and dynamic roles in regulating the differentiation potential of hMSCs and maintaining pluripotency. STEM CELLS 2012;30:876–887

Aging-Related Genes in Mesenchymal Stem Cells: A Mini-Review

Adult stem cells in mammalian organs play pivotal roles in the maintenance and repair of these organs throughout the life of the adult and maintain the proper homeostasis of a tissue or organ. Among the adult stem cells described to date, mesenchymal stem cells (MSCs) are highlighted for clinical applications because MSCs have many advantages for cell therapy, including multilineage differentiation, homing, immune modulation and wound-healing effects. However, as the aging of MSCs leads to an age-associated decline in their number and function, it is important to clarify the age-associated factors and regulatory mechanism associated with the MSC aging process. In this review, we amass and discuss the recent data related to age-associated genes in MSCs. In particular, the activities of epigenetic regulatory factors, including histone acetylase and DNA methyltransferase, modulate gene expression and crosstalk with each other during the MSC senescence process. p16INK4A and high-mobility group A2 play important age-associated roles in the regulation of MSC stemness, and lamin A- and prelamin A-dependent nuclear abnormalities have significant biological relevance in MSC aging. Taken together, the information described here, including the epigenetic regulatory factors, transcription factors and cell signaling, could be used toward the development of treatments for MSC aging and related defects.

Human Umbilical Cord Blood Mesenchymal Stem Cell‐Derived PGE2 and TGF‐β1 Alleviate Atopic Dermatitis by Reducing Mast Cell Degranulation

Mesenchymal stem cell (MSC) is a promising tool for the therapy of immune disorders. However, their efficacy and mechanisms in treating allergic skin disorders are less verified. We sought to investigate the therapeutic efficacy of human umbilical cord blood‐derived MSCs (hUCB‐MSCs) against murine atopic dermatitis (AD) and to explore distinct mechanisms that regulate their efficacy. AD was induced in mice by the topical application of Dermatophagoides farinae. Naïve or activated‐hUCB‐MSCs were administered to mice, and clinical severity was determined. The subcutaneous administration of nucleotide‐binding oligomerization domain 2 (NOD2)‐activated hUCB‐MSCs exhibited prominent protective effects against AD, and suppressed the infiltration and degranulation of mast cells (MCs). A β‐hexosaminidase assay was performed to evaluate the effect of hUCB‐MSCs on MC degranulation. NOD2‐activated MSCs reduced the MC degranulation via NOD2‐cyclooxygenase‐2 signaling. In contrast to bone marrow‐derived MSCs, hUCB‐MSCs exerted a cell‐to‐cell contact‐independent suppressive effect on MC degranulation through the higher production of prostaglandin E2 (PGE2). Additionally, transforming growth factor (TGF)‐β1 production from hUCB‐MSCs in response to interleukin‐4 contributed to the attenuation of MC degranulation by downregulating FcεRI expression in MCs. In conclusion, the subcutaneous application of NOD2‐activated hUCB‐MSCs can efficiently ameliorate AD, and MSC‐derived PGE2 and TGF‐β1 are required for the inhibition of MC degranulation. Stem Cells 2015;33:1254–1266

MicroRNA-141-3p plays a role in human mesenchymal stem cell aging by directly targeting ZMPSTE24

Summary Human mesenchymal stem cell (hMSC) aging may lead to a reduced tissue regeneration capacity and a decline in physiological functions. However, the molecular mechanisms controlling hMSC aging in the context of prelamin A accumulation are not completely understood. In this study, we demonstrate that the accumulation of prelamin A in the nuclear envelope results in cellular senescence and potential downstream regulatory mechanisms responsible for prelamin A accumulation in hMSCs. We show for the first time that ZMPSTE24, which is involved in the post-translational maturation of lamin A, is largely responsible for the prelamin A accumulation related to cellular senescence in hMSCs. Direct binding of miR-141-3p to the 3′UTR of ZMPSTE24 transcripts was confirmed using a 3′UTR-luciferase reporter assay. We also found that miR-141-3p, which is overexpressed during senescence as a result of epigenetic regulation, is able to decrease ZMPSTE24 expression levels, and leads to an upregulation of prelamin A in hMSCs. This study provides new insights into mechanisms regulating MSC aging and may have implications for therapeutic application to reduce age-associated MSC pool exhaustion.

HMGA2 regulates the in vitro aging and proliferation of human umbilical cord blood-derived stromal cells through the mTOR/p70S6K signaling pathway ☆

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. However, there is currently no information available on the functional significance and molecular mechanisms of the cellular in vitro aging and proliferation of human umbilical cord blood-derived stromal cells (hUCBSCs). In the present study, we evaluated the direct effects of HMGA2 on the cellular aging and proliferation of hUCBSCs and investigated potential regulatory mechanisms responsible for the corresponding functions. We found that the overexpression of HMGA2 enhanced proliferation and reduced or even reversed the in vitro aging process of hUCBSCs. 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 hUCBSCs. From the molecular/cellular functional analysis of microarray data, we found that HMGA2 overexpression induced a PI3K/Akt/mTOR/p70S6K cascade, which in turn suppressed the expression of p16(INK4A) and p21(CIP1/WAF1) in hUCBSCs. These results provide novel insights into the mechanism by which HMGA2 regulates the in vitro aging and proliferation of hUCBSCs.

A p38 MAPK-Mediated Alteration of COX-2/PGE2 Regulates Immunomodulatory Properties in Human Mesenchymal Stem Cell Aging

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 this study, we evaluated the effect of replicative senescence on the immunomodulatory ability of hMSCs in vitro and in vivo. Late-passage hMSCs showed impaired suppressive effect on mitogen-induced mononuclear cell proliferation. Strikingly, 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. We 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. In conclusion, our data indicate that the immunomodulatory ability of hMSCs gradually declines with consecutive passages via a p38-mediated alteration of COX-2 and PGE2 levels.

Growth arrest and forced differentiation of human primary glioblastoma multiforme by a novel small molecule

Glioblastoma multiforme is the most common malignant brain tumor in adults, with an average survival of less than one year due to its resistance to therapy. Recent studies reported that GBM initiates from CD133-expressing cancer stem cells (CSC). However, the efficacy of CSC targeting is limited. A newly developed approach in cancer treatment is the forced differentiation of cancer cells. Here, we show that the treatment of the novel small molecule, CG500354, into CD133-expressing human primary GBM cells induces growth arrest by cell cycle regulators, p53, p21, p27 and phase-specific cyclins, and neural differentiation, as confirmed by neural progenitor/precursor markers, nestin, GFAP and Tuj1. When GBM-derived cells caused the tumors in NOD/SCID mice, CG500354 induced GBM-derived cells differentiation into Tuj1 and GFAP expressing cells. We next demonstrated that CG500354 plays a tumor-suppressive role via cAMP/CREB signaling pathway. CG500354 increases not only the extracellular cAMP level but also the protein level of PKA and CREB. Additionally, both mimetic substances, Forskolin and Rolipram, revealed comparable results with CG500354. Our findings indicate that induction of growth arrest and neural differentiation via cAMP/CREB signaling pathway by CG500354 treatment suggests the novel targeting of PDE4D in the development of new drugs for brain tumor therapy.

miR-543 and miR-590-3p regulate human mesenchymal stem cell aging via direct targeting of AIMP3/p18.

Previously, AIMP3 (aminoacyl-tRNAsynthetase-interacting multifunctional protein-3) was shown to be involved in the macromolecular tRNA synthetase complex or to act as a tumor suppressor. In this study, we report a novel role of AIMP3/p18 in the cellular aging of human mesenchymal stem cells (hMSCs). We found that AIMP3/p18 expression significantly increased in senescent hMSCs and in aged mouse bone marrow-derived MSCs (mBM-MSCs). AIMP3/p18 overexpression is sufficient to induce the cellular senescence phenotypes with compromised clonogenicity and adipogenic differentiation potential. To identify the upstream regulators of AIMP3/p18 during senescence, we screened for potential epigenetic regulators and for miRNAs. We found that the levels of miR-543 and miR-590-3p significantly decreased under senescence-inducing conditions, whereas the AIMP3/p18 protein levels increased. We demonstrate for the first time that miR-543 and miR-590-3p are able to decrease AIMP3/p18 expression levels through direct binding to the AIMP/p18 transcripts, which further compromised the induction of the senescence phenotype. Taken together, our data demonstrate that AIMP3/p18 regulates cellular aging in hMSCs possibly through miR-543 and miR-590-3p.

bFGF enhances the IGFs-mediated pluripotent and differentiation potentials in multipotent stem cells

It has widely been reported that basic fibroblast growth factor (bFGF) promotes proliferation of human stem cells and contributes to the maintenance of their self-renewal capability through repeated replications. In contrast to embryonic stem cells (ESCs), the effects of growth factors on adult stem cells are poorly understood. In human umbilical cord blood-derived multipotent stem cells (hUCB-MSCs), bFGF is associated with an increased number of proliferating cells. Furthermore, expression levels of ESC markers were increased after treatment with bFGF. bFGF also increased the expression of FGFR, which in turn increased expression of insulin-like growth factor (IGFs). Since IGFs exert autocrine and paracrine effects on stem cells, bFGF-mediated release of IGFs from hUCB-MSCs might enhance FGFR1 and IGF1R expression in neighboring cells. These receptors could subsequently regulate the effects of bFGF and IGFs in adult stem cells. These results suggest that positive feedback regulation of bFGF and IGFs leads to proliferation of hUCB-MSCs.

PGE2 maintains self-renewal of human adult stem cells via EP2-mediated autocrine signaling and its production is regulated by cell-to-cell contact.

Mesenchymal stem cells (MSCs) possess unique immunomodulatory abilities. Many studies have elucidated the clinical efficacy and underlying mechanisms of MSCs in immune disorders. Although immunoregulatory factors, such as Prostaglandin E2 (PGE2), and their mechanisms of action on immune cells have been revealed, their effects on MSCs and regulation of their production by the culture environment are less clear. Therefore, we investigated the autocrine effect of PGE2 on human adult stem cells from cord blood or adipose tissue, and the regulation of its production by cell-to-cell contact, followed by the determination of its immunomodulatory properties. MSCs were treated with specific inhibitors to suppress PGE2 secretion, and proliferation was assessed. PGE2 exerted an autocrine regulatory function in MSCs by triggering E-Prostanoid (EP) 2 receptor. Inhibiting PGE2 production led to growth arrest, whereas addition of MSC-derived PGE2 restored proliferation. The level of PGE2 production from an equivalent number of MSCs was down-regulated via gap junctional intercellular communication. This cell contact-mediated decrease in PGE2 secretion down-regulated the suppressive effect of MSCs on immune cells. In conclusion, PGE2 produced by MSCs contributes to maintenance of self-renewal capacity through EP2 in an autocrine manner, and PGE2 secretion is down-regulated by cell-to-cell contact, attenuating its immunomodulatory potency.