Won Serk Kim

Hypoxia‐enhanced wound‐healing function of adipose‐derived stem cells: Increase in stem cell proliferation and up‐regulation of VEGF and bFGF

Adipose‐derived stem cells (ADSCs) have been shown to induce wound‐healing effects. Because inflammation near the wound area induces oxygen deficiency, it is interesting to elucidate the effect of hypoxia on the function of ADSCs. In this work, we asked: (1) does hypoxia alter the wound‐healing function of ADSCs? and (2) what are the major factors responsible for the alteration in the wound‐healing function? Effect of hypoxia on the proliferation of ADSCs was first examined that hypoxia (2% O2) enhanced the proliferation of ADSCs in either the presence of serum or in the absence of serum. The conditioned medium of ADSCs harvested under hypoxia (hypoCM) significantly promoted collagen synthesis and the migration of human dermal fibroblasts, compared with that in normoxia (norCM). In the animal studies, hypoCM significantly reduced the wound area compared with norCM. Furthermore, mRNA and protein measurements showed that hypoxia up‐regulated growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Inhibition of VEGF and bFGF using neutralizing antibodies reversed the migration of the wounded human dermal fibroblasts and the healing of wounds in animal experiment. Collectively, these results suggest that hypoxia increases the proliferation of ADSCs and enhances the wound‐healing function of ADSCs, at least partly, by up‐regulating the secretion of VEGF and bFGF.

Protective role of adipose-derived stem cells and their soluble factors in photoaging

As individuals age, the skin undergoes changes, such as irregular pigmentation, thinning and loss of elasticity, that are due to both genetic and environmental factors. These changes may worsen, progressing to precancerous and cancerous diseases. Various medical treatments and topical cosmeceuticals have been used to treat some symptoms of photoaging, however, the results have been less than satisfactory. Mesenchymal stem cells within the stromal-vascular fraction of subcutaneous adipose tissue, adipose-derived stem cells (ADSCs), display multi-lineage developmental plasticity and secrete various growth factors that control and manage the damaged neighboring cells. Recently, the production and secretion of growth factors has been reported as an essential function of ADSCs, and diverse regenerative effects of ADSCs have been demonstrated in the skin. For example, conditioned medium from ADSCs (ADSC-CM) stimulated both collagen synthesis and migration of dermal fibroblasts, which improved the wrinkling and accelerated wound healing in animal models. ADSC-CM also inhibited melanogenesis in B16 melanoma cells, and protected dermal fibroblasts from oxidative stress induced by chemicals and UVB irradiation. Therefore, ADSCs and soluble factors show promise for the treatment of photoaging, and this review introduces recent research developments of the ADSCs and ADSC-derived secretory factors regarding this issue.

Hypoxia induces adipocyte differentiation of adipose‐derived stem cells by triggering reactive oxygen species generation

Generation of reactive oxygen species (ROS) by NADPH oxidase 4 (Nox4) induces the proliferation and migration of adipose‐derived stem cells (ASCs). However, the functional role of mitochondrial ROS (mtROS) generation in ASCs is unknown. Therefore, we have investigated whether hypoxia induces the differentiation of ASCs via ROS generation. We also have tried to identify the cellular mechanisms of ROS generation underlying adipocyte differentiation. Hypoxia (2%) and ROS generators, such as antimycin and rotenone, induced adipocyte differentiation, which was attenuated by an ROS scavenger. Although Nox4 generates ROS and regulates proliferation of ASCs, Nox4 inhibition or Nox4 silencing did not inhibit adipocyte differentiation; indeed fluorescence intensity of mito‐SOX increased in hypoxia, and treatment with mito‐CP, a mtROS scavenger, significantly reduced hypoxia‐induced adipocyte differentiation. Phosphorylation of Akt and mTOR was induced by hypoxia, while inhibition of these molecules prevented adipocyte differentiation. Thus hypoxia induces adipocyte differentiation by mtROS generation, and the PI3K/Akt/mTOR pathway is involved.

Relationships between Lymph Node Metastasis and Expression of CD31, D2-40, and Vascular Endothelial Growth Factors A and C in Papillary Thyroid Cancer

Objectives To investigate the relationships between lymph node metastasis (LNM) and expression of CD31, D2-40 and vascular endothelial growth factors (VEGF)-A and -C in patients with papillary thyroid cancer (PTC). Methods Paraffin-embedded thyroid tissues of 72 patients were evaluated, which included 25 patients with thyroid nodular hyperplasia (TNH), 24 PTC patients without LNM, and 23 PTC patients with LNM. Three pathologists, who were blinded to the patients clinical information, assessed the immunohistochemical staining results. The amount of expression was scored as high (>25% of cells stained) or low (0-25%). Results A higher level of VEGF-A expression was observed in the PTC groups regardless of LNM when compared to the group with TNH (91.3%, 79.2%, 4.0%, respectively). VEGF-C expression in the PTC with LNM group was significantly higher than the other two groups (P<0.05). No difference in microvessel density (MVD) scores was observed using CD31 among the three groups. The lymphatic vessel density (LVD) score using D2-40 was significantly higher in patients having PTC with LNM than the other groups (P<0.05). Conclusion VEGF-C and D2-40 were more highly expressed in patients having PTC with LNM than in patients having PTC without LNM or in those having TNH. Analysis of VEGF-C level and LVD using D2-40 may be helpful in the diagnosis of PTC and the evaluation of LNM potential in patients with PTC.

Isolation of adipose-derived stem cells by using a subfractionation culturing method

Objective: Adipose-derived stem cells (ASCs) isolated from subcutaneous adipose tissue have been tested in clinical trials. However, ASCs isolated by enzyme digestion and centrifugation are heterogeneous and exhibit wide variation in regenerative potential and clinical outcomes. Therefore, we developed a new method for isolating clonal ASCs (cASCs) that does not use enzyme digestion or centrifugation steps. Research design and methods: In addition to cell surface markers and differentiation potential, we compared the mitogenic, paracrine and hair growth-promoting effects of ASCs isolated by the gradient centrifugation method (GCM) or by the new subfractionation culturing method (SCM). Results: We selected three cASCs isolated by SCM that showed high rates of proliferation. The cell surface markers expressed by ASCs isolated by GCM or SCM were very similar, and SCM-isolated ASCs could potentially differentiate into different cell lineages. However, cASC lines exhibited better mitogenic and paracrine effects than ASCs isolated by GCM. The expression of Diras3, Myb, Cdca7, Mki67, Rrm2, Cdk1 and Ccna2, which may play a key role in cASC proliferation, was upregulated in cASCs. In addition, cASCs exhibited enhanced hair growth-promoting effects in dermal papilla cells and animal experiments. Conclusions: SCM generates a highly homogeneous population of ASCs via a simple and effective procedure that can be used in therapeutic settings.

An update on niche composition, signaling and functional regulation of the adipose-derived stem cells

Introduction: The self-renewal and differentiation of stem cells are controlled by both intrinsic factors and the surrounding microenvironment, which is known as the stem cell niche. Although the niches of adipose-derived stem cells (ASCs) are composed of diverse factors within the adipose tissue, the mechanisms by which niches are maintained, regulated and harmonized to support the ASCs are just beginning to be discovered. Areas covered: This review introduces the recent advances in the anatomic nature of the dynamic in vivo niches of ASCs. Additionally, new findings concerning the signaling pathways involved in the self-renewal, proliferation, differentiation and paracrine mechanisms will be described. Finally, we suggest optimized methods for expanding ASCs in vitro by mimicking the niche factors to enhance the regenerative potential of ASCs. Expert opinion: Fibroblast growth factor 2 is a self-renewal factor that can expand the lifespan of ASCs in long-term culture and platelet-derived growth factor-B/D has most potent mitogenic effects on short-term ASC expansion. Reactive oxygen species donors and stimulators of the phosphoinositide 3-kinase/protein kinase B and MAPK pathways can be used to increase the production yield of ASCs. Additionally, hypoxia can increase the proliferation of ASCs and priming under hypoxic conditions enhances the regenerative potential of ASCs.

Lysophosphatidic acid increases the proliferation and migration of adipose‑derived stem cells via the generation of reactive oxygen species

Phospholipid derivatives, such as lysophosphatidic acid (LPA), exhibit mitogenic effects on mesenchymal stem cells; however, the molecular mechanism underlying this stimulation has yet to be identified. The aims of the present study were as follows: To evaluate the stimulatory effects of LPA on the proliferation and migration of adipose‑derived stem cells (ASCs); to study the association between reactive oxygen species (ROS) and LPA signaling in ASCs; and to investigate the microRNAs upregulated by LPA treatment in ASCs. The results of the present study demonstrated that LPA increased the proliferation and migration of ASCs, and acted as a mitogenic signal via extracellular signal‑regulated kinases 1/2 and the phosphoinositide 3‑kinase/Akt signaling pathways. The LPA1 receptor is highly expressed in ASCs, and pharmacological inhibition of it by Ki16425 significantly attenuated the proliferation and migration of ASCs. In addition, LPA treatment generated ROS via NADPH oxidase 4, and ROS were able to function as signaling molecules to increase the proliferation and migration of ASCs. The induction of ROS by LPA treatment also upregulated the expression of miR‑210. A polymerase chain reaction array assay demonstrated that the expression levels of adrenomedullin and Serpine1 were increased following treatment with LPA. Furthermore, transfection with Serpine1‑specific small interfering RNA attenuated the migration of ASCs. In conclusion, the present study is the first, to the best of our knowledge, to report that ROS generation and miR‑210 expression are associated with the LPA‑induced stimulation of ASCs, and that Serpine1 mediates the LPA‑induced migration of ASCs. These results further suggest that LPA may be used for ASC stimulation during stem cell expansion.

The pivotal role of PDGF and its receptor isoforms in adipose-derived stem cells.

Platelet-derived growth factor (PDGF) is one of the growth factors that reportedly regulates cell growth and division of mesenchymal cells. Although PDGF isoforms and their receptors reportedly play a pivotal role in mesenchymal stem cell regulation, there is a paucity of literature reviewing the role of PDGF in adipose-derived stem cells (ASCs). Therefore, we summarized previous reports on the expression and functional roles of PDGF and its receptor isoforms in this review. In addition, we examined findings pertaining to underlying molecular mechanisms and signaling pathways with special focus on PDGF-D/PDGFRβ. ASCs only express PDGF-A, -C, -D, PDGFRα, and PDGFRβ. PDGFRα expression decreases with adipocyte lineage, while PDGFRβ inhibits white adipocyte differentiation. In addition, PDGFRβ induces proliferation, migration, and angiogenesis and up-regulates the expression of paracrine factors in ASCs. Although PDGF-B and -D mediate their functions mainly by PDGFRβ and ROS generation, there are many differences between them in terms of regulating ASCs. PDGF-D is endogenous, generates ROS via the mitochondrial electron transport system, and regulates the autocrine loop of ASCs in vivo. Furthermore, PDGF-D has stronger mitogenic effects than PDGF-B.

Hypoxia Suppresses Spontaneous Mineralization and Osteogenic Differentiation of Mesenchymal Stem Cells via IGFBP3 Up-Regulation

Hypoxia has diverse stimulatory effects on human adipose-derived stem cells (ASCs). In the present study, we investigated whether hypoxic culture conditions (2% O2) suppress spontaneous mineralization and osteogenic differentiation of ASCs. We also investigated signaling pathways and molecular mechanisms involved in this process. We found that hypoxia suppressed spontaneous mineralization and osteogenic differentiation of ASCs, and up-regulated mRNA and protein expression of Insulin-like growth factor binding proteins (IGFBPs) in ASCs. Although treatment with recombinant IGFBPs did not affect osteogenic differentiation of ASCs, siRNA-mediated inhibition of IGFBP3 attenuated hypoxia-suppressed osteogenic differentiation of ASCs. In contrast, overexpression of IGFBP3 via lentiviral vectors inhibited ASC osteogenic differentiation. These results indicate that hypoxia suppresses spontaneous mineralization and osteogenic differentiation of ASCs via intracellular IGFBP3 up-regulation. We determined that reactive oxygen species (ROS) generation followed by activation of the MAPK and PI3K/Akt pathways play pivotal roles in IGFBP3 expression under hypoxia. For example, ROS scavengers and inhibitors for MAPK and PI3K/Akt pathways attenuated the hypoxia-induced IGFBP3 expression. Inhibition of Elk1 and NF-κB through siRNA transfection also led to down-regulation of IGFBP3 mRNA expression. We next addressed the proliferative potential of ASCs with overexpressed IGFBP3, but IGFBP3 overexpression reduced the proliferation of ASCs. In addition, hypoxia reduced the osteogenic differentiation of bone marrow-derived clonal mesenchymal stem cells. Collectively, our results indicate that hypoxia suppresses the osteogenic differentiation of mesenchymal stem cells via IGFBP3 up-regulation.

Mitochondria-Targeted Vitamin E Protects Skin from UVB-Irradiation

Mitochondria-targeted vitamin E (MVE) is designed to accumulate within mitochondria and is applied to decrease mitochondrial oxidative damage. However, the protective effects of MVE in skin cells have not been identified. We investigated the protective effect of MVE against UVB in dermal fibroblasts and immortalized human keratinocyte cell line (HaCaT). In addition, we studied the wound-healing effect of MVE in animal models. We found that MVE increased the proliferation and survival of fibroblasts at low concentration (i.e., nM ranges). In addition, MVE increased collagen production and downregulated matrix metalloproteinase1. MVE also increased the proliferation and survival of HaCaT cells. UVB increased reactive oxygen species (ROS) production in fibroblasts and HaCaT cells, while MVE decreased ROS production at low concentration. In an animal experiment, MVE accelerated wound healing from laser-induced skin damage. These results collectively suggest that low dose MVE protects skin from UVB irradiation. Therefore, MVE can be developed as a cosmetic raw material.