The Effects of Human Bone Marrow-Derived Mesenchymal Stem Cell Conditioned Media Produced with Fetal Bovine Serum or Human Platelet Lysate on Skin Rejuvenation Characteristics.

BACKGROUND AND OBJECTIVES: Human mesenchymal stem cell-conditioned medium (MSC-CM) is produced using mesenchymal stem cell culture technology and has various benefits for the skin, including wrinkle removal, skin regeneration, and increased antioxidant activity. Its popularity is thus increasing in the field of functional cosmetics. METHODS AND RESULTS: In this study, we analyzed the effects of fetal bovine serum-supplemented MSC-CM (FBSMSC-CM) and human platelet lysate-supplemented MSC-CM (hPL-MSC-CM) on skin rejuvenation characteristics. We found that the concentrations of important growth factors (VEGF, TGF-ß1, and HGF) and secretory proteins for skin regeneration were significantly higher in hPL-MSC-CM than in FBS-MSC-CM. Furthermore, the capacity for inducing proliferation of human dermal fibroblast (HDF) and keratinocytes, the migration ability of HDF, extracellular matrix (ECM) production such as collagen and elastin was higher in hPL-MSC-CM than that in FBSMSC-CM. CONCLUSIONS: These results support the usefulness and high economic value of hPL-MSC-CM as an alternative source of FBS-MSC-CM in the cosmetic industry for skin rejuvenation.

Culturing at Low Cell Density Delays Cellular Senescence of Human Bone Marrow-Derived Mesenchymal Stem Cells in Long-Term Cultures.

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSCs) have immense therapeutic potential for treating intractable and immune diseases. They also have applications in regenerative medicine in which distinct treatments do not exist. Thus, MSCs are gaining attention as important raw materials in the field of cell therapy. Importantly, the number of MSCs in the bone marrow is limited and they are present only in small quantities. Therefore, mass production of MSCs through long-term culture is necessary to use them in cell therapy. However, MSCs undergo cellular senescence through repeated passages during mass production. In this study, we explored methods to prolong the limited lifetime of MSCs by culturing them with different seeding densities. METHODS AND RESULTS: We observed that in long-term cultures, low-density (LD, 50 cells/cm2) MSCs showed higher population doubling level, leading to greater fold increase, than high-density (HD, 4,000 cells/cm2) MSCs. LD-MSCs suppressed the expression of aging-related genes. We also showed that reactive oxygen species (ROS) were decreased in LD-MSCs compared to that in HD-MSCs. Further, proliferation potential increased when ROS were inhibited in HD-MSCs. CONCLUSIONS: The results in this study suggest that MSC senescence can be delayed and that life span can be extended by controlling cell density in vitro. These results can be used as important data for the mass production of stem cell therapeutic products.

rhBMP-2 Pre-Treated Human Periodontal Ligament Stem Cell Sheets Regenerate a Mineralized Layer Mimicking Dental Cementum.

The periodontal complex consisting of alveolar bone, cementum, and periodontal ligaments (PDL) supports human teeth through the systematic orchestration of mineralized tissues and fibrous tissues. Importantly, cementum, the outermost mineralized layer of dental roots, plays an essential role by bridging the inner ligaments from the dental root to the alveolar bone. When the periodontal complex is damaged, the regeneration of each component of the periodontal complex is necessary; however, it is still challenging to achieve complete functional regeneration. In this study, we tried to control the regeneration of cementum and PDL by using a human PDL stem cell (hPDLSC) sheet engineering technology with the pretreatment of recombinant human BMP-2 (rhBMP-2). Isolated hPDLSCs obtained from extracted human teeth were pretreated with rhBMP-2 for in vitro osteogenic differentiation and grafted on the micro/macro-porous biphasic calcium phosphate (MBCP) blocks, which represent dental roots. The MBCPs with hPDLSC sheets were implanted in the subcutaneous layer of immune-compromised mice, and rhBMP-2 pretreated hPDLSC sheets showed higher mineralization and collagen ligament deposition than the no-pretreatment group. Therefore, the rhBMP-2-hPDLSC sheet technique could be an effective strategy for the synchronized regeneration of two different tissues: mineralized tissue and fibrous tissues in periodontal complexes.

Comparative study on metabolite level in tissue-specific human mesenchymal stem cells by an ultra-performance liquid chromatography quadrupole time of flight mass spectrometry.

Mesenchymal stem cells (MSCs) are a promising therapeutic option for cell-based therapy due to their immunomodulatory and regenerative properties. They can be isolated from various adult tissues, including bone marrow, fat, dental tissue, and glandular tissue. Although they share common characteristics, little is known about the biological differences between MSC populations derived from different tissues. In this study, we used MS to compare the endogenous metabolite level in the human MSCs originating from the bone marrow, adipose tissue, periodontal ligaments, and salivary glands. Using an optimized metabolomics technique, we verified that human MSCs exhibit differences in the endogenous metabolite level depending on their source material, while the multivariate analysis showed that 5 lysophosphatidylcholines and 3 lysophosphatidylethanolamines can serve as markers for the discrimination between MSC sources and may be related to differences in their differentiation capacity. These results may significantly contribute to further mechanistic studies on the MSCs and provide novel insights into the properties and optimal usage of MSCs from different tissues.

ICOSL expression in human bone marrow-derived mesenchymal stem cells promotes induction of regulatory T cells.

Mesenchymal stem cells (MSCs) can modulate lymphocyte proliferation and function. One of the immunomodulatory functions of MSCs involves CD4+CD25+FoxP3+ regulatory T cells (Tregs), which negatively regulate inflammatory responses. MSC-mediated Treg induction is supposed to be regulated by mechanisms requiring both soluble and cell contact-dependent factors. Although the involvement of soluble factors has been revealed, the contact-dependent mechanisms in MSC-mediated Treg induction remain unclear. We attempted to identify molecule(s) other than secreted factors that are responsible for MSC-mediated Treg induction and to uncover the underlying mechanisms. Under in vitro Treg-inducing conditions, ICOSL expression in MSCs coincided with Treg induction in co-cultures of MSCs with CD4+ T cells. When cultured in a transwell plate, MSCs failed to induce Tregs. Neutralization or knockdown of ICOSL significantly reduced Tregs and their IL-10 release. ICOSL overexpression in MSCs promoted induction of functional Tregs. ICOSL-ICOS signaling promoted Treg differentiation from CD4+ T cells through activation of the phosphoinositide 3-kinase-Akt pathway. MSCs primed with Interleukin-1ß significantly induced Tregs through ICOSL upregulation. We demonstrated that the Treg-inducing activity of MSCs is proportionate to their basal ICOSL expression. This study provides evidence that ICOSL expression in human MSCs plays an important role in contact-dependent regulation of MSC-mediated Treg induction.

Galectin-9 is Involved in Immunosuppression Mediated by Human Bone Marrow-derived Clonal Mesenchymal Stem Cells.

Bone marrow-derived mesenchymal stem cells (MSCs) have immunomodulatory properties and can suppress exaggerated pro-inflammatory immune responses. Although the exact mechanisms remain unclear, a variety of soluble factors are known to contribute to MSC-mediated immunosuppression. However, functional redundancy in the immunosuppressive properties of MSCs indicates that other uncharacterized factors could be involved. Galectin-9, a member of the ß-galactoside binding galectin family, has emerged as an important regulator of innate and adaptive immunity. We examined whether galectin-9 contributes to MSC-mediated immunosuppression. Galectin-9 was strongly induced and secreted from human MSCs upon stimulation with pro-inflammatory cytokines. An in vitro immunosuppression assay using a knockdown approach revealed that galectin-9-deficient MSCs do not exert immunosuppressive activity. We also provided evidence that galectin-9 may contribute to MSC-mediated immunosuppression by binding to its receptor, TIM-3, expressed on activated lymphocytes, leading to apoptotic cell death of activated lymphocytes. Taken together, our findings demonstrate that galectin-9 is involved in MSC-mediated immunosuppression and represents a potential therapeutic factor for the treatment of inflammatory diseases.

Manufacture of Clinical-Grade Human Clonal Mesenchymal Stem Cell Products from Single Colony Forming Unit-Derived Colonies Based on the Subfractionation Culturing Method.

Stem cell products derived from mesenchymal stem cells (MSCs) have been widely used in clinical trials, and a few products have been already commercialized. However, the therapeutic effects of clinical-grade MSCs are still controversial owing to mixed results from recent clinical trials. A potential solution to overcome this hurdle may be to use clonal stem cells as the starting cell material to increase the homogeneity of the final stem cell products. We have previously developed an alternative isolation and culture protocol for establishing a population of clonal MSCs (cMSCs) from single colony forming unit (CFU)-derived colonies. In this study, we established a good manufacturing practice (GMP)-compatible procedure for the clinical-grade production of human bone marrow-derived cMSCs based on the subfractionation culturing method. We optimized the culture procedures to expand and obtain a clonal population of final MSC products from single CFU-derived colonies in a GMP facility. The characterization results of the final cMSC products met our preset criteria. Animal toxicity tests were performed in a good laboratory practice facility, and showed no toxicity or tumor formation in vivo. These tests include single injection toxicity, multiple injection toxicity, biodistribution analysis, and tumorigenicity tests in vivo. No chromosomal abnormalities were detected by in situ karyotyping using oligo-fluorescence in situ hydridization (oligo-FISH), providing evidence of genetic stability of the clinical-grade cMSC products. The manufacture and quality control results indicated that our GMP methodology could produce sufficient clonal population of MSC products from a small amount of bone marrow aspirate to treat a number of patients.

Establishment and characterization of mesenchymal stem cell-like clonal stem cells from mouse salivary glands.

Successful therapy for radiation-induced salivary gland (SG) hypofunction is currently unavailable; however, tissue-specific stem cells are expected to be promising candidates for SG regeneration. Here, we present our method for the establishment of single cell-derived clonal stem cells from mouse SGs and describe their characteristics. Salivary gland-derived clonal stem cells (SGSCs) were isolated and expanded in vitro by a modified subfractionation culture method. The properties of SGSCs were examined with respect to their marker expression, gene expression, differentiation potential, and in vitro immunosuppressive activity relative to bone marrow-derived mesenchymal stem cells (BM-MSCs). SGSCs appeared to largely share the characteristics of BM-MSCs based on their marker expression, whereas they differentially expressed some genes, including AQP5, E-Cadherin, Laminin, ZO-1, and COL4. SGSCs showed the ability to differentiate into fat, bone, and cartilage cell types, as well as into α-amylase-producing and hepatocyte-like cells after appropriate induction. The in vitro immunosuppressive activity of SGSCs was found to be more potent than that of BM-MSCs. These results showed that SGSCs possess the properties of MSCs with some differential gene expression and they are salivary-specific stem cells with both epithelial and mesenchymal properties. The biological functions of SGSCs and their relevance to SG epithelial progenitor cells require further investigation.

Mesenchymal stem cells' interaction with skin: wound-healing effect on fibroblast cells and skin tissue.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the ability to secrete growth factors. Because wound healing is associated with fibroblast cells and extracellular matrix (ECM) in the dermis and epidermis, we used fibroblast cells to resolve the question of whether or not MSCs regulate wound healing in vitro via a regenerative function. Using a cell proliferation assay, we demonstrated that conditioned media (CM) obtained from MSCs significantly enhanced the cell survival ability of fibroblast cells. Moreover, by measurement of mRNA and protein, we observed that CM also promoted the production or secretion of collagen, elastin, and fibronectin. To better understand the effects of ECM-related wound healing, we measured the level of collagen-degradative enzyme (matrix metalloprotease-1), and observed that CM suppressed matrix metalloprotease-1 expression. For the determination of oxidative stress, which has an influence on wound healing, we performed the superoxide dismutase and glutathione peroxidase assays; our results suggested that CM inhibited the oxidative stress of fibroblast cells. In order to widely investigate the wound-healing effects of MSCs, we performed in vivo experiments, and observed that MSCs stimulated wound healing. In summary, the results of this study suggest that MSCs inhibit the loss of fibroblast cells and ECM, and accumulation of oxidative stress. We found that MSCs stimulate wound healing in vitro and in vivo, suggesting that MSCs have the potential to enhance wound healing.

Anti-angiogenic activity of a homoisoflavanone from Cremastra appendiculata.

A homoisoflavanone, 5,7-dihydroxy-3-(3-hydroxy-4-methoxybenzyl)-6-methoxychroman-4-one ( 1), was isolated from the bulb of Cremastra appendiculata (D. Don) Makino (Orchidaceae) as a potent inhibitor of angiogenesis. It inhibited basic fibroblast growth factor (bFGF)-induced in vitro angiogenesis and in vivo angiogenesis of the chorioallantoic membrane (CAM) of chick embryo without showing any toxicity.