Mesenchymal stem cells (MSCs) have been recognized as a therapeutic tool for various diseases due to its unique ability for tissue regeneration and immune regulation. However, poor survival during in vitro expansion and after being administrated in vivo limits its clinical uses. Accordingly, protocols for enhancing cell survivability is critical for establishing an efficient cell therapy is needed. CDDOMe is a synthetic C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid, which is known to stimulate nuclear factor erythroid 2-related factor 2 (Nrf2)- antioxidant response element (ARE) pathway. Herein, report that CDDO-Me promoted the proliferation of MSCs and increased colony forming units (CFU) numbers. No alteration in differentiation into tri-lineage mesodermal cells was found after CDDO-Me treatment. We observed that CDDO-Me treatment reduced the cell death induced by oxidative stress, demonstrated by the augment in the expression of Nrf2-downstream genes. Lastly, CDDO-Me led to the nuclear translocation of NRF2. Our data indicate that CDDO-Me can enhance the functionality of MSCs by stimulating cell survival and increasing viability under oxidative stress.

Herbal medicine has been the basis for medical treatments through much of human history, and such traditional medicine is still widely practiced today. Modern medicine makes use of many plant-derived compounds as the basis for pharmaceutical drugs. In traditionally, Achyranthes aspera, Safflower (Carthamus tinctorius) seed and Acanthopanax senticosus have been used for the treatment and prevention of bone-related diseases. In this study, we investigated the pharmacological effect of mixture of Achyranthes aspera, Safflower (Carthamus tinctorius) seed and Acanthopanax senticosus and the other herbs. Two types of enzymes were used to enhance the extraction components of amino acid, mineral content, free sugar, and flavor recovery in extracting natural herbal mixtures(NME). We evaluated regulation of osteogenic differentiation in human bone marrow mesenchymal stem cells using alkaline phosphatase staining, alizarin red S staining and RT-PCR. The CCK-8 assay indicated that NME had no cytotoxicity but increased cell survival. In addition, NME promoted the mineralization and expression of osteogenic differention marker genes in human bone marrow mesenchymal stem cells. Therefore, NME has an effect of promoting proliferation and osteogenic differentiation of human mesenchymal stem cell.

본 연구는 사람의 다양한 세포주를 이용하여 활성산소종(과산화수소수)이 세포의 노화에 미치는 영향을 비교 조사하였다. 여러 농도의 과산화수소수에 세포주를 일주일 동안 배양하여 MTT 방법으로 과산화수소수에 대한 세포 성장의 반억제농도를 구하였다. 그 결과, 50대에서 유래하는 피부 섬유아세포와 10대의 노화 유도 피부 섬유아세포와 비교하여 10대에서 유래하는 피부 섬유아세포에서 과산화수소수에 대한 반억제농도의 값이 유의적으로 더 높았고, 10대의 피부 섬유아세포보다는 10대의 여러 조직 기원하는 성체줄기세포에서 반억제농도의 값이 유의적으로 더 높게 관찰되었다. 또한, 50 ppm 과산화수소수를 1주일 동안 처리한 후, 50대의 피부 섬유아세포에서 다른 세포주에 비해 세포 성장이 현저히 억제되었고, 노화 관련 베타-갈락토시다아제의 활성이 증가되는 것을 관찰하였다. 또한, 활성산소의 세포 독성을 중화시키는 두 유전자, 글루타티온 과산화효소(GPX)와 카탈라아제(CAT)의 발현을 각 세포주에서 조사하였을 때, CAT의 발현은 모든 세포주에서 대체로 낮았지만, GPX 유전자의 발현이 50 대의 피부 섬유아세포보다 10대의 피부 섬유아세포와 성체줄기세포에서 현저히 높게 발현되는 것을 관찰하였다. 이상의 결과에서 활성산소는 세포 노화를 유도하고, GPX의 발현이 높은 10대의 피부 섬유아세포와 줄기세포보다는 50대의 피부 섬유아세포와 노화된 피부 섬유아세포에서 활성산소종에 대해 더 큰 민감성을 가지고 있는 것을 알 수 있었다.

Stem cells have special properties, such as self-renewal, proliferation, and the multilineage differentiation. Generally, stem cells are categorized into embryonic stem cells (ESCs), adult stem cells (ASCs), and induced pluripotent stem cells (iPSCs). Mesenchymal stem cells (MSCs) are a type of ASCs with a multipotent property. MSCs are easily isolated from various tissues and organs in the human body and can differentiation into multiple lineages, such as bone, cartilage, fat, and muscles. Compared to ESCs and iPSCs, MSCs possess less proliferation and differentiation capacities, therefore, a much scientific concern is concerned toward promoting the proliferation and the differentiation potency of MSCs. There are various methods to achieve this goal such as the treatment of various types of small molecules or culturing on specific peptides. Producing of high-quality MSCs with enhanced proliferation and differentiation capacities will definitely be a useful tool for stem cell-mediated tissue regeneration and the further clinical application.

The trans-differentiation potential of mesenchymal stem cells (MSCs) is employed, but there is little understanding of the cell source-dependent trans-differentiation potential of MSCs into corneal epithelial cells. In the present study, we induced trans-differentiation of MSCs derived from umbilical cord matrix (UCM-MSCs) and from dental tissue (D-MSCs), and we comparatively evaluated the in vitro trans-differentiation properties of both MSCs into corneal epithelial-like cells. Specific cell surface markers of MSC (CD44, CD73, CD90, and CD105) were detected in both UCM-MSCs and D-MSCs, but MHCII and CD119 were significantly lower (P < 0.05) in UCM-MSCs than in D-MSCs. In UCM-MSCs, not only expression levels of Oct3/4 and Nanog but also proliferation ability were significantly higher (P < 0.05) than in D-MSCs. In vitro differentiation abilities into adipocytes and osteocytes were confirmed for both MSCs. UCM-MSCs and D-MSCs were successfully trans-differentiated into corneal epithelial cells, and expression of lineage-specific markers (Cytokeratin-3, -8, and -12) were confirmed in both MSCs using immunofluorescence staining and qRT-PCR analysis. In particular, the differentiation capacity of UCM-MSCs into corneal epithelial cells was significantly higher (P < 0.05) than that of D-MSCs. In conclusion, UCM-MSCs have higher differentiation potential into corneal epithelial-like cells and have lower expression of CD119 and MHC class II than D-MSCs, which makes them a better source for the treatment of corneal opacity.

Although stem cells are used as important cell therapies in regenerative medicine, the electrophysiological problems that arise in the expansion of cells have not been known much. This study was conducted to investigate the functional expression of inward rectifying K+ current (IKir) using a patch-clamp technique, and the change in the resting membrane potential and the membrane capacitance were investigated in mesenchymal stem cells derived from human umbilical vein (hUC-MSC). The IKir plays an important role in regulating the resting membrane potential in many cells and is known to contribute to the maintenance of intracellular K+ concentration. In this study, electrophysiologically recorded current exhibited typical IKir characteristics. The current shifted along the K+ equilibrium potential (Ek) with the extracellular K+ concentration change. In addition, IKir was blocked by the divalent Ba2+ in a dose-dependent manner. The frequency of functional expression of IKir changed with number of passages (P2: 5.3% vs P8: 77.8% vs P12: 34.5%). There was no significant change in the resting membrane potential of hUC-MSC (P2: -21.0 mV, P8: -20.1 mV and P12: -21.9 mV). However, the capacitance of the cell membrane was significantly changed after P9 (P2: 8.9 pF vs P9: 16.9 pF) compared to P2. All the results suggest that changes in electrophysiological distribution of IKir as the passages increase may cause changes in K+ permeability even in cell proliferation and differentiation, suggesting a possible physiological role in maintaining cell homeostasis and resting membrane potential (RMP).

Fucoidan has been extensively studied as medicinal materials due to its biological activities including osteoblastic differentiation effect. However, osteoblastic effect by fucoidan is unknown in alveolar bone marrow derived mesenchymal stem cells (ABM-MSCs). The present study was undertaken to evaluate the effect of fucoidan on Osteoblastic differentiation in ABM-MSCs and explore its mechanism. Cell proliferation was analyzed by crystal violet staining. Osteoblast differentiation was determined by alkaline phosphatase activity, calcium accumulation assay and gene expression of osteoblast markers. We found that fucoidan induced cell proliferation of ABM-MSCs. Furthermore, fucoidan increased the ALP activity, calcium accumulation, and osteoblast specific genes such as Runx2, type I collagen alpha 1. Moreover, fucoidan induces the expression of asporin and bone morphogenic protein (BMP)-2 and asporin. Based on these results, these finding indicate that fucoidan induces osteoblast differentiation in ABM-MSCs and partially enhanced the mRNA expression of BMP-2 and asporin.

Bioactive peptides function effectively with a minimal amount compared to proteins. Recently SPARC related modular calcium binding 1 (SMOC1) has been implicated in regulating osteoblast differentiation and limb and eye development. In this study we synthesized a peptide covering 16 amino acids derived from the extracellular calcium binding (EC) domain of SMOC1, and its effects on proliferation and osteoblast differentiation of human bone marrow mesenchymal stem cells were examined. Treatment of SMOC1 peptide did not modulate proliferation of BMSCs. However, mineralization of BMSCs was significantly increased with a dose dependent manner. Consistently expression of osteoblast differentiation marker genes including type 1 collagen and osteocalcin was also dose dependently increased. Taken together, these results suggest that peptide derived from the EC domain of SMOC1 recapitulates at least partially osteogenic function of SMOC1.

Objective. To investigate the effects of the hypoxia inducible factor-1 (HIF-1) activation–mimicking agent cobalt chloride (CoCl2) on the osteogenic differentiation of human mesenchy-mal stem cells (hMSCs) and elucidate the underlying mole-cular mechanisms. Study design. The dose and exposure periods for CoCl2 in hMSCs were optimized by cell viability assays. After confirmation of CoCl2-induced HIF-1α and vas-cular endothelial growth factor expression in these cells by RT-PCR, the effects of temporary preconditioning with CoCl2 on hMSC osteogenic differentiation were evaluated by RT- PCR analysis of osteogenic gene expression, an alkaline phos-phatase (ALP) activity assay and by alizarin red S staining. Results. Variable CoCl2 dosages (up to 500 µM) and exposure times (up to 7 days) on hMSC had little effect on hMSC survival. After CoCl2 treatment of hMSCs at 100 µM for 24 or 48 hours, followed by culture in osteogenic differentiating media, several osteogenic markers such as Runx-2, osteocal-cin and osteopontin, bone sialoprotein mRNA expression level were found to be up-regulated. Moreover, ALP acti-vity was increased in these treated cells in which an accele-rated osteogenic capacity was also verified by alizarin red S staining. Conclusions. The osteogenic differentiation poten-tial of hMSCs could be preserved and even enhanced by CoCl2 treatment.

Human umbilical cord is easy to obtain because it is discarded after birth, so that ethical issues can be avoided. Chondrogenesis studies using MSCs from bone marrow, cord blood, and adipose have indicated that TGFβ3 and BMP6 stimulate chondrogenesis. Therefore, we investigated chondrogenesis of hUC-MSCs on TGFβ3, BMP6, and combination of the two growth factors. We initiated chondrogenesis of cells by application of physical forces to form 3D cell clusters. After initiation, we designated four experimental groups for differentiation of cells, as follows: control, 10 ng/mL TGFβ3, 100 ng/mL BMP6, and the combination of 5 ng/mL TGFβ3 and 50 ng/mL BMP6. For analysis of chondrogenesis, GAG contents, mRNA expression, histological analysis and immunohistochemistry (IHC) were performed. For analysis of GAG contents, GAG assay was performed and RT-PCR was performed for determination of chondrogenic markers. Histological analysis was performed through safranin O, alcian blue, and IHC was performed using collagen type I and II. GAG contents were increased 184% by TGFβ3, 147% by BMP6, and 189% by the combination of TGFβ3 and BMP6, compared to control. The growth factors improved collagen II and aggrecan expression; in particular, TGFβ3 and BMP6 showed a synergistic effect, compared to only TGFβ3 or BMP6 treated. The results of histological and IHC analysis indicated that chondrogenic differentiation in TGFβ3 and the combination of TGFβ3 and BMP6 showed more cartilage deposition. In conclusion, TGFβ3 and BMP6 differentiated hUC-MSCs into chondrogenic clusters of the combination treatment of the two growth factors showed more efficient chondrogenic ability.

The use of high throughput screening (HTS) in drug development is principally for the selection new drug candidates or screening of chemical toxicants. This system minimizes the experimental environment and allows for the screening of candidates at the same time. Umbilical cord-derived stem cells have some of the characteristics of fetal stem cell and have several advantages such as the ease with which they can be obtained and lack of ethical issues. To establish a HTS system, optimized conditions that mimic typical cell culture conditions in a minimal space such as 96 well plates are needed for stem cell growth. We have thus established a novel HTS system using human umbilical cord derived-mesenchymal stem cells (hUC-MSCs). To determine the optimal cell number, hUC-MSCs were serially diluted and seeded at 750, 500, 200 and 100 cells per well on 96 well plates. The maintenance efficiencies of these dilutions were compared for 3, 7, 9, and 14 days. The fetal bovine serum (FBS) concentration (20, 10, 5 and 1%) and the cell numbers (750, 500 and 200 cells/well) were compared for 3, 5 and 7 days. In addition, we evaluated the optimal conditions for cell cycle block. These four independent optimization experiments were conducted using an MTT assay. In the results, the optimal conditions for a HTS system using hUC-MSCs were determined to be 300 cell/well cultured for 8 days with 1 or 5% FBS. In addition, we demonstrated that the optimal conditions for a cell cycle block in this culture system are 48 hours in the absence of FBS. In addition, we candidates using our HTS system which demonstrates the feasibility if using hUC-MSCs for this type of screen. Moreover, the four candidate compounds can be tested for stem cell research application.

Human mesenchymal stem cell (hMSCs) isolated from human adult bone marrow have self-renewal capacity and can differentiate into multiple cell types in vitro and in vivo. A number of studies have now demonstrated that MSCs can differentiate into various neuronal populations. Due to their autologous characteristics, replacement therapy using MSCs is considered to be safe and does not involve immunological complications. The basic helix-loop-helix (bHLH) transcription factor Olig2 is necessary for the specification of both oligodendrocytes and motor neurons during vertebrate embryogenesis. To develop an efficient method for inducing neuronal differentiation from MSCs, we attempted to optimize the culture conditions and combination with Olig2 gene overexpression. We observed neuron-like morphological changes in the hMSCs under these induction conditions and examined neuronal marker expression in these cells by RTPCR and immunocytochemistry. Our data demonstrate that the combination of Olig2 overexpression and neuron-specific conditioned medium facilitates the neuronal differentiation of hMSCs in vitro. These results will advance the development of an efficient stem cell-mediated cell therapy for human neurodegenerative diseases.

The purpose of this study was to examine the effects of vi tamin D3 and 1'etinoic acid(RA) on the human mesenchymal stem ce!ls(MSC) g1'owth and osteogenic differentiations. Cell proliferation, mineralization, cell cycle, expression of cell cycle regu l atOJγ proteins and markers fo1' osteogenic differenatiaiton were determined by MTI assay, mineralization assay, flow cytomet1'Y‘ and Western blot analysis, respectively. Cell viability was dec1'ease by each vitamin D3 and RA added to MSC. it was more decrease by vitamin D3 and RA. Mineralized nodule formation revealed similar expression pattern with positive cont rol group at vitamin D3 and RA mixed add to MSC. At vitamin D3 and RA mixed add to MSC after 7 days of incubation was increase G1 s tage. after 21 days of incubation was inhibit cell cycle prog1'ess by inc1'ease of sub-G1 Treatment vitamin D3 to MSC inhibits p53 and p21, but inc1'ease pRb. RA inhibit p53, but increase p21 and pRb, vitamin D3 plus RA group was same as added RA group. so two vitamin was effect to inhibited cell growth each different mechanism. Expression of BMP-2 protein was prominent in osteogonic supplement treated g1'oup of MSC at 2 weeks cultivation days, but vi tamin D3 treatment decreased BMP-2 expression rather than in (+) control group. BSP protein was notably increased in the OS compa red to positive controls at 2 weeks cultivation, but similar to that of vitamin D3 group t1'eatment group and was least expressed in plus RA mixed group, at 3 weeks, BSP expression was similar to 1'esult of 2 weeks Collectively, these results shows that vitamin D3 and RA have diffe1'ential effects on the MSCs g1'owth and differ entia tion 211

The present study was conducted to investigate gelatinolytic activities in HAM and to determine whether there are any changes in gelatinolytic activity profiles when the cells are cultured in hepatogenic medium. Placenta was obtained during caesarean section of the volunteers, with informed consent. HAM were isolated from amniotic membrane using collagenase type A HAM were cultured in hepatogenic medium for 3 weeks and the conditioned media were obtained at day 7, 14 and 21. The zymographic pattern of gelatinolytic activity of the HAM did not undergo a change during passages. When the HAM were cultured in a fibronectin-coated dishes in a hepatogenic medium, there was no significant difference of the gelatinase pattern between before and after culture. However, when bFGF was added to the culture, a dramatic increase of 62kDa and 59kDa gelatinases was observed. Interestingly, when ITS instead of FN was present, HAM-conditioned medium also showed a similar increase of both gelatinases. Immunoblotting analysis demonstrated that both 62kDa and 59kDa gelatinases were the active form of MMP-2 resulting from the turnover of MMP-2 proform. Futher study will be necessary to determine the relationship between bFGF and active MMP-2 during hepatogenesis of HAM.

The use of human mesenchymal stem cells (hMSCs) in cell-based therapy has attracted extensive interest in the field of regenerative medicine, and it shows applications to numerous incurable diseases. hMSCs show several superior properties for therapeutic use compared to other types of stem cells. Different cell types are discussed in terms of their advantages and disadvantages, with focus on the characteristics of hMSCs. hMSCs can proliferate readily and produce differentiated cells that can substitute for the targeted affected tissue. To maximize the therapeutic effects of hMSCs, a substantial number of these cells are essential, requiring extensive ex vivo cell expansion. However, hMSCs have a limited lifespan in an in vitro culture condition. The senescence of hMSCs is a double-edged sword from the viewpoint of clinical applications. Although their limited cell proliferation potency protects them from malignant transformation after transplantation, senescence can alter various cell functions including proliferation, differentiation, and migration, that are essential for their therapeutic efficacy. Numerous trials to overcome the limited lifespan of mesenchymal stem cells are discussed.