Achaete-scute family bHLH transcription factor 2 activation promotes hepatoblastoma progression.

Achaete-scute family bHLH transcription factor 2 (ASCL2) is highly expressed in hepatoblastoma (HB) tissues, but its role remains unclear. Thus, biological changes in the HB cell line HepG2 in response to induced ASCL2 expression were assessed. ASCL2 expression was induced in HepG2 cells using the Tet-On 3G system, which includes doxycycline. Cell viability, proliferation activity, mobility, and stemness were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony-formation, migration, invasion, and sphere-formation assays. Quantitative reverse-transcription polymerase chain reaction was used to assess the expression of markers for proliferation (CCND1 and MYC), epithelial-mesenchymal transition (EMT; SNAI1, TWIST1, and ZEB1), mesenchymal-epithelial transition (CDH1), and stemness (KLF4, POU5F1, and SOX9). Compared with the non-induced HepG2 cells, cells with induced ASCL2 expression showed significant increases in viability, colony number, migration area (%), and sphere number on days 7, 14, 8, and 7, respectively, and invasion area (%) after 90 h. Furthermore, induction of ASCL2 expression significantly upregulated CCND1, MYC, POU5F1, SOX9, and KLF4 expression on days 2, 2, 3, 3, and 5, respectively, and increased the ratios of SNAI1, TWIST1, and ZEB1 to CDH1 on day 5. ASCL2 promoted the formation of malignant phenotypes in HepG2 cells, which may be correlated with the upregulation of the Wnt signaling pathway-, EMT-, and stemness-related genes. ASCL2 activation may therefore be involved in the progression of HB.

Polymethylmethacrylate-hydroxyapatite antibacterial and antifungal activity against oral bacteria: An in vitro study.

Objective: Reconstruction of alveolar bone defects resulting from aging, trauma, ablative surgery or pathology, remains a significant clinical challenge. The objective of this study was to investigate the antibacterial and antifungal activities of mixed polymethylmethacrylate-hydroxyapatite (PMMA-HA) against oral microorganisms. Our findings could provide valuable insights into the prospective application of PMMA-HA as a synthetic bone graft material to manage alveolar bone defects via tissue engineering. Methods: HA powder was obtained from the Center for Ceramics in Indonesia and PMMA granules were obtained from HiMedia Laboratories; these were prepared in 20:80, 30:70, and 40:60 ratios. The antibacterial diffusion method was then performed against Staphylococcusaureus, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Fusobacterium nucleatum, while the antifungal diffusion method was used to test against Candida albicans. Standardized protocols were used for microbial culturing and inhibition zones were measured with digital calipers. Statistical analyses included one-way ANOVA and Kruskal-Wallis tests, supplemented by post-hoc Tukey HSD tests. Results: A PMMA-HA scaffold with a 20:80 ratio demonstrated the highest antibacterial activity against S. aureus, A. actinomycetemcomitans, P. gingivalis, and F. nucleatum. This was followed by the 30:70 and 40:60 ratios in terms of antibacterial activity. Statistical significance was achieved with p < 0.05 in comparison to controls. However, none of the PMMA-HA ratios showed antifungal activity against C. albicans. Conclusion: PMMA-HA scaffolds have significant activity against bacteria, but not against fungi.

The Effect of Mixed Polymethylmethacrylate and Hydroxyapatite on Viability of Stem Cell from Human Exfoliated Deciduous Teeth and Osteoblast.

OBJECTIVES: Stem cell from human exfoliated deciduous teeth (SHED) has great potential for bone tissue engineering and cell therapy for regenerative medicine. It has been combined with biomaterials such as mixed of polymethylmethacrylate (PMMA) and hydroxyapatite (HA) as candidates for synthetic bone graft biomaterial. The aim of this study was to analyze the toxicity test of mixed PMMA-HA scaffold seeded with SHED and osteoblast in vitro. MATERIALS AND METHODS: SHED was isolated from the pulp of noncarious deciduous teeth and osteoblast cells were cultured, and exposed to PMMA-HA scaffolds with three concentration groups: 20/80, 30/70, and 40/60 for 24 hours. Cytotoxicity test was performed by MTT assay to cell viability. STATISTICAL ANALYSIS: Data were analyzed using IBM SPSS Statistics 25, one-way analysis of variance followed by least significant difference test, considering the level of significance p-value less than 0.05 RESULTS: The percentage of SHED's viability was best in the PMMA-HA group with concentrations of 20/80, followed by 30/70, and 40/60 with 87.03, 75.33, and 65.79%, respectively. The percentage of osteoblast cell's viability was best in the PMMA-HA group with concentrations of 20/80, followed by 30/70, and 40/60 with 123.6, 108.36, and 93.48%, respectively. CONCLUSIONS: Mixed PMMA-HA was not toxic for the SHED and osteoblast. This characteristic is the initial requirement to be proposed as an alternative material for healing alveolar bone defects. In vivo animal research is mandatory to confirm the use of PMMA-HA on the alveolar defect model.

Oncogenic Role of ADAM32 in Hepatoblastoma: A Potential Molecular Target for Therapy.

Outcomes of pediatric hepatoblastoma (HBL) have improved, but refractory cases still occur. More effective and safer drugs are needed that are based on molecular mechanisms. A disintegrin and metalloproteases (ADAMs) are expressed with high frequency in various human carcinomas and play an important role in cancer progression. In this study, we analyzed expression of ADAMs in HBL with a cDNA microarray dataset and found that the expression level of ADAM32 is particularly high. To investigate the role of ADAM32 in cancer, forced expression or knockdown experiments were conducted with HepG2 and HBL primary cells. Colony formation, cell migration and invasion, and cell viability were increased in HepG2 expressing ADAM32, whereas knockdown of ADAM32 induced a decrease in these cellular functions. Quantitative RT-PCR demonstrated an association between ADAM32 expression and the expression of genes related to cancer stem cells and epithelial-mesenchymal transition (EMT), suggesting a role of ADAM32 in cancer stemness and EMT. Furthermore, knockdown of ADAM32 increased cisplatin-induced apoptosis, and this effect was attenuated by a caspase-8 inhibitor, suggesting that ADAM32 plays a role in extrinsic apoptosis signaling. We conclude that ADAM32 plays a crucial role in progression of HBL, so it might be a promising molecular target in anticancer therapy.

Study of Alveolar Bone Remodeling Using Deciduous Tooth Stem Cells and Hydroxyapatite by Vascular Endothelial Growth Factor Enhancement and Inhibition of Matrix Metalloproteinase-8 Expression in vivo.

Background: Periodontitis progression is characterized by alveolar bone loss, and its prevention is a major clinical problem in periodontal disease management. Matrix metalloproteinase-8 (MMP-8) has been shown to adequately monitor the treatment of chronic periodontitis patients as gingival crevicular fluid MMP-8s were positively associated with the severity of periodontal disease. Moreover, modulating the vascular endothelial growth factor (VEGF) levels in bones could be a good way to improve bone regeneration and cure periodontitis as VEGF promotes endothelial cell proliferation, proteolytic enzyme release, chemotaxis, and migration; all of which are required for angiogenesis. Purpose: The aim of this study was to determine the effect of hydroxyapatite incorporated with stem cells from exfoliated deciduous teeth (SHED) in Wistar rats' initial alveolar bone remodeling based on the findings of MMP-8 and VEGF expressions. Methods: A hydroxyapatite scaffold (HAS) in conjunction with SHED was transplanted into animal models with alveolar mandibular defects. A total of 10 Wistar rats (Rattus norvegicus) were divided into two groups: HAS and HAS + SHED. Immunohistochemistry staining was performed after 7 days to facilitate the examination of MMP-8 and VEGF expressions. Results: The independent t-test found significant downregulation of MMP-8 and upregulation VEGF expressions in groups transplanted with HAS in conjunction with SHED compared with the HAS group (p < 0.05). Conclusion: The combination of SHED with HAS on alveolar bone defects may contribute to initial alveolar bone remodeling as evident through the assessments of MMP-8 and VEGF expressions.

The Identification of Marker Genes for Predicting the Osteogenic Differentiation Potential of Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) have the potential to differentiate into a variety of mature cell types and are a promising source of regenerative medicine. The success of regenerative medicine using MSCs strongly depends on their differentiation potential. In this study, we sought to identify marker genes for predicting the osteogenic differentiation potential by comparing ilium MSC and fibroblast samples. We measured the mRNA levels of 95 candidate genes in nine ilium MSC and four fibroblast samples before osteogenic induction, and compared them with alkaline phosphatase (ALP) activity as a marker of osteogenic differentiation after induction. We identified 17 genes whose mRNA expression levels positively correlated with ALP activity. The chondrogenic and adipogenic differentiation potentials of jaw MSCs are much lower than those of ilium MSCs, although the osteogenic differentiation potential of jaw MSCs is comparable with that of ilium MSCs. To select markers suitable for predicting the osteogenic differentiation potential, we compared the mRNA levels of the 17 genes in ilium MSCs with those in jaw MSCs. The levels of 7 out of the 17 genes were not substantially different between the jaw and ilium MSCs, while the remaining 10 genes were expressed at significantly lower levels in jaw MSCs than in ilium MSCs. The mRNA levels of the seven similarly expressed genes were also compared with those in fibroblasts, which have little or no osteogenic differentiation potential. Among the seven genes, the mRNA levels of IGF1 and SRGN in all MSCs examined were higher than those in any of the fibroblasts. These results suggest that measuring the mRNA levels of IGF1 and SRGN before osteogenic induction will provide useful information for selecting competent MSCs for regenerative medicine, although the effectiveness of the markers is needed to be confirmed using a large number of MSCs, which have various levels of osteogenic differentiation potential.

Genetic Markers Can Predict Chondrogenic Differentiation Potential in Bone Marrow-Derived Mesenchymal Stromal Cells.

The precise predictions of the differentiation direction and potential of mesenchymal stromal cells (MSCs) are an important key to the success of regenerative medicine. The expression levels of fate-determining genes may provide tools for predicting differentiation potential. The expression levels of 95 candidate marker genes and glycosaminoglycan (GAG) contents after chondrogenic induction in 10 undifferentiated ilium and 5 jaw MSC cultures were determined, and their correlations were analyzed. The expression levels of eight genes before the induction of chondrogenic MSC differentiation were significantly correlated with the GAG levels after induction. Based on correlation patterns, the eight genes were classified into two groups: group 1 genes (AURKB, E2F1, CDKN2D, LIF, and ACLY), related to cell cycle regulation, and group 2 genes (CD74, EFEMP1, and TGM2), involved in chondrogenesis. The expression levels of the group 2 genes were significantly correlated with the ages of the cell donors. The expression levels of CDKN2D, CD74, and TGM2 were >10-fold higher in highly potent MSCs (ilium MSCs) than in MSCs with limited potential (jaw MSCs). Three-dimensional (3D) scatter plot analyses of the expression levels of these genes showed reduced variability between donors and confirmed predictive potential. These data suggest that group 2 genes are involved in age-dependent decreases in the chondrogenic differentiation potential of MSCs, and combined 3D analyses of the expression profiles of three genes, including two group 2 genes, were predictive of MSC differentiation potential.

Serum-Free Medium Enhances the Immunosuppressive and Antifibrotic Abilities of Mesenchymal Stem Cells Utilized in Experimental Renal Fibrosis.

Serum used in culture medium brings risks of immune reactions or infections and thus may hinder using ex vivo expanded mesenchymal stem cells (MSCs) for medical treatment. Here, we cultured MSCs in a serum-free medium (SF-MSCs) and in a medium containing 10% fetal bovine serum (10%MSCs) and investigated their effects on inflammation and fibrosis. MSC-conditioned medium suppressed transforming growth factor-ß1-induced phosphorylation of Smad2 in HK-2 cells, with no significant difference between the two MSCs. This finding suggests that the direct antifibrotic effect of SF-MSCs is similar to that of 10%MSCs. However, immunohistochemistry revealed that renal fibrosis induced by unilateral ureteral obstruction in rats was more significantly ameliorated by the administration of SF-MSCs than by that of 10%MSCs. Coculture of MSCs and monocytic THP-1 cell-derived macrophages using a Transwell system showed that SF-MSCs significantly induced polarization from the proinflammatory M1 to the immunosuppressive M2 phenotype macrophages, suggesting that SF-MSCs strongly suppress the persistence of inflammation. Furthermore, the gene expression of tumor necrosis factor-α-induced protein 6 (TSG-6), which inhibits the recruitment of inflammatory cells, was higher in SF-MSCs than in 10%MSCs, and TSG-6 knockdown in SF-MSCs attenuated the anti-inflammatory responses in unilateral ureteral obstruction rats. These findings imply that SF culture conditions can enhance the immunosuppressive and antifibrotic abilities of MSCs and the administration of ex vivo expanded SF-MSCs has the potential to be a useful therapy for preventing the progression of renal fibrosis. Stem Cells Translational Medicine 2018;7:893-905.

Characterization of human dental pulp cells grown in chemically defined serum-free medium.

Dental pulp cells (DPCs) are promising candidates for use as transplantable cells in regenerative medicine. However, ex vivo expansion of these cells typically requires culture media containing fetal bovine serum, which may cause infection and immunological reaction following transplantation. In addition, the proliferation and differentiation of DPCs markedly depend upon serum batches. Therefore, the present study examined whether DPCs could be expanded under serum-free conditions. DPCs obtained from four donors were identified to proliferate actively in the serum-free medium, STK2, when compared with those cells in control medium (Dulbecco's modified Eagle's medium containing 10% serum). The high proliferative potential with STK2 was maintained through multiple successive culture passages. DNA microarray analyses demonstrated that the gene expression profile of DPCs grown in STK2 was similar to that of cells grown in the control medium; however, a number of genes related to cell proliferation, including placental growth factor and inhibin-ßE, were upregulated in the STK2 cultures. Following induction of osteogenesis, DPCs grown in STK2 induced alkaline phosphatase activity and calcification at higher levels compared with the control medium cultures, indicating maintenance of differentiation potential in STK2. This serum-free culture system with DPCs may have applications in further experimental studies and as a clinical strategy in regenerative medicine.

DEC2 is a negative regulator for the proliferation and differentiation of chondrocyte lineage-committed mesenchymal stem cells.

Differentiated embryo chondrocyte 2 (DEC2) is a basic helix-loop-helix-Orange transcription factor that regulates cell differentiation in various mammalian tissues. DEC2 has been shown to suppress the differentiation of mesenchymal stem cells (MSCs) into myocytes and adipocytes. In the present study, we examined the role of DEC2 in the chondrogenic differentiation of human MSCs. The overexpression of DEC2 exerted minimal effects on the proliferation of MSCs in monolayer cultures with the growth medium under undifferentiating conditions, whereas it suppressed increases in DNA content, glycosaminoglycan content, and the expression of several chondrocyte-related genes, including aggrecan and type X collagen alpha 1, in MSC pellets in centrifuge tubes under chondrogenic conditions. In the pellets exposed to chondrogenesis induction medium, DEC2 overexpression downregulated the mRNA expression of fibroblast growth factor 18, which is involved in the proliferation and differentiation of chondrocytes, and upregulated the expression of p16INK4, which is a cell cycle inhibitor. These findings suggest that DEC2 is a negative regulator of the proliferation and differentiation of chondrocyte lineage-committed mesenchymal cells.

High glucose promotes TGF-ß1 production by inducing FOS expression in human peritoneal mesothelial cells.

BACKGROUND: High glucose (HG) induces production of transforming growth factor-beta1 (TGF-ß1), but the mechanism remains elusive. The aim of this study was to determine the gene(s) involved in HG-induced TGF-ß1 production in human peritoneal mesothelial cells (HPMCs). METHODS: Microarray analysis was performed following a 3-h preincubation of HPMCs in 4 or 0.1% glucose medium. Transcriptional genes were selected using Gene Ontology analysis for biological processes, including regulation of transcription and DNA-dependent. The effects of small interfering RNA (siRNA) treatments on the up-regulation of TGF-ß1 mRNA were assessed by quantitative real-time polymerase chain reaction (qPCR). Finally, enzyme-linked immunosorbent assay (ELISA) was performed to determine which gene(s) contribute to the production of TGF-ß1 protein in the medium. RESULTS: Microarray analysis revealed that the expression of 51 genes increased by more than 3-fold. Gene ontology analysis identified 13 genes for further study. qPCR confirmed mRNA amplification for 9 of the 13 genes. Furthermore, HG-induced up-regulation of TGF-ß1 mRNA was attenuated by the siRNA of 4 genes: MDS1 and EVI1 complex locus (MECOM), FBJ murine osteosarcoma viral oncogene homolog B (FOSB), FBJ murine osteosarcoma viral oncogene homolog (FOS) and activating transcription factor 3 (ATF3). ELISA showed that siRNA treatment of FOS, but not MECOM, FOSB or ATF3, suppressed the increase of TGF-ß1 protein in the medium. CONCLUSIONS: FOS is a downstream effector of HG stimulation in HPMCs that contributes to TGF-ß1 production, suggesting that blocking FOS expression may be a therapeutic target for peritoneal fibrosis.

Characteristic expression of MSX1, MSX2, TBX2 and ENTPD1 in dental pulp cells.

Dental pulp cells (DPCs) are a promising source of transplantable cells in regenerative medicine. However, DPCs have not been fully characterized at the molecular level. The aim of the present study was to distinguish DPCs from various source-derived mesenchymal stem cells (MSCs), fibroblasts (FBs) and other cells by the expression of several DPC-characteristic genes. DPCs were isolated from human pulp tissues by the explant method or the enzyme digestion method, and maintained with media containing 10% serum or 7.5% platelet-rich plasma. RNA was isolated from the cells and from dental pulp tissue specimens. The mRNA levels were determined by DNA microarray and quantitative polymerase chain reaction analyses. The msh homeobox 1, msh homeobox 2, T-box 2 and ectonucleoside triphosphate diphosphohydrolase 1 mRNA levels in DPCs were higher than that of the levels identified in the following cell types: MSCs derived from bone marrow, synovium and adipose tissue; and in cells such as FBs, osteoblasts, adipocytes and chondrocytes. The enhanced expression in DPCs was consistently observed irrespective of donor age, tooth type and culture medium. In addition, these genes were expressed at high levels in dental pulp tissue in vivo. In conclusion, this gene set may be useful in the identification and characterization of DPCs in basic studies and pulp cell-based regeneration therapy.

Age-dependent decrease in the chondrogenic potential of human bone marrow mesenchymal stromal cells expanded with fibroblast growth factor-2.

BACKGROUND AIMS: Human bone marrow mesenchymal stromal cells are useful in regenerative medicine for various diseases, but it remains unclear whether the aging of donors alters the multipotency of these cells. In this study, we examined age-related changes in the chondrogenic, osteogenic and adipogenic potential of mesenchymal stromal cells from 17 donors (25-81 years old), including patients with or without systemic vascular diseases. METHODS: All stem cell lines were expanded with fibroblast growth factor-2 and then exposed to differentiation induction media. The chondrogenic potential was determined from the glycosaminoglycan content and the SOX9, collagen type 2 alpha 1 (COL2A1) and aggrecan (AGG) messenger RNA levels. The osteogenic potential was determined by monitoring the alkaline phosphatase activity and calcium content, and the adipogenic potential was determined from the glycerol-3-phosphate dehydrogenase activity and oil red O staining. RESULTS: Systemic vascular diseases, including arteriosclerosis obliterans and Buerger disease, did not significantly affect the trilineage differentiation potential of the cells. Under these conditions, all chondrocyte markers examined, including the SOX9 messenger RNA level, showed age-related decline, whereas none of the osteoblast or adipocyte markers showed age-dependent changes. CONCLUSIONS: The aging of donors from young adult to elderly selectively decreased the chondrogenic potential of mesenchymal stromal cells. This information will be useful in stromal cell-based therapy for cartilage-related diseases.

Feasibility and limitations of the round robin test for assessment of in vitro chondrogenesis evaluation protocol in a tissue-engineered medical product.

Tissue-engineered medical products (TEMPs) should be evaluated before implantation. Therefore, it is indispensable to establish evaluation protocols in regenerative medicine. Whether or not such evaluation protocols are reasonable is generally verified through a 'round robin' test. However, the round robin test for TEMPs intrinsically includes a deficiency, because 'identical' specimens can not be prepared for TEMPs. The aim of the study was to assess the feasibility and limitations of the round robin test for TEMPs by using a prepared evaluation protocol. We adopted tissue-engineered cartilage constructs as delivered specimens and a protocol of measuring sGAG content as an evaluation protocol proposed to ISO TC150/SC7, which is an invasive, but usually applied, method, although non-invasive methods are keenly required in evaluating TEMPs. The results showed that: (a) the coefficient of variation (CV) of the measured sGAG contents in intralaboratory tests was ~5% at most; (b) the CV of sGAG content in the scheme where each participating laboratory measured different constructs was comparable with that in the scheme where each participating laboratory measured one half of a construct along with the organizing laboratory; (c) the CV caused by factors other than the specimen was ~15%, comparable to that in reproducible experiments in biomedical fields. Based on these results, the study concludes that a round robin test for a TEMP could be valuable, under the condition that the delivered TEMPs are sufficiently reproducible so that the CV of the measured values is < 5% in the organizing laboratory.

Impact of zinc fingers and homeoboxes 3 on the regulation of mesenchymal stem cell osteogenic differentiation.

We propose zinc fingers and homeoboxes 3 (ZHX3) as new osteogenic markers for mesenchymal stem cells (MSC). ZHX3 mRNA expression was upregulated within 1-6 h after incubation of MSCs in the osteogenic induction medium, and reached maximum levels after 24 h of incubation. Two to 4 days later, ZHX3 mRNA levels had decreased sharply. Maximal mRNA levels were 3- to 5-fold higher than those in the undifferentiated state. In contrast, Runt-related transcription factor2 (RUNX2) mRNA expression was downregulated at 2-4 h after incubation, and levels were only enhanced 1.4-fold after 12 and 24 h of incubation. Further, Osterix mRNA levels increased only 1.6-fold after 4 and 24 h of incubation. Thus, ZHX3 expression may be a better marker of MSC osteogenic differentiation than RUNX2 or Osterix expression at the initial stage of differentiation. Knockdown of ZHX3 using 2 distinct small interfering RNA (siRNA) oligonucleotides had little effect on cell morphology or on MSC proliferation, regardless of the differentiation state of the cells. However, ZHX3 siRNAs suppressed Osterix, but not RUNX2 mRNA expression, within 1 h of osteogenic differentiation, and this suppression was sustained for at least 24 h. The 2 ZHX3 siRNAs also suppressed alkaline phosphatase induction and matrix mineralization (assessed using alizarin red staining), and, further, suppressed the calcium content of the cultures at a later stage of differentiation (days 6-21). The effects of ZHX3 siRNAs on the osteogenic differentiation were comparable to those of RUNX2 and Osterix siRNAs. These findings suggest that ZHX3 is involved in the switch from the undifferentiated state of MSC to an osteogenic program, and that ZHX3 may be useful as an early osteogenic differentiation marker.

Selection of common markers for bone marrow stromal cells from various bones using real-time RT-PCR: effects of passage number and donor age.

Bone marrow stromal cells (BMSCs) are valuable in tissue engineering and cell therapy, but the quality of the cells is critical for the efficacy of therapy. To test the quality and identity of transplantable cells, we identified the molecular markers that were expressed at higher levels in BMSCs than in fibroblasts. Using numerous BMSC lines from tibia, femur, ilium, and jaw, together with skin and gum fibroblasts, we compared the gene expression profiles of these cells using DNA microarrays and low-density array cards. The differentiation potential of tibia and femur BMSCs was similar to that of iliac BMSCs, and different from jaw BMSCs, but all BMSC lines had many common markers that were expressed at much higher levels in BMSCs than in fibroblasts; several BMSC markers showed discrete expression patterns between jaw and other BMSCs. The common markers are probably useful in routine tests, but their efficacy may depend upon the passage number or donor age. In our study the passage number markedly altered the expression levels of several markers, while donor age had little effect on them. Considering the effects of in vivo location of BMSCs and passage, magnitude of increase in expression levels, and interindividual differences, we identified several reliable markers -- LIF, IGF1, PRG1, MGP, BMP4, CTGF, KCTD12, IGFBP7, TRIB2, and DYNC1I1 -- among many candidates. This marker set may be useful in a routine test for BMSCs in tissue engineering and cell therapy.

Comprehensive analysis of chemotactic factors for bone marrow mesenchymal stem cells.

To understand which growth factors/cytokines can affect migration of mesenchymal stem cells (MSCs) to injured tissues, we compared the effects of many (26) growth factors/cytokines on the migration activity of rabbit and human MSCs using a microchemotaxis chamber. Among them, platelet-derived growth factor (PDGF)-BB, PDGF-AB, epidermal growth factor (EGF), HB-EGF, transforming growth factor (TGF-alpha), insulin growth factor (IGF-I), hepatocyte growth factor (HGF), fibroblast growth factor (FGF-2), and thrombin consistently enhanced the migration of rabbit and human MSCs at appropriate concentrations. PDGF-BB showed the greatest effect on migration. Various combinations of these factors further enhanced the migration of MSCs, whereas combinations of factors that shared common cell-surface receptors did not induce the additive stimulation. On the other hand, some combinations, including that of FGF-2 or thrombin with PDGF-BB, suppressed the migration activity of MSCs. These findings suggest that combinations of growth factors are important to eliciting the maximal chemotactic effect. The factors that induced the migration of MSCs also enhanced their proliferation, suggesting that migration and proliferation can take place simultaneously. The above factors were also effective in stimulating the migration of fibroblasts, but thrombin alone selectively enhanced the migration of MSCs, suggesting that thrombin is useful to stimulate migration of MSCs without migration of fibroblasts.