Differential Expression of STATs and Its Function in Osteogenesis of Mesenchymal Stem Cells / 대한정형외과연구학회지

PURPOSE: To investigate the role of STAT5 during osteogenesis differentiation of human mesenchymal stem cells(hMSC). MATERIALS AND METHODS: To assess the expression pattern of STATs during osteogenic differentiation, we performed western blot analysis and RT-PCR. By RNA interference, we have performed effect of STAT5A and STAT5B in osteogenesis. As a result of Luciferase assay, the promoter activity of DLX5 was decreased by STAT5A. RESULTS: To assess the expression pattern of STATs during osteogenic differentiation, we have performed western blot and RT-PCR after 14 day differentiation period. STAT1, 2, 3 and 4 showed no change in expression level during differentiation, while STAT5A and STAT5B displayed steady increase compared to the control. When STAT5A was knock down, the level of osteogenesis was increased. The transcriptional activity of DLX5 was decreased about 70% by STAT5A. CONCLUSION: The expression of STAT5A and STAT5B increased during osteogenesis of hMSC. Also, we shown that STAT5A regulated the transcriptional activity of DLX5. These results indicate that STAT5A acts as a pivotal transcription factor in osteogenesis of hMSC.

Duration and Magnitude of Extracellular Signal-Regulated Protein Kinase Phosphorylation Determine Adipogenesis or Osteogenesis in Human Bone Marrow-Derived Stem Cells

PURPOSE: Imbalances between osteogenic and adipogenic differentiation leads to diseases such as osteoporosis. The aim of our study was to demonstrate the differences in extracellular signal-regulated kinase (ERK) phosphorylation during both adipogenesis and osteogenesis of human bone marrow-derived stem cells (BMSCs). MATERIALS AND METHODS: Using troglitazone, GW9662 and U0126, we investigated their role in hBMSC differentiation to adipogenic and osteogenic fates. RESULTS: ERK1/2 inhibition by U0126 suppressed proliferator-activated receptor (PPAR)gamma expression and lipid accumulation, while it decreased the mRNA expression of adipogenic genes (lipoprotein lipase, PPARgamma, and adipocyte protein) and osteogenic genes (type I collagen and osteopontin). ERK phosphorylation was transient and decreased during adipogenesis, whereas it occurred steadily during osteogenesis. Troglitazone, a PPARgamma agonist, induced adipogenesis by inhibiting ERK phosphorylation even in an osteogenic medium, suggesting that ERK signaling needs to be shut off in order to proceed with adipose cell commitment. Cell proliferation was greatly increased in osteogenesis but was not changed during adipogenesis, indicating that ERK might play different roles in cellular proliferation and differentiation between the two committed cell types. CONCLUSION: The duration and magnitude of ERK activation might be a crucial factor for the balance between adipogenesis and osteogenesis in human bone marrow-derived stem cells.

Duration and Magnitude of Extracellular Signal-Regulated Protein Kinase Phosphorylation Determine Adipogenesis or Osteogenesis in Human Bone Marrow-Derived Stem Cells

PURPOSE: Imbalances between osteogenic and adipogenic differentiation leads to diseases such as osteoporosis. The aim of our study was to demonstrate the differences in extracellular signal-regulated kinase (ERK) phosphorylation during both adipogenesis and osteogenesis of human bone marrow-derived stem cells (BMSCs). MATERIALS AND METHODS: Using troglitazone, GW9662 and U0126, we investigated their role in hBMSC differentiation to adipogenic and osteogenic fates. RESULTS: ERK1/2 inhibition by U0126 suppressed proliferator-activated receptor (PPAR)gamma expression and lipid accumulation, while it decreased the mRNA expression of adipogenic genes (lipoprotein lipase, PPARgamma, and adipocyte protein) and osteogenic genes (type I collagen and osteopontin). ERK phosphorylation was transient and decreased during adipogenesis, whereas it occurred steadily during osteogenesis. Troglitazone, a PPARgamma agonist, induced adipogenesis by inhibiting ERK phosphorylation even in an osteogenic medium, suggesting that ERK signaling needs to be shut off in order to proceed with adipose cell commitment. Cell proliferation was greatly increased in osteogenesis but was not changed during adipogenesis, indicating that ERK might play different roles in cellular proliferation and differentiation between the two committed cell types. CONCLUSION: The duration and magnitude of ERK activation might be a crucial factor for the balance between adipogenesis and osteogenesis in human bone marrow-derived stem cells.

The Role of microRNAs Involved in Mesenchymal Stem Cell Differentiation / 대한정형외과연구학회지

Over the past decade, microRNA has been emerging as a key regulator involved in various biological and physiological process including cell cycle, tissue development and pathogenesis. The effort to decipher the complicated machinery behind microRNA function is underway yet the complete understanding is far from being reached. However, various groups have made successful identification of the role of a specific microRNA involved in a specific process. This article summarizes the microRNAs identified in one such specific area, namely, differentiation of mesenchymal stem cell. Characterized by its unique potential to differentiate into three separate tissue cell types, adipocyte, chondrocyte and osteoblast, mesenchymal stem cells have been of immense interest for its possible applicability in regenerative medicine. Despite this interest, however, the study of microRNA function in MSCs differentiation remains rudimentary. The aim of this paper is to summarize the currently identified, experimentally validated and accepted by the scientific community as novel, microRNAs involved in MSC differentiation.

Clonal Heterogeneity of Human Bone Marrow-Derived Stromal Cells / 대한정형외과연구학회지

PURPOSE: The isolated human bone marrow-derived stromal cells (BMSCs) were expanded and showed multipotentiality for differentiation to both adipocytes and osteoblasts. We selected the multiple clones and characterized their proliferation potentials, surface markers and differentiation potentials. MATERIALS AND METHODS: BMSCs were isolated from bone marrow. After forming colonies, colonies were selected and cultured. We performed MTT assay and FACS. Cultured colonies were differentiated into adipocyte and osteoblast. RESULTS: BMSCs were isolated and plated in tissue-culture dishes. The number of clones was about 30. Clones were divided into three groups. Group I was round and small in size, and showed rapid proliferation. Group II showed similar pattern in cell size and proliferation with group I clones during early passage. But, after 4 passages, the clones became flat and larger cells and showed slower proliferation. Finally, group III clones were flat and large cells and replicated slowly in early passage. Group I was fibroblastic in morphology, but Group II changed to flat and larger cells after passage 4. FACS and proliferation assay were performed at passage 5. Group I and Group II cells differed in their expression of the cell-surface epitopes, CD29 and CD105. Group I cells were differentiated into adipocytes better than Group II cells. CONCLUSION: In this study, these results indicated that cell surface markers were expressed differently for clones having different differentiation properties. These clonal BMSCs may also be used for the identification of lineage-determining factors.

Transdifferentiation from Adipogenic bone Marrow Derived Stromal Cell to Osteoblast / 대한정형외과연구학회지

PURPOSE: The inhibition of marrow adipogenesis with a concomitant increase in osteogenesis could be a therapeutic target by preventing the increase of adipocyte formation or diverting the preexisting adipocytes. The changes of morphology and gene expression, and the role of Mitogen-Activated Protein Kinase (MAPK) was evaluated in the process of transdifferentiation from adipogenic MSC to osteogenic MSC. MATERIALS AND METHODS: Human mesenchymal stem cells were isolated from bone marrow and cultured in adipogenic medium in the presence of 25 muM troglitazone for 14 days. Then cells were cultured in osteogenic medium for 28 days. To find out the mechanisms responsible for transdifferentiation, we performed RT-PCR, several stains, alkaline phosphatase activity, and western blot analysis. RESULTS: In RT-PCR, when adipogenic mesenchymal stem cells were cultured in osteogenic medium, mRNA levels of the adipogenic genes (LPL, aP2, and PPAR-nu) were decreased and those of the osteogenic genes (collagen type I and osteocalcin) were increased. The adipogenic MSC in osteogenic medium for 28 days exhibited a fibroblastic appearance and were stained positively with von Kossa and alkaline phosphatase. In western blot analysis, we found that ERK phosphorylation was inhibited in the early stage of adipogenesis. CONCLUSION: In this study, adipogenic mesenchymal stem cells were shown to differentiate into the osteogenic MSC under appropriate culture conditions. The evidence of the transdifferentiation of human mesenchymal stem cells suggests that it could be a therapeutic target in osteoporosis.