Conditioned Medium from Bone Marrow Mesenchymal Stem Cells Transiently Retards Osteoblast Differentiation by Downregulating Runx2

Abstract

Mesenchymal stem cells (MSCs) are attractive candidates for cell therapy and regenerative medicine because of their potential for proliferation and multilineage differentiation. MSCs secrete various cytokines, acting as trophic mediators to regulate neighboring cells. Osteoblasts are the cells directly responsible for forming new bone, and they are the final target of many osteogenic regulators. However, the induction effect of MSCs on osteoblasts is still unknown. In this study, we isolated osteoblasts from rat calvariae and investigated their proliferation and differentiation under induction of varied concentrations of MSC-conditioned medium (MSC-CM). Cells in the MSC-CM groups showed a reduction in cell proliferation at 3–6 days, and a decrease in the expression of osteocalcin and osteopontin at 3 days, with low levels of alkaline phosphatase activity. The expression of osteogenic markers went back to normal at 7 days. In order to evaluate the molecular mechanisms underlying this suppression, levels of two osteoblastic transcription factors, runt-related transcription factor 2 (Runx2) and osterix (Osx), were detected at both mRNA and protein levels. The results indicated that MSC-CM significantly inhibited Runx2 expression in a concentration-dependent manner. However, the effect was not due to the inhibition of Osx, for Osx was not significantly altered. This work demonstrates that MSCs may suppress osteoblast proliferation and transiently retard osteoblast differentiation by downregulating Runx2. These results highlight the need to take into account the paracrine effect of MSCs when using them in regenerative medicine for the repair of bone defects.

Glimepiride Promotes Osteogenic Differentiation in Rat Osteoblasts via the PI3K/Akt/eNOS Pathway in a High Glucose Microenvironment

“…ALP is an early osteoblast marker and Runx2 is a critical transcription factor for osteoblastic differentiation [22], [23], We found that hyperglycemia interfered with the differentiation of rat osteoblasts, and glimepiride increased the expression of these marker genes at two different glucose concentrations. However, we showed that inhibition of PI3K P85 by LY294002 reduced ALP, Runx2 and OCN mRNA expression in osteoblasts.…”

“…Furthermore, we extracted mRNA from both groups of osteoblasts cultured with basic media containing 5.5 mM or 16.5 mM glucose for 7 days and performed real-time PCR to determine the expression levels of osteoblast marker genes, including Runx2 (Runt-related transcription factor 2) (Fig. 4A), a critical transcription factor for osteoblastic differentiation [23], and ALP (Fig. 4B), an early osteoblast marker [24].…”

Low Osteogenic Differentiation Potential of Placenta-Derived Mesenchymal Stromal Cells Correlates with Low Expression of the Transcription Factors Runx2 and Twist2

“…differences in Runx2 expression in these cells. Another reason for the differences in Runx2 expression may be due to fluctuations of Runx2 during the cell cycle [50]. It has been shown that proliferating cells may contain more Runx2 [49].…”

“…However, experimental evidence from other laboratories provided evidence that a transient serum deprivation before induction of differentiation, which also causes a retardation of proliferation, could facilitate the differentiation of MSC in vitro [53]. In addition, supernatants from MSC transiently suppressed maturation of osteoblasts by downregulating expression of Runx2 [50]. Therefore, differences in the release of osteogenic factors from bmMSC in primary culture or during initiation of differentiation could account for their prominent osteogenic potential.…”

Prospect of Mesenchymal Stem Cells Conditioned Medium in Tissue Regeneration

“…Mesenchymal stem cells (MSCs) are an appealing candidate for tissue regeneration due to their ability to differentiate into many cell types (1). However, the use of stem cells has several problems such as complicated safety and quality management and high cost and difficult cell handling (2).…”