Synergistic effect of Indian hedgehog and bone morphogenetic protein-2 gene transfer to increase the osteogenic potential of human mesenchymal stem cells

Abstract

IntroductionTo stimulate healing of large bone defects research has concentrated on the application of mesenchymal stem cells (MSCs).MethodsIn the present study, we induced the overexpression of the growth factors bone morphogenetic protein 2 (BMP-2) and/or Indian hedgehog (IHH) in human MSCs by adenoviral transduction to increase their osteogenic potential. GFP and nontransduced MSCs served as controls. The influence of the respective genetic modification on cell metabolic activity, proliferation, alkaline phosphatase (ALP) activity, mineralization in cell culture, and osteogenic marker gene expression was investigated.ResultsTransduction had no negative influence on cell metabolic activity or proliferation. ALP activity showed a typical rise-and-fall pattern with a maximal activity at day 14 and 21 after osteogenic induction. Enzyme activity was significantly higher in groups cultured with osteogenic media. The overexpression of BMP-2 and especially IHH + BMP-2 resulted in a significantly higher mineralization after 28 days. This was in line with obtained quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses, which showed a significant increase in osteopontin and osteocalcin expression for osteogenically induced BMP-2 and IHH + BMP-2 transduced cells when compared with the other groups. Moreover, an increase in runx2 expression was observed in all osteogenic groups toward day 21. It was again more pronounced for BMP-2 and IHH + BMP-2 transduced cells cultured in osteogenic media.ConclusionsIn summary, viral transduction did not negatively influence cell metabolic activity and proliferation. The overexpression of BMP-2 in combination with or without IHH resulted in an increased deposition of mineralized extracellular matrix, and expression of osteogenic marker genes. Viral transduction therefore represents a promising means to increase the osteogenic potential of MSCs and the combination of different transgenes may result in synergistic effects.

Hedgehog signaling is involved in the BMP9-induced osteogenic differentiation of mesenchymal stem cells

“…For example, Shh signaling induces osteoblast differentiation by interacting with BMP2 (26,40). Ihh and the BMP2 gene synergistically increase the osteogenic potential of human MSCs (43). Shh signaling, acting as a negative effector of BMP signaling, suppresses osteo/dentinogenic differentiation in stem cells from apical papilla (44).…”

The human arthritic hip joint is a source of mesenchymal stromal cells (MSCs) with extensive multipotent differentiation potential

“…Stainings were performed as reported previously in order to detect mineralization of the extracellular matric (ECM) using Alizarin Red S (1%; Sigma-Aldrich) [23], while immunohistochemical stainings were used to reveal extracellular collagen type I (Col I) formations. The adipogenic differentiation was detected through staining of lipid drops using Oil RedO (all Sigma-Aldrich) as described previousely [24].…”

“…The frozen pellets were sectioned, placed on SuperFrost ® cryosection slides (Thermo Fischer Scientific GmbH) and fixed with 3% ice-cold acetone (Carl Roth GmbH). Alizarin Red S, Oil redO and alcian blue stainings were performed as outlined previously [23,24,26]. In addition, immunohistochemical stainings were performed on monolayers and pellets using the following antibodies: Col I -monoclonal anti Col Iα1 (5 μg/mL; Abcam pls, Cambridge, Great Britain); Col IIpolyclonal Col IIα1 antibodies (5 μg/mL; Acris Antibodies GmbH, Herford, Germany); Col X -polyclonal Col X antibodies (5 μg/mL; Abcam pls).…”

The human arthritic hip joint is a source of mesenchymal progenitor cells (MPCs) with extensive multipotent differentiation potential

“…Cell cultures were stored at 37°C, 5% CO 2 and medium changes were performed every 3 to 4 days (d). After the cells showed confluent growth osteogenesis and adipogenesis were induced as described in previous studies [24,25]. Other wells and chambers were covered with standard culture medium to maintain controls for comparison to the differentiated MPCs.…”

“…To detect mineralization of the extracellular matric (ECM) Alizarin Red S (1%; Sigma-Aldrich) stainings were performed as reported previously [25], while immunohistochemical stainings were used to reveal extracellular collagen type I (Col I) formations. The adipogenic differentiation was detected through staining of lipid drops using Oil RedO (all Sigma-Aldrich) as described previousely [26].…”