Evaluation of human DPSCs expressing BMP2/7 heterodimer in a doxycycline inducible manner
According to the literature, BMP2/7 is a stronger inducer of osteoblastic differentiation than BMP2 or BMP7- homodimers. Our aim was to establish modified human DPSC cells derived from dental pulp tissue and expressing BMP2/7 heterodimer in a doxycycline-inducible manner and to evaluate the osteogenic potential of the genetically modified cells. Lentiviral transduction was used to introduce the Tet-ON--regulated transgene--containing vector to the cells, and the heterodimer was co-expressed together with a GFP marker protein allowing the determination of the success rate of the transduction by fluorescence imaging and flow cytometric methods. Endogenous heterodimers were detected at mRNA and protein levels by RNAseq, qPCR and western blot, while secreted heterodimers were detected by BMP2- and BMP7 specific ELISA assays. Osteogenic differentiation induced by BMP2/7 was monitored by the measurement of alkaline phosphatase (ALP) activity, mineralization and the measurement of the expression level of RUNX2 and ALPL genes involved in the regulation of osteoblast directed differentiation. Our results showed that the transduced cells were able to express the BMP2/7 heterodimer as a result of doxycycline induction on a dose dependent manner. Although ALP activity did not change, increased calcium deposition could be measured in the extracellular matrix, as a result of heterodimer expression which correlates with the results obtained by RNA sequencing. Based on our results BMP2/7 expressing DPSCs could be used in the treatment of bone defects where heterodimers could have not only autocrine effect but also paracrine effect on other stem/preosteoblast cells recruited to the bone defect area.