Identification of genes responsible for maintenance of differentiation capability in dental pulp stem cells

Stem cells exist in various tissues, including dental follicles and dental pulps. Adult stem cells (ASC) can be isolated from patients for autologous transplantation, which eliminates the risk of immune rejection with low or no tumorigenesis. However, one of the challenges is that ASC progressively lose their differentiation ability when cultured in vitro. This prevents expansion of large quantities of high-potential stem cells for therapeutics, especially for stem cells with limited tissue source, such as dental pulp stem cells (DPSC). The goal of this study is to define possible molecular regulation causing loss of differentiation. To achieve this goal, we determined that DPSC at passages 3 and 5 (early passage) possessed strong differentiation capability, and such differentiation capability is completely lost at passage 11 (late passage). Using whole-genome microarray to compare the transcriptomes, we found that the expression of 34 genes were decreased for more than 10-fold in p11 DPSC when compared to p3. After confirming gene expression with RT-PCR, heat shock protein B8 (HspB8) and the GIPC PDZ domain-containing family (Gipc2) were selected for siRNA knockdown study. Knockdown of HspB8 in early-passage DPSC resulted in the cells losing differentiation, but knockdown of Gipc2 had no effect, suggesting that HspB8 plays an important role in maintaining DPSC differentiation. To further study HspB8, we constructed 2 vectors, one containing the coding sequence (CDS) and 3’ untranslated region (3’UTR) and another containing only the CDS. Transfection of the vectors into early passage DPSC dramatically increased both HspB8 mRNA and protein. However, transfection of the vectors into the late passage DPSC resulted in overexpression of HspB8 mRNA, but increase of HspB8 protein was seen only in CDS transfection. Given that 3’UTR of mRNA is the major target region for microRNAs (miRNAs), the results indicate that miRNAs are responsible for down-regulation of HspB8 in long-term culture of DPSCs. We conclude that high-level HspB8 expression is essential for differentiation of DPSC, and down-regulation of HspB8 in cultured DPSC is likely due to increased expression of miRNAs. These are novel findings regarding HspB8 and miRNAs on the regulation of stem cell fate