Roles of Heat Shock Protein-B8 and MicroRNAs in Maintaining Differentiation Capability of Dental Pulp Stem Cells

Objectives: One of the major hurdles in applications of adult stem cells is that they lose stemness during in vitro expansion. Dental pulp stem cells (DPSCs) possess strong differentiation potential at early-passages and lose such potential at late-passages at in vitro culture. We found that reduction of heat shock protein-B8 (HspB8) expression is associated with loss of differentiation in DPSCs. MicroRNAs have emerged as major regulators of stem cell fates. Our objectives were to determine if HspB8 plays a role in maintaining differentiation capability of DPSCs, and if microRNAs involve in regulating HspB8 expression and differentiation of DPSCs.
Methods: DPSCs isolated from rat 1^st molars were cultured to obtain different passages of cells. HspB8 expression in the early-passage DPSCs was silenced by siRNAs to determine its effect on differentiation. The coding sequence (CDS) of HspB8 with or without 3’-untranslated region (3’UTR) was cloned and transfected into the early- and late-passage DPSCs. Expression of HspB8 was determined in the transfected cells. HspB8-targeting microRNAs highly expressed in late-passage DFSCs were cloned and transfected into the early-passage DPSCs to evaluate their effect on differentiation of the cells.
Results: Early-passage DPSCs lost differentiation capability when HspB8 expression was knocked down. Transfection of DPSCs with the vector containing HspB8 CDS and 3’UTR dramatically increased HspB8 mRNA in both early- and late-passage DPSCs; however, increase of HspB8 protein was seen only in the early-passage DPSCs. In contrast, transfection of DPSCs with the vector containing HspB8 CDS resulted in overexpression of mRNA and protein in both early- and late-passages, suggesting binding of microRNAs to HspB8 3’UTR to inhibit the translation of HspB8 mRNA into protein in the late-passage DPSCs. When HspB8 targeting-microRNAs were transfected into the early-passage DPSCs, the differentiation capability of the cells was reduced.
Conclusions: MicroRNAs are involved in maintaining differentiation capability of DPSCs through regulating HspB8 expression.