Induced Reprogramming of Human Dental Pulp Stem Cells by Cell Penetrating Peptide Based-proteins

Objectives: Most human induced pluripotent stem cells (iPSCs) are made by viral vectors which can integrate the reprogramming factors into the host genomes and increase the risk of tumor formation. To overcome these limitations, we performed the generation of iPSCs from human dental pulp stem cells (hDPSCs) using recombinant proteins, Klf4, Oct4, Sox2, c-Myc, Lin28.
Methods: The recombinant proteins were fused in the pET-21b based-frame included a cell penetrating peptide, termed low molecular weight protamine (LMWP) for cellular penetration, even nuclear. The plasmid was introduced into Escherichia coli (BL21 strain) and induced by isopropyl 1-thio-β-D-galactoside (IPTG). The recombinant proteins were purified using Ni-NTA affinity chromatography. The hDPSCs were treated with purified proteins every 5 days during 20 days, and cultured on feeder layer in human embryonic stem cell (hESC) cultured medium until the colony forms.
Results: Purified proteins was observed by Commassie stained SDS-PAGE analysis and Western blot. The penetrating ability of recombinant proteins was investigated by western blotting and confocal microscopy. The generated colonies were positively stained by alkaline phosphatase (ALP) and fluorescence labeled-antibodies which can show their pluripotency. These data could describe that the purified recombinant cell-penetrating proteins are capable of reprogramming hDPSCs to iPSCs.
Conclusions: Cell-permeable reprogramming proteins could not only be used as safety reagents for reprogramming of human dental derived cells, but also replaced previous methods which have potent risks with above strategy.