Development of cationic nanoparticles with Sox2, Klf4, Oct4 and c-Myc proteins in induced pluripotent stem cells’ generation

Induced pluripotent stem cells (iPS cells) are artificial stem cells, generated by somatic cell reprogramming. In 2006, Shinya Yamanaka and Kazutoshi Takahashi investigated 24 candidates that are specifically expressed in embryonic stem cells (ES cells) as pluripotent-correlated genes, they finally found out that Klf4, Sox2, Oct4 and c-Myc, which are known as Yamanaka factors, are able to derive iPS cells from adult fibroblasts. Since then many DNA-dependent reprogramming methods have been developed, and these methods have the same problem, which hinder the clinical application of this type of iPS cells. The problem of DNA-dependent methods is uncontrollable genome integration during reprogramming process, so the following researcher focused on DNA-free reprogramming vectors, such as proteins, microRNA and mRNA. Those DNA-free methods won’t modify host genome and therefore those methods are more promising in regenerative medicine area.
Based on these reasons, I used proteins as DNA-free reprogramming vectors to generate iPS cells. Proteins are easy to overexpress, purify and store up, but without specific peptide sequence or protein carrier, most proteins are unable to cross cell membrane, and hence I cooperated with Dr. Yi-You Huang and Dr. Ming-Ju Chou using their gelatin–polyethyleneimine (gelatin-PEI) nanoparticles as my protein carrier. On the other hand, gelatin-PEI nanoparticles have many primary and secondary amines work as proton sponge, which provide high efficiency of endosomal escape.
In my research, the uptake efficiency experiment performed by flow cytometry showed that HS68 cells are able to uptake almost 90% of gelatin-PEI nanoparticle when particle concentration reach 50ug/ml. Besides, cell viability assay showed that although HS68 population will decrease after 24 hours particle application, HS68 cells will repopulate after three days, indicating that repeating protein delivery is possible.
On the other hand, I also showed that both enhanced green fluorescent proteins (eGFPs) and Yamanaka four factors can be overexpressed in Escherichia coli expression system, and these proteins were able to be purified by Ni-NTA affinity columns. Furthermore, after I mixed gelatin-PEI nanoparticles and eGFPs with HS68 cells, these particles are able to transport our model protein eGFPs into HS68 cell line, and transfection process won’t cause severe cell death.
Overall, these data showed that gelatin-PEI nanoparticles are able to carry our model proteins, eGFPs, into human foreskin fibroblasts. I suppose that we can combine proteins and nanoparticles as reprogramming vectors, and this might be a new method to generate iPS cells