Reprogramming Adipose Tissue-Derived Mesenchymal Stem Cells into Pluripotent Stem Cells by a Mutant Adeno-Associated Viral Vector

Abstract

Induced pluripotent stem (iPS) cells have great potential for personalized regenerative medicine. Although several different methods for generating iPS cells have been reported, improvement of safety and efficiency is imperative. In this study, we tested the feasibility of using a triple tyrosine mutant AAV2 (Y444 + 500 + 730F) vector, designated AAV2.3m, to generate iPS cells. We developed a polycistronic rAAV2.3m vector expressing three reprogramming factors, Klf4, Oct4, and Sox2, and then used this vector to infect mouse adipose-derived mesenchymal stem cells (AT-MSCs) to induce the generation of iPS cells. We demonstrated that (1) the triple tyrosine mutant AAV2 vector is able to reprogram mouse adult adipose tissue-derived stem cells into the pluripotent state. Those rAAV2.3m-derived iPS (rAAV2.3m-iPS) cells express endogenous pluripotencyassociated genes including Oct4, Sox2, and SSEA-1, and form teratomas containing multiple tissues in vivo; (2) c-myc, an oncogene, is dispensable in rAAV2.3m-mediated cellular reprogramming; and (3) transgene expression is undetectable after reprogramming, whereas vector DNA is detectable, indicating that transgenes are silenced. These results indicated the rAAV vector may have some advantages in generating iPS cells.

AAV Biology, Infectivity and Therapeutic Use from Bench to Clinic

“…For instance, the common practice is to reprogram patient-derived fibroblasts into a specific cell type that is affected by a disease. Some attempts have been performed to reprogram fibroblasts of patients suffering from retinal diseases into iPSC and finally differentiate the pluripotent stem cells into retinal pigment epithelial (RPE) cells that manifest the diseased phenotype [68,69]. Following the validation of iPSC and then RPE cells, a panel of rAAV serotypes could be tested for their efficiency to transduce the cells and the most effective ones could be chosen for delivering the healthy gene copy in order to re-establish normal cellular phenotype [70].…”

“…Even though the approach resulted in the generation of pluripotent stem cells from mouse embryonic or adult fibroblast cultures, still the efficiency was extremely low, the presence of c-Myc oncogene significantly increased the incidence of tumorigenicity and the use of retrovirus posed the threat of integration into the genome. Several new strategies have been developed, including the use of rAAV [69,71]. Considering the advantages of using rAAV for gene delivery, such as long-term transgene expression for efficient reprogramming of mature cells as well as safety and efficacy as a gene delivery vehicle in the clinic; researchers have attempted their use in the reprogramming of fully differentiated fibroblasts as well as adipose-derived mesenchymal stem cells.…”

A new alternative for corneal endothelial regeneration using autologous dental pulp stem cells

“…This difference could be due to the later development in time of the DPSC, which are isolated from third molars, compared to broblasts, which are differentiated cells. In fact, DPSC endogenously express some pluripotent markers [32], which has been described to enhance the reprogramming process [33][34][35]. Therefore, the generation of iPSC seems to be easily performed and with higher pluripotent expression when using DPSC compared to broblasts.…”

Pluripotent stem cells to hepatocytes, the journey so far

“…Due to the ease of isolation and maintenance for reprogramming, the most popular source is fibroblasts. Other cell types, including keratinocytes (25), mesenchymal stem cells (26), adipose stem cells (27), hair follicular cells (28), neural stem cells (29) and urinary cells (30), have also been used successfully. A more desirable source of starting material is human peripheral blood, which can be readily obtained through non-invasive routine clinical procedures.…”