ABSTRACT
Takahashi and Yamanaka established the first technique in which transcription factors related to pluripotency are incorporated into the genome of somatic cells to enable reprogramming of these cells. The expression of these transcription factors enables a differentiated somatic cell to reverse its phenotype to an embryonic state, generating induced pluripotent stem cells (iPSCs). iPSCs from canine fetal fibroblasts were produced through lentiviral polycistronic human and mouse vectors (hOSKM/mOSKM), aiming to obtain pluripotent stem cells with similar features to embryonic stem cells (ESC) in this animal model. The cell lines obtained in this study were independent of LIF or any other supplemental inhibitors, resistant to enzymatic procedure (TrypLE Express Enzyme), and dependent on bFGF. Clonal lines were obtained from slightly different protocols with maximum reprogramming efficiency of 0.001%. All colonies were positive for alkaline phosphatase, embryoid body formation, and spontaneous differentiation and expressed high levels of endogenous OCT4 and SOX2. Canine iPSCs developed tumors at 120 days post-injection in vivo. Preliminary chromosomal evaluations were performed by FISH hybridization, revealing no chromosomal abnormality. To the best of our knowledge, this report is the first to describe the ability to reprogram canine somatic cells via lentiviral vectors without supplementation and with resistance to enzymatic action, thereby demonstrating the pluripotency of these cell lines.
Subject(s)
Fetus/cytology , Fibroblasts/drug effects , Fibroblasts/physiology , Leukemia Inhibitory Factor/pharmacology , Pluripotent Stem Cells/physiology , Animals , Dogs , Fibroblasts/cytology , Gene Expression Regulation/physiology , Mice , Mice, Inbred BALB C , Mice, Nude , Polymerase Chain Reaction/veterinaryABSTRACT
The aim of this study was to further clarify the mechanisms involved in inducing pluripotency using canine foetal fibroblast cells. The two pluripotency-related transcription factors, OCT4 and SOX2, coupled to a fluorescent reporter gene were transduced, individually or in combination, using a lentiviral system. Stable transgenic cell lineages were obtained and canine cells showed to be highly responsive to the integration and expression of human SOX2 and OCT4, also depending on the amount of virus used for incubation. Such positive results are essential for the establishment of pluripotency induction through the incorporation of known transcription factors into the genome of somatic cells.
