Generation of LIF-independent induced pluripotent stem cells from canine fetal fibroblasts

• Published in: Theriogenology Vol. 92; pp. 75 - 82
• Main Authors: Gonçalves, N.J.N., Bressan, F.F., Roballo, K.C.S., Meirelles, F.V., Xavier, P.L.P., Fukumasu, H., Williams, C., Breen, M., Koh, S., Sper, R., Piedrahita, J., Ambrósio, C.E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.2017
• Subjects: Animals; Canine; Cellular reprogramming; Dogs; Fetus - cytology; Fibroblasts - cytology; Fibroblasts - drug effects; Fibroblasts - physiology; Gene Expression Regulation - physiology; iPSC; Leukemia Inhibitory Factor - pharmacology; Mice; Mice, Inbred BALB C; Mice, Nude; Pluripotency; Pluripotent Stem Cells - physiology; Polymerase Chain Reaction - veterinary; Stem cells; Cellular reprogramming; Pluripotency; iPSC; Stem cells; Canine
• ISSN: 0093-691X; 1879-3231
• DOI: 10.1016/j.theriogenology.2017.01.013

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. Overview of the steps for obtaining the iPS cells. From the somatic cell chosen, genic factors are added, and after the formation of colonies, they must be characterized as positive for alkaline phosphatase, embryoid body formation, gene markers for immunofluorescence and real-time PCR, in vivo tumor formation and the possible abnormalities in chromosomes, resulting from reprogramming process. [Display omitted] •iPSCs cells were generated by canine fetal fibroblast.•IPSCs were generated without LIF and resistant to enzymatic passages.•The strains were positive for all characterization testes and capable to forming tumor mass after 120 days.•The evaluated strains showed no chromosomal changes.