Non-viral generation of marmoset monkey iPS cells by a six-factor-in-one-vector approach

Debowski K, Warthemann R, Lentes J, Salinas-Riester G, Dressel R, Langenstroth D, Gromoll J, Sasaki E, Behr R.
Source: PLoS ONE
Publication Date: (2015)
Issue: 10(3): 1-21
Research Area:
Stem Cells
Gene Expression
Cells used in publication:
Fibroblast, dermal, macaque
Species: monkey
Tissue Origin:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
transfection or marmoset monkey fibroblasts of a newborn with piggyback transposase/transposon system: CAG promotor driven SOX2; OCT4; KLF4; c-MYC; LIN28; NANOG; + transposase plasmid Nucleofector 2b: Basic Nucleofector Kit for Primary Mammalian Fibroblasts and program V-013; 4D Nucleofector X-unit: P2 solution and program CA-137 After nucleoefection cells are plated on irradiated MEF in M10 media +puromycin (5 days) then switch to ESM+20% Knockserum replacement+ Valproic acid.
Groundbreaking studies showed that differentiated somatic cells of mouse and human origin could be reverted to a stable pluripotent state by the ectopic expression of only four proteins. The resulting pluripotent cells, called induced pluripotent stem (iPS) cells, could be an alternative to embryonic stem cells, which are under continuous ethical debate. Hence, iPS cell-derived functional cells such as neurons may become the key for an effective treatment of currently incurable degenerative diseases. However, besides the requirement of efficacy testing of the therapy also its long-term safety needs to be carefully evaluated in settings mirroring the clinical situation in an optimal way. In this context, we chose the long-lived common marmoset monkey (Callithrix jacchus) as a non-human primate species to generate iPS cells. The marmoset monkey is frequently used in biomedical research and is gaining more and more preclinical relevance due to the increasing number of disease models. Here, we describe, to our knowledge, the first-time generation of marmoset monkey iPS cells from postnatal skin fibroblasts by non-viral means. We used the transposon-based, fully reversible piggyback system. We cloned the marmoset monkey reprogramming factors and established robust and reproducible reprogramming protocols with a six-factor-in-one-construct approach. We generated six individual iPS cell lines and characterized them in comparison with marmoset monkey embryonic stem cells. The generated iPS cells are morphologically indistinguishable from marmoset ES cells. The iPS cells are fully reprogrammed as demonstrated by differentiation assays, pluripotency marker expression and transcriptome analysis. They are stable for numerous passages (more than 80) and exhibit euploidy. In summary, we have established efficient non-viral reprogramming protocols for the derivation of stable marmoset monkey iPS cells, which can be used to develop and test cell replacement therapies in preclinical settings.