Comparison of American mink embryonic stem and induced pluripotent stem cell transcriptomes

Abstract

BackgroundRecently fibroblasts of many mammalian species have been reprogrammed to pluripotent state using overexpression of several transcription factors. This technology allows production of induced pluripotent stem (iPS) cells with properties similar to embryonic stem (ES) cells. The completeness of reprogramming process is well studied in such species as mouse and human but there is not enough data on other species. We produced American mink (Neovison vison) ES and iPS cells and compared these cells using transcriptome analysis.ResultsWe report the generation of 10 mink ES and 22 iPS cell lines. The majority of the analyzed cell lines had normal diploid chromosome number. The only ES cell line with XX chromosome set had both X-chromosomes in active state that is characteristic of pluripotent cells. The pluripotency of ES and iPS cell lines was confirmed by formation of teratomas with cell types representing all three germ layers. Transcriptome analysis of mink embryonic fibroblasts (EF), two ES and two iPS cell lines allowed us to identify 11831 assembled contigs which were annotated. These led to a number of 6891 unique genes. Of these 3201 were differentially expressed between mink EF and ES cells. We analyzed expression levels of these genes in iPS cell lines. This allowed us to show that 80% of genes were correctly reprogrammed in iPS cells, whereas approximately 6% had an intermediate expression pattern, about 7% were not reprogrammed and about 5% had a "novel" expression pattern. We observed expression of pluripotency marker genes such as Oct4, Sox2 and Rex1 in ES and iPS cell lines with notable exception of Nanog.ConclusionsWe had produced and characterized American mink ES and iPS cells. These cells were pluripotent by a number of criteria and iPS cells exhibited effective reprogramming. Interestingly, we had showed lack of Nanog expression and consider it as a species-specific feature.

Derivation of ringed seal (Phoca hispida) induced multipotent stem cells

“…There also was a weak expression of Nanog (data not shown). We were not able to determine its expression level as qualitative PCR was performed, though we previously showed a very low level of Nanog expression in American mink pluripotent stem cells (Menzorov et al, 2015). Importantly, we were not able to amplify human KLF4 transgene (Fig.…”

“…To produce iPS cells from the ringed seal fibroblasts we used lentiviral vectors LeGO (http://www.lentigo-vectors.de/vectors.htm) with EGFP and human reprogramming transcription factors: OCT4, SOX2, C-MYC and KLF4, courtesy of Dr. Sergei L. Kiselev, Moscow. We used previously the published protocol (Menzorov et al, 2015) with minor modifications. Lentiviruses were produced in the Phoenix cell line using Lipofectamine 3000 (Thermo Fisher Scientific, USA).…”

Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression

“…To introduce EGFP into Phoenix cell line we used LeGO-G2 vector (Addgene, 25917) according to previously described protocol (Menzorov et al 2015).…”

Generation of American mink induced pluripotent stem cells: a protocol

“…В эксперименте можно получить десятки колоний ИПСК. Морфология ИПСК должна со-ответствовать плюрипотентным клеткам американской норки (Sukoyan et al, 1993;Menzorov et al, 2015). На рис.…”