Transposon mediated reprogramming of buffalo fetal fibroblasts to induced pluripotent stem cells in feeder free culture conditions

Commonly, induced pluripotent stem (iPS) cells are generated by viral transduction of four core reprogramming genes, but recent evidences suggest that slightly different combination of transcription factors improve the efficiency and quality of generated iPS cells. However, vectors like retro- and l...

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Bibliographic Details
Published in:Research in veterinary science 2019-04, Vol.123, p.252-260
Main Authors: Kumar, Deepak, Anand, Taruna, Vijayalakshmy, Kennady, Sharma, Papori, Rajendran, Rasika, Selokar, Naresh L., Yadav, P.S., Kumar, Dharmendra
Format: Article
Language:English
Subjects:
Alkaline phosphatase
Animals
Biomarkers
Buffalo
Cell culture
Deoxyribonucleic acid
DNA
Efficiency
Electroporation
Fetuses
Fibroblasts
Gene expression
Genes
Genetic engineering
Genetic modification
Genomes
Insertional mutagenesis
iPS
KLF4 protein
Laboratories
Morphology
Oct-4 protein
piggyBac
Pluripotency
Reprogramming
Stem cell transplantation
Stem cells
Transcription factors
Transgenic animals
Transposase
Transposon
Transposons
Veterinary medicine
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Summary:Commonly, induced pluripotent stem (iPS) cells are generated by viral transduction of four core reprogramming genes, but recent evidences suggest that slightly different combination of transcription factors improve the efficiency and quality of generated iPS cells. However, vectors like retro- and lentiviral may cause insertional mutagenesis due to its integrating ability. Hence, alternate methods with safety concerns are needed to be investigated. Therefore, the present study was undertaken to reprogram buffalo fibroblasts using non-viral piggyBac (PB) transposon mediated transfer of six transcription factors. To generate buffalo iPS cells, fibroblasts were isolated from buffalo fetus at passage 2. The cells were co-electroporated with a PB transposon having CAGGS promoter driven cassette of Oct4, Sox2, Klf4, cMyc, Nanog, and Lin28 transcription factors separated by self-cleaving 2A peptide and a helper plasmid pCMV-PB transposase. After 12–14 days post electroporation, fibroblast cells morphology was observed to change to round structures which formed loose aggregates of cells on day 18. Putative iPS cell colonies were propagated in feeder free system and characterized through expression of pluripotency markers such as alkaline phosphatase, SSEA-1, SSEA-4, SSEA-5, TRA-1-81, Oct4, Nanog and Sox2 and endogenous genes supported the stemness property of the generated cells. These cells differentiated in vitro to form embryoid bodies and were found to express three germ layers markers. In conclusion, generation of buffalo iPS cells using transposon system provides insights into viral-free iPS technology which will facilitate genetic modification of the buffalo genome and help in the production of transgenic animals using genetically modified iPS cells. •Reprogramed buffalo fibroblasts using piggyBac mediated nucleofection of six transcription factors.•Generated buffalo iPS cells maintained high expression of endogenous pluripotent markers.•Generated buffalo iPS cells were able to form embryoid bodies, expressed three germ layer markers.•Viral-free iPS facilitates genetic modification and production of transgenic animals using modified iPS cells.
ISSN:0034-5288
1532-2661
DOI:10.1016/j.rvsc.2019.01.015