Establishment of Transgene-Free Porcine Induced Pluripotent Stem Cells.
J Vanessa ConradJaime A NeiraMargaret RusteikaSusanne MeyerDennis O CleggLi-Fang ChuPublished in: Current protocols (2024)
Although protocols to generate authentic transgene-free mouse and human induced pluripotent stem cells (iPSCs) are now well established, standard methods for reprogramming porcine somatic cells still suffer from low efficiency and transgene retention. The Basic Protocol describes reprogramming procedures to establish transgene-free porcine iPSCs (PiPSCs) from porcine fibroblasts. This method uses episomal plasmids encoding POU5F1, SOX2, NANOG, KLF4, SV40LT, c-MYC, LIN28A, and microRNA-302/367, combined with an optimized medium, to establish PiPSC lines. Support protocols describe the establishment and characterization of clonal PiPSC lines, as well as the preparation of feeder cells and EBNA1 mRNA. This optimized, step-by-step approach tailored to this species enables the efficient derivation of PiPSCs in ∼4 weeks. The establishment of transgene-free PiPSCs provides a new and valuable model for studies of larger mammalian species' development, disease, and regenerative biology. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Reprogramming of porcine fibroblasts with episomal plasmids Support Protocol 1: Preparation of mouse embryonic fibroblasts for feeder layer Support Protocol 2: Preparation of in vitro-transcribed EBNA1 mRNA Support Protocol 3: Establishment of clonal porcine induced pluripotent stem cell (PiPSC) lines Support Protocol 4: PiPSC characterization: Genomic DNA PCR and RT-PCR Support Protocol 5: PiPSC characterization: Immunostaining.
Keyphrases
- induced pluripotent stem cells
- randomized controlled trial
- stem cells
- induced apoptosis
- escherichia coli
- endothelial cells
- epstein barr virus
- transcription factor
- systematic review
- cell cycle arrest
- extracellular matrix
- mesenchymal stem cells
- endoplasmic reticulum stress
- high resolution
- mass spectrometry
- cell proliferation
- bone marrow
- binding protein
- cell therapy
- multidrug resistant
- cell death
- diabetic rats
- gestational age
- case control