Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Abstract

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs) 1 . While improvements in several gene delivery methods have been described [2][3][4][5][6][7][8][9] , transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor 11 , trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells. Video LinkThe video component of this article can be found at https://www.jove.com/video/3110/ ProtocolOur protocol begins with a method to adapt human iPSCs to feeder-free cultures, followed by protocols for transfecting human iPSCs using GeneJuice (EMD) and nucleofection of human iPSCs using an AMAXA nuclefector device.Note: The following procedures are performed in a sterile laminar flow hood. All media and solutions are equilibrated to 37°C or room temperature before starting unless otherwise specified.

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“…The H9 hESCs (WiCell) were stably transfected with an OCT4-eGFP (octamer-binding transcription factor 4-enhanced green fluorescence protein) reporter plasmid as previously described [11]. OCT4 was a marker for undifferentiated cells.…”

The effects of electrospun substrate-mediated cell colony morphology on the self-renewal of human induced pluripotent stem cells

“…Additionally, the induced cells were transfected with a GFPlabeled OCT4 reporter [36]. The cells were adapted to Geltrex ® -coated cell culture dishes (Life Technologies, Grand Island, NY) in mTeSR1 medium (StemCell Technologies, Vancouver, Canada) at 37 C and 5% CO 2 .…”

Effects of magnesium on growth and proliferation of human embryonic stem cells

“…The H9 hESCs (WiCell) were stably transfected with an octamer-binding transcription factor-green fluorescent protein (OCT4-GFP) reporter plasmid as previously described [2]. H9-OCT4 ESCs were maintained in a T-25 flask (Falcon) under feeder-free conditions using Geltrex TM matrix (Invitrogen) and 1 mL of mTeSR®1 media (STEMCELL Technologies).…”