Interrogating ground state rhesus macaque and human pluripotent stem cells
Pather, Sarshan Rubintheran
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Iterative chemical screening has resulted in optimized culture conditions to support the self-renewal of human and non-human primate pluripotent stem cells (PSCs) in a mouse embryonic stem cell-like ground state of pluripotency. While all protocols employ leukemia inhibitory factor (LIF) supplemented with MEK/ERK and GSK3-β inhibitors (LIF 2i), many indispensably require additional anti-apoptotic small molecules and primed growth factors. The finding of a minimal cocktail of LIF 2i plus axin-stabilizing XAV939 (termed LIF 3i) that efficiently reverts select myeloid progenitor hiPSC lines to a ground state advances the possibility that among heterogeneous PSCs, many lines are epigenetically more amenable to naïve reversion than others. Here, I report characterization of LIF 3i-permissive human naïve PSCs by examining transcriptome microarray data and OCT3/4 enhancer DNA methylation profiles. Additionally, I report an optimized feeder-dependent culture system to support naïve-like self-renewal in non-permissive rhesus macaque embryonic stem cells (rESCs) derived from in vitro fertilization (IVF), somatic cell nuclear transfer (SCNT), and parthenogenesis. Use of a potent VEGF receptor inhibitor, sunitinib malate, together with LIF 3i or LIF 2i, promoted naïve-like ground state self-renewal in rESCs. Efficient derivation and careful interrogation of human and non-human primate naïve PSCs before in vitro differentiation, plasmid-based gene targeting, or blastocyst complementation experiments is a necessary process to advance naïve pluripotency in stem cell biology and regenerative medicine.