Publication date: 25th July 2016
For maintaining pluripotency, mouse embryonic stem cells (mESCs) are typically grown on mitotically inactivated mouse embryonic fibroblasts (MEFs). While the role of MEF conditioned media (MEFCM) and leukemia inhibitory factor (LIF) in regulating mESC pluripotency has led to culturing of mESCs on LIF/MEFCM supplemented gelatin-coated substrates, the role of physical interactions between MEFs and mESCs in regulating mESC pluripotency remains to be fully understood. Here, we address this question by characterizing the physicochemical properties of MEF derived matrices (MEFDMs), and probing their role in regulating mESC fate. We show that MEFDM composition and stiffness—dictated by MEF contractility—regulates mESC pluripotency by modulating mESC contractility. While mESC pluripotency is retained on softer MEFDMs, the robust mechanoadaptation on stiff MEFDMs drives loss of pluripotency. Restoration of mESC pluripotency on stiff MEFDMs by inhibition of mESC contractility by blebbistatin and LIF independently, demonstrates the role of mechanoadaptation in regulating mESC pluripotency and illustrates the mechano-inhibitory function of LIF. Long-term culture of mESCs on MEFDMs under LIF-free conditions triggers loss of pluripotency, and induces stiffness-dependent expression of the osteogenic transcription factor Runx2. Maintenance of genomic integrity (euploidy) on MEFDMs but not on gelatin-coated substrates, combined with the ability of MEFDMs in supporting LIF-free expansion and differentiation of mESCs, illustrates the suitability of MEFDMs for clinical and regenerative medicine applications.
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