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Published on: May 30, 2012
Biophysical regulation of mouse embryonic stem cell fate and genomic integrity by feeder derived matrices
Lakshmi Kavitha Sthanam1, Amlan Barai1, Anuj Rastogi1
1Department of Biosciences & Bioengineering, IIT Bombay, Mumbai, 400076, India.
Abstract:
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 through integrin-mediated mechanoadaptation. While baseline mESC pluripotency is maintained at early time points, activation of mESC contractility by LPA leads to drop in pluripotency levels. In contrast, addition of blebbistatin and LIF independently increases pluripotency by suppressing mechanoadaptation, highlighting the role of mechanoadaptation in regulating pluripotency and illustrating the role of LIF as a mechano-inhibitor in mESCs. Long-term culture of mESCs on MEFDMs under LIF-free conditions triggers loss of pluripotency, and induces ligand-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.
Insights
Mouse embryonic stem cells (mESCs) maintain pluripotency through physical interactions with MEF-derived matrices (MEFDMs). These matrices regulate mESC contractility and fate, offering potential for regenerative medicine.
Area of Science:
- Stem Cell Biology
- Biomaterials Science
- Mechanobiology
Background:
- Mouse embryonic stem cells (mESCs) require specific conditions for pluripotency maintenance, typically involving feeder cells (MEFs) and specific media (MEFCM, LIF).
- The physical cues and interactions between mESCs and their microenvironment, particularly MEF-derived matrices (MEFDMs), are less understood compared to biochemical factors.
- Understanding these physical interactions is crucial for developing improved methods for mESC culture and application.
Purpose of the Study:
- To investigate the role of physical interactions between mESCs and MEF-derived matrices (MEFDMs) in regulating mESC pluripotency and fate.
- To characterize the physicochemical properties of MEFDMs and their influence on mESC behavior.
- To explore the potential of MEFDMs for clinical and regenerative medicine applications.
Main Methods:
- Characterization of the physicochemical properties (composition, stiffness) of MEF-derived matrices (MEFDMs).
- Culture of mESCs on MEFDMs and assessment of pluripotency markers and cell contractility.
- Manipulation of mESC contractility using agents like LPA and blebbistatin, and assessment of pluripotency.
- Long-term culture of mESCs on MEFDMs under LIF-free conditions to evaluate pluripotency maintenance and differentiation potential.
- Comparison of MEFDMs with traditional gelatin-coated substrates for mESC culture and genomic integrity.
Main Results:
- MEFDM stiffness and composition, regulated by MEF contractility, modulate mESC pluripotency via integrin-mediated mechanoadaptation.
- Activation of mESC contractility (e.g., by LPA) reduces pluripotency, while suppression (e.g., by blebbistatin or LIF) enhances it.
- LIF acts as a mechano-inhibitor, suppressing mechanoadaptation and maintaining pluripotency.
- Long-term LIF-free culture on MEFDMs leads to loss of pluripotency and induction of osteogenic differentiation (Runx2 expression).
- MEFDMs maintain genomic integrity (euploidy) and support LIF-free expansion and differentiation, unlike gelatin substrates.
Conclusions:
- Physical interactions and mechanoadaptation play a critical role in regulating mESC pluripotency.
- MEF-derived matrices (MEFDMs) provide a biomimetic environment that supports mESC pluripotency and differentiation.
- MEFDMs show significant promise for clinical applications in regenerative medicine due to their ability to support LIF-free expansion and maintain genomic stability.
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