The Biophysical Micro-Environment’s Influence on Cell Fate Decisions During Macrophage Activation and Somatic Cell Reprogramming
- Author(s): Reitz, Zachary Joseph Weber
- Advisor(s): Downing, Timothy L
- et al.
Cells are known to sense and respond to their mechanical microenvironment in profound ways. Various evidence has implicated the adhesome, a body of adhesion associated proteins, in several cell fate decisions, including differentiation and proliferation. Furthermore, recent work has demonstrated that substrate topography can modulate the epigenetic state of fibroblasts, facilitate cell reprogramming, and temper inflammatory activation. However, the effectors responsible for such phenomena remain poorly understood. Here we explore the effect of biomaterial design, adhesion, and intracellular forces of the epigenetic and transcriptional regulation of cell state during macrophage activation and somatic cell reprogramming.
Regarding macrophage activation, we relied entirely on murine bone marrow-derived macrophages (BMDMs). To study the effect of adhesion on BMDM behavior, we utilized microcontact printing to produce fibronectin patterned substrates ontop of which we seeded BMDMs. Next, we induced macrophage activation using lipopolysaccharide (LPS) and interferon-γ (INFγ) and found patterned substrates reduce inflammatory gene expression and histone-3 acetylation (H3Ac) while also increasing cellular elongation. We incorporated these results into a bioinformatic reanalysis of transcriptional and epigenetic data derived from a study of H3Ac protein binding during macrophage polarization. This analysis allowed us to identify epigenetic mechanisms linking the adhesome and inflammatory gene expression.
In our investigation of somatic cell reprogramming, we found extracellular matrix binding (ECM) and mechanosensitive ion channel activation reduce reprogramming efficiency. In addition to these results, we discovered that 104 adhesome genes are dynamically regulated during the reprogramming process. Subsequently, we performed an shRNA knockdown of each of these genes and found over 90% of our knockdowns increased the number of TRA-1-60 positive pluripotent colonies. Our knockdown of SHROOM3, a gene associated with apical cellular constriction, increased reprogramming efficiency by 27-fold. Further investigation into SHROOM3 identified a mechano-sensitive critical state transition, which may be necessary for successful reprogramming.
These observations establish adhesome gene expression and mechanical signaling as influential regulators of cell fate during macrophage activation and reprogramming. Moreover, our findings may guide future attempts to regulate cell state and fate using biophysical stimuli. They may also help shed light on cell behavior in various disease states involving cancer and inflammation.