Chancellor's Professor and Chair UCLA Department of Bioengineering, United States
Introduction: Cell reprogramming affords the capacity to convert mature cells to alternative fates, which represents a major advancement in modern biology and has wide applications in regenerative medicine, disease modeling, and drug screening. By employing exogenous transcription factors, somatic cells can be directly reprogrammed into distantly related cell types, such as neurons and cardiomyocytes. While biochemical factors have been widely recognized as regulatory signals of cell reprogramming, how biophysical factors, e.g., mechanical properties of cell-adhesive matrices, regulate cell reprogramming are not well understood. Since extracellular matrix (ECM) has complex mechanical properties, including viscoelasticity, nonlinear elasticity, and plasticity, engineering synthetic matrices with tunable mechanical properties to investigate the mechanical regulation of cell reprogramming will provide crucial insights of cell fate determination during development and tissue regeneration. Therefore, we investigate how matrix viscoelasticity regulates cell reprogramming by employing hydrogels with independently tunable stiffness and viscoelasticity.
Materials and Methods: We used covalent and ionic crosslinking of alginate hydrogel to independently control the stiffness and viscoelasticity respectively, and adjusted the concentration of crosslinkers and the molecular weight of alginate polymers to fabricate matrix with defined mechanical properties. Mechanical characterization was performed to confirm the elastic moduli and stress relaxation properties of hydrogels by using three interconnected methods including atomic force microscopy (AFM), rheology measurement, and compression tests. Adult mouse fibroblasts transduced with doxycycline (DOX)-inducible lentiviral vectors containing three reprogramming factors (i.e., Brn2, Ascl1, and Myt1l, BAM) were then seeded onto hydrogels. Neuron-specific class III β-tubulin (Tuj1) expression of cells after one week will be examined to compare the reprogramming efficiency of fibroblasts on hydrogels with different properties. Mechanical regulation of epigenetic changes was examined by the analysis of histone modifications and chromatin accessibility.
Results, Conclusions, and Discussions: We constructed alginate hydrogels with different stiffness and stress relaxation behaviors by tuning the concentration of covalent and ionic crosslinkers (Figure 1a). Rheology measurement and compression test results demonstrated that covalently crosslinked alginate hydrogels do not exhibit stress relaxation in response to deformation, while ionically crosslinked gels have time-dependent mechanical dynamics (Figure 1b-c). To determine the effect of matrix stress relaxation on the direct conversion of fibroblasts into neurons, BAM-transduced fibroblasts were cultured on alginate hydrogels of tunable mechanical properties. After 7 days, cultures were fixed and immunostained for Tuj1 to identify iN cells and determine the reprogramming efficiency. We found that matrices with stress relaxation behaviors significantly enhanced the reprogramming efficiency compared to matrices without stress relaxation (Figure 1d-e), especially at the low stiffness that facilitates neural maturation. Epigenetic analysis suggested that viscoelastic matrices promoted a more open chromatin structure for cell reprogramming.
By employing hydrogels that have similar viscoelastic properties to native tissue and ECM, our findings provide mechanistic insights of how mechanical cues regulate cell reprogramming, and will facilitate the development of innovative materials for cell engineering in vitro and in vivo.
