Abstract
Biophysical cues influence many aspects of cell behavior. Stiffness of the extracellular matrix is probed by cells and transduced into biochemical signals through mechanotransduction protein networks, strongly influencing stem cell behavior. Cellular stemness is intimately related with mechanical properties of the cell, like intracellular contractility and stiffness, which in turn are influenced by the microenvironment. Pluripotency is associated with soft and low-contractility cells. Hence, we postulated that soft cell culture substrates, presumably inducing low cellular contractility and stiffness, increase the reprogramming efficiency of mesenchymal stem/stromal cells (MSCs) into induced pluripotent stem cells (iPSCs). We demonstrate that soft substrates (1.5 or 15 kPa polydimethylsiloxane – PDMS) caused modulation of several cellular features of MSCs into a phenotype closer to pluripotent stem cells (PSCs). MSCs cultured on soft substrates presented more relaxed nuclei, lower maturation of focal adhesions and F-actin assembling, more euchromatic and less heterochromatic nuclear DNA regions, and increased expression of pluripotency-related genes. These changes correlate with the reprogramming of MSCs, with a positive impact on the kinetics, robustness of colony formation and reprogramming efficiency. Additionally, substrate stiffness influences several phenotypic features of iPS cells and colonies, and data indicates that soft substrates favor full iPSC reprogramming.
Highlights
Cellular microenvironment biophysical cues strongly influence stem/progenitor cell behavior by regulating processes like migration[1,2,3], proliferation[4,5,6], differentiation[7,8,9,10] and maintenance of pluri/multipotency[11,12,13,14]
The results suggest that soft substrates allow mesenchymal stem/stromal cells (MSCs) to acquire a more relaxed state, which correlates with the enhanced formation of induced pluripotent stem cells (iPSCs)-like colonies at earlier time-points when compared with cells on a stiff substrate (Fig. 4C)
We have shown that MSC reprogramming to iPSCs is faster on a softer matrix
Summary
Cellular microenvironment biophysical cues strongly influence stem/progenitor cell behavior by regulating processes like migration[1,2,3], proliferation[4,5,6], differentiation[7,8,9,10] and maintenance of pluri/multipotency[11,12,13,14]. Nuclear rigidity increases with the differentiation state of the cell, pluripotent stem cells (PSCs), multipotent cells and fully differentiated cells present www.nature.com/scientificreports/. The nuclei of pluripotent cells present more euchromatic regions than progenitor[23] and fully differentiated[24] cells. This seems intimately related to mechanical signals and intracellular stiffness and contractility, since the nucleus is mechanically coupled to cytoskeletal elements by the LINC (Linker of Nucleoskeleton to Cytoskeleton) complex[25]. The pluripotent state is associated with intracellular and nuclear softness and an overall relaxed state of the cytoskeleton and the cell in general. Since intracellular contractility of mesenchymal stem/stromal cells (MSCs) scales with substrate stiffness[29], we postulated that by using soft cell culture substrates, low intracellular contractility and stiffness should be achieved and reprogramming efficiency into iPSCs should increase
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