Investigating the Spatial Distribution of Integrin β₁ in Patterned Human Mesenchymal Stem Cells Using Super-Resolution Imaging

Abstract

Lineage commitment of human mesenchymal stem cells (hMSCs) could be directed through micro/nanopatterning of the extracellular matrix (ECM) between cells and substrate. Integrin receptors, integrator of the ECM and cell cytoskeleton, function as molecular bridges linking cells to different biophysical cues translated from patterned ECM. Here we report the distinct recruitment of active integrin β1 (ITG-β1) in hMSCs when they were committed toward the cardiomyogenic lineage on a micropatterned surface. In addition, a systematic study of the distribution of ITG-β1 was performed on focal adhesions (FAs) using a direct stochastic optical reconstruction microscopy (dSTORM) technique, a super-resolution imaging technique to establish the relationship between types of integrin expression and its distribution pattern that are associated with cardiomyogenic differentiation of hMSCs. We ascertained that elongated FAs of ITG-β1 expressed in patterned hMSCs were more prominent than FAs expressed in unpatterned hMSCs. However, there was no significant difference observed between the widths of FAs from both experimental groups. It was found in patterned hMSCs that the direction of FA elongation coincides with cell orientation. This phenomenon was however not observed in unpatterned hMSCs. These results showed that the biophysical induction methods like FAs patterning could selectively induce hMSCs lineage commitment via integrin-material interaction.

Role of Cytoskeletal Tension in the Induction of Cardiomyogenic Differentiation in Micropatterned Human Mesenchymal Stem Cell

“…Previous studies have illustrated the role of elongated FAs in cardiomyogenic differentiation of cells . In addition, we investigated the dominant role of elongated ITG‐β 1 FA in myocardial lineage commitment of similar patterned hMSCs . Thus, based on previous findings and our current observations we postulated that micropatterning promoted the formation of elongated ITG‐β 1 FA which in turn induced the development of aligned actomyosin stress fibers without crosslinking.…”

“…Recently, we showed that active ITG‐β 1 FAs were predominantly expressed in hMSCs cultured on fibronectin coated substrates as compared to other types of ITG‐β . In addition, in another study, it was observed that ITG‐β 1 activation through increased fibronectin density enhanced the cell traction forces .…”

“…Briefly, PDMS stamps were covered with fibronectin dissolved in phosphate buffer saline (PBS) solution (PAA) (50 μg mL −1 ) for 1 h and then blow dried with pressurized nitrogen gas. Cell micropatterning was done according to the procedure described earlier . For that purpose, fibronectin inked stamps were mounted onto amino‐silanized glass coverslips.…”

Modulating Human Mesenchymal Stem Cell Plasticity Using Micropatterning Technique

“…Cell micropatterning was performed as previously described [22] . Briefly, polydimethylsiloxane (PDMS) elastomeric patterned stamps bearing 20 µm elevated lanes and 40 µm spacing were fabricated and utilized to ink the human plasma derived fibronectin (BD Biosciences) onto glutaraldehyde (Sigma) treated cover glasses [18] .…”

Simple and Rapid Bioink Jet Printing for Multiscale Cell Adhesion Islands

“…Most of them involve the contact of a stamp or a writing head with a substrate . In spite of their merits, they all lead to significant drawbacks in terms of repeatability, resolution, and costs . Conversely, the droplet‐based techniques involve contact‐free procedures and offer several advantages in terms of simplicity, versatility, and control of deposition .…”