RESUMO
The intricate interplay between biochemical and physical cues dictates pluripotent stem cell (PSC) differentiation to form various tissues. While biochemical modulation has been extensively studied, the role of biophysical microenvironments in early lineage commitment remains elusive. Here, we introduce a novel 3D cell culture system combining electrospun nanofibers with microfabricated polydimethylsiloxane (PDMS) patterns. This system enables the controlled formation of semispherical human induced pluripotent stem cell (hiPSC) colonies, facilitating the investigation of local mechanical stem cell niches on mechano-responsive signaling and lineage specification. Our system unveiled spatially organized RhoA activity coupled with actin-myosin cable formation, suggesting mechano-dependent hiPSC behaviors. Nodal network analysis of RNA-seq data revealed RhoA downstream regulation of YAP signaling, DNA histone modifications, and patterned germ layer specification. Notably, altering colony morphology through controlled PDMS microwell shaping effectively modulated the spatial distribution of mechano-sensitive mediators and subsequent differentiation. This study provides a cell culture platform to decipher the role of biophysical cues in early embryogenesis, offering valuable insights for material design in tissue engineering and regenerative medicine applications.
RESUMO
After decades of research, fully functional skin regeneration is still a challenge. Skin is a multilayered complex organ exhibiting a cascading healing process affected by various mechanisms. Specifically, nutrients, oxygen, and biochemical signals can lead to specific cell behavior, ultimately conducive to the formation of high-quality tissue. This biomolecular exchange can be tuned through scaffold engineering, one of the leading fields in skin substitutes and equivalents. The principal objective of this investigation was the design, fabrication, and evaluation of a new class of three-dimensional fibrous scaffolds consisting of poly(ε-caprolactone) (PCL)/calcium alginate (CA), with the goal to induce keratinocyte differentiation through the action of calcium leaching. Scaffolds fabricated by electrospinning using a PCL/sodium alginate solution were treated by immersion in a calcium chloride solution to replace alginate-linked sodium ions by calcium ions. This treatment not only provided ion replacement, but also induced fiber crosslinking. The scaffold morphology was examined by scanning electron microscopy and systematically assessed by measurements of the pore size and the diameter, alignment, and crosslinking of the fibers. The hydrophilicity of the scaffolds was quantified by contact angle measurements and was correlated to the augmentation of cell attachment in the presence of CA. The in vitro performance of the scaffolds was investigated by seeding and staining fibroblasts and keratinocytes and using differentiation markers to detect the evolution of basal, spinous, and granular keratinocytes. The results of this study illuminate the potential of the PCL/CA scaffolds for tissue engineering and suggest that calcium leaching out from the scaffolds might have contributed to the development of a desirable biological environment for the attachment, proliferation, and differentiation of the main skin cells (i.e., fibroblasts and keratinocytes).
