Substrate stiffness has been indicated as an important factor to control stem cell fate, including proliferation and differentiation. To optimize the stiffness for the differentiation process from h-iPSCs (human induced pluripotent stem cells) into h-iCSCs (human corneal stromal cells derived from h-iPSCs) and the phenotypic maintenance of h-iCSCs in vitro, h-iPSCs were cultured on matrigel-coated tissue culture plate (TCP) (106 kPa), matrigel-coated polydimethylsiloxane (PDMS) 184 (1250 kPa), and matrigel-coated PDMS 527 (4 kPa) before they were differentiated to h-iCSCs. Immunofluorescence staining, quantitative real-time polymerase chain reaction (RT-qPCR), and western blot demonstrated that the stiffer substrate TCP promoted the h-iCSCs' differentiation from h-iPSCs. On the contrary, softer PDMS 527 was more effective to maintain the phenotype of h-iCSCs cultured in vitro. Finally, we cultured h-iCSCs on PDMS 527 until P3 and seeded them on a biomimetic collagen membrane to form the single-layer and multiple-layer bioengineered corneal stroma with high transparency properties and cell survival rate. In conclusion, the study is helpful for differentiating h-iPSCs to h-iCSCs and corneal tissue engineering by manipulating stiffness mechanobiology.
Keywords: bioengineered corneal stroma; h-iPSCs; keratocytes; mechanical force; polydimethylsiloxane.