Embodiments of the presently disclosed subject matter provide biomimetic cell culture substrates comprising highly tunable patterned polymer nanofiber matrices capable of modulating expression of critical self-renewal factors and markers of cell-cell interaction to maintain stemness of human mesenchymal stem cells in vitro. Embodiments of the presently-disclosed subject matter also provide scalable, highly repeatable methods of making biomimetic cell culture substrates by hot pressing thermoplastic polymer films into femtosecond laser-ablated nanopore molds to form patterned polymer nanofiber matrices on flat thermoplastic substrates.