Y-shaped DNA as a dynamic self-assembly nanomaterial for phenotype-specific regulation of stem cell differentiation on the gene level

While genetic engineering has offered new strategies for regulating stem cell differentiation, the efficacy varies in cells with different phenotypes or lineage commitments, leading to inconsistent differentiation outcomes and uncertainty in regenerative medicine. To address this issue, we employ a Y-shaped DNA (Y-DNA) as a nanomaterial to phenotype-specifically regulate differentiation of human mesenchymal stem cells (hMSCs). Y-DNA is composed of three DNA strands with complementary sequences and different roles. The Y-DNA designed in the present study can be uniquely activated by miR-106a-5p, a microRNA preferentially expressed in adipogenesis-biased hMSCs. Upon activation, the Y-DNA disassembles, releasing an antisense oligonucleotide that inhibits expression of cofilin, which serves as a key regulator to enhance adipogenic differentiation, and thus, prevents hMSCs from undergoing osteogenic differentiation. The key regulatory role of cofilin in hMSC differentiation is verified at the single-cell level on arginine–glycine–aspartate microislands under the nonfouling background of poly(ethylene glycol) hydrogels. Our strategy effectively redirects these cells towards osteogenic differentiation by inhibiting adipogenic differentiation, demonstrating dose dependence with high specificity, selectivity, and low toxicity. hMSCs cultured in a dual induction medium (a mixture of adipogenic medium and osteogenic medium) show enhanced osteogenic differentiation after transfection with the nanostructured Y-DNA. This approach addresses the challenge of cell heterogeneity in bone regeneration, offering a promising solution for precise control over stem cell fate. The ability of Y-DNA to specifically target cells with a propensity for adipogenic differentiation and to reprogram their lineage commitment has significant implications for the field of regenerative medicine, particularly in applications requiring enhanced purity of cell differentiation outcomes. © The Author(s) 2025.