Liu Wengang, Liu Ruili, Chu Lok Ting, Wang Xinlei, Wu Jianpeng, Ding Jiandong, Chen Ting Hsuan
Department of Biomedical Engineering, City University of Hong Kong, Hong Kong Special Administrative Region 999077, China.
State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, Shanghai 200438, China.
Regen Biomater. 2025 May 14;12:rbaf043. doi: 10.1093/rb/rbaf043. eCollection 2025.
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.
虽然基因工程为调节干细胞分化提供了新策略，但在具有不同表型或谱系定向的细胞中，其功效有所不同，导致分化结果不一致，给再生医学带来了不确定性。为解决这一问题，我们采用Y形DNA（Y-DNA）作为纳米材料，以表型特异性地调节人间充质干细胞（hMSC）的分化。Y-DNA由三条具有互补序列且作用不同的DNA链组成。本研究设计的Y-DNA可被miR-106a-5p独特激活，miR-106a-5p是一种在倾向于脂肪生成的hMSC中优先表达的微小RNA。激活后，Y-DNA解体，释放出一种反义寡核苷酸，该反义寡核苷酸抑制cofilin的表达，cofilin作为增强脂肪生成分化的关键调节因子，从而阻止hMSC进行成骨分化。在聚乙二醇水凝胶的非污染背景下，在精氨酸-甘氨酸-天冬氨酸微岛上，在单细胞水平验证了cofilin在hMSC分化中的关键调节作用。我们的策略通过抑制脂肪生成分化有效地将这些细胞重定向为成骨分化，显示出剂量依赖性，具有高特异性、选择性和低毒性。在双诱导培养基（脂肪生成培养基和成骨培养基的混合物）中培养的hMSC在用纳米结构的Y-DNA转染后，成骨分化增强。这种方法解决了骨再生中细胞异质性的挑战，为精确控制干细胞命运提供了一个有前景的解决方案。Y-DNA特异性靶向倾向于脂肪生成分化的细胞并重新编程其谱系定向的能力对再生医学领域具有重要意义，特别是在需要提高细胞分化结果纯度的应用中。
