Abstract
Human mesenchymal stem cells (hMSCs) are widely used in the field of regenerative medicine, especially in bone tissue engineering. However, their differentiation is influenced by several factors, such as intracellular signaling pathways and cell heterogeneity. If not regulated appropriately, the suboptimal differentiation effect has impeded its clinical application. Therefore, further research could be complemented on how to precisely regulate their differentiation and how to purify hMSCs with osteogenesis bias.Firstly, using gold micropatterned poly(ethylene glycol) (PEG) hydrogels, we measured the velocity of nucleus displacement of hMSCs. Interestingly, higher velocity positively correlates with enhanced adipogenic differentiation at the early stage (1 day) than that of cells with osteogenic commitment. Previous studies revealed cofilin as an important regulator in osteogenic differentiation and actin stabilization. By employing siRNA and gene overexpression techniques, our findings demonstrated that hMSCs with silenced cofilin gene exhibited a notable decrease in velocity of nucleus displacement and an enhanced potential for osteogenesis. Conversely, cells overexpressing cofilin displayed increased nucleus displacement and a tendency towards adipogenesis. These results emphasize the crucial regulatory role of cofilin during the early stages of hMSC differentiation.
Second, based on these findings, using miR-106a-5p with distinct expression levels in hMSCs displaying diverse differentiation tendencies as a trigger, we designed a phenotype-specific Y-shaped DNA nanostructure. This unique Y-DNA nanostructure can be specifically activated by the microRNAs highly expressed in cells with non-osteogenic commitment such as miR-106a-5p found in adipogenesis-biased hMSCs. Upon activation, the nanostructure undergoes disassembly and releases antisense oligonucleotide (ASO) to inhibit the expression of cofilin protein. The nanostructure exhibits concentration dependence, high specificity, high selectivity, and low toxicity. The hMSCs cultured in a dual induction medium demonstrated a significant enhancement in osteogenic differentiation after being transfected with this nanostructure. This methodology offers a promising approach to overcome the cell heterogeneity obstacle in bone regeneration applications.
Third, we conducted single-cell RNA-sequencing analyses to explore different transcriptomic profiles of early differentiated (1-d adipogenic (AM) and osteogenic (OM) sole induction) and undifferentiated (GM) human bone morrow derived MSCs (hBMMSCs). By analyzing genes associated with cofilin phosphorylation and dephosphorylation signal pathway, we identified potential genes correlated to cell membrane surface proteins. Flow cytometry analysis revealed a significant increase in CD266 expression in 1-d OM hBMMSCs compared to cells induced for 1-d AM cells. Furthermore, cells with high CD266 expression, sorted from GM and 1-d OM cells, exhibited enhanced osteogenic ability compared to cells with low CD266 expression. Remarkably, this marker demonstrated that similar trends exist in human adipose-derived MSCs, indicating the universality of surface marker for identifying cells with enhanced osteogenic potential. This discovery provides a novel surface marker for the purification of hMSCs.
In summary, the Y-shaped DNA material proposed for targeted regulation of hMSCs differentiation and the surface marker CD266 used for enhancing and purification of osteogenic biased hMSCs, which provides exciting potential in the field of regenerative medicine applications.
| Date of Award | 30 Aug 2024 |
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| Original language | English |
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| Supervisor | Ting Hsuan CHEN (Supervisor) |