Ortega-Pineda Lilibeth, Guilfoyle Elizabeth, Rincon-Benavides Maria Angelica, Anaparthi Amrita Lakshmi, Lemmerman Luke R, Cuellar-Gaviria Tatiana Z, Lawrence William, Buss Jill L, Deng Binbin, Blackstone Britani N, Salazar-Puerta Ana, McComb David W, Powell Heather, Gallego-Perez Daniel, Higuita-Castro Natalia
Department of Biomedical Engineering, The Ohio State University, Columbus, OH.
Biomedical Science Graduate Program, The Ohio State University, Columbus, OH.
Adv Nanobiomed Res. 2023 Oct;3(10). doi: 10.1002/anbr.202200173. Epub 2023 Sep 5.
Direct nuclear reprogramming has the potential to enable the development of β cell replacement therapies for diabetes that do not require the use of progenitor/stem cell populations. However, despite their promise, current approaches to β cell-directed reprogramming rely heavily on the use of viral vectors. Here we explored the use of extracellular vesicles (EVs) derived from human dermal fibroblasts (HDFs) as novel non-viral carriers of endocrine cell-patterning transcription factors, to transfect and transdifferentiate pancreatic ductal epithelial cells (PDCs) into hormone-expressing cells. Electrotransfection of HDFs with expression plasmids for , , and () led to the release of EVs loaded with at the gene, mRNA, and protein level. Exposing PDC cultures to -loaded EVs led to successful transfection and increased expression in PDCs, which ultimately resulted in endocrine cell-directed conversions based on the expression of insulin/c-peptide, glucagon, and glucose transporter 2 (Glut2). These findings were further corroborated in a mouse model following intraductal injection of - vs sham-loaded EVs. Collectively these findings suggest that dermal fibroblast-derived EVs could potentially serve as a powerful platform technology for the development and deployment of non-viral reprogramming-based cell therapies for insulin-dependent diabetes.
直接核重编程有潜力推动糖尿病β细胞替代疗法的发展，这类疗法无需使用祖细胞/干细胞群体。然而，尽管前景广阔，但目前β细胞定向重编程方法严重依赖病毒载体的使用。在此，我们探索了源自人皮肤成纤维细胞（HDFs）的细胞外囊泡（EVs）作为内分泌细胞模式转录因子的新型非病毒载体，用于转染胰腺导管上皮细胞（PDCs）并将其转分化为表达激素的细胞。用编码 、 和 （）的表达质粒对HDFs进行电穿孔，导致在基因、mRNA和蛋白质水平上释放载有 的EVs。将PDC培养物暴露于载有 的EVs导致成功转染，并增加了PDCs中 的表达，最终基于胰岛素/C肽、胰高血糖素和葡萄糖转运蛋白2（Glut2）的表达实现了内分泌细胞定向转化。在小鼠模型中，通过导管内注射载有 的EVs与假载EVs后，这些发现得到了进一步证实。总体而言，这些发现表明，真皮成纤维细胞衍生的EVs可能作为一种强大的平台技术，用于开发和应用基于非病毒重编程的胰岛素依赖型糖尿病细胞疗法。
