Bioengineering Graduate Program, University of Kansas, Lawrence, KS, USA.
Department of Plastic Surgery, University of Kansas Medical Center, 3901 Rainbow Blvd., Mail Stop: 3051, Kansas City, KS, USA.
Tissue Eng Regen Med. 2023 Oct;20(6):993-1013. doi: 10.1007/s13770-023-00565-6. Epub 2023 Jul 29.
The secretome of adipose-derived mesenchymal stem cells (ASCs) offers a unique approach to understanding and treating wounds, including the critical process of epidermal regeneration orchestrated by keratinocytes. However, 2D culture techniques drastically alter the secretory dynamics of ASCs, which has led to ambiguity in understanding which secreted compounds (e.g., growth factors, exosomes, reactive oxygen species) may be driving epithelialization.
A novel tissue-mimetic 3D hydrogel system was utilized to enhance the retainment of a more regenerative ASC phenotype and highlight the functional secretome differences between 2D and 3D. Subsequently, the ASC-secretome was stratified by molecular weight and the presence/absence of extracellular vesicles (EVs). The ASC-secretome fractions were then evaluated to assess for the capacity to augment specific keratinocyte activities.
Culture of ASCs within the tissue-mimetic system enhanced protein secretion ~ 50%, exclusively coming from the > 100 kDa fraction. The ASC-secretome ability to modulate epithelialization functions, including migration, proliferation, differentiation, and morphology, resided within the "> 100 kDa" fraction, with the 3D ASC-secretome providing the greatest improvement. 3D ASC EV secretion was enhanced two-fold and exhibited dose-dependent effects on epidermal regeneration. Notably, ASC-EVs induced morphological changes in keratinocytes reminiscent of native regeneration, including formation of stratified cell sheets. However, only 3D-EVs promoted collective cell sheet migration and an epithelial-to-mesenchymal-like transition in keratinocytes, whereas 2D-EVs contained an anti-migratory stimulus.
This study demonstrates how critical the culture environment is on influencing ASC-secretome regenerative capabilities. Additionally, the critical role of EVs in modulating epidermal regeneration is revealed and their translatability for future clinical therapies is discussed.
脂肪间充质干细胞（ASCs）的分泌组为了解和治疗伤口提供了一种独特的方法，包括角质形成细胞协调的表皮再生的关键过程。然而，二维培养技术极大地改变了 ASCs 的分泌动力学，这导致了对哪些分泌化合物（如生长因子、外泌体、活性氧）可能驱动上皮化的理解存在歧义。
利用新型组织模拟 3D 水凝胶系统增强了更具再生能力的 ASC 表型的保留，并突出了 2D 和 3D 之间功能分泌组的差异。随后，根据分子量和细胞外囊泡（EVs）的存在/不存在对 ASC 分泌组进行分层。然后评估 ASC 分泌组部分，以评估其增强特定角质形成细胞活性的能力。
在组织模拟系统中培养 ASCs 可使蛋白分泌增加约 50%，仅来自>100 kDa 级分。ASC 分泌组调节上皮化功能（包括迁移、增殖、分化和形态）的能力存在于>100 kDa 级分中，3D ASC 分泌组提供了最大的改善。3D ASC EV 的分泌增加了两倍，并表现出剂量依赖性的表皮再生作用。值得注意的是，ASC-EVs 诱导角质形成细胞形态发生变化，类似于天然再生，包括形成分层细胞片。然而，只有 3D-EVs 促进了角质形成细胞的集体细胞片迁移和上皮-间充质样转化，而 2D-EVs 含有抗迁移刺激。
本研究表明培养环境对影响 ASC 分泌组再生能力的重要性。此外，还揭示了 EVs 在调节表皮再生中的关键作用，并讨论了它们在未来临床治疗中的转化潜力。
