Campos Jonas, Sampaio-Marques Belém, Santos Diogo, Barata-Antunes Sandra, Ribeiro Miguel, Serra Sofia C, Pinho Tiffany S, Canto-Gomes João, Marote Ana, Cortez Margarida, Silva Nuno A, Michael-Titus Adina T, Salgado António J
Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057, Braga, Portugal.
ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal.
Tissue Eng Regen Med. 2025 Jan;22(1):113-128. doi: 10.1007/s13770-024-00679-5. Epub 2024 Nov 4.
Priming strategies that improve the functionality of MSCs may be required to address issues limiting successful clinical translation of MSC therapies. For conditions requiring high trophic support such as brain and spinal cord injuries, priming MSCs to produce higher levels of trophic factors may be instrumental to facilitate translation of current MSC therapies. We developed and tested a novel molecular priming paradigm using docosahexaenoic acid (DHA) to prime adipose tissue-derived mesenchymal stromal cells (ASCs) to enhance the secretome neuroregulatory potential.
Comprehensive dose-response and time-course assays were carried to determine an optimal priming protocol. Secretome total protein measurements were taken in association with cell viability, density and morphometric assessments. Cell identity and differentiation capacity were studied by flow cytometry and lineage-specific markers. Cell growth was assessed by trypan-blue exclusion and senescence was probed over time using SA-β-gal, morphometry and gene expression. Secretomes were tested for their ability to support differentiation and neurite outgrowth of human neural progenitor cells (hNPCs). Neuroregulatory proteins in the secretome were identified using multiplex membrane arrays.
Priming with 40 µM DHA for 72 h significantly enhanced the biosynthetic capacity of ASCs, producing a secretome with higher protein levels and increased metabolic viability. DHA priming enhanced ASCs adipogenic differentiation and adapted their responses to replicative senescence induction. Furthermore, priming increased concentrations of neurotrophic factors in the secretome promoting neurite outgrowth and modulating the differentiation of hNPCs.
These results provide proof-of-concept evidence that DHA priming is a viable strategy to improve the neuroregulatory profile of ASCs.
可能需要采用能够改善间充质干细胞（MSC）功能的预处理策略，以解决限制MSC疗法成功临床转化的问题。对于诸如脑和脊髓损伤等需要高营养支持的病症，预处理MSC以产生更高水平的营养因子可能有助于促进当前MSC疗法的转化。我们开发并测试了一种新型分子预处理模式，使用二十二碳六烯酸（DHA）预处理脂肪组织来源的间充质基质细胞（ASC），以增强其分泌组的神经调节潜能。
进行了全面的剂量反应和时间进程分析，以确定最佳预处理方案。在进行细胞活力、密度和形态学评估的同时，测定分泌组总蛋白含量。通过流式细胞术和谱系特异性标志物研究细胞身份和分化能力。通过台盼蓝排斥法评估细胞生长情况，并使用SA-β-半乳糖苷酶、形态学和基因表达随时间检测衰老情况。测试分泌组支持人类神经祖细胞（hNPC）分化和神经突生长的能力。使用多重膜阵列鉴定分泌组中的神经调节蛋白。
用40µM DHA预处理72小时可显著增强ASC的生物合成能力，产生蛋白质水平更高且代谢活力增强的分泌组。DHA预处理增强了ASC的成脂分化能力，并使其对复制性衰老诱导的反应得到调整。此外，预处理增加了分泌组中神经营养因子的浓度，促进神经突生长并调节hNPC的分化。
这些结果提供了概念验证证据，表明DHA预处理是改善ASC神经调节特性的可行策略。
