Faghih Hossein, Javeri Arash, Taha Masoumeh Fakhr
Department of Stem Cells and Regenerative medicine, Institute for Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
PLoS One. 2025 Sep 2;20(9):e0320367. doi: 10.1371/journal.pone.0320367. eCollection 2025.
The extracellular matrix (ECM) plays essential roles in regulating various aspects of nervous system development. The ECM can be obtained through decellularization techniques, which preserve the native structure of tissue while removing cells and genetic material. Despite recent advancements in decellularization methods, removing cells from brain tissue remains challenging due to its delicate mechanical structure. Moreover, previous studies have not specifically evaluated the impacts of decellularized brain ECM on dopaminergic specification of stem cells. Here, we decellularized rat brain sections using a combination of chemical and enzymatic factors. Successful decellularization of sections was confirmed by DAPI, Haematoxylin and Eosin and Masson's trichrome staining, laminin immunostaining, DNA content analysis, and scanning electron microscopy. The sections were then recellularized with human adipose tissue-derived stem cells (hADSCs) and subjected to dopaminergic differentiation using a combination of growth factors. Some ADSCs were also differentiated in gelatin-coated tissue culture plates, employing a conventional two-dimensional culture method. After 12 days, the differentiated cells in both conditions expressed certain neuronal markers, especially those related to dopaminergic differentiation. However, GLI1, VMAT2, GIRK2, and TH genes, as well as NEFL, FOXA2, LMX1A, and TH proteins were upregulated in the ADSCs differentiated on decellularized sections. Furthermore, DDC and CALB were exclusively expressed by the ADSCs on decellularized brain sections. Overall, our findings indicated the significance of decellularized brain ECM to serve as an effective bioscaffold for dopaminergic differentiation of hADSCs. This highlights the importance of decellularization techniques for the advancement of midbrain tissue engineering and regenerative medicine for Parkinson's disease in the future.
细胞外基质（ECM）在调节神经系统发育的各个方面发挥着重要作用。ECM可通过脱细胞技术获得，该技术在去除细胞和遗传物质的同时保留组织的天然结构。尽管脱细胞方法最近取得了进展，但由于脑组织机械结构脆弱，从脑组织中去除细胞仍然具有挑战性。此外，先前的研究尚未具体评估脱细胞脑ECM对干细胞多巴胺能分化的影响。在这里，我们使用化学和酶因素的组合对大鼠脑切片进行脱细胞处理。通过DAPI、苏木精和伊红以及Masson三色染色、层粘连蛋白免疫染色、DNA含量分析和扫描电子显微镜证实切片成功脱细胞。然后用人类脂肪组织来源的干细胞（hADSCs）对切片进行再细胞化处理，并使用生长因子组合进行多巴胺能分化。一些ADSCs也在明胶包被的组织培养板中采用传统的二维培养方法进行分化。12天后，两种条件下分化的细胞均表达了某些神经元标记物，尤其是与多巴胺能分化相关的标记物。然而，在脱细胞切片上分化的ADSCs中，GLI1、VMAT2、GIRK2和TH基因以及NEFL、FOXA2、LMX1A和TH蛋白上调。此外，DDC和CALB仅由脱细胞脑切片上的ADSCs表达。总体而言，我们的研究结果表明脱细胞脑ECM作为hADSCs多巴胺能分化的有效生物支架的重要性。这突出了脱细胞技术对未来帕金森病中脑组织工程和再生医学发展的重要性。
