Roy Vincent, Bienjonetti Isabella, Paquet Alexandre, Gros-Louis François
Department of Surgery, Faculty of Medicine, Laval University, Quebec City, QC, Canada.
Division of Regenerative Medicine, CHU de Quebec research Center, Laval University, Quebec City, QC, Canada.
Biotechnol J. 2025 Mar;20(3):e202400594. doi: 10.1002/biot.202400594.
Studying neurological disorders in vitro remains challenging due to the complexity of the human brain and the limited availability of primary neural cells. Tissue engineering enables the development of three-dimensional (3D) cell culture systems by generating a self-produced extracellular matrix (ECM) substrate. Culturing cells within this ECM substrate is known to more effectively mimic physiological conditions compared to traditional monolayer cultures. In this study, we analyzed the proteome and matrisome of 3D cultured dermal fibroblasts embedded in a self-produced ECM. Interestingly, in silico analysis predicted strong activation of neurogenesis-associated functions in this tissue-engineered 3D model. We showed that ECM proteins typically linked to neuronal development and maintenance were also expressed by dermal fibroblasts. Coculturing dermal fibroblasts with induced pluripotent stem cell (iPSC)-derived motor neurons notably enabled long-lasting culture periods while minimizing neuronal death, all without the need for costly media supplements. Furthermore, fibroblast-conditioned media enhanced neuronal survival. Although we demonstrated that the dermal fibroblast-derived ECM provided a rich matrix of proteins and signaling molecules that support neuronal growth and survival, the ECM alone seems insufficient to sustain the neuronal networks. These findings suggest that 3D cultured patient-derived dermal fibroblasts generate a neuro-supportive microenvironment and could serve as a cost-effective and less invasive alternative to brain biopsies for modeling complex neurological disorders. This approach offers a promising platform for studying such neural growth and survival and exploring therapeutic strategies for neurological diseases.
由于人类大脑的复杂性以及原代神经细胞的可用性有限，体外研究神经疾病仍然具有挑战性。组织工程通过生成自我产生的细胞外基质（ECM）底物，促进了三维（3D）细胞培养系统的发展。与传统的单层培养相比，在这种ECM底物中培养细胞能更有效地模拟生理条件。在本研究中，我们分析了嵌入自我产生的ECM中的3D培养真皮成纤维细胞的蛋白质组和基质组。有趣的是，计算机分析预测了这种组织工程3D模型中神经发生相关功能的强烈激活。我们发现，通常与神经元发育和维持相关的ECM蛋白也由真皮成纤维细胞表达。将真皮成纤维细胞与诱导多能干细胞（iPSC）衍生的运动神经元共培养，显著延长了培养周期，同时将神经元死亡降至最低，而且无需昂贵的培养基补充剂。此外，成纤维细胞条件培养基可提高神经元存活率。虽然我们证明了真皮成纤维细胞衍生的ECM提供了丰富的蛋白质和信号分子基质，支持神经元生长和存活，但仅靠ECM似乎不足以维持神经元网络。这些发现表明，3D培养的患者来源的真皮成纤维细胞产生了一个神经支持性微环境，可作为一种经济有效且侵入性较小的替代脑活检的方法，用于模拟复杂的神经疾病。这种方法为研究此类神经生长和存活以及探索神经疾病的治疗策略提供了一个有前景的平台。
