Three-dimensional cell-cell interactions promote direct reprogramming of patient fibroblasts into functional and transplantable neurons

Direct reprogramming of somatic cells into induced neurons (iNs) has become an attractive strategy for the generation of patient-specific neurons for disease modeling and regenerative neuroscience. To this end, adult human dermal fibroblasts (hDFs) present one of the most relevant cell sources. However, iNs generated from adult hDFs using traditional two-dimensional (2D) cultures are difficult to maintain long-term in vitro and face challenges in survival upon transplantation into the adult brain. These limitations impose considerable constraints on the biomedical applications and translational potential of iN technology. Here, we present a platform for direct in vitro reprogramming of adult hDFs inside three-dimensional suspension microcultures (3D-iNs) that eliminates some of the major bottlenecks in the direct neuronal reprogramming field. We show that the 3D environment favors neuronal over fibroblast cellular identity to yield more robust conversion into functional neurons with extended culturing span. The 3D reprogramming approach also provides a platform for fusion of different neuronal populations into induced assembloids. Importantly, unlike conventional iNs, 3D-iNs can be gently harvested and transplanted into the adult rodent brain to reproducibly generate functionally integrated neuron-rich grafts. Due to its simplicity, versatility, and robustness, our approach could readily be adapted as a culturing platform used for a broad range of in vitro and in vivo studies to improve disease modeling, drug screening, and other biomedical applications. Bulk RNA-seq of fibroblasts, reprogrammed cells in 2D and 3D *************************************************************** Due to privacy concerns we are unable to submit raw data and has hence only uploaded count matrices. ***************************************************************

将体细胞直接重编程为诱导神经元（induced neurons, iNs）现已成为获取患者特异性神经元以开展疾病建模与再生神经科学研究的极具吸引力的策略。为此，成人皮肤成纤维细胞（adult human dermal fibroblasts, hDFs）是目前最具应用价值的细胞来源之一。然而，利用传统二维（two-dimensional, 2D）培养体系从成人hDFs诱导生成的iNs，难以在体外长期维持，且移植入成体大脑后存活率面临严峻挑战。这些局限性极大限制了iN技术的生物医学应用与转化潜力。本研究开发了一种可在三维悬浮微培养体系中直接体外重编程成人hDFs的平台，所获得的三维诱导神经元（3D-iNs）可破解直接神经元重编程领域的多项核心瓶颈。研究证实，三维培养环境更倾向于维持神经元而非成纤维细胞的细胞身份，可实现更高效的功能神经元转化，并延长体外培养周期。该三维重编程策略还可作为将不同神经元群体融合为诱导类组装体（induced assembloids）的研究平台。尤为重要的是，与传统iNs不同，3D-iNs可被温和收获并移植入成体啮齿类动物大脑，可稳定生成具备功能整合性的神经元富集移植物。由于该方法具备操作简便、通用性强且结果稳定的优势，可轻松适配多种体外与体内研究场景，用于优化疾病建模、药物筛选及其他生物医学应用。本研究对成纤维细胞、二维培养及三维培养中的重编程细胞开展了批量RNA测序（bulk RNA-seq）。
****************************************************************
因隐私顾虑，我们无法提交原始数据，因此仅上传了计数矩阵（count matrices）。
****************************************************************