1. Academic Validation
  2. Soft substrates promote direct chemical reprogramming of fibroblasts into neurons

Soft substrates promote direct chemical reprogramming of fibroblasts into neurons

  • Acta Biomater. 2022 Aug 27;S1742-7061(22)00527-X. doi: 10.1016/j.actbio.2022.08.049.
Ziran Xu 1 Yan Li 2 Pengdong Li 3 Yingying Sun 4 Shuang Lv 5 Yin Wang 6 Xia He 7 Jinying Xu 8 Zhixiang Xu 9 Lisha Li 10 Yulin Li 11
Affiliations

Affiliations

  • 1 The Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun 130021, China. Electronic address: [email protected].
  • 2 Division of Orthopedics and Biotechnology, Department for Clinical Intervention and Technology (CLINTEC), Karolinska Institute, Stockholm, Sweden. Electronic address: [email protected].
  • 3 The Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun 130021, China; The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People's Hospital, Qingyuan 511518, Guangdong, China. Electronic address: [email protected].
  • 4 The Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun 130021, China; Department of Stomatology, The First Hospital of Jilin University, Changchun 130021, China. Electronic address: [email protected].
  • 5 The Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun 130021, China. Electronic address: [email protected].
  • 6 The Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun 130021, China. Electronic address: [email protected].
  • 7 The Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun 130021, China; Department of Pathology, Shanxi Bethune Hospital, Taiyuan 030032, China. Electronic address: [email protected].
  • 8 The Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun 130021, China; Department of Burns Surgery, The First Hospital of Jilin University, Changchun 130000, China. Electronic address: [email protected].
  • 9 The Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun 130021, China. Electronic address: [email protected].
  • 10 The Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun 130021, China. Electronic address: [email protected].
  • 11 The Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun 130021, China. Electronic address: [email protected].
Abstract

Fibroblasts can be directly reprogrammed via a combination of small molecules to generate induced neurons (iNs), bypassing intermediate stages. This method holds great promise for regenerative medicine; however, it remains inefficient. Recently, studies have suggested that physical cues may improve the direct reprogramming of fibroblasts into neurons, but the underlying mechanisms remain to be further explored, and the physical factors reported to date do not exhibit the full properties of the extracellular matrix (ECM). Previous in vitro studies mainly used rigid polystyrene dishes, while one of the characteristics of the native in-vivo environment of neurons is the soft nature of brain ECM. The reported stiffness of brain tissue is very soft ranging between 100 Pa and 3 kPa, and the effect of substrate stiffness on direct neuronal reprogramming has not been explored. Here, we show for the first time that soft substrates substantially improved the production efficiency and quality of iNs, without needing to co-culture with glial cells during reprogramming, producing more glutamatergic neurons with electrophysiological functions in a shorter time. Transcriptome sequencing indicated that soft substrates might promote glutamatergic neuron reprogramming through integrins, actin Cytoskeleton, Hippo signalling pathway, and regulation of mesenchymal-to-epithelial transition, and competing endogenous RNA network analysis provided new targets for neuronal reprogramming. We demonstrated that soft substrates may promote neuronal reprogramming by inhibiting microRNA-615-3p-targeting Integrin subunit beta 4. Our findings can aid the development of regenerative therapies and help improve our understanding of neuronal reprogramming. STATEMENT OF SIGNIFICANCE: First, we have shown that low stiffness promotes direct reprogramming on the basis of small molecule combinations. To the best of our knowledge, this is the first report on this type of method, which may greatly promote the progress of neural reprogramming. Second, we found that MicroRNA (miR)-615-3p may interact with Integrin subunit beta 4 (ITGB4), and the soft substrates may promote neural reprogramming by inhibiting miR-615-3p targeting ITGB4. We are the first to report on this mechanism. Our findings will provide more functional neurons for subsequent basic and clinical research in neurological regenerative medicine, and will help to improve the overall understanding of neural reprogramming. This work also provides new ideas for the design of medical biomaterials for nerve regeneration.

Keywords

Actin cytoskeleton; Human fibroblasts; ITGB4; Neuronal reprogramming; Small molecules; Soft substrates; YAP.

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