Theranostics 2021; 11(2):684-699. doi:10.7150/thno.46750

Research Paper

Expansion of murine and human olfactory epithelium/mucosa colonies and generation of mature olfactory sensory neurons under chemically defined conditions

Wenwen Ren1,4#, Li Wang1#, Xiujuan Zhang1#, Xiaoyu Feng2#, Liujing Zhuang3, Nan Jiang3, Rui Xu2, Xuewen Li2, Ping Wang3, Xicai Sun1✉, Hongmeng Yu1✉, Yiqun Yu1,2✉

1. Department of Otolaryngology, Eye, Ear, Nose and Throat Hospital, Shanghai Key Clinical Disciplines of Otorhinolaryngology, Fudan University, Shanghai, China 200031.
2. School of Life Sciences, Shanghai University, Shanghai, China 200444.
3. Biosensor National Special Laboratory, Key Laboratory for Biomedical Engineering of Education Ministry, Department of Biomedical Engineering, Zhejiang University, Hangzhou, China 310027.
4. Institutes of Biomedical Sciences, Fudan University, Shanghai, China 200031.
#These authors contributed equally to this work.

This is an open access article distributed under the terms of the Creative Commons Attribution License ( See for full terms and conditions.
Ren W, Wang L, Zhang X, Feng X, Zhuang L, Jiang N, Xu R, Li X, Wang P, Sun X, Yu H, Yu Y. Expansion of murine and human olfactory epithelium/mucosa colonies and generation of mature olfactory sensory neurons under chemically defined conditions. Theranostics 2021; 11(2):684-699. doi:10.7150/thno.46750. Available from

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Olfactory dysfunctions, including hyposmia and anosmia, affect ~100 million people around the world and the underlying causes are not fully understood. Degeneration of olfactory sensory neurons and incapacity of globose basal cells to generate olfactory sensory neurons are found in elder people and patients with smell disorders. Thus, olfactory stem cell may function as a promising tool to replace inactivated globose basal cells and to generate sensory neurons.

Methods: We established clonal expansion of cells from the murine olfactory epithelium as well as colony growth from human olfactory mucosa using Matrigel-based three-dimensional system. These colonies were characterized by immunostaining against olfactory epithelium cellular markers and by calcium imaging of responses to odors. Chemical addition was optimized to promote Lgr5 expression, colony growth and sensory neuron generation, tested by quantitative PCR and immunostaining against progenitor and neuronal markers. The differential transcriptomes in multiple signaling pathways between colonies under different base media and chemical cocktails were determined by RNA-Seq.

Results: In defined culture media, we found that VPA and CHIR99021 induced the highest Lgr5 expression level, while LY411575 resulted in the most abundant yield of OMP+ mature sensory neurons in murine colonies. Different base culture media with drug cocktails led to apparent morphological alteration from filled to cystic appearance, accompanied with massive transcriptional changes in multiple signaling pathways. Generation of sensory neurons in human colonies was affected through TGF-β signaling, while Lgr5 expression and cell proliferation was regulated by VPA.

Conclusion: Our findings suggest that targeting expansion of olfactory epithelium/mucosa colonies in vitro potentially results in discovery of new source to cell replacement-based therapy against smell loss.

Keywords: Olfactory epithelium/mucosa, three-dimensional culture, colony, Lgr5, olfactory sensory neurons