Basic Study
Copyright ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Jun 26, 2023; 15(6): 589-606
Published online Jun 26, 2023. doi: 10.4252/wjsc.v15.i6.589
Single cell RNA sequencing reveals mesenchymal heterogeneity and critical functions of Cd271 in tooth development
Yan-Yan Zhang, Feng Li, Xiao-Ke Zeng, Yan-Hui Zou, Bing-Bing Zhu, Jia-Jia Ye, Yun-Xiao Zhang, Qiu Jin, Xin Nie
Yan-Yan Zhang, Feng Li, Xiao-Ke Zeng, Yan-Hui Zou, Bing-Bing Zhu, Jia-Jia Ye, Yun-Xiao Zhang, Qiu Jin, Xin Nie, School & Hospital of Stomatology, Wenzhou Medical University, Wenzhou 325027, Zhejiang Province, China
Author contributions: Zhang YY conceived experiments, interpreted data and wrote a manuscript; Li F, Zeng XK and Zou YH performed the experiment; Zhu BB, Ye JJ, Zhang YX, and Jin Q provided a critical insight into this study; Nie X supervised this study; All authors read and approved the final version for submission.
Supported by National Natural Science Foundation of China (General Program), No. 31870971; and Medical Health Science and Technology Project of Zhejiang Province, No. 2023KY155.
Institutional animal care and use committee statement: All animal experiments were performed according to the protocols approved by the Medical Ethics Committee of Wenzhou Medical University, No. wydw2019-0224.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Data sharing statement: The datasets analyzed for this study can be found in the NCBI SRA: 506PRJNA891206. https://www.ncbi.nlm.nih.gov/sra/PRJNA891206.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Xin Nie, PhD, Professor, School & Hospital of Stomatology, Wenzhou Medical University, Xueyuan West Road, Lucheng District, Wenzhou 325027, Zhejiang Province, China. dr.xinnie@qq.com
Received: February 9, 2023
Peer-review started: February 9, 2023
First decision: April 10, 2023
Revised: April 18, 2023
Accepted: May 5, 2023
Article in press: May 5, 2023
Published online: June 26, 2023
Processing time: 137 Days and 8.9 Hours
Abstract
BACKGROUND

Accumulating evidence suggests that the maxillary process, to which cranial crest cells migrate, is essential to tooth development. Emerging studies indicate that Cd271 plays an essential role in odontogenesis. However, the underlying mechanisms have yet to be elucidated.

AIM

To establish the functionally heterogeneous population in the maxillary process, elucidate the effects of Cd271 deficiency on gene expression differences.

METHODS

p75NTR knockout (Cd271-/-) mice (from American Jackson laboratory) were used to collect the maxillofacial process tissue of p75NTR knockout mice, and the wild-type maxillofacial process of the same pregnant mouse wild was used as control. After single cell suspension, the cDNA was prepared by loading the single cell suspension into the 10x Genomics Chromium system to be sequenced by NovaSeq6000 sequencing system. Finally, the sequencing data in Fastq format were obtained. The FastQC software is used to evaluate the quality of data and CellRanger analyzed the data. The gene expression matrix is read by R software, and Seurat is used to control and standardize the data, reduce the dimension and cluster. We search for marker genes for subgroup annotation by consulting literature and database; explore the effect of p75NTR knockout on mesenchymal stem cells (MSCs) gene expression and cell proportion by cell subgrouping, differential gene analysis, enrichment analysis and protein-protein interaction network analysis; understand the interaction between MSCs cells and the differentiation trajectory and gene change characteristics of p75NTR knockout MSCs by cell communication analysis and pseudo-time analysis. Last we verified the findings single cell sequencing in vitro.

RESULTS

We identified 21 cell clusters, and we re-clustered these into three subclusters. Importantly, we revealed the cell–cell communication networks between clusters. We clarified that Cd271 was significantly associated with the regulation of mineralization.

CONCLUSION

This study provides comprehensive mechanistic insights into the maxillary- process-derived MSCs and demonstrates that Cd271 is significantly associated with the odontogenesis in mesenchymal populations.

Keywords: Cd271; Mesenchymal stem cells; Single cell RNA sequencing; Osteogenesis; Mineralization; Tooth development

Core Tip: Our study reveals the following findings: (1) High cellular heterogeneity and molecular details; (2) Significant functional and signaling differences between cell types; (3) Novel subclusters of mesenchymal stem cells; and (4) Crucial cell-cell interactions of mesenchymal subpopulations. We provided new insights into the biological features of mesenchymal stem cells at the single cell level. Our findings contribute to thorough exploration of the mechanism of Cd271 in regulating odontogenesis and osteogenesis which add to the theory of tooth development.