Wnt signaling pathway is involved in differentiation of embryonic stem cells into thymic epithelial progenitor cells together with autoimmune regulators

doi:10.12307/2024.132

Chinese Journal of Tissue Engineering Research ›› 2024, Vol. 28 ›› Issue (13): 1996-2001.doi: 10.12307/2024.132

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Wnt signaling pathway is involved in differentiation of embryonic stem cells into thymic epithelial progenitor cells together with autoimmune regulators

Wen Tinghao1, Li Yuandi1, He Keke1, Song Wenqian1, Wang Xianbin1, Gao Jie1, 2, Su Min1, 3, Hu Rong1, 3

1Department of Histology and Embryology, Basic Medical College, Guizhou Medical University, Guiyang 550025, Guizhou Province, China; 2Translational Medicine Research Center, Guizhou Medical University, Guiyang 550025, Guizhou Province, China; 3National Joint Local Engineering Laboratory for Cell Engineering and Biomedicine Technique, Guizhou Province Key Laboratory of Regenerative Medicine, Key Laboratory of Adult Stem Cell Translational Research (Chinese Academy of Medical Sciences), Guizhou Medical University, Guiyang 550025, Guizhou Province, China

Received:2023-02-17 Accepted:2023-04-07 Online:2024-05-08 Published:2023-08-28
Contact: Hu Rong, MD, Professor, Department of Histology and Embryology, Basic Medical College, Guizhou Medical University, Guiyang 550025, Guizhou Province, China; National Joint Local Engineering Laboratory for Cell Engineering and Biomedicine Technique, Guizhou Province Key Laboratory of Regenerative Medicine, Key Laboratory of Adult Stem Cell Translational Research (Chinese Academy of Medical Sciences), Guizhou Medical University, Guiyang 550025, Guizhou Province, China
About author:Wen Tinghao, Master, Department of Histology and Embryology, Basic Medical College, Guizhou Medical University, Guiyang 550025, Guizhou Province, China
Supported by:
National Natural Science Foundation of China, No. 82171343 (to SM); National Natural Science Foundation of China, No. 82060151 (to SM); Guizhou Provincial Science and Technology Plan Project, No. [2020]4Y230 (to SM); Guizhou Provincial Science and Technology Plan Project, No. [2020]4103 (to SM); Guizhou Provincial Graduate Research Fund, No. YJSKYJJ[2021]150 (to WXB)

Abstract

Abstract: BACKGROUND: Autoimmune regulator gene (Aire) and Wnt signaling pathway play an important role in the maintenance and differentiation of mouse embryonic stem cell pluripotency. However, whether the Wnt signal and Aire are involved in the differentiation of embryonic stem cells to thymic epithelial progenitor cells remains poorly understood.
OBJECTIVE: To investigate the relationship of the Wnt signaling pathway and Aire with the differentiation of embryonic stem cells.
METHODS: A two-step differentiation method was used to induce mouse embryonic stem cells to differentiate into endoderm and then into thymic epithelial progenitor cells. Mouse embryonic stem cells were infected with Aire shRNA lentivirus, and monoclonal stable strains were screened by puromycin. Mouse embryonic stem cells were collected on days 0, 3 and 10 of the directed induction of differentiation after the induced differentiation by the two-step differentiation method. Cellular immunofluorescence, flow cytometry, western blot assay, and real-time qPCR were used to detect the expression changes of related genes and proteins.
RESULTS AND CONCLUSION: (1) Immunofluorescence staining showed positive expression of SSEA1 and OCT4 on day 0 of targeted induction of differentiation. (2) Immunofluorescence staining showed double-positive expression of SOX17 and FOXA2 on day 3 of targeted induction of differentiation. (3) Flow cytometry results showed positive expression of EPCAM1, K5 and K8 on day 10 of targeted induction of differentiation. (4) Compared with undifferentiated mouse embryonic stem cells, the expressions of Wnt7a, β-catenin, and Gsk-3β proteins were elevated, and the expression level of Aire protein was decreased in induced differentiated thymic epithelial progenitor cells. (5) Compared with undifferentiated mouse embryonic stem cells, the expressions of Wnt7a, β-catenin, Gsk-3β and Aire mRNA were elevated in thymic epithelial progenitor cells. (6) Compared with normal cultured mouse embryonic stem cells and their ultimately differentiated thymic epithelial progenitor cells, the expression levels of Wnt7a, β-catenin and Gsk-3β proteins were reduced in mouse embryonic stem cells with knockdown of Aire genes and their final differentiated thymic epithelial progenitor cells. In conclusion, the Wnt signaling pathway and Aire are jointly involved in the process of targeted induction of differentiation of mouse embryonic stem cells into mouse thymic epithelial progenitor cells.

Key words: Wnt signaling pathway, autoimmune regulator, embryonic stem cell, thymic epithelial progenitor cell, endoderm cell

CLC Number:

Wen Tinghao, Li Yuandi, He Keke, Song Wenqian, Wang Xianbin, Gao Jie, Su Min, Hu Rong. Wnt signaling pathway is involved in differentiation of embryonic stem cells into thymic epithelial progenitor cells together with autoimmune regulators[J]. Chinese Journal of Tissue Engineering Research, 2024, 28(13): 1996-2001.

Figures/Tables 6

References

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Wnt signaling pathway is involved in differentiation of embryonic stem cells into thymic epithelial progenitor cells together with autoimmune regulators

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