Enhanced neural differentiation of neural stem cells by sustained release of Shh from TG2 gene-modified EMSC co-culture in vitro


As a promising cell therapy, neural crest-derived ectoderm mesenchymal stem cells (EMSCs) secrete high amounts of extracellular matrix (ECM) and neurotrophic factors, promoting neural stem cell (NSC) differentiation into neuronal lineages and aiding tissue regeneration. Additionally, the forced overexpression of secreted proteins can increase the therapeutic efficacy of the secretome. Tissue transglutaminase (TG2) is a ubiquitously expressed member of the transglutaminase family of calcium-dependent crosslinking enzymes, which can stabilize the ECM, inducing smart or living biomaterial to stimulate differentiation and enhance the neurogenesis of NSCs. In this study, we examined the neuronal differentiation of NSCs induced by TG2 gene-modified EMSCs (TG2-EMSCs) in a co-culture model directly. Two weeks after initiating differentiation, levels of the neuronal markers, tubulin beta 3 class III and growth-associated protein 43, were higher in NSCs in the TG2-EMSC co-culture group and those of the astrocytic marker glial fibrillary acidic protein were lower, compared with the control group. These results were confirmed by immunofluorescence, and laminin, fibronectin and sonic hedgehog (Shh) contributed to this effect. The results of western blot analysis and the enzyme-linked immunoassay showed that after TG2-EMSCs were co-cultured for 2 weeks, they expressed much higher levels of Shh than the control group. Moreover, the sustained release of Shh was observed in the TG2-EMSC co-culture group. Overall, our findings indicate that EMSCs can induce the differentiation of NSCs, of which TG2-EMSCs can promote the differentiation of NSCs compared with EMSCs.

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Data availability

The data used to support the findings of this study are available from the corresponding author upon request.


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This study was supported by the National Natural Science Foundation of China (Grants No. 81571830). This study was also supported by the Special fund project for the development of health science and technology in Nanjing (Grants No. YKK19129) and Jiangsu University Clinical/Medical Science and Technology Development Fund Projectproject number (Grants No. JYL20180040).

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Correspondence to Naiyan Lu.

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Shi, W., Bian, L., Lv, D. et al. Enhanced neural differentiation of neural stem cells by sustained release of Shh from TG2 gene-modified EMSC co-culture in vitro. Amino Acids 53, 11–22 (2021). https://doi.org/10.1007/s00726-020-02918-0

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  • Tissue transglutaminase 2
  • Shh
  • EMSC
  • NSC
  • Differentiation