Atoh1 overexpression promotes Guinea pig bone marrow mesenchymal stem cells to differentiate into neural stem cell

Abstract

Sensorineural hearing loss (SNHL) is a prevalent condition in otolaryngology. A key obstacle is finding effective strategies for regenerating damaged cochlear hair cells in adult animals. A practical and reliable approach has been developed to create a superior cell source for stem cell transplantation in the inner ear to treat SNHL. Atoh1 is involved in the differentiation of neurons, intestinal secretory cells, and mechanoreceptors including auditory hair cells, and thus plays an important role in neurogenesis. Lentivirus-mediated transfection of bone marrow mesenchymal stem cells (BMSCs) was utilized to achieve stable expression of the essential transcription factor Atoh1, which is crucial for developing auditory hair cells without compromising cell survival. By manipulating the induction conditions through altering the cell growth environment using anti-adherent culture, the synergistic impact of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) was effectively applied to significantly improve the differentiation efficiency of bone marrow-derived mesenchymal stem cells (BMSC) into neural stem cells (NSCs) following Atoh1 transfection, thereby reducing the induction time. The study indicated that the newly proposed transdifferentiation method effectively transformed BMSCs into NSCs in a controlled environment, presenting a potential approach for stem cell transplantation to promote hair cell regeneration.

Keywords: Atoh1; Bone marrow mesenchymal stem cells; Hair cell regeneration; Neural stem cells.

Fig. 1

Expression of lentivirus overexpressing Atoh1 gene after infection with BMSCs. (a) Morphological findings of BMSCs infected by lentivirus overexpressing Atoh1 gene. All scale bars: 50 μm. (b) Infection efficiency of lentivirus overexpressing Atoh1. *p ＜ 0.05,**p ＜ 0.01,n = 5. (c) Expression of Atoh1 mRNA in BMSCs infected with lentivirus. *p ＜ 0.05,**p ＜ 0.01,n = 3. (d–e) Expression of ATOH1 protein in BMSCs infected with lentivirus and WB strip. *p ＜ 0.05,**p ＜ 0.01,n = 3. The data obtained from the above experiments are expressed as mean and standard deviation. One-way ANOVA followed by Tukey's post hoc test was used for statistical analysis.

Fig. 2

Morphological findings on different days after subgroup induction of BMSCs into NSCs. Scale bars: Trial-D1, NC-D1, 20 μm, others, 50 μm.

Fig. 3

CCK-8 detection and immunofluorescence results after BMSCs induction to NSCs. (a) Cell proliferation on different days after BMSCs induction to NSCs. (b) Quantitative analysis of the nestin. (c) Immunofluorescence results of Nestin after bone marrow mesenchymal stem cells are induced into NSCs. All scale bars: 50 μm. The data obtained from the above experiments are expressed as mean and standard deviation. One-way ANOVA followed by Tukey's post hoc test was used for statistical analysis. *p ＜ 0.05,**p ＜ 0.01,n = 3.