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Bmi1 regulates neural differentiation of mesenchymal stem cells through the Wnt3a‑RhoA signaling pathway to repair ischemic brain injury in rats

  • Authors:
    • Kunling Chen
    • Hongjie Zhou
    • Jie Zhang
    • Yiwei Zhang
    • Xiaobing Dou
    • Qin Yu
    • Liping Zhou
  • View Affiliations / Copyright

    Affiliations: School of Life Science, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, P.R. China
    Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 155
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    Published online on: July 24, 2025
       https://doi.org/10.3892/ijmm.2025.5596
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Abstract

Ischemic brain injury (IBI) is characterized by high morbidity, disability and mortality rates; however, it lacks effective clinical treatments. Mesenchymal stem cells (MSCs), as pluripotent stem cells with self‑renewal capacity and multilineage differentiation potential, have emerged as a promising therapeutic strategy for neurological disorders. In the present study, in vitro experiments were performed using the Wnt signaling agonist Wnt3a and the B lymphoma Mo‑MLV insertion region 1 homolog (Bmi1) small molecule inhibitor PTC209 to treat MSCs, and the roles and regulatory mechanisms of the Bmi1 and Wnt3a‑RhoA signaling pathways on the neural differentiation of MSCs were explored by MTT assay, immunofluorescence analysis and western blotting. In vivo experiments were also performed by establishing a rat model of middle cerebral artery occlusion (MCAO), transplanting different MSCs into the rat brain tissues after in vitro labeling, and comparing ischemic brain damage in each group of rats by Neurological Severity Score scoring, grasp assay, triphenyltetrazolium chloride staining, hematoxylin and eosin staining, and assessing neurological recovery via immunofluorescence and western blot analysis. The in vivo study aimed to assess the roles of the Bmi1 and Wnt3a‑RhoA signaling pathways in brain injury repair in MCAO rats and the mechanism. Specifically, recombinant Wnt3a cytokine was administered to upregulate the Wnt3a‑RhoA pathway, whereas the small‑molecule inhibitor PTC209 was utilized to suppress Bmi1 expression. The findings suggested that Bmi1 modulates the neural differentiation of MSCs through its regulatory effects on Wnt3a and RhoA expression, thereby influencing the reparative potential of MSCs in ischemic brain tissue. These findings highlight the therapeutic relevance of targeting Wnt3a‑RhoA activation and Bmi1 inhibition in MSC‑based interventions for IBI.
View Figures

Figure 1

Effects of B lymphoma Mo-MLV
insertion region 1 homolog and Wnt3a on the proliferation of MSCs.
(A) Morphology of primary MSCs inoculated into culture flasks.
Morphology of MSCs in primary culture for (B) 3 and (C) 7 days. (D)
Morphological characterization of third-generation MSCs. Effects of
different concentrations of (E) Wnt3a and (F) PTC209 on the
proliferation of MSCs. **P<0.01 vs. 0 μM
group. MSCs, mesenchymal stem cells.

Figure 2

Role of Bmi1 in neural
differentiation of MSCs. (A and B) Morphology of third-generation
MSCs induced to neurally differentiate for 3 h; scale bars, (A) 100
μm and (B) 50 μm. (C) Effects of Wnt3a and Bmi1 on
the expression of the neural stem cell marker Nestin. (D) Effects
of Wnt3a and Bmi1 on the expression of Olig2. (E) Effects of Wnt3a
and Bmi1 on the expression of GFAP. BME, β-mercaptoethanol; Bmi1, B
lymphoma Mo-MLV insertion region 1 homolog; GFAP, glial fibrillary
acidic protein; MSCs, mesenchymal stem cells.

Figure 3

Effects of Wnt3a and B lymphoma
Mo-MLV insertion region 1 homolog on neuronal marker expression.
*P<0.05, **P<0.01 vs. NC;
##P<0.01 vs. BME. BME, β-mercaptoethanol; NC, normal
control.

Figure 4

Expression of Bmi1 and Wnt3a-RhoA
signaling pathway-related proteins in each group.
*P<0.05, **P<0.01 vs. NC;
##P<0.01 vs. BME. BME, β-mercaptoethanol; Bmi1, B
lymphoma Mo-MLV insertion region 1 homolog; NC, normal control.

Figure 5

Detection of CM-Dil fluorescence
labeling of MSCs in vivo and in vitro. (A)
Experimental flowchart. (B) Morphology of third-generation MSCs.
(C) CM-Dil in vitro labeling of MSCs. Results of (D) CM-Dil
fluorescence detection and (E) DAPI staining in slices of the CA1
hippocampal region of brain tissue. (F) Results of co-localization
of (D) and (E). (G) Results of co-localization of CM-Dil and DAPI
staining in the CA1 hippocampal region. Arrows show MSCs
transplanted into brain tissue. H&E, hematoxylin and eosin; IF,
immunofluorescence; MCAO, middle cerebral artery occlusion; MSCs,
mesenchymal stem cells; SD, Sprague-Dawley; TTC,
triphenyltetrazolium chloride; WB, western blotting.

Figure 6

Role of B lymphoma Mo-MLV insertion
region 1 homolog in MSC transplantation to repair pathological
injury in a rat model of MCAO. (A) Neurological Severity Score
results. (B) Grip measurement results. **P<0.01 vs.
Sham; #P<0.05 and ##P<0.01 vs. MCAO.
(C) Triphenyltetrazolium chloride staining results for each group.
**P<0.01 vs. Sham; ##P<0.01 vs. MCAO,
※P<0.05 vs. MSCs. (D) Pathological staining of the
CA1 area in the hippocampal region of rat brain tissue in each
group. Black arrows indicate degenerated and atrophied neuronal
cells, and red arrows indicate tissue interstitial spaces. MCAO,
middle cerebral artery occlusion; MSCs, mesenchymal stem cells.

Figure 7

Effects of downregulation of B
lymphoma Mo-MLV insertion region 1 homolog on neurological recovery
in rats. Number of (A) NeuN-positive cells and (B) GFAP-positive
cells in the CA1 region of the hippocampus in each group of rats.
**P<0.01 vs. Sham; ##P<0.01 vs. MCAO;
▲P<0.05, ▲▲P<0.01 vs. MSCs. GFAP, glial
fibrillary acidic protein; MCAO, middle cerebral artery occlusion;
MSCs, mesenchymal stem cells.

Figure 8

NeuN and GFAP protein expression in
each group. For GFAP and NeuN, all visible bands were included in
the semi-quantitative analysis. **P<0.01 vs. Sham;
##P<0.01 vs. MCAO; ▲P<0.05,
▲▲P<0.01 vs. MSCs. GFAP, glial fibrillary acidic
protein; MCAO, middle cerebral artery occlusion; MSCs, mesenchymal
stem cells.

Figure 9

The mechanism underlying the effects
of Bmi1 on MSC transplantation-induced ischemic brain injury
repair. In vitro experiments used Wnt3a and PTC209 to treat
MSCs to explore the roles and regulatory mechanisms of the Bmi1 and
Wnt3a-Rhoa signaling pathways in the neural differentiation of
MSCs. In vivo experiments were conducted to investigate the
role of the Bmi1 and Wnt3a-RhoA signaling pathway in brain injury
repair in MCAO rats and its mechanism. Different MSCs were
transplanted into rat brain tissues after in vitro labeling,
after which, the ischemic brain injury in each group was compared
and the recovery of neurological function was assessed. Bmi1, B
lymphoma Mo-MLV insertion region 1 homolog; H&E, hematoxylin
and eosin; IF, immunofluorescence; MSCs, mesenchymal stem cells;
NSS, Neurological Severity Score; WB, western blotting.
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Copy and paste a formatted citation
Spandidos Publications style
Chen K, Zhou H, Zhang J, Zhang Y, Dou X, Yu Q and Zhou L: Bmi1 regulates neural differentiation of mesenchymal stem cells through the Wnt3a‑RhoA signaling pathway to repair ischemic brain injury in rats. Int J Mol Med 56: 155, 2025.
APA
Chen, K., Zhou, H., Zhang, J., Zhang, Y., Dou, X., Yu, Q., & Zhou, L. (2025). Bmi1 regulates neural differentiation of mesenchymal stem cells through the Wnt3a‑RhoA signaling pathway to repair ischemic brain injury in rats. International Journal of Molecular Medicine, 56, 155. https://doi.org/10.3892/ijmm.2025.5596
MLA
Chen, K., Zhou, H., Zhang, J., Zhang, Y., Dou, X., Yu, Q., Zhou, L."Bmi1 regulates neural differentiation of mesenchymal stem cells through the Wnt3a‑RhoA signaling pathway to repair ischemic brain injury in rats". International Journal of Molecular Medicine 56.4 (2025): 155.
Chicago
Chen, K., Zhou, H., Zhang, J., Zhang, Y., Dou, X., Yu, Q., Zhou, L."Bmi1 regulates neural differentiation of mesenchymal stem cells through the Wnt3a‑RhoA signaling pathway to repair ischemic brain injury in rats". International Journal of Molecular Medicine 56, no. 4 (2025): 155. https://doi.org/10.3892/ijmm.2025.5596
Copy and paste a formatted citation
x
Spandidos Publications style
Chen K, Zhou H, Zhang J, Zhang Y, Dou X, Yu Q and Zhou L: Bmi1 regulates neural differentiation of mesenchymal stem cells through the Wnt3a‑RhoA signaling pathway to repair ischemic brain injury in rats. Int J Mol Med 56: 155, 2025.
APA
Chen, K., Zhou, H., Zhang, J., Zhang, Y., Dou, X., Yu, Q., & Zhou, L. (2025). Bmi1 regulates neural differentiation of mesenchymal stem cells through the Wnt3a‑RhoA signaling pathway to repair ischemic brain injury in rats. International Journal of Molecular Medicine, 56, 155. https://doi.org/10.3892/ijmm.2025.5596
MLA
Chen, K., Zhou, H., Zhang, J., Zhang, Y., Dou, X., Yu, Q., Zhou, L."Bmi1 regulates neural differentiation of mesenchymal stem cells through the Wnt3a‑RhoA signaling pathway to repair ischemic brain injury in rats". International Journal of Molecular Medicine 56.4 (2025): 155.
Chicago
Chen, K., Zhou, H., Zhang, J., Zhang, Y., Dou, X., Yu, Q., Zhou, L."Bmi1 regulates neural differentiation of mesenchymal stem cells through the Wnt3a‑RhoA signaling pathway to repair ischemic brain injury in rats". International Journal of Molecular Medicine 56, no. 4 (2025): 155. https://doi.org/10.3892/ijmm.2025.5596
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