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. 2024 Jul 12;19(7):e0304809.
doi: 10.1371/journal.pone.0304809. eCollection 2024.

The differentiation of Lgr5+ progenitor cells on nanostructures of self-assembled silica beads

Wenjun Cai  1 , Zhichun Huang  1 , Baobin Sun  1 , Ling Lu  1 , Xiaoqiong Ding  1 , Feng Tao  1
Affiliations

The differentiation of Lgr5+ progenitor cells on nanostructures of self-assembled silica beads

Wenjun Cai et al. PLoS One. .

Abstract

Supporting cells(SCs) have been demonstrated to be a reliable source for regenerating hair cells(HCs). Previous research has reported that Lgr5+ SCs can regenerate HCs both in vitro and in vivo. However, there is limited knowledge about the impact of the material on Lgr5+ cells. In this study, Lgr5+ cells were isolated from neonatal Lgr5-EGFP-CreERT2 transgenic mice by flow cytometry and then plated on self-assembled silica beads (SB). Lgr5+ cell differentiation was observed by immunofluorescence. We found that in the direct differentiation assay, the SB group generated more hair cells than the control group(*p < 0.05). Especially in the SB group, Lgr5+ progenitors generated significantly more Myo7a+ HCs outside of the colony than in the control group(**p < 0.01). In the sphere differentiation assay, we found that the diameter of spheres in the SB group was significantly larger compared to those of the control group(**p < 0.01). However, the difference in the ratio of myo7a+ cell counts was not obvious(P>0.05). The experiment proved that the self-assembled silica beads could promote the differentiation of Lgr5+ progenitors in vitro. Our findings implicate that nanostructures of self-assembled silica beads can be used as vectors for stem cell research in the inner ear.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. SEM images.
(A)SEM images of the nanostructures assembled with SB-500. (B) SEM image of Lgr5+ progenitors cultured on SB-500.
Fig 2
Fig 2. The images of flow cytometry.
Fig 3
Fig 3. Experiment process.
We used the Lgr5-EGFP-CreERT2 transgenic mice to get the basilar membrane of Cochlea and separated Lgr5+ progenitors. Then in direct differentiation assay, we cultured the sorted Lgr5+ cells at 50 cells/μl and added EdU from day 4 to 7. The total culture time was 10 days. In the sphere differentiation assay, we cultured the sorted Lgr5+ cells into spheres and then plated the spheres into silica beads. The total culture time was 15 days.
Fig 4
Fig 4. Immunofluorescence figures in the direct differentiation assay.
(A)In the SB group, Lgr5+ progenitors generated a large number of Myo7a+ cells on the inside of the colony, and some of them were labeled with EdU. (B)In the SB group, Lgr5+ progenitors generated more Myo7a+ cells on the outside of the colony. (C)In the control group, Lgr5+ progenitors generated few number of Myo7a+ cells on the inside of the colony, and some of the cells were labeled with EdU. (D)In the control group, Lgr5+ progenitors generated few Myo7a+ cells on the outside of the colony. (E)Both total and outside of the colony, in the SB group Lgr5+ progenitors formed more Myo7a+ cells compared with the control group. (F)The number of colonies in each well per 5,000 cells. The Lgr5+ progenitors formed more colonies in the SB group than in the control group.
Fig 5
Fig 5. Live cell workstation images.
(A) shows the Lgr5+ progenitor cells. (B) shows the sphere formed by Lgr5+ progenitor cells.
Fig 6
Fig 6. Immunofluorescence figure in the sphere differentiation assay.
(A、B)In the sphere differentiation assay, Lgr5+ progenitors formed much the same Myo7a+ cells in the SB group and control group. (C)In the sphere differentiation assay, the diameter of spheres in the SB group was larger than the control group. (D)In the sphere differentiation assay, the ratio of myo7a+ cell counts in the sphere was not obvious between the two groups.
See this image and copyright information in PMC

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Grants and funding

This work was supported by grants from the nanjing medical science and technology development project (YKK16274). There was no additional external funding received for this study.