Int J Stem Cells.  2021 Nov;14(4):465-474. 10.15283/ijsc21078.

Sox9 Is Crucial for Mesenchymal Stem Cells to Enhance Cutaneous Wound Healing

• Bie Q
• Zhai R
• Chen Y
• Li Y
• Xie N
• Wang B
• Yuan P
• Zhou X
• Cong H
• Chang X
• Xiong H
• Zhang B

• KMID: 2522564
• DOI: http://doi.org/10.15283/ijsc21078

Abstract

Background and Objectives
Human umbilical cord mesenchymal stem cells (HUC-MSCs) are promising candidates for cell-based therapy in regenerative medicine or other diseases due to their superior characteristics, including higher proliferation, faster self-renewal ability, lower immunogenicity, a noninvasive harvest procedure, easy expansion in vitro, and ethical access, compared with stem cells from other sources.
Methods and Results
In the present study, we knocked down the expression of SOX9 in HUC-MSCs by lentivirus interference and found that knockdown of SOX9 inhibited the proliferation and migration of HUC-MSCs and influenced the expression of cytokines (IL-6 and IL-8), growth factors (GM-CSF and VEGF) and stemness-related genes (OCT4 and SALL4). In addition, the repair effect of skin with burn injury in rats treated with HUC-MSCs transfected with sh-control was better than that rats treated with HUC-MSCs transfected with shSOX9 or PBS, and the accessory structures of the skin, including hair follicles and glands, were greater than those in the other groups. We found that knockdown of the expression of SOX9 obviously inhibited the expression of Ki67, CK14 and CK18.
Conclusions
In conclusion, this study will provide a guide for modifying HUC-MSCs by bioengineering technology in the future.

Keyword

HUC-MSCs; SOX9; Skin; Regenerative medicine

Figure

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  Fig. 1 Human umbilical cord mesenchymal stem cells. (A) HUC-MSCs growth from human umbilical cord tissue, which were observed on 3∼4 days for culture by inverted microscope. (B) HUC-MSCs fusion reached 80% at one week of culture.

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  Fig. 2 The proliferation and migration abilities of HUC-MSCs transfected with shSOX9 or shControl. (A) CCK-8 Kit detected the proliferation of HUC-MSCs transfected with shSOX9 or shControl. (B) Count the cell number of HUC-MSCs transfected with shSOX9 or shControl at different times. (C) Cloning ability of HUC-MSCs transfected with shSOX9 or shControl. (D) Statistical analysis of the clone number according to figure subpart C (n=3; ***p<0.001). (E) The migration of HUC-MSCs transfected with shSOX9 or shControl. (F) Statistical analysis of the migration number according to figure subpart E (n=3; ***p<0.001).

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  Fig. 3 The expression of cytokines and stemness related genes mRNA in HUC-MSCs transfected with shSOX9 or shControl. qRT-PCR was performed to detect the expression of IL-8 (A), IL-6 (B), GM-CSF (C), VEGF (D)，OCT4 (E), SALL4 (F) (n=3; *p<0.05; **p<0.01; ***p<0.001; ns: no statistical difference).

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  Fig. 4 Effects of knockdown SOX9 in HUC-MSCs on repairing in wound injury model of rat. (A) The scar of rat in wound injury model treated with PBS or HUC-MSCs transfected with shcontrol or shsox9 at different timepoints day 1, 5, 9, 12, 15 (n=3). (B) Statistical analysis of scar area in back of rat treated with PBS or HUC-MSCs transfected with shcontrol or shsox9 at different timepoints from day 1, 5, 9, 12, 15 (n=3, ns: no statistical difference).

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  Fig. 5 Evaluate scar healing in rat treated with HUC-MSCs transfected with shSOX9 or shcontrol and PBS. HE staining the wound repair skin tissues of rat treated with PBS or HUC-MSCs transfected with shcontrol or shsox9 (n=3). (B) Statistical analysis the thickness of epidermis and dermis (n=3, ns: no statistical difference). (C) Immunofluorescence staining the expression of CK14, CK18 and ki67 in the wound repair skin tissues of rat treated with PBS or HUC-MSCs transfected with shcontrol or shsox9 (n=3).

Reference

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