Promotion of skin wound healing using hypoimmunogenic epidermal cell sheets

Abstract

Objective: The physiological process of wound healing is dynamic, continuous, and intricate. Nowadays, full-thickness burn wounds are treated by autologous skin transplantation. Unfortunately, when substantial burns develop, there are fewer donor sites accessible, making it difficult to satisfy the requirement for large-scale skin transplants and increasing the risk of patient mortality. This study investigated the possibility of using a newly created hypoimmunogenic epidermal cell sheet to heal skin wounds.

Methods: Transfection with lentivirus was used to generate Keratinocytes (KCs) that overexpress Indoleamine 2,3-Dioxygenase (IDO). Western blotting and quantitative polymerase chain reaction were used to measure IDO levels. To evaluate the function of IDO⁺ keratinocytes, CCK-8 and Transwell assays were performed. In cell sheet induction media, KCs and Fibroblasts (FBs) were cultured to yield epidermal cell sheets. The full-thickness skin excisions of BALB/c mice were transplanted with epidermal cell sheets. To assess the tumorigenicity of IDO⁺ keratinocytes, BALB/c nude mouse xenograft models were also used. CD3 and CD31 immunofluorescence labeling of wound tissue on day 12 to identify T lymphocyte infiltration and capillary development. ELISA measurement of IL-1 and TNF-α concentrations.

Results: IDO ⁺ keratinocytes dramatically enhanced the expression levels of IDO mRNA and protein, as well as the amount of kynurenine in the conditioned media of IDO⁺ keratinocytes, compared to the Control and NC groups. CD8⁺ T cell apoptosis was considerably greater in the IDO group than in the Control and NC groups. Nevertheless, the proliferation and migratory capabilities of IDO⁺ keratinocytes were not substantially different from those of the Control and NC groups. In vitro cultivation of the hypoimmunogenic epidermal cell sheet was effective. In vivo transplantation experiments demonstrated that IDO⁺ epidermal cell sheets can effectively promote wound healing without tumorigenicity, and IDO⁺ epidermal cell sheets may promote wound healing by decreasing the expression levels of inflammatory factors (TNF and IL-1) in wound tissue, decreasing CD3⁺ T lymphocytes, and increasing infiltration and new capillaries in wound tissue.

Conclusion: In this study, we successfully constructed the hypoimmunogenic epidermal cell sheet and demonstrated that the hypoimmunogenic epidermal cell sheet could accelerate wound healing.

Keywords: Allogenic skin substitute; Epidermal cell sheet; Indoleamine 2,3-dioxygenase; Skin; Wound healing.

Fig. 1

Features of keratinocytes overexpressing IDO. a. Quantitative reverse transcription polymerase chain reaction was used to detect the gene expression of IDO. (∗∗∗P < 0.001, n = 3). b. Expression of IDO protein was evaluated in the Control, NC and IDO group by Western blot analysis (∗∗∗P < 0.001, n = 3). c. Kynurenine levels in IDO transfected keratinocytes conditioned medium. Conditioned medium was collected from the same number of infected and non-infected cells at 72 h post-transfection. Kynurenine levels were determined (∗∗∗P < 0.001, n = 3). d. CCK8 assay showed the proliferative ability of Control, NC and IDO group (n = 3). e. Transwell test was used to analyze cell migration, and the number of positive cells was counted. (Scale bar, 10 μm n = 3). f. Analysis of apoptosis of CD8⁺ T lymphocytes by flow cytometry (∗∗∗P < 0.001, n = 3).

Fig. 2

Keratinocytes overexpressing IDO were not tumorigenic. a. The tumorigenicity test of Control, NC and IDO cells in nude mice (the area indicated by the red arrow on day 0 is the cell injection site, and the area marked by the red arrow on day 40 is subcutaneous tumor on day 40 after cell injection, ∗∗∗P < 0.001, n = 5) b. HE staining of tumors in the NC, IDO and HepG2 group. NC and IDO group showed normal skin tissue, while there was a large number of infiltrated neutrophils in the HepG2 group was observed, as shown by the yellow arrow; the local cells were loosely arranged, and the cytoplasm was vacuolated, as shown by the red arrow; a large number of tumor cells in the tissue increased atypia, and the nucleus was obviously irregular, as indicated by the black arrow (Scale bar, 100 μm. n = 5). c. Immunohistochemical staining revealed CK5-positive cells in tumor tissues in the NC, IDO and HepG2 groups (Scale bar, 100 μm. n = 5).

Fig. 3

Characteristics of IDO + keratinocyte cell sheets. a. Preparation of Epidermal Cell Sheets. (n = 5). b. Quantitative reverse transcription polymerase chain reaction was used to detect the IDO expression of cell sheets (∗∗∗P < 0.001, n = 3). c. Expression of IDO protein was evaluated in cell sheets by Western blot analysis (∗∗∗P < 0.001, n = 3). d. HE staining of cell sheets in the Control, NC and IDO group (Scale bar, 10 μm n = 5). e. IDO immunohistochemical staining of cell sheets in the Control, NC and IDO group. (Scale bar, 10 μm n = 5). f. Analysis of apoptosis of CD8⁺ T lymphocytes by flow cytometry (∗P < 0.05, ∗∗P < 0.01, n = 3).

Fig. 4

IDO ⁺ keratinocyte cell sheets promoting wound healing. a. Representative images showing wound healing on days 0, 4, 8 and 12 in Control, NC and IDO groups. Wound healing rates on days 4, 8 and 12 in Control, NC and IDO groups (∗P < 0.05 and ∗∗P < 0.01, n = 5). b. H&E staining of skin tissues in Control, NC and IDO groups on 4, 8 and 12 days, on day 12 after wounding, the epidermis in the IDO groups was closer to physiological conditions (Scale bar, 100 μm. n = 5). c. Expression of IDO mRNA and protein were evaluated in the Control, NC and IDO group by qPCR and Western blot analysis (∗∗∗P < 0.001, n = 3). d. Immunohistochemical staining for Human-specific IDO protein on day 12 wound tissue (n = 5).

Fig. 5

Mechanism of IDO + epidermal cell sheet promoting wound healing. a. Immunofluorescence staining for CD3 on day 12 wound tissue (n = 5). b. Immunofluorescence staining for CD31 on day 12 wound tissue (n = 5). c. Quantitative analysis of capillary-like structures shown in (b) (∗∗∗P < 0.001, n = 5). d. Quantitative analysis of CD3 T lymphocyte infiltration shown in (a) (∗∗∗P < 0.001, n = 5). e. The expression of IL-1 and TNF-a in the grinding supernatant of wound tissue on the 12th day detected by ELISA (∗∗∗P < 0.001, n = 5).

Fig. 6

a. Quantitative reverse transcription polymerase chain reaction was used to detect the gene expression of COL4A1 and LAMA1. (n = 3). b. Expression of COL4A1 and LAMA1 protein was evaluated in the Control, NC and IDO group by Western blot analysis (n = 3).