Transcriptomic and in vivo approaches introduced human iPSC-derived microvesicles for skin rejuvenation

Abstract

The skin undergoes the formation of fine lines and wrinkles through the aging process; also, burns, trauma, and other similar circumstances give rise to various forms of skin ulcers. Induced pluripotent stem cells (iPSCs) have become promising candidates for skin healing and rejuvenation due to not stimulating inflammatory responses, low probability of immune rejection, high metabolic activity, good large-scale production capacity and potentials for personalized medicine. iPSCs can secrete microvesicles (MVs) containing RNA and proteins responsible for the normal repairing process of the skin. This study aimed to evaluate the possibility, safety and effectiveness of applying iPSCs-derived MVs for skin tissue engineering and rejuvenation applications. The possibility was assessed using the evaluation of the mRNA content of iPSC-derived MVs and the behavior of fibroblasts after MV treatment. Investigating the effect of microvesicle on stemness potential of mesenchymal stem cells was performed for safety concerns. In vivo evaluation of MVs was done in order to investigate related immune response, re-epithelialization and blood vessel formation to measure effectiveness. Shedding MVs were round in shape distributed in the range from 100 to 1000 nm in diameter and positive for AQP3, COL2A, FGF2, ITGB, and SEPTIN4 mRNAs. After treating dermal fibroblasts with iPSC-derived MVs, the expressions of collagens Iα1 and III transcripts (as the main fibrous extracellular matrix (ECM) proteins) were upregulated. Meanwhile, the survival and proliferation of MV treated fibroblasts did not change significantly. Evaluation of stemness markers in MV treated MSCs showed negligible alteration. In line with in vitro results, histomorphometry and histopathology findings also confirmed the helpful effect of MVs in skin regeneration in the rat burn wound models. Conducting more investigations on hiPSCs-derived MVs may lead to produce more efficient and safer biopharmaceutics for skin regeneration in the pharmaceutical market.

Figure 1

Overview of the procedure of isolating MVs from human iPSCs and the effects of hiPSCs-derived MVs both in vitro and in vivo. After treating dermal fibroblasts with MVs, the in vitro analysis showed that the rate of collagen expression had been increased, and the survival and proliferation alterations were not considerable. Within in vivo trials, the wounds created on the dorsal skin of the rats were treated by lotions containing Eucerin and different amounts of MVs. The in vivo analysis showed an increased rate of epidermis layer regeneration and decreased rate of inflammation in the wound site of mice treated with higher amounts of MVs. MVs, microvesicles; iPSCs, induced pluripotent stem cells; hiPSCs, human induced pluripotent stem cells; ECM, extracellular matrix.

Figure 2

Microvesicle characterization; (A) The population homogeneity and size range of isolated MVs obtained by DLS. (B) TEM image of a single isolated MV demonstrated a rounded shape.

Figure 3

Evaluation of the apoptosis and necrosis of skin fibroblasts under MVs treatment. (A) representative images of cultivated MV + and MV- fibroblasts in DMEM/F12 medium on days 0, 3, and 5, scale bars 100 µm (B) The rate of apoptosis and necrosis in fibroblasts characterized by Annexin V/PI dual staining flow cytometry on days 0, 3, and 5 and MVs treated groups. For further evaluation, a scattered plot chart is drawn. Q1: necrotic cells (Annexin V-/PI +), Q2: late apoptotic cells (Annexin V + /PI +), Q3: early apoptotic cells (Annexin V + /PI-), Q4: viable cells (Annexin V-/PI-). (C) The percentage of viable, early apoptotic, late apoptosis, and necrotic cells on days 0, 3, and 5 and MVs treated groups using a bar chart. Data are statistically significant in the live group on day 0 and day3/MVs- (**P-value < 0.01) and also in late apoptotic MVs treated groups on day 3 and day 5 compared to day 0. (D) On days 3 and 5 after MVs treatment cell absorbance at 570 nm had no significant differences. (E) Investigation of survival of MVs + and MVs- fibroblasts by Trypan blue assay on days 0, 3, and 5. MVs treatment did not affect fibroblast viability significantly. Data are presented as means ± SD of at least three independent experiments. Data presents no significance in any group. *P < 0.05 was considered a significant difference compared to the control group (i.e., day 0).

Figure 4

Evaluating transcriptional changes of some stemness markers in MVs treated cells. (A) Skin ECM marker genes (collagen I and III) mRNA expression in treated fibroblasts; Both markers were significantly upregulated under MVs treatment. (B) Stemness gene markers (Sox9, Oct4, and Nanog) mRNA expression in treated MSCs; Results suggested that MVs down-regulated Sox9 and Oct4, and up-regulated Nanog transcription. Data are presented as means ± SD of at least three independent experiments. *P < 0.05 and **P < 0.01 was considered significant differences compared to the control.

Figure 5

(A) Photographs were taken from rat skins in different groups and days to evaluate the changes in wound-healing. Group B: a wound created on the dorsal skin with pure Eucerin lotion (untreated group), Group C: a wound created on the dorsal skin with 0.5% MVs + Eucerin lotion treatment, and Group D: a wound created on the dorsal skin with 1% MVs + Eucerin lotion treatment. (B) The diagram represents the percentages of wound-healing in the study groups over a period of 18 days, *P < 0.05.

Figure 6

H&E-stained microscopic sections of healed incisions on day 18 of treatments in different groups. Thin arrows: infiltration of inflammatory cells, black thick arrows: crusty scab, white arrows: epithelial layer, Arrowhead: neo-vascularization. Group A as control group is not shown. Group B: a wound created on the dorsal skin with pure Eucerin lotion (untreated group), Group C: a wound created on the dorsal skin with 0.5% MVs + Eucerin lotion treatment, and Group D: a wound created on the dorsal skin with 1% MVs + Eucerin lotion treatment, scale bars are drawn on the images.