The Stimulation of Macrophages by Systematical Administration of GM-CSF Can Accelerate Adult Wound Healing Process.

Skin wound repair remains a major challenge in clinical care, and various strategies have been employed to improve the repair process. Recently, it has been reported that macrophages are important for the regeneration of various tissues and organs. However, their influence on wound repair is unclear. Here, we aimed to explore whether macrophages would participate in the wound healing process and to explore new possibilities of treatment for skin defects. We firstly created a mouse full-thickness skin defect model to observe the distribution of macrophages in the regenerating tissue and then detected the influence of macrophages on skin defect repair in both macrophage-depletion and macrophage-mobilization models. We found that the number of macrophages increased significantly after skin defect and persisted during the process of wound repair. The regeneration process was significantly prolonged in macrophage-depleted animals. RT-qPCR and ELISA assays further demonstrated that the expression of growth factors was perturbed in the regenerating tissue. The activation of macrophages by granulocyte-macrophage colony-stimulating factor (GM-CSF) injection could significantly improve wound healing, accompanied with an upregulation of the expression of various growth factors. In conclusion, the current study demonstrated that macrophages are critical for skin regeneration and that GM-CSF exhibited therapeutic potential for wound healing.

Novel hemostatic biomolecules based on elastin-like polypeptides and the self-assembling peptide RADA-16.

BACKGROUND: Safe and effective hemostatic materials are important for reducing mortality resulting from excessive hemorrhage. In this work, new biomaterials with hemostatic effects were created by fusing the gene coding for RADA-16, a self-assembling peptide with the sequence RADARADARADARADA, to the 3'-end of the open reading frame (ORF) encoding elastin-like polypeptides through gene recombination. RESULTS: The fusion proteins, termed 36R, 60R and 96R, were solubly over-expressed in Escherichia coli BL21 (DE3) based on genetic manipulation of the high-efficiency prokaryotic expression vector pET28a (+) and bacterial transformation. Western Blot analysis showed that the over-expressed proteins were the target fusion proteins. The target proteins 36R with 94.72% purity, 60R with 96.91% purity and 96R with 96.37% purity were prepared using an inverse phase transition cycle at 65 °C followed by His-tag affinity chromatography. The proliferation results of the mouse fibroblast cell line L929 and hippocampus neuron cell line HT22 indicated that the fusion proteins did not cause obvious cell toxicity. The lyophilized spongy film of the purified 36R, 60R and 96R could stop the hemorrhage of a 2 × 2 mm bleeding wound in the mouse liver after 27.21 ± 1.92 s, 18.65 ± 1.97 s and 15.85 ± 1.21 s, respectively. The hemostasis time was 21.23 ± 1.84 s for rat-tail collagen and 14.44 ± 1.33 s for RADA-16 lyophilized on gauze. The hemostatic time of three treated groups were all significantly superior to that of the negative control without any hemostasis treatment, which spontaneously stopped bleeding after 37.64 ± 1.34 s. Statistical analysis showed that the spongy film with purified 96R exhibited an exciting hemostatic effect that was superior to rat-tail collagen and close to that of RADA-16 lyophilized on gauze. CONCLUSIONS: These results revealed that the fusion proteins achieved by gene recombination technology could serve as a promising hemostatic material.

PD-L1 expression levels in mesenchymal stromal cells predict their therapeutic values for autoimmune hepatitis.

BACKGROUND: Autoimmune hepatitis is a chronic inflammatory hepatic disorder with no effective treatment. Mesenchymal stromal cells (MSCs) have emerged as a promising treatment owing to their unique advantages. However, their heterogeneity is hampering use in clinical applications. METHODS: Wharton's jelly derived MSCs (WJ-MSCs) were isolated from 58 human donors using current good manufacturing practice conditions. Gene expression profiles of the WJ-MSCs were analyzed by transcriptome and single-cell RNA-sequencing (scRNA-seq), and subsequent functional differences were assessed. Expression levels of programmed death-ligand 1 (PD-L1) were used as an indicator to screen WJ-MSCs with varied immunomodulation activities and assessed their corresponding therapeutic effects in a mouse model of concanavalin A-induced autoimmune hepatitis. RESULTS: The 58 different donor-derived WJ-MSCs were grouped into six gene expression profile clusters. The gene in different clusters displayed obvious variations in cell proliferation, differentiation bias, trophic factor secretion, and immunoregulation. Data of scRNA-seq revealed four distinct WJ-MSCs subpopulations. Notably, the different immunosuppression capacities of WJ-MSCs were positively correlated with PD-L1 expression. WJ-MSCs with high expression of PD-L1 were therapeutically superior to WJ-MSCs with low PD-L1 expression in treating autoimmune hepatitis. CONCLUSION: PD-L1 expression levels of WJ-MSCs could be regarded as an indicator to choose optimal MSCs for treating autoimmune disease. These findings provided novel insights into the quality control of MSCs and will inform improvements in the therapeutic benefits of MSCs.