Customized Proteinaceous Nanoformulation for In Vivo Chemical Reprogramming.

Sweat gland (SwG) regeneration is crucial for the functional rehabilitation of burn patients. In vivo chemical reprogramming that harnessing the patient's own cells in damaged tissue is of substantial interest to regenerate organs endogenously by pharmacological manipulation, which could compensate for tissue loss in devastating diseases and injuries, for example, burns. However, achieving in vivo chemical reprogramming is challenging due to the low reprogramming efficiency and an unfavorable tissue environment. Herein, this work has developed a functionalized proteinaceous nanoformulation delivery system containing prefabricated epidermal growth factor structure for on-demand delivery of a cocktail of seven SwG reprogramming components to the dermal site. Such a chemical reprogramming system can efficiently induce the conversion of epidermal keratinocytes into SwG myoepithelial cells, resulting in successful in situ regeneration of functional SwGs. Notably, in vivo chemical reprogramming of SwGs is achieved for the first time with an impressive efficiency of 30.6%, surpassing previously reported efficiencies. Overall, this proteinaceous nanoformulation provides a platform for coordinating the target delivery of multiple pharmacological agents and facilitating in vivo SwG reprogramming by chemicals. This advancement greatly improves the clinical accessibility of in vivo reprogramming and offers a non-surgical, non-viral, and cell-free strategy for in situ SwG regeneration.

A volar skin excisional wound model for in situ evaluation of multiple-appendage regeneration and innervation.

Background: Promoting rapid wound healing with functional recovery of all skin appendages is the main goal of regenerative medicine. So far current methodologies, including the commonly used back excisional wound model (BEWM) and paw skin scald wound model, are focused on assessing the regeneration of either hair follicles (HFs) or sweat glands (SwGs). How to achieve de novo appendage regeneration by synchronized evaluation of HFs, SwGs and sebaceous glands (SeGs) is still challenging. Here, we developed a volar skin excisional wound model (VEWM) that is suitable for examining cutaneous wound healing with multiple-appendage restoration, as well as innervation, providing a new research paradigm for the perfect regeneration of skin wounds. Methods: Macroscopic observation, iodine-starch test, morphological staining and qRT-PCR analysis were used to detect the existence of HFs, SwGs, SeGs and distribution of nerve fibres in the volar skin. Wound healing process monitoring, HE/Masson staining, fractal analysis and behavioral response assessment were performed to verify that VEWM could mimic the pathological process and outcomes of human scar formation and sensory function impairment. Results: HFs are limited to the inter-footpads. SwGs are densely distributed in the footpads, scattered in the IFPs. The volar skin is richly innervated. The wound area of the VEWM at 1, 3, 7 and 10 days after the operation is respectively 89.17% ± 2.52%, 71.72% ± 3.79%, 55.09 % ± 4.94% and 35.74% ± 4.05%, and the final scar area accounts for 47.80% ± 6.22% of the initial wound. While the wound area of BEWM at 1, 3, 7 and 10 days after the operation are respectively 61.94% ± 5.34%, 51.26% ± 4.89%, 12.63% ± 2.86% and 6.14% ± 2.84%, and the final scar area accounts for 4.33% ± 2.67% of the initial wound. Fractal analysis of the post-traumatic repair site for VEWM vs human was performed: lacunarity values, 0.040 ± 0.012 vs 0.038 ± 0.014; fractal dimension values, 1.870 ± 0.237 vs 1.903 ± 0.163. Sensory nerve function of normal skin vs post-traumatic repair site was assessed: mechanical threshold, 1.05 ± 0.52 vs 4.90 g ± 0.80; response rate to pinprick, 100% vs 71.67% ± 19.92%, and temperature threshold, 50.34°C ± 3.11°C vs 52.13°C ± 3.54°C. Conclusions: VEWM closely reflects the pathological features of human wound healing and can be applied for skin multiple-appendages regeneration and innervation evaluation.

Small molecules facilitate single factor-mediated sweat gland cell reprogramming.

BACKGROUND: Large skin defects severely disrupt the overall skin structure and can irreversibly damage sweat glands (SG), thus impairing the skin's physiological function. This study aims to develop a stepwise reprogramming strategy to convert fibroblasts into SG lineages, which may provide a promising method to obtain desirable cell types for the functional repair and regeneration of damaged skin. METHODS: The expression of the SG markers cytokeratin 5 (CK5), cytokeratin 10 (CK10), cytokeratin 18 (CK18), carcino-embryonic antigen (CEA), aquaporin 5 (AQP5) and α-smooth muscle actin (α-SMA) was assessed with quantitative PCR (qPCR), immunofluorescence and flow cytometry. Calcium activity analysis was conducted to test the function of induced SG-like cells (iSGCs). Mouse xenograft models were also used to evaluate the in vivo regeneration of iSGCs. BALB/c nude mice were randomly divided into a normal group, SGM treatment group and iSGC transplantation group. Immunocytochemical analyses and starch-iodine sweat tests were used to confirm the in vivo regeneration of iSGCs. RESULTS: EDA overexpression drove HDF conversion into iSGCs in SG culture medium (SGM). qPCR indicated significantly increased mRNA levels of the SG markers CK5, CK18 and CEA in iSGCs, and flow cytometry data demonstrated (4.18 ± 0.04)% of iSGCs were CK5 positive and (4.36 ± 0.25)% of iSGCs were CK18 positive. The addition of chemical cocktails greatly accelerated the SG fate program. qPCR results revealed significantly increased mRNA expression of CK5, CK18 and CEA in iSGCs, as well as activation of the duct marker CK10 and luminal functional marker AQP5. Flow cytometry indicated, after the treatment of chemical cocktails, (23.05 ± 2.49)% of iSGCs expressed CK5+ and (55.79 ± 3.18)% of iSGCs expressed CK18+, respectively. Calcium activity analysis indicated that the reactivity of iSGCs to acetylcholine was close to that of primary SG cells [(60.79 ± 7.71)% vs. (70.59 ± 0.34)%, ns]. In vivo transplantation experiments showed approximately (5.2 ± 1.1)% of the mice were sweat test positive, and the histological analysis results indicated that regenerated SG structures were present in iSGCs-treated mice. CONCLUSION: We developed a SG reprogramming strategy to generate functional iSGCs from HDFs by using the single factor EDA in combination with SGM and small molecules. The generation of iSGCs has important implications for future in situ skin regeneration with SG restoration.

Sweat Gland Organoids Originating from Reprogrammed Epidermal Keratinocytes Functionally Recapitulated Damaged Skin.

Restoration of sweat glands (SwGs) represents a great issue in patients with extensive skin defects. Recent methods combining organoid technology with cell fate reprogramming hold promise for developing new regenerative methods for SwG regeneration. Here, a practical strategy for engineering functional human SwGs in vitro and in vivo is provided. First, by forced expression of the ectodysplasin-A in human epidermal keratinocytes (HEKs) combined with specific SwG culture medium, HEKs are efficiently converted into SwG cells (iSwGCs). The iSwGCs show typical morphology, gene expression pattern, and functions resembling human primary SwG cells. Second, by culturing the iSwGCs in a special 3D culturing system, SwG organoids (iSwGOs) that exhibit structural and biological features characteristic of native SwGs are obtained. Finally, these iSwGOs are successfully transplanted into a mouse skin damage model and they develop into fully functioning SwGs in vivo. Regeneration of functional SwG organoids from reprogrammed HEKs highlights the great translational potential for personalized SwG regeneration in patients with large skin defects.

Pleiotropic Roles of CXCR4 in Wound Repair and Regeneration.

Wound healing is a multi-step process that includes multiple cellular events such as cell proliferation, cell adhesion, and chemotactic response as well as cell apoptosis. Accumulating studies have documented the significance of stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor 4 (CXCR4) signaling in wound repair and regeneration. However, the molecular mechanism of regeneration is not clear. This review describes various types of tissue regeneration that CXCR4 participates in and how the efficiency of regeneration is increased by CXCR4 overexpression. It emphasizes the pleiotropic effects of CXCR4 in regeneration. By delving into the specific molecular mechanisms of CXCR4, we hope to provide a theoretical basis for tissue engineering and future regenerative medicine.