Plant-Derived Extracellular Vesicles: A New Revolutionization of Modern Healthy Diets and Biomedical Applications.

Plant-derived extracellular vesicles (PDEVs) have recently emerged as a promising area of research due to their potential health benefits and biomedical applications. Produced by various plant species, these EVs contain diverse bioactive molecules, including proteins, lipids, and nucleic acids. Increasing in vitro and in vivo studies have shown that PDEVs have inherent pharmacological activities that affect cellular processes, exerting anti-inflammatory, antioxidant, and anticancer activities, which can potentially contribute to disease therapy and improve human health. Additionally, PDEVs have shown potential as efficient and biocompatible drug delivery vehicles in treating various diseases. However, while PDEVs serve as a potential rising star in modern healthy diets and biomedical applications, further research is needed to address their underlying knowledge gaps, especially the lack of standardized protocols for their isolation, identification, and large-scale production. Furthermore, the safety and efficacy of PDEVs in clinical applications must be thoroughly evaluated. In this review, we concisely discuss current knowledge in the PDEV field, including their characteristics, biomedical applications, and isolation methods, to provide an overview of the current state of PDEV research. Finally, we discuss the challenges regarding the current and prospective issues for PDEVs. This review is expected to provide new insights into healthy diets and biomedical applications of vegetables and fruits, inspiring new advances in natural food-based science and technology.

Low-glucose culture environment can enhance the wound healing capability of diabetic adipose-derived stem cells.

BACKGROUND: Application of autologous adipose-derived stem cells (ASC) for diabetic chronic wounds has become an emerging treatment option. However, ASCs from diabetic individuals showed impaired cell function and suboptimal wound healing effects. We proposed that adopting a low-glucose level in the culture medium for diabetic ASCs may restore their pro-healing capabilities. METHODS: ASCs from diabetic humans and mice were retrieved and cultured in high-glucose (HG, 4.5 g/L) or low-glucose (LG, 1.0 g/L) conditions. Cell characteristics and functions were investigated in vitro. Moreover, we applied diabetic murine ASCs cultured in HG or LG condition to a wound healing model in diabetic mice to compare their healing capabilities in vivo. RESULTS: Human ASCs exhibited decreased cell proliferation and migration with enhanced senescence when cultured in HG condition in vitro. Similar findings were noted in ASCs derived from diabetic mice. The inferior cellular functions could be partially recovered when they were cultured in LG condition. In the animal study, wounds healed faster when treated with HG- or LG-cultured diabetic ASCs relative to the control group. Moreover, higher collagen density, more angiogenesis and cellular retention of applied ASCs were found in wound tissues treated with diabetic ASCs cultured in LG condition. CONCLUSIONS: In line with the literature, our study showed that a diabetic milieu exerts an adverse effect on ASCs. Adopting LG culture condition is a simple and effective approach to enhance the wound healing capabilities of diabetic ASCs, which is valuable for the clinical application of autologous ASCs from diabetic patients.

Timing Does Matter: Nerve-Mediated HDAC1 Paces the Temporal Expression of Morphogenic Genes During Axolotl Limb Regeneration.

Sophisticated axolotl limb regeneration is a highly orchestrated process that requires highly regulated gene expression and epigenetic modification patterns at precise positions and timings. We previously demonstrated two waves of post-amputation expression of a nerve-mediated repressive epigenetic modulator, histone deacetylase 1 (HDAC1), at the wound healing (3 days post-amputation; 3 dpa) and blastema formation (8 dpa onward) stages in juvenile axolotls. Limb regeneration was profoundly inhibited by local injection of an HDAC inhibitor, MS-275, at the amputation sites. To explore the transcriptional response of post-amputation axolotl limb regeneration in a tissue-specific and time course-dependent manner after MS-275 treatment, we performed transcriptome sequencing of the epidermis and soft tissue (ST) at 0, 3, and 8 dpa with and without MS-275 treatment. Gene Ontology (GO) enrichment analysis of each coregulated gene cluster revealed a complex array of functional pathways in both the epidermis and ST. In particular, HDAC activities were required to inhibit the premature elevation of genes related to tissue development, differentiation, and morphogenesis. Further validation by Q-PCR in independent animals demonstrated that the expression of 5 out of 6 development- and regeneration-relevant genes that should only be elevated at the blastema stage was indeed prematurely upregulated at the wound healing stage when HDAC1 activity was inhibited. WNT pathway-associated genes were also prematurely activated under HDAC1 inhibition. Applying a WNT inhibitor to MS-275-treated amputated limbs partially rescued HDAC1 inhibition, resulting in blastema formation defects. We propose that post-amputation HDAC1 expression is at least partially responsible for pacing the expression timing of morphogenic genes to facilitate proper limb regeneration.

A double fluorescence chimeric limb regeneration model reveals muscle fiber reconnection during axolotl limb regeneration / Modelo de regeneración quimérica de doble fluorescencia del miembro revela la reconexión de la fibra muscular durante la regeneración del miembro axolotl

SUMMARY: Axolotl limb regeneration is a fascinating characteristic that has attracted attention for several decades. Our previous studies on axolotl limb regeneration indicated that the satellite cells in the remnant muscles move distally into the blastema to regenerate new muscles that are separated by a gap from remnant muscles. Thereafter, the regenerative muscle fibers start to reconnect with remnant ones. In this study, the reconnection at the individual muscle fiber level was elucidated to test the hypothesis that this reconnection happens synchronously among involved muscles. Three pairs of EGFP+ mid-bud stage blastemas were transplanted onto freshly amputated stumps of RFP+ axolotls at the same thigh position to generate double fluorescence chimeric regenerative hindlimbs. These regenerative limbs were harvested very late far beyond they had reached the late differentiation stage. Fluorescence imaging of these limbs in cross sections revealed that in the proximal remnant part of the muscle fiber, reconnection occurred at a different pace among the muscles. In the major thigh muscle gracilis, the reconnection started from the periphery before it was completed. Furthermore, RFP+ muscle fibers contributed to muscle regeneration in the distal regenerative parts. Intriguingly, this red cell contribution was limited to ventral superficial muscles of the calf. This kind of double fluorescence chimeric limb regeneration model may help increase the understanding of the patterning of axolotl limb regeneration in late stages.

RESUMEN: La regeneración del miembro de Axolotl es una característica fascinante que ha llamado la atención durante varias décadas. Nuestros estudios previos sobre la regeneración del miembro del Axolotl indicaron que las células satélite en los músculos remanentes se mueven distalmente hacia el blastema para regenerar nuevos músculos que están separados por una brecha de músculos remanentes. A partir de entonces, las fibras musculares regenerativas comienzan a reconectarse con las restantes. En este estudio, se aclaró la reconexión a nivel de fibra muscular individual para probar la hipótesis de que esta reconexión ocurre sincrónicamente entre los músculos involucrados. Se trasplantaron tres pares de blastemas EGFP+ en la etapa de yema media en tocones recién amputados de axolotls RFP+ en la misma posición del muslo para generar miembros posteriores regenerativos quiméricos de fluorescencia doble. Estos miembros regenerativos se cosecharon muy tarde mucho más allá de haber alcanzado la etapa de diferenciación tardía. Las imágenes de fluorescencia de estos miembros en secciones transversales revelaron que en la parte remanente proximal de la fibra muscular, la reconexión se produjo a un ritmo diferente entre los músculos. En el músculo grácil, la reconexión comenzó desde la periferia antes de completarse. Además, las fibras musculares RFP+ contribuyeron a la regeneración muscular en las partes regenerativas distales. Curiosamente, esta contribución de glóbulos rojos se limitó a los músculos superficiales ventrales de la pantorrilla. Este tipo de modelo de regeneración quimérica de doble fluorescencia del miembro puede ayudar a aumentar la comprensión del patrón de la regeneración del miembro del Axolotl en etapas tardías.

Nerve-mediated expression of histone deacetylases regulates limb regeneration in axolotls.

Axolotls have amazing abilities to regenerate their lost limbs. Nerve and wound epidermis have great impacts on this regeneration. Histone deacetylases (HDACs) have been shown to play roles in the regeneration of amphibian tails and limbs. In this study, a bi-phasic up-regulation of HDAC1 was noted before early differentiation stage of axolotl limb regeneration. Limb regeneration was delayed in larvae incubated with an HDAC inhibitor MS-275. Local injection of MS-275 or TSA, another HDAC inhibitor, into amputation sites of the juveniles did not interfere with wound healing but more profoundly inhibited local HDAC activities and blastema formation/limb regeneration. Elevation of HDAC1 expression was more apparent in wound epidermis than in mesenchyme. Prior denervation prohibited this elevation and limb regeneration. Supplementation of nerve factors BMP7, FGF2, and FGF8 in the stump ends after amputation on denervated limbs not only enabled HDAC1 up-regulation but also led to more extent of limb regeneration. In conclusion, nerve-mediated HDAC1 expression is required for blastema formation and limb regeneration.

Diffusion tensor tractography reveals muscle reconnection during axolotl limb regeneration.

Axolotls have amazing ability to regenerate their lost limbs. Our previous works showed that after amputation the remnant muscle ends remained at their original location whilst sending satellite cells into the regenerating parts to develop into early muscle fibers in the late differentiation stage. The parental and the newly formed muscle fibers were not connected until very late stage. The present study used non-invasive diffusion tensor imaging (DTI) to monitor weekly axolotl upper arm muscles after amputation of their upper arms. DTI tractography showed that the regenerating muscle fibers became visible at 9-wpa (weeks post amputation), but a gap was observed between the regenerating and parental muscles. The gap was filled at 10-wpa, indicating reconnection of the fibers of both muscles. This was confirmed by histology. The DTI results indicate that 23% of the muscle fibers were reconnected at 10-wpa. In conclusion, DTI can be used to visualize axolotls' skeletal muscles and the results of muscle reconnection were in accordance with our previous findings. This non-invasive technique will allow researchers to identify the timeframe in which muscle fiber reconnection takes place and thus enable the study of the mechanisms underlying this reconnection.

Re-epithelialization of large wound in paedomorphic and metamorphic axolotls.

Axolotls (Ambystoma mexicanum) may heal their skin wounds scar-free in both paedomorphs and metamorphs. In previous studies on small punch skin wounds, rapid re-epithelialisation was noted in these two axolotl morphs. However, large wound size in mammals may affect wound healing. In this study, large circumferential full thickness excision wounds on the hind limbs were created on juvenile paedomorphic and metamorphic axolotls. The results showed re-epithelialisation was more quickly initiated in paedomorphs than in metamorphs after wounding. The migrating rate of epidermis on the wound bed was faster in paedomorphs than in metamorphs and thus completion of re-epithelialisation was faster in paedomorphs than in metamorphs. Within these re-epithelialisation periods, neither basement membrane nor dermis was reformed. Epidermal cell proliferation was detected by EdU-labelling technique. In the normal unwounded skin, epidermal proliferation rate was higher in paedomorphs than in metamorphs. After wounding, the epidermal proliferation rate was significantly lower in the migrating front on the wound bed than in the normal skin in paedomorphs. The EdU-labelling rate between normal skin and migration front was not different in metamorphs. Lacking of more proliferating epidermal cells on the wound bed indicated that the new epidermis here derived rather from migrating epidermal cells than from cell proliferation in situ. In conclusion, re-epithelialisation in the large wound might be fully completed in both morphs despite it was initiated earlier and with faster rate in paedomorphs than in metamorphs. The new epidermis on the wound bed derived mainly from cell migration than by cell proliferation in the re-epithelialisation period. J. Morphol. 278:228-235, 2017. © 2016 Wiley Periodicals,Inc.

Cooperative regulation of substrate stiffness and extracellular matrix proteins in skin wound healing of axolotls.

Urodele amphibians (Ambystoma mexicanum), unique among vertebrates, can regenerate appendages and other body parts entirely and functionally through a scar-free healing process. The wound epithelium covering the amputated or damaged site forms early and is essential for initiating the subsequent regenerative steps. However, the molecular mechanism through which the wound reepithelializes during regeneration remains unclear. In this study, we developed an in vitro culture system that mimics an in vivo wound healing process; the biomechanical properties in the system were precisely defined and manipulated. Skin explants that were cultured on 2 to 50 kPa collagen-coated substrates rapidly reepithelialized within 10 to 15 h; however, in harder (1 GPa) and other extracellular matrices (tenascin-, fibronectin-, and laminin-coated environments), the wound epithelium moved slowly. Furthermore, the reepithelialization rate of skin explants from metamorphic axolotls cultured on a polystyrene plate (1 GPa) increased substantially. These findings afford new insights and can facilitate investigating wound epithelium formation during early regeneration using biochemical and mechanical techniques.