Advances in stem cells treatment of diabetic wounds: A bibliometric analysis via CiteSpace.

Diabetes is a chronic medical condition that may induce complications such as poor wound healing. Stem cell therapies have shown promise in treating diabetic wounds with pre-clinical and clinical studies. However, little bibliometric analysis has been carried out on stem cells in the treatment of diabetic wounds. In this study, we retrieved relevant papers published from January 1, 2003, to December 31, 2023, from Chinese and English databases. CiteSpace software was used to analyze the authors, institutions, and keywords by standard bibliometric indicators. Our analysis findings indicated that publications on stem cells in the treatment of diabetic wounds kept increasing. The most prolific author was Qian Cai (n = 7) and Mohammad Bayat (n = 16) in Chinese and English databases, respectively. Institutions distribution analysis showed that Chinese institutions conducted most publications, and the most prolific institution was the Chinese People's Liberation Army General Hospital (n = 9) and Shahid Beheshti University of Medical Sciences (n = 17) in Chinese and English databases, respectively. The highest centrality keyword in Chinese and English databases was "wound healing" (0.54) and "in vitro" (0.13), respectively. There were 8 and 11 efficient and convincing keyword clusters produced by a log-likelihood ratio in the Chinese and English databases, respectively. The strongest burst keyword was "exosome" (strength 3.57) and "endothelial progenitor cells" (strength 7.87) in the Chinese and English databases, respectively. These findings indicated a direction for future therapies and research on stem cells in the treatment of diabetic wounds.

KAT2A-mediated succinylation modification of notch1 promotes the proliferation and differentiation of dental pulp stem cells by activating notch pathway.

BACKGROUND: Dental pulp stem cells (DPSCs) are a kind of undifferentiated dental mesenchymal stem cells with strong self-renewal ability and multi-differentiation potential. This study aimed to investigate the regulatory functions of succinylation modification in DPSCs. METHODS: DPSCs were isolated from the dental pulp collected from healthy subjects, and then stem cell surface markers were identified using flow cytometry. The osteogenic differentiation ability of DPSCs was verified by alkaline phosphatase (ALP) and alizarin red staining methods, while adipogenic differentiation was detected by oil red O staining. Meanwhile, the mRNA of two desuccinylases (SIRT5 and SIRT7) and three succinylases (KAT2A, KAT3B, and CPT1A) in DPSCs before and after mineralization induction were detected using quantitative real-time PCR. The cell cycle was measured by flow cytometry, and the expression of bone-specific genes, including COL1a1 and Runx2 were evaluated by western blotting and were combined for the proliferation and differentiation of DPSCs. Co-immunoprecipitation (co-IP) and immunofluorescence were combined to verify the binding relationship between proteins. RESULTS: The specific markers of mesenchymal stem cells were highly expressed in DPSCs, while the osteogenic differentiation ability of isolated DPSCs was confirmed via ALP and alizarin red staining. Similarly, the oil red O staining also verified the adipogenic differentiation ability of DPSCs. The levels of KAT2A were found to be significantly upregulated in mineralization induction, which significantly decreased the ratio of G0/G1 phase and increased S phase cells; converse results regarding cell cycle distribution were obtained when KAT2A was inhibited. Moreover, overexpression of KAT2A promoted the differentiation of DPSCs, while its inhibition exerted the opposite effect. The elevated KAT2A was found to activate the Notch1 signaling pathway, which succinylated Notch1 at the K2177 site to increase their corresponding protein levels in DPSCs. The co-IP results showed that KAT2A and Notch1 were endogenously bound to each other, while inhibition of Notch1 reversed the effects of KAT2A overexpression on the DPSCs proliferation and differentiation. CONCLUSION: KAT2A interacted directly with Notch1, succinylating the Notch1 at the K2177 site to increase their corresponding protein levels in DPSCs. Similarly, KAT2A-mediated succinylation modification of Notch1 promotes the DPSCs proliferation and differentiation, suggesting that targeting KAT2A and Notch1 may contribute to tooth regeneration.

Stem cell­mediated modulation of pyroptosis contributes to tissue repair in noninfective inflammatory­related diseases (Review).

Pyroptosis, a programmed cell death marked by lytic and inflammatory characteristics, plays a crucial role in non­infectious inflammation­related diseases but can lead to detrimental outcomes when dysregulated. Stem cells have emerged as key players in modulating pyroptosis through paracrine signaling, offering a novel avenue for tissue repair and regeneration. The present review delved into previous studies elucidating the intricate interplay between stem cells and pyroptosis, emphasizing the potential of stem cell­based therapies in regulating pyroptotic pathways. The exploration of this dynamic interaction holds promise for developing strategies to harness stem cells for effective tissue repair and regeneration in the context of inflammation­related diseases.

An adapted protocol to derive microglia from stem cells and its application in the study of CSF1R-related disorders.

BACKGROUND: Induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial function in health and disease. Yet, since differentiation and survival of iMGL are highly reliant on colony-stimulating factor 1 receptor (CSF1R) signaling, it is difficult to use iMGL to study microglial dysfunction associated with pathogenic defects in CSF1R. METHODS: Serial modifications to an existing iMGL protocol were made, including but not limited to changes in growth factor combination to drive microglial differentiation, until successful derivation of microglia-like cells from an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines, the quality of the new iMGL protocol was validated through cell yield assessment, measurement of microglia marker expression, transcriptomic comparison to primary microglia, and evaluation of inflammatory and phagocytic activities. Similarly, molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL. RESULTS: The newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to cultured primary human microglia and with higher scavenging and inflammatory competence at ~ threefold greater yield compared to the original protocol. Using this protocol, decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally, ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 (P2RY12) expression, a heightened capacity to internalize myelin, as well as heightened inflammatory response to Pam3CSK4. Poor P2RY12 expression was confirmed to be a consequence of CSF1R haploinsufficiency, as this feature was also observed following CSF1R knockdown or inhibition in mature control iMGL, and in CSF1RWT/KO and CSF1RWT/E633K iMGL compared to their respective isogenic controls. CONCLUSIONS: We optimized a pre-existing iMGL protocol, generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol, we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant, with preliminary characterization pointing toward functional alterations in migratory, phagocytic and inflammatory activities.

Extracellular Caspase-1 induces hair stem cell migration in wounded and inflamed skin conditions.

The wound-healing process is a paradigm of the directed migration of various pools of stem cells from their niche to the site of injury where they replenish damaged cells. Two decades have elapsed since the observation that wounding activates multipotent hair follicle stem cells to infiltrate the epidermis, but the cues that coax these cells out of their niche remain unknown. Here, we report that Caspase-1, a protein classically known as an integral component of the cytosolic inflammasome, is secreted upon wounding and has a non-canonical role in the extracellular milieu. Through its caspase activation recruitment domain (CARD), Caspase-1 is sufficient to initiate the migration of hair follicle stem cells into the epidermis. Uncovering this novel function of Caspase-1 also facilitates a deeper understanding of the mechanistic basis of the epithelial hyperplasia found to accompany numerous inflammatory skin diseases.

SALL4 in gastrointestinal tract cancers: upstream and downstream regulatory mechanisms.

Effective therapeutic targets and early diagnosis are major challenges in the treatment of gastrointestinal tract (GIT) cancers. SALL4 is a well-known transcription factor that is involved in organogenesis during embryonic development. Previous studies have revealed that SALL4 regulates cell proliferation, survival, and migration and maintains stem cell function in mature cells. Additionally, SALL4 overexpression is associated with tumorigenesis. Despite its characterization as a biomarker in various cancers, the role of SALL4 in GIT cancers and the underlying mechanisms are unclear. We describe the functions of SALL4 in GIT cancers and discuss its upstream/downstream genes and pathways associated with each cancer. We also consider the possibility of targeting these genes or pathways as potential therapeutic options for GIT cancers.

Stem Cell-Derived Exosomes: An Advanced Horizon to Cancer Regenerative Medicine.

Cancer research has made significant progress in recent years, and extracellular vesicles (EVs) based cancer investigation reveals several facts about cancer. Exosomes are a subpopulation of EVs. In the present decade, exosomes is mostly highlighted for cancer theranostic research. Tumor cell derived exosomes (TEXs) promote cancer but there are multiple sources of exosomes that can be used as cancer therapeutic agents (plant exosomes, stem cell-derived exosomes, modified or synthetic exosomes). Stem cells based regenerative medicine faces numerous challenges, such as promote tumor development, cellular reprogramming etc., and therefore addressing these complications becomes essential. Stem cell-derived exosomes serves as an answer to these problems and offers a better solution. Global research indicates that stem cell-derived exosomes also play a dual role in the cellular system by either inhibiting or promoting cancer. Modified exosomes which are genetically engineered exosomes or surface modified exosomes to increase the efficacy of the therapeutic properties can also be considered to target the above concerns. However, the difficulties associated with the exosomes include variations in exosomes heterogenity, isolation protocols, large scale production, etc., and these have to be managed effectively. In this review, we explore exosomes biogenesis, multiple stem cell-derived exosome sources, drug delivery, modified stem cells exosomes, clinical trial of stem cells exosomes, and the related challenges in this domain and future orientation. This article may encourage researchers to explore stem cell-derived exosomes and develop an effective and affordable cancer therapeutic solution.

Organ Frame Elements or Free Intercellular Gel-Like Matrix as Necessary Conditions for Building Organ Structures during Regeneration.

Over the past decades, an unimaginably large number of attempts have been made to restore the structure of mammalian organs after injury by introducing stem cells into them. However, this procedure does not lead to full recovery. At the same time, it is known that complete regeneration (restitution without fibrosis) is possible in organs with proliferating parenchymal cells. An analysis of such models allows to conclude that the most important condition for the repair of histological structures of an organ (in the presence of stem cells) is preservation of the collagen frame structures in it, which serve as "guide rails" for proliferating and differentiating cells. An alternative condition for complete reconstruction of organ structures is the presence of a free "morphogenetic space" containing a gel-like matrix of the embryonic-type connective tissue, which exists during embryonal development of organs in mammals or during complete regeneration in amphibians. Approaches aimed at preserving frame structures or creating a "morphogenetic space" could radically improve the results of organ regeneration using both local and exogenous stem cells.

HD-Zip II transcription factors control distal stem cell fate in Arabidopsis roots by linking auxin signaling to the FEZ/SOMBRERO pathway.

In multicellular organisms, specialized tissues are generated by specific populations of stem cells through cycles of asymmetric cell divisions, where one daughter undergoes differentiation and the other maintains proliferative properties. In Arabidopsis thaliana roots, the columella - a gravity-sensing tissue that protects and defines the position of the stem cell niche - represents a typical example of a tissue whose organization is exclusively determined by the balance between proliferation and differentiation. The columella derives from a single layer of stem cells through a binary cell fate switch that is precisely controlled by multiple, independent regulatory inputs. Here, we show that the HD-Zip II transcription factors (TFs) HAT3, ATHB4 and AHTB2 redundantly regulate columella stem cell fate and patterning in the Arabidopsis root. The HD-Zip II TFs promote columella stem cell proliferation by acting as effectors of the FEZ/SMB circuit and, at the same time, by interfering with auxin signaling to counteract hormone-induced differentiation. Overall, our work shows that HD-Zip II TFs connect two opposing parallel inputs to fine-tune the balance between proliferation and differentiation in columella stem cells.

Identification of cells of leukemic stem cell origin with non-canonical regenerative properties.

Despite most acute myeloid leukemia (AML) patients entering remission following chemotherapy, outcomes remain poor due to surviving leukemic cells that contribute to relapse. The nature of these enduring cells is poorly understood. Here, through temporal single-cell transcriptomic characterization of AML hierarchical regeneration in response to chemotherapy, we reveal a cell population: AML regeneration enriched cells (RECs). RECs are defined by CD74/CD68 expression, and although derived from leukemic stem cells (LSCs), are devoid of stem/progenitor capacity. Based on REC in situ proximity to CD34-expressing cells identified using spatial transcriptomics on AML patient bone marrow samples, RECs demonstrate the ability to augment or reduce leukemic regeneration in vivo based on transfusion or depletion, respectively. Furthermore, RECs are prognostic for patient survival as well as predictive of treatment failure in AML cohorts. Our study reveals RECs as a previously unknown functional catalyst of LSC-driven regeneration contributing to the non-canonical framework of AML regeneration.

Dental Pulp Stem Cell-Derived Exosomes Promote Sciatic Nerve Regeneration via Optimizing Schwann Cell Function.

Repair strategies for injured peripheral nerve have achieved great progresses in recent years. However, the clinical outcomes remain unsatisfactory. Recent studies have found that exosomes secreted by dental pulp stem cells (DPSC-exos) have great potential for applications in nerve repair. In this study, we evaluated the effects of human DPSC-exos on improving peripheral nerve regeneration. Initially, we established a coculture system between DPSCs and Schwann cells (SCs) in vitro to assess the effect of DPSC-exos on the activity of embryonic dorsal root ganglion neurons (DRGs) growth in SCs. We extracted and labeled human DPSC-exos, which were subsequently utilized in uptake experiments in DRGs and SCs. Subsequently, we established a rat sciatic nerve injury model to evaluate the therapeutic potential of DPSC-exos in repairing sciatic nerve damage. Our findings revealed that DPSC-exos significantly promoted neurite elongation by enhancing the proliferation, migration, and secretion of neurotrophic factors by SCs. In vivo, DPSC-exos administration significantly improved the walking behavior, axon regeneration, and myelination in rats with sciatic nerve injuries. Our study underscores the vast potential of DPSC-exos as a therapeutic tool for tissue-engineered nerve construction.

Genetic reprogramming with stem cells regenerates glomerular epithelial podocytes in Alport syndrome.

Glomerular filtration relies on the type IV collagen (ColIV) network of the glomerular basement membrane, namely, in the triple helical molecules containing the α3, α4, and α5 chains of ColIV. Loss of function mutations in the genes encoding these chains (Col4a3, Col4a4, and Col4a5) is associated with the loss of renal function observed in Alport syndrome (AS). Precise understanding of the cellular basis for the patho-mechanism remains unknown and a specific therapy for this disease does not currently exist. Here, we generated a novel allele for the conditional deletion of Col4a3 in different glomerular cell types in mice. We found that podocytes specifically generate α3 chains in the developing glomerular basement membrane, and that its absence is sufficient to impair glomerular filtration as seen in AS. Next, we show that horizontal gene transfer, enhanced by TGFß1 and using allogenic bone marrow-derived mesenchymal stem cells and induced pluripotent stem cells, rescues Col4a3 expression and revive kidney function in Col4a3-deficient AS mice. Our proof-of-concept study supports that horizontal gene transfer such as cell fusion enables cell-based therapy in Alport syndrome.

Expression profiling of stemness markers in testicular germline stem cells from neonatal and adult Swiss albino mice during their transdifferentiation in vitro.

BACKGROUND: Spermatogonial stem cells (SSCs) were considered to be stem cells with limited potencies due to their existence in adult organisms. However, the production of spermatogonial stem cell colonies with broader differentiation capabilities in primary germ cell cultures from mice of select genetic backgrounds (C57BL6/Tg14, ddY, FVB and 129/Ola) indicated that SSCs from these strains were pluripotent. METHODS: We established primary cultures of SSCs from neonatal and adult Swiss 3T3 Albino mice. Stemness of SSC colonies were evaluated by performing real-time PCR and immunofluorescence analysis for a panel of chosen stemness markers. Differentiation potentials of SSCs were examined by attempting the generation of embryoid bodies and evaluating the expression of ectodermal, mesodermal and endodermal markers using immunofluorescence and real-time PCR analysis. RESULTS: Spermatogonial stem cells from neonatal and mature mice testes colonised in vitro and formed compact spermatogonial stem cell colonies in culture. The presence of stem cell markers ALPL, ITGA6 and CD9 indicated stemness in these colonies. The differentiation potential of these SSC colonies was demonstrated by their transformation into embryoid bodies upon withdrawal of growth factors from the culture medium. SSC colonies and embryoid bodies formed were evaluated using immunofluorescence and real-time PCR analysis. Embryoid body like structures derived from both neonatal and adult mouse testis were quite similar in terms of the expression of germ layer markers. CONCLUSION: These results strongly suggest that SSC-derived EB-like structures could be used for further differentiation into cells of interest in cell-based therapeutics.

Administration of adipose-derived stem cells extracellular vesicles in a murine model of spinal muscular atrophy: effects of a new potential therapeutic strategy.

BACKGROUND: Spinal Muscular Atrophy (SMA) is an autosomal-recessive neuromuscular disease affecting children. It is caused by the mutation or deletion of the survival motor neuron 1 (SMN1) gene resulting in lower motor neuron (MN) degeneration followed by motor impairment, progressive skeletal muscle paralysis and respiratory failure. In addition to the already existing therapies, a possible combinatorial strategy could be represented by the use of adipose-derived mesenchymal stem cells (ASCs) that can be obtained easily and in large amounts from adipose tissue. Their efficacy seems to be correlated to their paracrine activity and the production of soluble factors released through extracellular vesicles (EVs). EVs are important mediators of intercellular communication with a diameter between 30 and 100 nm. Their use in other neurodegenerative disorders showed a neuroprotective effect thanks to the release of their content, especially proteins, miRNAs and mRNAs. METHODS: In this study, we evaluated the effect of EVs isolated from ASCs (ASC-EVs) in the SMNΔ7 mice, a severe SMA model. With this purpose, we performed two administrations of ASC-EVs (0.5 µg) in SMA pups via intracerebroventricular injections at post-natal day 3 (P3) and P6. We then assessed the treatment efficacy by behavioural test from P2 to P10 and histological analyses at P10. RESULTS: The results showed positive effects of ASC-EVs on the disease progression, with improved motor performance and a significant delay in spinal MN degeneration of treated animals. ASC-EVs could also reduce the apoptotic activation (cleaved Caspase-3) and modulate the neuroinflammation with an observed decreased glial activation in lumbar spinal cord, while at peripheral level ASC-EVs could only partially limit the muscular atrophy and fiber denervation. CONCLUSIONS: Our results could encourage the use of ASC-EVs as a therapeutic combinatorial treatment for SMA, bypassing the controversial use of stem cells.

Revolutionizing Stroke Recovery: Unveiling the Promise of Stem Cell Therapy.

Stem cells, renowned for their unique regenerative capabilities, present significant hope in treating stroke, a major cause of disability globally. This review offers a detailed analysis of stem cell applications in stroke (ischemic and hemorrhagic) recovery. It examines therapies based on autologous (patient-derived), allogeneic (donor-derived), and Granulocyte-Colony Stimulating Factor (G-CSF) based stem cells, focusing on cell types such as Mesenchymal Stem/Stromal Cells (MSCs), Bone Marrow Mononuclear Stem Cells (BMMSCs), and Neural Stem/Progenitor Cells (NSCs). The paper compiles clinical trial data to evaluate their effectiveness and safety and addresses the ethical concerns of these innovative treatments. By explaining the mechanisms of stem cell-induced neurological repair, this review underscores stem cells' potential in revolutionizing stroke rehabilitation and suggests avenues for future research.

Isolation and characterization of feline endometrial mesenchymal stem cells.

BACKGROUND: Recently, there has been a growing interest in stem cells for human medicine. Limited feline endometrial mesenchymal stem cell (fEM-MSC) research in veterinary medicine necessitates reporting for future feline disease research and therapy. OBJECTIVES: This study aimed to isolate fEM-MSCs from feline endometrial tissues and evaluate their morphology, proliferative ability, differentiation ability, and immunophenotype. METHODS: Feline endometrial tissues were obtained from the ovariohysterectomies of healthy cats and isolated using an enzymatic method. The morphology and proliferative ability of the isolated cells were assessed using a doubling time (DT) assay from passages 3 to 6 (P3 - P6). We measured pluripotency gene expressions of cells in P2 using quantitative real-time polymerase chain reaction (qRT-PCR). To investigate MSC characteristics, a trilineage differentiation assay was conducted in P4, and cells in P4 were immunophenotyped using flow cytometry. RESULTS: fEM-MSCs showed a typical spindle-shaped morphology under a microscope, and the DT was maintained from P3 to P6. fEM-MSCs could differentiate into adipocytes, osteoblasts, and chondrocytes, and expressed three pluripotency markers (OCT4, SOX2, and NANOG) by qRT-PCR. Immunophenotypic analysis showed that the fEM-MSCs were CD14 -, CD34 -, CD45 -, CD9+, and CD44+. CONCLUSIONS: In this study, the feline endometrium was a novel source of MSCs, and to the best of our knowledge, this is the first report on the isolation method and characteristics of fEM-MSCs.

Chemically modified microRNA delivery via DNA tetrahedral frameworks for dental pulp regeneration.

Dental pulp regeneration is a promising strategy for addressing tooth disorders. Incorporating this strategy involves the fundamental challenge of establishing functional vascular networks using dental pulp stem cells (DPSCs) to support tissue regeneration. Current therapeutic approaches lack efficient and stable methods for activating DPSCs. In the study, we used a chemically modified microRNA (miRNA)-loaded tetrahedral-framework nucleic acid nanostructure to promote DPSC-mediated angiogenesis and dental pulp regeneration. Incorporating chemically modified miR-126-3p into tetrahedral DNA nanostructures (miR@TDNs) represents a notable advancement in the stability and efficacy of miRNA delivery into DPSCs. These nanostructures enhanced DPSC proliferation, migration, and upregulated angiogenesis-related genes, enhancing their paracrine signaling effects on endothelial cells. This enhanced effect was substantiated by improvements in endothelial cell tube formation, migration, and gene expression. Moreover, in vivo investigations employing matrigel plug assays and ectopic dental pulp transplantation confirmed the potential of miR@TDNs in promoting angiogenesis and facilitating dental pulp regeneration. Our findings demonstrated the potential of chemically modified miRNA-loaded nucleic acid nanostructures in enhancing DPSC-mediated angiogenesis and supporting dental pulp regeneration. These results highlighted the promising role of chemically modified nucleic acid-based delivery systems as therapeutic agents in regenerative dentistry and tissue engineering.

Effect of graphene oxide/ poly-L-lactic acid composite scaffold on the biological properties of human dental pulp stem cells.

BACKGROUND: Tissue engineering has attracted recent attention as a promising bone repair and reconstruction approach. Dental pulp stem cells (DPSCs) are pluripotent and can differentiate into bone cells with the correct environment and substrate. Therefore, suitable scaffold materials are essential for fabricating functional three-dimensional (3D) tissue and tissue regeneration. Composite scaffolds consisting of biodegradable polymers are very promising constructs. This study aims to verify the biological function of human DPSCs seeded onto composite scaffolds based on graphene oxide (GO) and poly-L-lactic acid (PLLA). METHODS: The surface morphology was observed under scanning electron microscopy (SEM). Chemical composition was evaluated with Fourier transform infrared (FTIR) spectroscopy. The biocompatibility of GO/PLLA scaffolds was assessed using phalloidin staining of cytoskeletal actin filaments, live/dead staining, and a CCK-8 assay. The effect of GO/PLLA scaffolds on cell osteogenic differentiation was detected through ALP staining, ALP activity assays, and alizarin red S staining, complemented by quantitative real-time PCR (qRT-PCR) analysis. RESULTS: Our data showed that GO and PLLA are successfully integrated and the GO/PLLA scaffolds exhibit favorable bioactivity and biocompatibility towards DPSCs. Additionally, it was observed that the 0.15% GO/PLLA scaffold group promoted DPSC proliferation and osteogenic differentiation by forming more calcium nodules, showing a higher intensity of ALP staining and ALP activity, and enhancing the expression levels of differentiation marker genes RUNX2 and COL1. CONCLUSIONS: These results demonstrate that the GO/PLLA scaffold is a feasible composite material suitable for cell culture and holds promising applications for oral bone tissue engineering.

Adipose-derived stem cell exosomes act as delivery vehicles of microRNAs in a dog model of chronic hepatitis.

Exosomes are nanosized extracellular vesicles secreted by all cell types, including canine adipose-derived stem cells (cADSCs). By mediating intercellular communication, exosomes modulate the biology of adjacent and distant cells by transferring their cargo. In the present work after isolation and characterization of exosomes derived from canine adipose tissue, we treated the same canine donors affected by hepatopathies with the previously isolated exosomes. We hypothesize that cADSC-sourced miRNAs are among the factors responsible for a regenerative and anti-inflammatory effect in the treatment of hepatopathies in dogs, providing the clinical veterinary field with an effective and innovative cell-free therapy. Exosomes were isolated and characterized for size, distribution, surface markers, and for their miRNomic cargo by microRNA sequencing. 295 dogs affected with hepatopathies were treated and followed up for 6 months to keep track of their biochemical marker levels. Results confirmed that exosomes derived from cADSCs exhibited an average diameter of 91 nm, and positivity to 8 known exosome markers. The administration of exosomes to dogs affected by liver-associated inflammatory pathologies resulted in the recovery of the animal alongside the normalization of biochemical parameters of kidney function. In conclusion, cADSCs-derived exosomes are a promising therapeutic tool for treating inflammatory disorders in animal companions.

[The role of ROS/JNK/caspase 3 axis in apoptosis induction by menthol-favored electronic cigarette liquid in human periodontal ligament stem cells].

PURPOSE: To explore the cytotoxic effect of a menthol-favored E-liquid on human periodontal ligament stem cells (hPDLSCs), as well as the underlying mechanism of electronic cigarette (E-cig)-induced cell apoptosis. METHODS: PDLSCs were isolated and cultured from periodontal ligament tissues of healthy premolars extracted for orthodontic reasons. Cells in passage 3 were used to detect the surface markers of stem cells by flow cytometry. Then the cells were exposed to different doses of menthol-favored E-liquid (at 59 mg/L nicotine concentration) in the culture median (the final nicotine concentrations were 0.1 µg/mL, 1.0 µg/mL, 10 µg/mL, 50 µg/mL, 0.1 mg/mL, 0.2 mg/mL and 0.5 mg/mL, respectively) for different period of times (24, 48 and 72 h). The cell viability was analyzed by CCK-8 assay. Cell apoptosis was evaluated by flow cytometry (7-AAD and Annexin V staining) and TUNEL assay. Reactive oxygen species (ROS) production was detected with fluorescence probe DCFH-DA by confocal microscopy and flow cytometry. The protein expression levels associated with ROS/JNK/caspase 3 axis(p-JNK, JNK, c-Jun, p-c-Jun, Bcl-2, Bax and cleaved-caspase 3) were analyzed by Western blot. Immunocytofluorescense staining was applied to evaluate the expression level of p-JNK. After addition of NAC, a ROS scavenger, and MAPK/JNK specific blocker SP600125, their effects on E-cig-induced cell apoptosis were evaluated. Statistical analysis was performed with Graph Pad 5.0 software package. RESULTS: Human PDLSCs were successfully isolated and cultured and flow cytometry assay showed the mesenchymal stem cell surface biomarkers (CD73, CD90 and CD105) were positively expressed. CCK8 assay indicated cell viability was significantly(P＜0.001) different among all concentration groups at various time points (24, 48 or 72 h), and the difference in apoptosis rate among all concentration groups was also statistically significant (P＜0.001). After exposure to E-liquid with nicotine concentration ≥50 µg/mL, cell viability was significantly reduced, and the proportion of apoptotic cells and the cellular ROS level was significantly increased in a dose-dependent manner as compared with the control group(0.0 mg/mL). Western blot assay showed E-cig exposure could promote MAPK/JNK phosphorylation in a dose-dependent and time-dependent manner. Either NAC or SP600125 could partially rescue the E-cig-induced cell apoptosis via reversing up-regulation of p-JNK and cleaved caspase 3. CONCLUSIONS: ROS/JNK/caspase 3 axis is involved in menthol-favored E-liquid-induced apoptosis of hPDLSCs.