Differentiation of intestinal stem cells toward goblet cells under systemic iron overload stress are associated with inhibition of Notch signaling pathway and ferroptosis.

Iron overload can lead to oxidative stress and intestinal damage and happens frequently during blood transfusions and iron supplementation. However, how iron overload influences intestinal mucosa remains unknown. Here, the aim of current study was to investigate the effects of iron overload on the proliferation and differentiation of intestinal stem cells (ISCs). An iron overload mouse model was established by intraperitoneal injection of 120 mg/kg body weight iron dextran once a fortnight for a duration of 12 weeks, and an iron overload enteroid model was produced by treatment with 3 mM or 10 mM of ferric ammonium citrate for 24 h. We found that iron overload caused damage to intestinal morphology with a 64 % reduction in villus height/crypt depth ratio, and microvilli injury in the duodenum. Iron overload mediated epithelial function by inhibiting the expression of nutrient transporters and enhancing the expression of secretory factors in the duodenum. Meanwhile, iron overload inhibited the proliferation of ISCs and regulated their differentiation into secretory mature cells, such as goblet cells, through inhibiting Notch signaling pathway both in mice and enteroid. Furthermore, iron overload caused oxidative stress and ferroptosis in intestinal epithelial cells. In addition, ferroptosis could also inhibit Notch signaling pathway, and affected the proliferation and differentiation of ISCs. These findings reveal the regulatory role of iron overload on the proliferation and differentiation of ISCs, providing a new insight into the internal mechanism of iron overload affecting intestinal health, and offering important theoretical basis for the scientific application of iron nutrition regulation.

GITRL impairs hepatocyte repopulation by liver progenitor cells to aggravate inflammation and fibrosis by GITR+CD8+ T lymphocytes in CDE Mice.

As an alternative pathway for liver regeneration, liver progenitor cells and their derived ductular reaction cells increase during the progression of many chronic liver diseases. However, the mechanism underlying their hepatocyte repopulation after liver injury remains unknown. Here, we conducted progenitor cell lineage tracing in mice and found that fewer than 2% of hepatocytes were derived from liver progenitor cells after 9 weeks of injury with a choline-deficient diet supplemented with ethionine (CDE), and this percentage increased approximately three-fold after 3 weeks of recovery. We also found that the proportion of liver progenitor cells double positive for the ligand of glucocorticoid-induced tumour necrosis factor receptor (GITRL, also called Tnfsf18) and SRY-related HMG box transcription 9 (Sox9) among nonparenchymal cells increased time-dependently upon CDE injury and reduced after recovery. When GITRL was conditionally knocked out from hepatic progenitor cells, its expression in nonparenchymal cells was downregulated by approximately fifty percent, and hepatocyte repopulation increased by approximately three folds. Simultaneously, conditional knockout of GITRL reduced the proportion of liver-infiltrating CD8+ T lymphocytes and glucocorticoid-induced tumour necrosis factor receptor (GITR)-positive CD8+ T lymphocytes. Mechanistically, GITRL stimulated cell proliferation but suppressed the differentiation of liver progenitor organoids into hepatocytes, and CD8+ T cells further reduced their hepatocyte differentiation by downregulating the Wnt/ß-catenin pathway. Therefore, GITRL expressed by liver progenitor cells impairs hepatocyte differentiation, thus hindering progenitor cell-mediated liver regeneration.

Icariin Regulates EMT and Stem Cell-Like Character in Breast Cancer through Modulating lncRNA NEAT1/TGFß/SMAD2 Signaling Pathway.

Metastases and drug resistance are the major risk factors associated with breast cancer (BC), which is the most common type of tumor affecting females. Icariin (ICA) is a traditional Chinese medicine compound that possesses significant anticancer properties. Long non-coding RNAs (lncRNAs) are involved in a wide variety of biological and pathological processes and have been shown to modulate the effectiveness of certain drugs in cancer. The purpose of this study was to examine the potential effect of ICA on epithelial mesenchymal transition (EMT) and stemness articulation in BC cells, as well as the possible relationship between its inhibitory action on EMT and stemness with the NEAT1/transforming growth factor ß (TGFß)/SMAD2 pathway. The effect of ICA on the proliferation (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony assays), EMT (Western blotting, immunofluorescence, and wound healing), and stemness (mammosphere formation assays, Western blotting) of BC cells were examined. According to the findings, ICA suppressed the proliferation, EMT, and stem cell-like in MDA-MB-231 cells, and exerted its inhibitory impact by downregulating the TGFß/SMAD2 signaling pathway. ICA could significantly downregulate the expression of lncRNA NEAT1, and silencing NEAT1 enhanced the effect of ICA in suppressing EMT and expression of different stem cell markers. In addition, silencing NEAT1 was found to attenuate the TGFß/SMAD2 signaling pathway, thereby improving the inhibitory impact of ICA on stemness and EMT in BC cells. In conclusion, ICA can potentially inhibit the metastasis of BC via affecting the NEAT1/TGFß/SMAD2 pathway, which provides a theoretical foundation for understanding the mechanisms involved in potential application of ICA for BC therapy.

Use of adipose derived stem cells accelerates the healing process in third-degree burns.

INTRODUCTION: Burns are defined as a traumatic injury, usually of thermal origin, that affects the epithelial and adjacent tissue and is classified according to the depth reached. Tissue repair involved in this type of injury is often a challenge both due to its severity and the multiplicity of complications. Regenerative medicine has focused on the use of low-level laser photobiomodulation therapy (LLLT) and adipose-derived stem cells (ADSC), especially in the early stages of the process, to promote better healing and shorten repair time. Therefore, aim of this study was to evaluate the action of LLLT (660 nm) and ADSC in the repair process of burned skin tissue and investigate the association of the techniques (LLLT and ADSC). MATERIALS AND METHODS: An in vivo study was carried out using 96 rats (Wister) with a scald burn model at a temperature of 95ºC, exposing the animal's back for 14 s. Animals were randomized into seven groups and three periods, five, 14 and 21 days. The groups included GC: Control group, ADSC-: Group treated with CD49d negative cells, ADSC+ : Group treated with positive CD49d cells, CULT: Group treated with conventional isolation cells, LLLT: Group treated only with LLLT Low Power Laser, ADSC-LLLT: Group treated with CD49d negative cells and LLLT. ADSC+LLLT: Group treated with positive CD49d cells and LLLT. The groups treated with LLLT (660 nm; 5 J/cm2) received irradiation three times a week, on alternate days for five, 14 and 21 days, according to the time of biopsy. ADSC-treated groups received one to three applications of the cells in a total volume of 1000 µL starting soon after the surgical debridement of the burn. Photographic monitoring was carried out at 5, 14 and 21 days after the beginning of the experiment to assess the degree of lesion contraction. Macroscopic, morphometric and histopathological analyzes were performed. RESULTS: We showed significant re-epithelialization as well as an improvement in the healing process in the ADSC+, LLLT and ADSC+LLLT groups. We observed effects in the reduction of the inflammatory phase, increase in angiogenesis, decrease in oedema, greater collagen deposition, and better organization of the extracellular matrix compared to the other treatments. Moreover, the immunomagnetic separation of ADSC cells through the expression of the CD49d protein proved to be a useful means to obtain a more homogeneous population of cells with a role in tissue regeneration compared to the ADSC- and CULT groups. CONCLUSION: In conclusion, the association of ADSC+ with LLLT was effective in accelerating the burn repair process, stimulating cell proliferation and formation of more normal skin tissue.

Effect of photobiomodulation therapy on TGF-ß release from dentin, migration and viability of dental pulp stem cells in regenerative endodontics treatment: An ex vivo study.

BACKGROUND AND AIM: Regenerative endodontic procedures (REPs) are oriented by the principles of tissue engineering, incorporating dental pulp stem cells (DPSC), crucial growth factors like Transforming growth factor-ß (TGF-ß1), and scaffolds to facilitate the regeneration of dental pulp tissues. The present study aimed to investigate the effect of photobiomodulation (PBM) therapy, using an 808 nm diode laser on cellular modulation mechanisms in REPs. METHOD AND MATERIAL: A total of 108 human dentin discs obtained from intact single root teeth were randomly assigned into six groups (n = 8): 1. Positive control (EDTA), 2. PBM-1 (3 J/cm2), 3. PBM-2 (5 J/cm2), 4. EDTA+PBM-1, 5. EDTA+PBM-2, and 6. Negative control (NaOCl). Then, an extract solution was prepared from each disc and the concentration of released TGF-ß1 from the discs was measured using enzyme-linked immunosorbent assay (ELISA). Moreover, the extract solution was added to DPSC culture medium to evaluate cell viability and migration through MTT assay and scratch test, respectively. RESULT: The group exposed to PBM-1 showed the highest cell viability, while treatment with EDTA and EDTA+PBM-2 decreased cellular viability. Also, the PBM-treated groups showed significantly higher release of TGF-ß1 compared to the negative control. EDTA and EDTA+PBM-1 showed the highest release among all the groups. No significant difference was found between EDTA and EDTA+PBM-1, as well as between PBM-1 and PBM-2. Moreover, the PBM-1 group exhibited the highest migration after 24 h, which was significantly greater than other groups, except for the PBM-2 group. CONCLUSION: According to the obtained data, 808 nm mediated-PBM (3 J/cm2), both independently and in conjunction with EDTA, enhanced the release of TGF-ß1 from dentin and improved cell viability and migration of DPSCs. It seems that, PBM under the specific parameters employed in this study, could be an effective adjunctive therapy in REPs.

Photobiomodulation Facilitates Rat Cutaneous Wound Healing by Promoting Epidermal Stem Cells and Hair Follicle Stem Cells Proliferation.

BACKGROUND: Cutaneous wound healing represents a common fundamental phenomenon requiring the participation of cells of distinct types and a major concern for the public. Evidence has confirmed that photobiomodulation (PBM) using near-infrared (NIR) can promote wound healing, but the  cells involved and the precise molecular mechanisms remain elusive. METHODS: Full-thickness skin defects with a diameter of 1.0 cm were made on the back of rats and randomly divided into the control group, 10 J, 15 J, and 30 J groups. The wound healing rate at days 4, 8, and 12 postoperatively was measured. HE and Masson staining was conducted to reveal the histological characteristics. Immunofluorescence staining was performed to label the epidermal stem cells (ESCs) and hair follicle stem cells (HFSCs). Western blot was performed to detect the expressions of proteins associated with ESCs and HFSCs. Cutaneous wound tissues were collected for RNA sequencing. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes analysis was performed, and the hub genes were identified using CytoHubba and validated by qRT-PCR. RESULTS: PBM can promote reepithelialization, extracellular matrix deposition, and wound healing, increase the number of KRT14+/PCNA+ ESCs and KRT15+/PCNA+ HFSCs, and upregulate the protein expression of P63, Krt14, and PCNA. Three hundred and sixty-six differentially expressed genes (DEGs) and 7 hub genes including Sox9, Krt5, Epcam, Cdh1, Cdh3, Dsp, and Pkp3 were identified. These DEGs are enriched in skin development, cell junction, and cadherin binding involved in cell-cell adhesion etc., while these hub genes are related to skin derived stem cells and cell adhesion. CONCLUSION: PBM accelerates wound healing by enhancing reepithelialization through promoting ESCs and HFSCs proliferation and elevating the expression of genes associated with stem cells and cell adhesion. This may provide a valuable alternative strategy to promote wound healing and reepithelialization by modulating the proliferation of skin derived stem cells and regulating genes related to cell adhesion.

Ginseng promotes the function of intestinal stem cells through the Wnt/ß-catenin signaling pathway in D-galactose-induced aging mice.

BACKGROUND: Intestinal stem cells (ISCs) are the reservoir source of various types of intestinal cells, and the decline of stem cell function in the gut may be a potential factor for aging-related disease. The present study aimed to explore the regulatory mechanisms of Panax ginseng C.A.Meyer (Araliaceae, Panax genus) that could restore gut aging by enhancing intestinal function and regulating ISCs in aging mice based on the Wnt/ß-catenin signaling pathway. METHODS: A total of 60 ICR male mice were randomly divided into control, model, metformin, and ginseng water decoction (GWD) 3.6, 1.8, and 0.9 g/kg groups. The aging model was induced by 1 % D-galactose (s.c. 0.1 mL/10 g) for 28 days. Moreover, GWD was given to aging mice intragastrically (i.g.) once a day for 28 successive days. The learning memory ability, pathological status, and function in the ileum tissue, the activity of digestive enzymes, and short-chain fatty acid (SCFA) content in the colon were evaluated, and the related mechanism was investigated. RESULTS: Ginseng can decrease the escape latency time and increase the swimming speed and the number of crossing platforms in aging mice. Moreover, the pathology of ileum tissue improved, the length of the intestinal villi increased, and the width of the villi and the depth of the crypts decreased. The activities of trypsin, α-amylase, and lipase increased in duodenal content and intestinal mucosa. In the colon, the content of SCFA, such as acetic acid, propionic acid and butyric acid, increased, indicating that ginseng significantly improves intestinal function impairment. The mRNA expressions and protein levels of ß-catenin, C-myc, GSK-3ß, Lgr5, and Olfm4 were upregulated in the ginseng group. CONCLUSIONS: Ginseng improves intestinal function and regulates the function of ISCs in order to protect intestinal health by activating the Wnt/ß-catenin signaling pathway in aging mice.

Inhibition of Autophagy in Renal Cells With Human, Urine-derived, Stem-cell Exosomes to Improve Diabetic Nephropathy.

Context: Clinicians can use stem cells to repair kidney injury. The kidneys' exosome secretions hold the secret to this therapeutic impact. Exosomes from urine-derived stem cells can prevent and treat glomerular damage that diabetes can cause, but the underlying process has remained a mystery. Objective: The study aimed to investigate the protective impact of exosomes from urine-derived stem cells (USCs) against diabetic nephropathy (DN) and to determine the mechanisms involved. Design: The research team performed an animal study. Setting: The study took place at the Affiliated Hospital of Jiujiang University in Jiujiang, Jiangxi, China. Animals: The animals were rats, SD male rats, weighing 200-220g, 40 animals, purchased from Weitong Lihua Experimental Animal Technology Co., Ltd. (certificate number: SCXK (Beijing) 2021-0006). Intervention: Except for a control group, the rats in the groups had induced DN. The five groups, with 10 rats each, were: (1) the negative control group, which received 0.2 ml of PBS solution; (2) the DN group, a second negative control group, which received 0.2 ml of PBS solution, (3) the inhibitor group, an intervention group that received 20 mg/kg of autophagy inhibitor; (4) the exosomes group, an intervention group that received 100 ug/kg of exosomes; and (5) the exosomes + inhibitor group, an intervention group that received 100 ug/kg of exosomes + 20 mg/kg of autophagy inhibitor. From week 8, for four weeks the team injected the inhibitor, exosomes, and exosomes + inhibitor groups with the appropriate treatments using the rats' tail veins. Outcome Measures: The research team: (1) examined the USCs in the exosomes of stem cells; (2) assessed the rats' weights and fasting blood glucose (FBG), using a blood glucose meter; (3) used Coomassie brilliant blue (CBB) staining to determine the amount of protein in the rats' urine and assessed their biochemical indexes; and (4) used Western blot (WB) and a quantitative polymerase chain reaction (Q-PCR) to detect autophagy and the signal transduction pathway. Results: Human exosomes from USCs alleviated injury in the rats that DN caused by reducing urinary-protein levels, serum creatinine (SCR), blood urea nitrogen (BUN), glomerular cell accumulation, and kidney weights. In rats with induced DN, the exosomes + inhibitor significantly reduced the activation of the mTOR signaling pathway, reduced the autophagy of their kidney cells, increased the protein expression of Bcl-2 in the kidney tissues, and lessened the damage to glomerular cells. Conclusions: Human urine-derived stem cell exosomes can significantly reduce the activation of the mTOR signaling pathway, reduce the autophagy of rats' kidney cells, increase the protein expression of LC3B in kidney tissues, and reduce the damage to glomerular cells. By blocking the mTOR signaling pathway, human urogenic exosomes can alleviate the signs and symptoms of DN.

Therapeutic mechanism of Liangxue-Guyuan-Yishen decoction on intestinal stem cells and tight junction proteins in gastrointestinal acute radiation syndrome rats.

On the basis of the previous research, the Traditional Chinese Medicine theory was used to improve the drug composition for gastrointestinal acute radiation syndrome (GI-ARS). The purpose of this study was to study the therapeutic mechanism of Liangxue-Guyuan-Yishen decoction (LGYD) on GI-ARS and to provide a new scheme for the treatment of radiation injury. Here, we investigated the effects of LGYD on intestinal stem cells (ISCs) in a GI-ARS rat model. Rat health and survival and the protective efficacy of LGYD on the intestines were analyzed. The active principles in LGYD were detected using liquid chromatography-mass spectrometry (LC-MS). ISC proliferation, intestinal epithelial tight junction (TJ) protein expression and regulatory pathways were explored using immunohistochemistry, western blotting (WB) and reverse transcription quantitative polymerase chain reaction (RT-qPCR), respectively. Involvement of the WNT and MEK/ERK pathways in intestinal recovery was screened using network pharmacology analysis and validated by WB and RT-qPCR. LGYD administration significantly improved health and survival in GI-ARS rats. Pathological analysis showed that LGYD ameliorated radiation-induced intestinal injury and significantly promoted LGR5+ stem cell regeneration in the intestinal crypts, upregulated TJ protein, and accelerated crypt reconstruction in the irradiated rats. LC-MS revealed ≥13 constituents that might contribute to LGYD's protective effects. Collectively, LGYD can promote crypt cell proliferation and ISCs after radiation damage, the above effect may be related to WNT and MEK/ERK pathway.

Electroacupuncture improves cardiac function after myocardial infarction by regulating the mobilization and migration of endogenous stem cells.

OBJECTIVE: The aim of this study was to explore the role and mechanisms of electroacupuncture (EA) in the regulation of chemokines in endogenous stem cell mobilization and myocardial regeneration after myocardial infarction (MI). METHODS: An MI model was constructed in adult male Sprague-Dawley rats by ligating the left anterior descending coronary artery. After 4 weeks of treatment, echocardiography was used to detect changes in cardiac function, and Masson's trichrome staining was used to detect collagen deposition. In addition, immunofluorescence staining was applied to examine von Willebrand factor (vWF)-positive vessels, the expression of cardiac troponin T (cTnT) and proliferation marker Ki67, and the number of c-kit-positive, C-X-C chemokine receptor type 4 (CXCR4)-positive, and Sca-1-positive endogenous stem cells in the infarcted area. In addition, the expression of stromal cell-derived factor (SDF)-1 and stem cell factor (SCF) was detected. RESULTS: EA increased the ejection fraction after MI, reduced collagen deposition and cellular apoptosis, and increased the number of blood vessels compared with an untreated model group. EA significantly promoted cellular proliferation, except for myocardial cells, and significantly increased the number of c-kit-, CXCR4- and Sca-1-positive stem cells. Moreover, the expression of SDF-1 and SCF in myocardial tissue in the EA group was significantly higher than that in the (untreated) MI group. CONCLUSIONS: EA appears to promote angiogenesis and reduce collagen deposition, thus improving the cardiac function of rats with MI. The underlying mechanism of action may involve endogenous stem cell mobilization mediated by SDF-1/CXCR4 and SCF/c-kit.

Adipose Tissue-Derived Stromal/Stem Cells Transplantation with Cholecalciferol Supplementation in Recent-Onset Type 1 Diabetes Patients: Twelve Months Follow-Up.

To evaluate safety and therapeutic effect along 12 months of allogenic adipose tissue-derived stromal/stem cells (ASCs) transplantation with cholecalciferol (VITD) in patients with recent-onset type 1 diabetes (T1D). Prospective, phase II, open trial, pilot study in which patients with recent onset T1D received ASCs (1xKgx106 cells) and VITD 2000UI/day for 12 months (group 1) and were compared to controls with standard insulin therapy (group 2). Adverse events, C-peptide area under the curve (CPAUC), insulin dose, HbA1c and frequency of FoxP3+ in CD4+ or CD8+ T-cells(flow cytometry) were evaluated at baseline(T0), after 3(T3), 6(T6) and 12 months(T12). Eleven patients completed follow up (7:group 1;4:group 2). Group 1 had lower insulin requirement at T3(0.24±0.18vs0.53±0.23UI/kg,p=0.04), T6(0.24±0.15vs0.66±0.33 UI/kg,p=0.04) and T12(0.39±0.15vs0.74±0.29 UI/Kg,p=0.04).HbA1c was lower at T6 (50.57±8.56vs72.25±10.34 mmol/mol,p=0.01), without differences at T12 (57.14±11.98 in group 1 vs. 73.5±14.57 mmol/min in group 2, p=0.16). CPAUC was not significantly different between groups at T0(p=0.07), higher in group 1 at T3(p=0.04) and T6(p=0.006), but similar at T12(p=0.23). IDAA1c was significantly lower in group 1 than group 2 at T3,T6 and T12 (p=0.006, 0.006 and 0.042, respectively). IDDA1c was inversely correlated to FoxP3 expression in CD4 and CD8+ T cells at T6 (p<0.001 and p=0.01, respectively). In group 1, one patient had recurrence of a benign teratoma that was surgically removed, not associated to the intervention. ASCs with VITD without immunosuppression were safe and associated lower insulin requirements, better glycemic control, and transient better pancreatic function in recent onset T1D, but the potential benefits were not sustained.

Baicalin can enhance odonto/osteogenic differentiation of inflammatory dental pulp stem cells by inhibiting the NF-κB and ß-catenin/Wnt signaling pathways.

BACKGROUND: Scutellaria baicalensis Georgi is a famous traditional Chinese medicine, which is widely used in treating fever, upper respiratory tract infection and other diseases. Pharmacology study showed it can exhibit anti-bacterial, anti-inflammation and analgesic effects. In this study, we investigated the effect of baicalin on the odonto/osteogenic differentiation of inflammatory dental pulp stem cells (iDPSCs). METHODS AND RESULTS: iDPSCs were isolated from the inflamed pulps collected from pulpitis. The proliferation of iDPSCs was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2,5-tetrazolium bromide (MTT) assay and flow cytometry. Alkaline phosphatase (ALP) activity assay, alizarin red staining, Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay were conducted to examine the differentiation potency along with the involvement of nuclear factor kappa B(NF-κB) and ß-catenin/Wnt signaling pathway. MTT assay and cell-cycle analysis demonstrated that baicalin had no influence on the proliferation of iDPSCs. ALP activity assay and alizarin red staining demonstrated that baicalin could obviously enhance ALP activity and calcified nodules formed in iDPSCs. RT-PCR and Western blot showed that the odonto/osteogenic markers were upregulated in baicalin-treated iDPSCs. Moreover, expression of cytoplastic phosphor-P65, nuclear P65, and ß-catenin in iDPSCs was significantly increased compared with DPSCs, but the expression in baicalin-treated iDPSCs was inhibited. In addition, 20 µM Baicalin could accelerate odonto/osteogenic differentiation of iDPSCs via inhibition of NF-κB and ß-catenin/Wnt signaling pathways. CONCLUSION: Baicalin can promote odonto/osteogenic differentiation of iDPSCs through inhibition of NF-κB and ß-catenin/Wnt pathways, thus providing direct evidence that baicalin may be effective in repairing pulp with early irreversible pulpitis.

Prosopis juliflora (Sw.) DC phytochemicals induce apoptosis and inhibit cell proliferation signaling pathways, EMT, migration, invasion, angiogenesis and stem cell markers in melanoma cell lines.

ETHNOPHARMACOLOGICAL RELEVANCE: Prosopis juliflora (Sw.), DC is a xerophytic plant species that extensively grow in Asia, Africa, Australia, and Brazil. From ancient time P. juliflora is being utilized in various folk remedies for example in wound healing, fever, inflammation, measles, excrescences, diarrhea and dysentery. Traditionally, gum, paste, and smoke obtained from the leaves and pods are applied for anticancer, antidiabetic, anti-inflammatory, and antimicrobial purposes. AIM OF THE STUDY: Our previous studies have demonstrated the promising potential of Prosopis Juliflora leaves methanol extract (PJLME) against breast cancer, and suggested its possible integration as a complementary medicine for the effective management of breast cancer. However, evidence against how PJLME mechanistically target the cancer proliferative pathways and other targets is poorly understood. The basic aim of the present study was to understand the anti-melanoma potential of PJLME against B16f10 cells with possible mechanisms of action. MATERIALS AND METHODS: MTT assay was used to determine cell viability. Wound and transwell migration assay was performed to check migration potential of cells after PJLME treatment, while clonogenic assay was carried out to understand its colony inhibition actvity. Flow cytometry was used to perform annexin V/PI assay (apoptosis assay), ROS assay, cell cycle analysis. In-vitro angiogenesis assay was performed to check formation of capillary like vascular structure after PJLME treatment. Apoptotic genes, signaling pathways markers, EMT markers and stem cell markers were determined by western blotting. In-vivo BALB/C mice xenograft model study was performed to check the effect of PJLME on in-vivo melanoma tumor growth. RESULTS: The experimental outcome of the present study has clearly demonstrated the inhibition of growth, migration, invasion, colony formation and apoptosis inducing potential of PJLME against mouse melanoma cancer cells. Treatment of B16F10 melanoma cells with PJLME resulted in arrest of cell cycle at G0/G1 phase. Annexin V-FITC/PI assay confirmed the apoptosis inducing potential of PJLME in B16F10 and A375 melanoma cells. Furthermore, Western blot experiments confirmed that the treatment of PJLME downregulates the expression of anti-apoptotic gene like Bcl2 and increase the expression profile of pro-apoptotic genes like Bax, Bad, and Bak in B16F10 melanoma cells. HUVEC (Human umbilical vein endothelial cells) tube formation assay clearly demonstrated the anti-angiogenic potential of PJLME. The study also revealed that PJLME has potential to inhibit the Akt and Erk signaling pathways which are participating in cancer cell proliferation, migration, invasion etc. The outcome of qRT-PCR and immunoblotting analysis clearly unveiled that PJLME treatment leads to downregulation of epithelial-mesenchymal transition (EMT) as well as stem cell markers. Finally, the in-vivo animal xenograft model study also revealed the anti-melanoma potential of PJLME by significantly inhibiting the B16F10 melanoma tumor growth in BALB/c mice model. The LC-ESI-MS/MS analysis of PJLME showed the presence of variety of bioactive molecules associated with anticancer effects. CONCLUSION: The outcome of the present investigation clearly demonstrated the anti-melanoma potential of PJLME against B16f10 melanoma cells. PJLME can be explored as an adjuvant or complementary therapy against melanoma cancer, however further studies are required to understand the clinical efficacy of PJLME. Nevertheless, it can be further explored as a promising resource for identification of novel anticancer candidate drug.

N-acetylcysteine promotes the proliferation of porcine adipose-derived stem cells during in vitro long-term expansion for cultured meat production.

Cultured meat is an efficient, safe and sustainable meat production technology. Adipose-derived stem cell (ADSC) is a promising cell type for cultured meat. In vitro, obtaining numerous of ADSCs is a pivotal step for cultured meat. In this research, we demonstrated that the proliferation and adipogenic differentiation of ADSCs significantly decreased during serial passage. Then, senescence ß-galactosidase (SA-ß-gal) staining showed that the positive rate of P9 ADSCs was 7.74-fold than P3 ADSCs. Subsequently, RNA sequencing (RNA-seq) was performed for P3 and P9 ADSCs and found that PI3K-AKT pathway was up-regulated, but cell cycle and DNA repair pathway were down-regulated in P9 ADSCs. Then, N-Acetylcysteine (NAC) was added during long-term expansion and showed that NAC enhanced the ADSCs proliferation and maintained adipogenic differentiation. Finally, RNA-seq was performed for P9 ADSCs cultured with or without NAC and showed that NAC restored the cell cycle and DNA repair pathway in P9 ADSCs. These results highlighted that NAC was an excellent supplement for large-scale expansion of porcine ADSCs for cultured meat.

Human Stem Cell Use in Androgenetic Alopecia: A Systematic Review.

Androgenetic alopecia is a condition that results in hair loss in both men and women. This can have a significant impact on a person's psychological well-being, which can lead to a decreased quality of life. We conducted a systematic review to evaluate the efficacy of using stem cells in androgenic alopecia. The search was conducted in MEDLINE via PubMed, Web of Science, and Scopus databases. The review was performed on data pertaining to the efficacy of using different types of stem cells in androgenic alopecia: quantitative results of stem cell usage were compared to the control treatment or, different types of treatment for female and male androgenetic alopecia. Of the outcomes, the density of hair was analyzed. Fourteen articles were selected for this review. During and after treatment with stem cells, no major side effects were reported by patients with alopecia. The use of stem cells in androgenic alopecia seems to be a promising alternative to the standard treatment or it could play the role of complementary therapy to improve the effect of primary treatment. However, these results should be interpreted with caution until they can be reproduced in larger and more representative samples.

The stereological, immunohistological, and gene expression studies in an infected ischemic wound in diabetic rats treated by human adipose-derived stem cells and photobiomodulation.

We investigated the impacts of photobiomodulation (PBM) and human allogeneic adipose-derived stem cells (ha-ADS) together and or alone applications on the stereological parameters, immunohistochemical characterizing of M1 and M2 macrophages, and mRNA levels of hypoxia-inducible factor (HIF-1α), basic fibroblast growth factor (bFGF), vascular endothelial growth factor-A (VEGF-A) and stromal cell-derived factor-1α (SDF-1α) on inflammation (day 4) and proliferation phases (day 8) of repairing tissues in an infected delayed healing and ischemic wound model (IDHIWM) in type 1 diabetic (DM1) rats. DM1 was created in 48 rats and an IDHIWM was made in all of them, and they were distributed into 4 groups. Group1 = control rats with no treatment. Group2 = rats received (10 × 100000 ha-ADS). Group3 = rats exposed to PBM (890 nm, 80 Hz, 3.46 J/cm2). Group4 = rats received both PBM and ha-ADS. On day 8, there were significantly higher neutrophils in the control group than in other groups (p < 0.01). There were substantially higher macrophages in the PBM + ha-ADS group than in other groups on days 4 and 8 (p < 0.001). Granulation tissue volume, on both days 4 and 8, was meaningfully greater in all treatment groups than in the control group (all, p = 0.000). Results of M1 and M2 macrophage counts of repairing tissue in the entire treatment groups were considered preferable to those in the control group (p < 0.05). Regarding stereological and macrophage phenotyping, the results of the PBM + ha-ADS group were better than the ha-ADS and PBM groups. Results of the tested gene expression of repairing tissue on inflammation and proliferation steps in PBM and PBM + ha-ADS groups were meaningfully better than the control and ha-ADS groups (p < 0.05). We showed that PBM, ha-ADS, and PBM plus ha-ADS, hastened the proliferation step of healing in an IDHIWM in rats with DM1 by regulation of the inflammatory reaction, macrophage phenotyping, and augmented granulation tissue formation. In addition PBM and PBM plus ha-ADS protocols hastened and increased mRNA levels of HIF-1α, bFGF, SDF-1α, and VEGF-A. Totally, in terms of stereological and immuno-histological tests, and also gene expression HIF-1α and VEGF-A, the results of PBM + ha-ADS were superior (additive) to PBM, and ha-ADS alone treatments.

Targeted expression of bgl23-D, a dominant-negative allele of ATCSLD5, affects cytokinesis of guard mother cells and exine formation of pollen in Arabidopsis thaliana.

MAIN CONCLUSION: Targeted expression of bgl23-D, a dominant-negative allele of ATCSLD5, is a useful genetic approach for functional analysis of ATCSLDs in specific cells and tissues in plants. Stomata are key cellular structures for gas and water exchange in plants and their development is influenced by several genes. We found the A. thaliana bagel23-D (bgl23-D) mutant showing abnormal bagel-shaped single guard cells. The bgl23-D was a novel dominant mutation in the A. thaliana cellulose synthase-like D5 (ATCSLD5) gene that was reported to function in the division of guard mother cells. The dominant character of bgl23-D was used to inhibit ATCSLD5 function in specific cells and tissues. Transgenic A. thaliana expressing bgl23-D cDNA with the promoter of stomata lineage genes, SDD1, MUTE, and FAMA, showed bagel-shaped stomata as observed in the bgl23-D mutant. Especially, the FAMA promoter exhibited a higher frequency of bagel-shaped stomata with severe cytokinesis defects. Expression of bgl23-D cDNA in the tapetum with SP11 promoter or in the anther with ATSP146 promoter induced defects in exine pattern and pollen shape, novel phenotypes that were not shown in the bgl23-D mutant. These results indicated that bgl23-D inhibited unknown ATCSLD(s) that exert the function of exine formation in the tapetum. Furthermore, transgenic A. thaliana expressing bgl23-D cDNA with SDD1, MUTE, and FAMA promoters showed enhanced rosette diameter and increased leaf growth. Taken together, these findings suggest that the bgl23-D mutation could be a helpful genetic tool for functional analysis of ATCSLDs and manipulating plant growth.

Lactoferrin Mediates Enhanced Osteogenesis of Adipose-Derived Stem Cells: Innovative Molecular and Cellular Therapy for Bone Repair.

A prospective source of stem cells for bone tissue engineering is adipose-derived stem cells (ADSCs), and BMP-2 has been proven to be highly effective in promoting the osteogenic differentiation of stem cells. Rarely has research been conducted on the impact of lactoferrin (LF) on ADSCs' osteogenic differentiation. As such, in this study, we examined the effects of LF and BMP-2 to assess the ability of LF to stimulate ADSCs' osteogenic differentiation. The osteogenic medium was supplemented with the LF at the following concentrations to culture ADSCs: 0, 10, 20, 50, 100, and 500 µg/mL. The Cell Counting Kit-8 (CCK-8) assay was used to measure the proliferation of ADSCs. Calcium deposition, alkaline phosphatase (ALP) staining, real-time polymerase chain reaction (RT-PCR), and an ALP activity assay were used to establish osteogenic differentiation. RNA sequencing analysis was carried out to investigate the mechanism of LF boosting the osteogenic development of ADSCs. In the concentration range of 0-100 µg/mL, LF concentration-dependently increased the proliferative vitality and osteogenic differentiation of ADSCs. At a dose of 500 µg/mL, LF sped up and enhanced differentiation, but inhibited ADSCs from proliferating. LF (100 and 500 µg/mL) produced more substantial osteoinductive effects than BMP-2. The PI3 kinase/AKT (PI3K/AKT) and IGF-R1 signaling pathways were significantly activated in LF-treated ADSCs. The in vitro study results showed that LF could effectively promote osteogenic differentiation of ADSCs by activating the PI3K/AKT and IGF-R1 pathways. In our in vitro investigation, an LF concentration of 100 µg/mL was optimal for osteoinduction and proliferation. Our study suggests that LF is an attractive alternative to BMP-2 in bone tissue engineering. As a bioactive molecule capable of inducing adipose stem cells to form osteoblasts, LF is expected to be clinically used in combination with biomaterials as an innovative molecular and cellular therapy to promote bone repair.

Role of curcumin and its nanoformulations in the treatment of neurological diseases through the effects on stem cells.

Curcumin from turmeric is a natural phenolic compound with a promising potential to regulate fundamental processes involved in neurological diseases, including inflammation, oxidative stress, protein aggregation, and apoptosis at the molecular level. In this regard, employing nanoformulation can improve curcumin efficiency by reducing its limitations, such as low bioavailability. Besides curcumin, growing data suggest that stem cells are a noteworthy candidate for neurodegenerative disorders therapy due to their anti-inflammatory, anti-oxidative, and neuronal-differentiation properties, which result in neuroprotection. Curcumin and stem cells have similar neurogenic features and can be co-administered in a cell-drug delivery system to achieve better combination therapeutic outcomes for neurological diseases. Based on the evidence, curcumin can induce the neuroprotective activity of stem cells by modulating their related signalling pathways. The present review is about the role of curcumin and its nanoformulations in the improvement of neurological diseases alone and through the effect on different categories of stem cells by discussing the underlying mechanisms to provide a roadmap for future investigations.

Intestinal Stem Cells Damaged by Deoxycholic Acid via AHR Pathway Contributes to Mucosal Barrier Dysfunction in High-Fat Feeding Mice.

High-fat exposure leads to impaired intestinal barrier function by disrupting the function of intestinal stem cells (ISCs); however, the exact mechanism of this phenomenon is still not known. We hypothesize that high concentrations of deoxycholic acid (DCA) in response to a high-fat diet (HFD) affect aryl hydrocarbon receptor (AHR) signalling in ISCs and the intestinal barrier. For this purpose, C57BL/6J mice feeding on a low-fat diet (LFD), an HFD, an HFD with the bile acid binder cholestyramine, and a LFD with the DCA were studied. We found that high-fat feeding induced an increase in faecal DCA concentrations. An HFD or DCA diet disrupted the differentiation function of ISCs by downregulating AHR signalling, which resulted in decreased goblet cells (GCs) and MUC2, and these changes were reversed by cholestyramine. In vitro experiments showed that DCA downregulated the differentiation function of ISCs, which was reversed by the AHR agonist 6-formylindolo [3,2-b]carbazole (FICZ). Mechanistically, DCA caused a reduction in indoleamine 2,3-dioxygenase 1 (IDO1) in Paneth cells, resulting in paracrine deficiency of the AHR ligand kynurenine in crypts. We demonstrated for the first time that DCA disrupts intestinal mucosal barrier function by interfering with AHR signalling in ISCs. Supplementation with AHR ligands may be a new therapeutic target for HFD-related impaired intestinal barrier function.