Background: Autistic Spectrum Disorder (ASD) is a common neurodevelopmental disorder and no effective treatment for the core symptoms is currently available. The present study is part of a larger clinical trial assessing the effects of cannabis oil on autism co-morbidities.Objectives: The aim of the present study was to assess the safety of a CBD-rich oil treatment in children and adolescents with ASD.Methods: Data from 59 children and young adults (ages 5–25 years) from a single-arm, ongoing, prospective, open-label, one center, phase III study was analyzed. Participants received the Nitzan Spectrum® Oil, with cannabis extracts infused in medium chain triglyceride (MCT) oil with a cannabidiol:THC ratio of 20:1, for 6 months. Blood analysis was performed before treatment initiation, and after 3 months. Complete blood count, glucose, urea, creatinine, electrolytes, liver enzymes , bilirubin, lipid profile, TSH, FT4, thyroid antibodies, prolactin, and testosterone measurements were performed at baseline, prior to starting treatment and at study midpoint, after 3 months of treatment.Results: 59 children  were followed for 18 ± 8 weeks (mean ±SD). The mean total daily dose was 7.88 ± 4.24 mg/kg body weight. No clinically significant differences were found in any of the analytes between baseline and 3 months follow up. Lactate dehydrogenase was significantly higher before treatment (505.36 ± 95.1 IU/l) as compared to its level after 3 months of treatment (470.55 ± 84.22 IU/L) (p = 0.003). FT4 was significantly higher after 3 months of treatment (15.54 ± 1.9) as compared to its level before treatment (15.07 ± 1.88) (p = 0.03), as was TSH [(2.34 ± 1.17) and (2.05 ± 1.02)] before and after 3 months of treatment, respectively (p = 0.01). However, all these values were within normal range. A comparison of the group with additional medications (n = 14) to those who received solely medical cannabis (n = 45) showed no difference in biochemical analysis, including liver enzymes, which remained stable, except for change in potassium level which was significantly higher in the group that did not receive additional medications (0.04 ± 0.37) compared to the group receiving concomitant drug therapy (-0.2 ± 0.33) (p = 0.04). A comparison of patients who received a high dose of the cannabis oil (upper quartile-16 patients), with those receiving a low dose (lower quartile—14 patients) showed no significant difference between the two groups, except for the mean change of total protein, which was significantly higher among patients receiving high dose of CBD (0.19 ± 2.74) compared to those receiving a low dose of CBD (1.71 ± 2.46 (p = 0.01), and mean change in number of platelets, that was significantly lower among patients who received high dose of CBD (13.46 ± 31.38) as compared to those who received low dose of CBD (29.64 ± 26.2) (p = 0.0007). However, both of these changes lack clinical significance.Conclusion: CBD-rich cannabis oil (CBD: THC 20:1), appears to have a good safety profile. Long-term monitoring with a larger number of participants is warranted. Autistic Spectrum Disorder (ASD) is a neurodevelopmental disorder that includes a wide range of complex developmental disabilities. The core symptoms of autism include impairments in social interaction and communication, as well as the presence of restricted and repetitive behaviors . DespiteOne of the newer treatment options being investigated for ASD, as well as an array of other conditions, is cannabidiol (CBD)-rich cannabis extract . ConditiCannabis contains a number of active compounds, including Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD) and terpenoids . CBD hasBoth CBD and THC bind to both the CB1/CB2 receptors in the human endocannabinoid system (ECS), with varying levels of affinity .The human endocannabinoid system, composed of endogenous, lipid-based retrograde neurotransmitters that bind to cannabinoid receptors and cannabinoid receptor proteins throughout the central and peripheral nervous system  is oftenThe majority of the available data on the safe use of CBD in children is on the treatment of epilepsy, specifically FDA-approved Epidiolex® . RecentlThe effect of medical cannabis on hormones such as prolactin, thyroid function and testosterone, should be is an under-explored area of study. In one double-blind study of 11 healthy adults, in which CBD or a placebo at doses of 300 mg or 600 mg were administered by injection, basal prolactin and growth hormone levels remained unchanged both after placebo and CBD. We have yet to identify literature which suggests CBD has any adverse effect on kidney function . As a reClinicalTrials.gov (NCT05212493).The aim of this prospective cohort was to assess the safety-related blood tests of children and young adults with ASD taking a CBD-rich cannabis oil-based product. Participants were recruited during the period spanning November 14 2019 to May 25 2021. Data from a baseline sample (pre-treatment) of 59 children and young adults (ages 5–25 years) from an ongoing prospective single-arm, open-label, one center, phase 3 study was analyzed. Eligibility for inclusion in the study included a diagnosis of ASD (DSM-5) by a pediatric neurologist or a pediatric psychiatrist in the community healthcare setting. In addition, patients were required to have at least one severe co-morbidity, such as problems with sleep, aggression/self-injury behaviors, anxiety, or irritability that existed for at least 6 months. Prior to enrollment, treatment and collection of data, written informed consent was obtained from the parents of all participants. Children and adolescents with a known genetic syndrome such as tuberous sclerosis, Fragile X syndrome, and Angelman syndrome were excluded, as were those currently receiving or who had received medical cannabis therapy in the past, had current psychosis, schizophrenia or schizo-affective disorder or had a first degree relative with these disorders. Children diagnosed with a metabolic illness, immune disorder or liver cancer, or epilepsy with clinical symptoms  were also excluded from participating. Ethical approval for the study was obtained from the local Helsinki committee (Number 281–17-ASF) and was registered with The treatment protocol was individualized for each patient using a personalized medicine approach. Participants received medical cannabis extract infused in MCT oil with a CBD:THC ratio of 20:1  for 6 months. Parents were instructed to start with one oral drop daily (each drop contains: 0.3 mg THC and 5.7 mg CBD) and increase the dosage gradually until they perceived improvements in their child’s behavior such as decreased irritability, aggressiveness, hyperactivity, and/or sleep disturbances. The amount and timing of doses during each day was tailored to individual needs of the child . Parents completed a bi-weekly phone interview where they reported compliance, behavior, symptoms, and side effects. The maximum dose did not exceed 10 mg/kg/day  of CBD and 0.5 mg/kg/day  of THC based upon previous findings . InformaMedical interview was performed and weight/height measurements collected. Primary outcome measures included blood analytes which included: complete blood count, glucose, urea, creatinine, electrolytes, liver enzymes (aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl transferase (GGT)), bilirubin, lipid profile, thyroid stimulating hormone (TSH), free T4, thyroid antibodies, prolactin, and testosterone. CBC was measured in whole blood and all other biochemistry parameters were measured in the serum using Cobas® 8000 analyzer (Roche Diagnostics). Blood tests were performed in the morning, at baseline, prior to starting treatment and at study midpoint, after 3 months of treatment. Secondary outcomes were change in safety tests both with varying doses of CBD as well as with concomitant drug usage.Baseline–All participants underwent a medical history and physical exam, completed questionnaires and underwent blood testing, growth measurements, EEG, Autism Diagnostic Observation Schedule (ADOS), Vineland adaptive behavior scales, and cognitive tests.Follow up—Biweekly during follow-up interviews by telephone were conducted throughout the study. At midpoint (3 months), an in person interview was conducted and blood work and growth indices were taken. After 6 months, growth measurements, ADOS, EEG, Cognitive and Vineland adaptive behavior scales were completed.t-test. Unpaired t-test was utilized in two secondary analyses. The first analysis examined the differences before and after CBD treatment in patients treated with co-medications versus patients who were not exposed to other medications; the second analysis compared the differences before and after treatment in patients exposed to high CBD dosage  versus patients exposed to low CBD dosage . Data was analyzed with “PANDAS” and “Scipy“ packages in python via the platform of Jupyter notebook. The significance levels were set at 0.05. were followed for 18 ± 8 weeks (mean ±SD).Baseline patient characteristics are presented in The average doses in the morning, noon, evening and the daily total doses were as follows: Morning dose mean—4.04 ± 1.71 drops body weight; Noon mean—2.02 ± 2.28; Evening mean—3.03 ± 1.84 and Total day—7.88 ± 4.24 drops body weight ).p = 0.5) however those values are all within the normal range.No clinical or statistically significant differences were found in any of the analytes between baseline and at 3 months follow up. No significant change was observed in complete blood count, including hemoglobin, red blood cells or leucocytes, before and after 3 months of treatment. Platelet levels were higher before treatment (1.63 ± 46.75) compared to after treatment (8.08 ± 26.68) (p = 0.003). Upon further examination of thyroid function measures, it appears that TSH values were tending to decrease while FT4 tended to increase. However, the changes in both measures remained within normal ranges. FT4 was significantly higher after 3 months of treatment (15.54 ± 1.9) as compared to its level before treatment (15.07 ± 1.88) (p = 0.03). TSH was higher before treatment (2.34 ± 1.17) as compared to the level after 3 months of treatment (2.05 ± 1.02) (p = 0.01).No statistically significant difference was observed in urea or creatinine before or after 3 months of treatment. No statistically significant difference was observed in liver enzymes , thyroid hormones, thyroid antibodies, prolactin, or other hormones . All of n = 14, Methylphenidate-4, Aripiprazole- 4, Melatonin—3, Risperdal 1, Roxetine-1, Seroquel-1), and those who received solely medical cannabis (n = 45) showed no difference in biochemical analysis, including liver enzymes, which remained stable, except of change in potassium level, which was significantly higher before treatment (0.04 ± 0.37) as compared to after 3 months of treatment (-0.2 ± 0.33) (p = 0.04). (p = 0.01), and the mean change in number of platelets that was significantly lower among patients who received high dose of CBD (13.46 ± 31.38) as compared to those who received low dose of CBD (29.64 ± 26.2) (p = 0.0007) . . We also 0.0007) . The meaThis study may support the long-term safety of medical cannabis use in children and young adults with ASD. Over the first 3 months of the study, a few statistically significant changes were observed including the level of LDH and thyroid hormones, however, these changes have no clinical significance. The remaining tests which assessed hematological, chemical, and endocrine function, were all within the normal range. In addition, no differences in safety test results were identified between children taking or not taking anti-psychotic medications together with CBD-rich cannabis oil, nor between those receiving high or low dose of medical cannabis.The recent literature presents a favorable safety profile of CBD in human trials. The majority of the available data comes from research on Epidiolex®, a 100% CBD product, in patients with epilepsy. A 2018 meta-analysis of four randomized controlled trials with patients with Lennox-Gastaut or Dravet syndrome showed that Epidiolex® was associated with a higher rate of increased serum aminotransferases compared to placebo . Thiele In a 2020 study on adults given 1500 mg of CBD, in the first 2–4 weeks after initiating treatment, seven (44%) participants experienced peak serum ALT values greater than the upper limit of normal. For five (31%) participants, the value exceeded five times the upper limit of normal, meeting international criteria for drug-induced liver injury  . InteresThere was no effect on liver function when cannabis was given with other medications, as was reported in previous studies on Epidiolex®. A possible explanation may be that our participants received a lower dosage of phytocannaboids and the medications they received did not have the same effect as was seen with Valporal in the Epidiolex® study. Although a significant change in the TSH, FT4, and LDH was observed, they were all within normal range.Several studies have reported on the pharmacokinetics and pharmacodynamics of cannabinoids. Two systematic reviews  reportedAlthough our study was open-label with no placebo group, the findings support the biochemical safety of the preparation used in the study. In order to further evaluate the safety of the preparation used, a comparison was made between patients who were at the upper quartile of cannabis dosage to patients who were at the lower quartile, and no clinical significant difference was observed .We also compared biochemistry analysis between patients who received other medications such as risperidone, methylphenidate, melatonin to those who received medical cannabis only , and thep = 0.01), and the mean change in number of platelets that was significantly lower among patients who received high dose of CBD (13.46 ± 31.38) as compared to those who received low dose of CBD (29.64 ± 26.2) (p = 0.0007) (A comparison of patients who received a high dose of the cannabis oil (upper quartile-16 patients receiving CBD 3.49—6.53 mg/kg body weight), with those receiving low dose (lower quartile—14 patients receiving CBD 0.7–1/- mg/kg body weight) showed no significant differences between these two groups with regard to biochemical analysis, except of the mean change of mean total protein, which was significantly higher among patients with high dose of CBD (0.19 ± 2.74) as compared to those with low dose of CBD (1.71 ± 2.46 ( 0.0007) . HoweverIt is possible, however, that due to the small number of patients receiving concomitant medications, no difference was observed.A theoretical limitation of our study might be the open-label method, however, the biochemistry parameters examined in our study are objective numbers which are not dependent on a double-blind study.CBD-rich cannabis oil (CBD: THC 20:1), as part of a monitored treatment program over 3 months, appears to have a good safety profile. Long-term monitoring with larger number of participants is needed.


Radiotherapy is an important modality in lung cancer treatment. Despite advances in treatment planning and dose delivery, patient benefit is still limited by in-field relapse and metastatic recurrence. Simultaneous application of cisplatinum-based chemotherapy leads to moderately improved outcomes, thus providing proof-of-concept for radiosensitization strategies in lung cancer. In an unbiased functional genetic screen for radiosensitization targets in lung cancer, we identified syntaxin 18, a protein involved in retrograde vesicular transport between the Golgi apparatus and endoplasmic reticulum, as mediator of radioresistance. Downregulation of endogenous syntaxin 18 specifically reduced clonogenic survival of radioresistant and radiosensitive lung cancer cells following X-radiation. Gene expression programs regulating DNA repair, mitotic checkpoints and mitosis were altered in isogenic cells with reduced syntaxin 18 expression. Functionally, this translated into impaired DNA damage-induced cell cycle checkpoints leading to cell death by mitotic catastrophe. Interestingly, downregulation of syntaxin 18 in lung cancer cells also impaired expression of markers of epithelial-mesenchymal-transition, and reduced migration and invasion capacity. These findings suggest that syntaxin 18 is a key player regulating genes responsible for controlling the growth of the primary tumor as well as metastases upon radiotherapy of lung cancer. They provide a promising lead for biologically rational radiosensitization strategies impacting on radiation-induced cell death as well as metastasis. Lung cancer is the leading cause of cancer-related deaths worldwide and 85% of patients are diagnosed with non-small-cell lung cancer (NSCLC). Of these, 70% are diagnosed with advanced or metastatic disease. Curative treatment options for patients with locally advanced NSCLC (WHO stage III) include radiotherapy, chemotherapy and surgery. While meta-analyses have confirmed that concurrent chemoradiotherapy produces superior survival outcomes , the radTo identify candidates for modulation of radiosensitivity in NSCLC, we used A549 human lung cancer cells that were lentivirally transduced to express a barcoded-shRNA library targeting more than 5000 genes enriched in signaling pathway targets Fig. . After XTo validate STX18 as a modulator of radiosensitivity, A549 and H460 cells were stably transduced with a shRNA targeting STX18. In three independent single cell clones, STX18 mRNA expression was significantly decreased  Fig. . ReducedTo identify possible targets of STX18, RNA profiling of A549 cells with and without STX18 knockdown was performed before and after irradiation with 10 Gy. Using gene set enrichment analysis (GSEA), we identified genes and pathways differently expressed between the control cell lines and two clonal cell lines. Comparison of A549 cells transfected with a control shRNA (A549-shScr) and A549-shSTX18 (K2 and K3) cells showed a significant enrichment of gene sets including DNA repair, epithelial-mesenchymal transition (EMT) and transforming growth factor β (TGFβ). All these gene sets were more highly expressed in the control cell line Fig. , howeverTo validate the involvement of STX18 in regulating cell cycle checkpoint associated proteins, expression and activity of ATM and ataxia telangiectasia and rad3 related (ATR) as well as their downstream targets checkpoint kinase (Chk) 2 and 1, respectively, were analyzed in A549 cells after irradiation with 10 Gy Fig.  while H4As indicated above, RNA profiling of A549 cells with high or low levels of STX18 and subsequent GSEA highlighted an enrichment of genes involved in EMT and TGFβ signaling in the former Fig. . To valiRadiotherapy represents a mainstay for treating NSCLC. In the last decades, significant technological progress in stereotactic body radiation therapy has led to improved targeting of the tumor, while sparing the surrounding healthy tissue. Thus, radiotherapy is now widely used for the ablation of NSCLC primary tumors in patients who are not suitable for surgery , 9. In lIt is well defined that after DNA damage, ATM/Chk2 and ATR/Chk1 stabilize p53 by phosphorylation, increasing its activity as transcriptional regulator and leading to cell cycle arrest in the G1 phase. This cell cycle checkpoint is absent in cells lacking p53 function , 18. SevIn addition to linking STX18 and the radiation response via major determinants of cell cycle regulation, RNA profiling of A549 cells also revealed that STX18 levels affect TGFβ-induced pathways and EMT. We showed that STX18 expression promoted a mesenchymal phenotype, with elevated expression of mesenchymal-associated markers in addition to increased migration and invasion abilities. This is in line with findings of others demonstrating the involvement of syntaxin 1 in glioblastoma cell invasion, while expression of syntaxin 6 expression was associated with increased migration of esophageal cancer cell , 26. It Altogether, our findings highlight a new link between vesicular transport, radioresistance and metastatic progression, which constitute two hallmarks of failure of radiotherapy in locally advanced NSCLC. The effects observed from cells with different levels of STX18 might hint at novel strategy to exploit cell cycle checkpoint deficiency for enhancing the efficacy of radiotherapy in NSCLC and simultaneously suppressing its metastatic potential.g for 5 min if not stated otherwise.All cell lines were purchased from ATCC  and authenticated by STR analysis. The absence of mycoplasma was tested regularly by PCR. A549 and NCI-H460 cells were cultured in RPMI 1640 ; HEK293FT and Phoenix-Eco cells were cultured in DMEM (Gibco). All cell media were supplemented with 10% FBS . All centrifuging steps were performed at 300 × Irradiation was performed using a RS320 X-ray irradiator  kindly provided by the working group of Prof. Stuschke . Cells were irradiated at 300 kV and 10 mA.FuGENE  was used to transfect cells at a ratio of 2:1 (ratio FuGENE to DNA), according to the manufacturer’s protocol. Lentiviral and retroviral particles were generated in HEK293FT and Phoenix-Eco cells, respectively. For transduction, 30,000 cells were seeded in six-well plates and incubated for 24 h with viral particles supplemented with 1 µg/mL polybrene  to enhance transduction efficiency. Cells were then selected with 1 µg/mL puromycin , or 800 µg/mL neomycin  for 7 days. Single cell clones were generated using limiting dilution. Lentiviral STX18 shRNA and scrambled controls were obtained from Sigma. For reintroduction of STX18, an empty vector (Mscv.PG.Neo) and a plasmid carrying STX18 (Mscv.STX18) were used. For sequences of shRNA, see Supplementary Table The DECIPHER shRNA Library Human module 1 , consisting of 27,500 pooled shRNA targeting 5043 signaling pathway genes was used to perform a screen to compare the shRNA distribution prior to and after irradiation of A549 cells. Briefly, HEK293FT were transfected with the shRNA library as described previously [RNA was isolated using High Pure RNA isolation Kit (Roche) and 1 µg of RNA was reverse transcribed  according to the manufacturer’s protocol. Gene expression was quantified by using SYBR Green as a fluorescent probe and primers specified in Supplementary Table RNA was isolated from cells using RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. 3’UTR mRNA-Seq library preparation and analysis were performed on an Illumina HiSeq2500. Transcript level quantification was performed using salmon v.0.14.1  against https://fiji.sc/ (Accessed on 20. January 2022)). Bands were normalized to respective loading controls and show the mean of three independent experiments. Means, standard deviation (SD) and statistical analysis are included in Supplementary Table Proteins were isolated using a 1% NP40  buffer containing cOmplete protease inhibitor cocktail  and phosphatase inhibitors cocktails 2 and 3 . 30 µg of proteins were loaded on a 4–15% gradient gel  and subsequently transferred onto a nitrocellulose membrane. Membranes were blocked, and antibodies diluted in 5% BSA  Tris-buffered saline with Tween-20. If not stated otherwise, all antibodies were diluted 1:1000. The following antibodies were used for immunoblotting: beta-Actin , STX18, p53 , Frizzled 5, E-cadherin, ZO-1, Vimentin, Zeb1 , Vinculin , phospho-ATR, ATR, phospho-Chk1, Chk1, phospho-ATM, ATM, phospho-Chk2, Chk2 , ATR . Secondary antibodies were HRP-conjugated . Western Blot densitometry was performed using the Fiji software -2,5-diphenyl-2H- tetrazolium bromide . 2000–4000 cells were seeded in a 96-well plate. After the indicated timepoint, 0.5 mg/mL MTT was added to the wells, and 4 h later, solubilization solution was added  and the plate was incubated overnight at 37 °C. The absorbance was then measured at 595 nm in a plate reader .To analyze the effect of irradiation on the cell cycle distribution, 40,000 cells were seeded in six-well plates. After adherence, cells were irradiated and 72 h later, cells were collected and incubated with Propidium/Iodide (PI) in the dark at 4 °C for at least 30 min. Cells were analyzed using a FACS Celesta .For determining levels of apoptosis upon irradiation, 40000 cells were seeded in six-well plates and collected 72 h after irradiation. For analysis after treatment, cells were pre-treated with 0.25 or 1 µM Berzosertib  or 0.25 or 1 µM Chir-124  for 2 h before irradiation with 6 or 10 Gy, and collected after 72 h. Staining was accomplished using Annexin V/PI  according to the manufacturer’s protocol. Cells were analyzed using a FACS Celesta (BD BioSciences).For assessment of anoikis, plates were first coated with poly(2-hydroxyethyl methacrylate)  and incubated overnight at room temperature. Seventy-two hours after seeding, cells were stained and measured as described.To determine survival upon irradiation, 150–10,000 cells were seeded in six-well plates. Adherent cells were irradiated with doses as indicated. After 10 days, colonies were fixed with 70% ethanol and stained with Coomassie brilliant blue. Colonies formed by more than 50 cells were counted. Plating efficiency and surviving fraction were calculated as follows: plating efficiency= number of colonies formed/number of cells seeded (untreated cells); surviving fraction = (number of colonies formed)/(number of cells seeded) × plating efficiency.Cells were harvested after irradiation and fixed in 70% ethanol at −20 °C overnight. After fixation, cells were resuspended in 0.25% Triton X100 in phosphate-buffered saline-Tween (PBS-T), prior to blocking in 1% BSA in PBS-T. Cells were then incubated 90 min with the H3pS10 antibody . After incubation with the secondary antibody coupled to Alexa Fluor 488 , PI (4 µg/ml) and RNAse (0.60 µg/mL) in PBS were added. Samples were analyzed with a flow cytometer  and the fraction of mitotic cells was determined by normalizing the number of H3pS10 and PI-positive cells to the total number of cells.To identify fragmented nuclei upon irradiation, 90,000 cells were seeded and irradiated the next day. Seventy hours later, cells were treated with 0.1 µg/mL colcemid (Gibco) for 2 h. Cells were then collected, centrifuged, and cells were incubated 5 min in 5 mL hypotonic solution (75 mM potassium chloride). After centrifugation, cells were resuspended in fixative solution (methanol and acetic acid mixed 3:1) and incubated overnight at 4 °C. After centrifugation (7 min), cells were washed twice in fixative solution, and resuspended in 2 drops of fixative solution. Samples were spread on each slide and left to dry overnight at 4 °C. Slides were then stained with Giemsa (Roth)  for 12 min, and rinsed with water. Picture recording and subsequent quantification were achieved using the Axio Scan.Z1 microscope . Abnormal nuclei were defined by fragmentation, aggregation, or formation of micronuclei. At least 1000 cells per sample were evaluated.To quantify DNA damage upon irradiation, 25,000 cells were seeded on 8-chamber slides . After fixation and permeabilization with 3% PFA, 0.5% Triton X-100 and 8% Sucrose in PBS, cells were blocked with 5% FBS, 5% NGS, 0.2% Triton X-100 in PBS. Next, samples were incubated with the γH2A.X antibody  for 1 h at room temperature followed by overnight at 4 °C. After washing and incubation with the secondary antibody , cells were stained with a DAPI/Hoechst33342 mix . Pictures were recorded using an Axio observer fluorescence microscope  and analyzed with the Focinator 2.0 software, which corrects for the area of the nucleus and allows for quick and semi-automated counting of γH2A.X in at least 50 cells .130,000 cells were seeded in a six-well plate the day before treatment. The following day, cells were treated with 5 Gy. After the desired timepoint, cells were collected and 1 200 cells were spread on the slide with low-melting point agarose . Slides were processed according to the manufacturer’s protocol. After drying, slides were stained in SYBR Green (Thermo Fisher Scientific) solution 1:5000 in water for 30 min at room temperature in the dark. Pictures were taken with an EVOS M5000 microscope (Thermo Fisher Scientific) and comet tail lengths were analyzed using ImageJ 1.51j8 . Comet tMigration/invasion assay was performed using the BioCoat© GFR Matrigel  plates. After overnight starvation in a medium containing reduced serum levels (0.5%), 40,000 cells were seeded in starvation medium on top of the inserts, which were placed in a 24-well plate filled with 10% FBS medium. After 24 h, cells that did not migrate or invade were removed, and migrated/invaded cells were fixed with 70% ethanol and stained with crystal violet . Pictures of migrating and invading cells were recorded using a Primovert microscope .p > 0.05. When significant results are shown, p-values are indicated as following: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns no statistical significance.On graphs, means ± SD are shown. Significance was estimated for experiments biologically repeated at least three times using GraphPad Prism 6. For comparison of groups involving one variable, one-way ANOVA was used with post hoc test. For comparison of groups involving two variables, two-way ANOVA was used. Both tests were used with Dunnett, Tukey or Sidak post-hoc tests, when applicable. If not stated differently, results can be considered not significant Supplementary FiguresSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Uncropped Western BlotsDataset 1Author ChecklistAuthor contributions


Methyl gallate is a polyphenolic compound found in many plants, and its antioxidant, antitumor, antibacterial, and anti-inflammatory effects have been extensively studied. More recently, antidepressant-like effects of methyl gallate have been demonstrated in some studies. In the present study, we examined the effects of methyl gallate on melanogenesis, including the tyrosinase inhibitory effect, the melanin content, and the molecular signaling pathways involved in this inhibition. The results showed that methyl gallate inhibited tyrosinase activity and significantly downregulated the expressions of melanin synthesis-associated proteins, including microphthalmia-associated transcription factor (MITF), tyrosinase, dopachrome tautomerase (Dct), and tyrosinase-related protein-1 (TRP1). In conclusion, our findings indicated that activation of MEK/ERK and PI3K/Akt promoted by methyl gallate caused downregulation of MITF and triggered its downstream signaling pathway, thereby inhibiting the production of melanin. In summary, methyl gallate showed significant inhibitory activity against melanin formation, implying that it may be a potential ingredient for application in skin-whitening cosmetics. Melanocytes are responsible for the production of melanin, which contributes to the colored pigmentation in the skin and hair. In the epidermis of the skin, ultraviolet irradiation promotes melanocytes to generate melanin, and the dispersal of melanin via melanosomes to keratinocytes protects the human skin from extensive sunburn. Nonetheless, aberrant regulation of melanogenesis leads to pigmentation disorders such as melasma, hyperpigmentation, age spots, and blemishes –3. Severβ [Melanogenesis is a tyrosinase-initiated pigmentation process that converts L-3,4-dihydroxyphenylalanine to dopaquinone, followed by oxidation to form melanin . The patβ , 13.β- and cAMP-dependent signaling in melanogenesis regulation [MITF is a key factor in determining PKC-gulation , 15. Morgulation , 17, whigulation , 19.In melanocyte cells, the mitogen-activated protein kinase (MAPK) family, including p38MAPK and extracellular signal-regulated kinase (ERK), is mostly involved in regulating MITF expression , 21. In in vitro cell-culture model, this study aims to identify the antimelanogenesis mechanism of methyl gallate in B16F10 melanocyte cells.Methyl gallate is a gallic acid derivative and has been shown in previous studies to possess several biological properties, such as antioxidant , anti-inβ-actin antibody were purchased from Sigma-Aldrich . PVDF (polyvinylidene difluoride) membranes were obtained from Millipore . Antibodies against ERK, p-ERK, TRP1, and Dct were obtained from ProteinTech group . Antibodies against tyrosinase, MITF, p-MITF, MC1R, RSK1, p-RSK1 CREB, and p-CREB were purchased from Epitomics . Antibodies against PI3K, p-PI3K, AKT, p-AKT, p38MAPK, and p-p38MAPK were purchased from Cell Signaling Technology .Methyl gallate, kojic acid, arbutin, protease inhibitor cocktail, MTT, 3,4-dihydroxyphenylalanine (L-DOPA), LY294002, PD98059, and goat antirabbit μg/mL streptomycin at 37°C with 5% CO2 atmosphere. Methyl gallate dissolved in DMSO and further diluted with DMEM to achieve the indicated final concentrations . Cells were cultured with different concentrations of methyl gallate and harvested after 24 h of incubation. Experiments were performed in triplicate and repeated multiple times.B16F10 mouse melanoma cells were purchased from the Taiwan Food Industry Research and Development Institute . The cells were cultured and maintained in Dulbecco's modified Eagle's medium  contained with 10% FBS, 100 U/mL penicillin, and 100 4 cells/well in 96 well plates. After treatment with various concentrations of methyl gallate, the cells were incubated at 37°C for 24 h. The cell viability was determined using 3--2,5-diphenyl tetrazolium bromide (MTT) solution  and then added to each well, and cells were incubated at 37°C for 4 h. After the removal of the MTT solution, 100 μl DMSO was added to the well and incubated for 10 min. Absorbance was determined at 595 nm using a microtiter plate ELISA reader.MTT cell viability assay was assessed to determine the viability of methyl gallate against B16F10 cells. Cells were seeded at 1 × 105 cells/well) were incubated in 24 well plates. In brief, B16F10 cells were treated with methyl gallate for 24 h. The cells were washed with PBS and then dissolved in 200 μl of 1N NaOH at 80°C for 2 h. The samples were centrifuged for 30 min at 12,000 rpm to collect the supernatant. The relative melanin content was determined by measuring the absorbance at 475 nm on a microtiter plate ELISA reader. The melanin content was determined using standard curves using synthetic melanin solutions (0∼200 μg/ml). Melanin production was calculated as the μg of melanin/μg of total proteins in a cell extract.B16F10 cells (1 × 106 cells/well) were incubated in 24 well plates. After treatment with different concentrations of methyl gallate  for 24 h, cells were washed with PBS and lysed with RIPA buffer containing protease inhibitor cocktail. The cell lysates were then centrifuged at 12,000 rpm for 15 min to collect the supernatant. The sodium phosphate buffer  reacted with an equal volume of 1 mg/ml L-DOPA. After incubation at 37°C for 2 h. Absorbance was then measured at 405 nm in an ELISA reader. Each measured result was expressed as the percentage change from the control [Tyrosinase activity was estimated as L-DOPA oxidase activity. B16F10 cells  for 30 min, followed by incubation in the same buffer containing 2 mM L-DOPA at 37°C.The tyrosinase activity staining was analyzed according to a previously described method . B16F10 μg) extracted from whole cells were loaded onto and separated by 12.5% SDS–PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). The membranes were blocked with PBST buffer (PBS buffer containing 0.05% Tween 20) containing 0.2% gelatin for 2 h at 4°C. Thereafter, the membranes were incubated with appropriate rabbit polyclonal antibodies against mouse tyrosinase, TRP-1, Dct, MC1R, MITF, p-MITF, CREB, p-CREB, p38MAPK, p-p38MAPK, ERK, p-ERK, MEK, p-MEK, PI3K, p-PI3K, AKT, p-AKT, RSK1, and p-RSK1 for overnight at 4°C. The membranes were washed three times in PBST and then probed with goat antirabbit horseradish peroxidase-conjugated antibody  for 1 h. All bands were visualized using ECL western blotting reagents (pierce). The western blot data were quantified with Image J 1.47 software (https://www.downloadcrew.com/article/28008-imagej).After treatment, B16F10 cells were lysed with RIPA buffer containing protease inhibitor cocktail. Equal protein amounts . Results with p < 0.05 were considered statistically significant.The results of the MTT assay were subjected by Student's μM, and the cell viability was measured by MTT assay. We also used kojic acid (200 μM) and arbutin (2 mM) as positive controls to compare the survival rates of B16F10 cells. The experimental results showed that the survival rate of the B16F10 cells was approximately 90% when 200 μM methyl gallate was added and approximately 80% when 600 μM methyl gallate was added and with 200 μM kojic acid and 2 mM arbutin separately. The cell viability was approximately 80% in each experiment , tyrosinase activity staining analysis showed that with the increase of methyl gallate concentration, the inhibition of tyrosine activity was greater  for treatment. After 24 hours of reaction, western blotting analysis was used to determine differences in protein expressions. From the experimental results, it was observed that the expressions of melanin-related proteins, such as MC1R, were decreased with increasing methyl gallate concentrations; decreases in the expressions of MITF, phosphorylated CREB, tyrosinase, TRP-1, and Dct were also observed with increasing methyl gallate concentrations , the activity of tyrosinase and the content of melanin in the cells were studied. It was found that the activity of tyrosinase and the melanin content decreased significantly by approximately 50% after methyl gallate treatment in B16F10 melanocytes . To confμM methyl gallate had no significant cytotoxicity against B16F10 melanocytes. In addition, methyl gallate treatment caused significant dose-dependent inhibition of intracellular tyrosinase and melanin contents  are used in the treatment or prevention of abnormal skin pigmentation [μM methyl gallate was higher than that with the treatment with 200 μM kojic acid and 2 mM arbutin, and the inhibition of melanin by 200 μM methyl gallate was better than that of 200 μM kojic acid and 2 mM arbutin. Methyl gallate is a potent compound that inhibits melanin and was proven to be more effective than kojic acid and arbutin in cell experiments.Methyl gallate at 200 entation , 30, TheIn addition to the cAMP regulatory pathway as the major signal transduction pathway, activation of MEK/ERK and PI3K/Akt signaling pathways  regulates melanin synthesis , 31. Theβ (GSK3β) [Study results have indicated that the MEK/ERK pathway regulates the phosphorylation of MITF. When the MEK/ERK pathway is activated, it will promote the phosphorylation of MITF, followed by ubiquitination, and degradation. The downstream expressions of melanin-related proteins such as tyrosinase, TRP1, and Dct are decreased, reducing melanin synthesis , 34. Act (GSK3β)  have beep-CREB expression decreased after treatment with methyl gallate, and p-MITF protein fragmentation increased with increasing concentrations of methyl gallate, as did the expressions of p-MEK and p-ERK. The protein expression levels of p-RSK1 and p-AKT increased significantly. It was speculated that methyl gallate may activate p-MITF via the MEK/ERK and PI3K/Akt signaling pathways to down-regulate tyrosinase, TRP-1, and DCT in B16F10 melanocytes [The phosphorylated active CREB further binds MITF, which in turn stimulates the transcription of the key melanogenic enzymes . CREB phanocytes .Previous studies have demonstrated that melanogenesis is mediated by the regulation of MITF activation via phosphorylation of p38 MAPK . ActivatWe used a MEK/ERK inhibitor (PD98059) and a PI3K/Akt inhibitor (LY294002) to verify whether methyl gallate activates the MEK/ERK and PI3K/Akt signaling pathways, which in turn affects tyrosinase activity and melanin synthesis and decreases the expressions of proteins involved in melanin synthesis. First, we added a MEK/ERK inhibitor (PD98059) and a PI3K/Akt inhibitor (LY294002) and recorded the activity of tyrosinase and the content of melanin. Then, we used immunostaining analysis to verify the melanin synthesis pathways of MEK/ERK and PI3K/Akt. The results suggested that methyl gallate does activate the MEK/ERK and PI3K/Akt signaling pathways, thereby reducing the expressions of tyrosinase and melanin-related proteins and inhibiting the synthesis of melanin .α-MSH-stimulated B16F10 cells [Nerya et al. isolated -Shogaol 10 cells .β (GSK3β) [The previous study showed that methyl gallate decreased melanin pigmentation in a concentration-dependent manner but did not directly inhibit tyrosinase activity. Further analysis showed that methyl gallate had no effect on extracellular signal-regulated kinase (ERK) activation but induced phosphorylation of glycogen synthase kinase 3 (GSK3β) . The resWe demonstrate that methyl gallate suppresses the tyrosinase activity in B16F10 cells and reveal the molecular mechanism involved in melanogenesis. Our data suggest that methyl gallate may regulate melanogenesis by restraining the production of MITF, tyrosinase, TRP1, and Dct, and the process is associated with the phosphorylation of PI3K/Akt or MEK/ERK. According to our results, methyl gallate reduces melanin synthesis and could be a useful agent for skin whitening; it may also have the potential for application in cosmeceuticals in the future.


Longitudinal bone growth, achieved through endochondral ossification, is accomplished by a cartilaginous structure, the physis or growth plate, comprised of morphologically distinct zones related to chondrocyte function: resting, proliferating and hypertrophic zones. The resting zone is a stem cell-rich region that gives rise to the growth plate, and exhibits regenerative capabilities in response to injury. We discovered a FoxA2+group of long-term skeletal stem cells, situated at the top of resting zone, adjacent the secondary ossification center, distinct from the previously characterized PTHrP+ stem cells. Compared to PTHrP+ cells, FoxA2+ cells exhibit higher clonogenicity and longevity. FoxA2+ cells exhibit dual osteo-chondro-progenitor activity during early postnatal development (P0-P28) and chondrogenic potential beyond P28. When the growth plate is injured, FoxA2+ cells expand in response to trauma, and produce physeal cartilage for growth plate tissue regeneration. The growth plate cartilage supports long bone growth. Here the authors identify FoxA2+ long term stem cells in the growth plate that are stratified with short term PTHrP+ cells, participate in production of hyaline cartilage, expand in response to trauma, and whose ablation impairs cartilage regeneration. Physeal injuries are a major cause of skeletal morbidity in growing children, manifesting as angular or shortened limb deformities, due to partial or complete arrest of GP function2. Clinically, GP injuries are classified according to the Salter–Harris (SH) system, predicated on fracture morphology involving the physis ± epiphysis and/or metaphysis: SH1) physeal shear separation; SH2) physis + metaphysis; SH3) physis + epiphysis; SH 4) physis + epiphysis + metaphysis; SH 5) crush injury to physis. SH1 fractures have the best prognosis, healing without disrupted growth. The cleavage plane is primarily through the HZ, structurally the weakest zone comprising the GP, owing to lack of calcification and limited collagenous matrix. Stiffened by a collagen matrix, the adjacent RZ and PZ are unaffected4. In contrast, SH 3–5 injuries disturb all zones, resulting in disturbed bone growth4. These clinical scenarios suggest that involvement of the RZ affects the ability of the GP to regenerate in response to injury. In rabbits, even when the PZ and HZ from ulnar GP were excised, the restoration of the GP was still possible, as long as the RZ remained intact6. As the name implies, the “resting zone” houses a quiescent progenitor cell population1. Several studies have identified in mouse7, rat8, and rabbit9 slow-cycling cells present in the RZ immediately adjacent to the secondary ossification center (SOC). In pulse-chase experiments, animals were administered [3H] thymidine or 5-bromo-2-deoxyuridine (BrdU) for a prolonged period sufficient to allow slow-cycling stem cells to complete one cycle. At the end of the chase, the label diminishes in proliferating cells, but remains undiluted in slow-cycling cells. While these reports demonstrate the presence of slow-cycling, potential progenitor cells at the RZ, their genetic signature remains undefined, as there is no biomarker to isolate and characterize these cells.Longitudinal bone growth is achieved by endochondral ossification, implemented by the physis or growth plate (GP), a cartilaginous structure located between the epiphysis and metaphysis at the ends of long bones forming the appendicular skeleton. Human GP is comprised of five morphologically distinct zones: resting (RZ), proliferation (PZ), hypertrophy (HZ), calcification (CZ), and ossification (OZ)col2CRE;ERT;R26-Confetti mouse line and functional perturbations, established that formation of the SOC triggered GP chondroprogenitors to undergo a radical shift in clonality and to acquire self-renewal capabilities10. However, since “Confetti” labeling is based on collagen type II expression, an uber-marker for all GP cells, there was no unique biomarker to isolate and/or characterize individual stem cell subgroups. Next, Mizuhashi et al. employed a PTHrPCre.ERT;Tomatof/+ mouse strain to reveal that PTHrP marked a subset of stem cells located at the bottom of the RZ11. Labeling, tracing and isolating RZ cells based on their PTHrP expression both in vivo and in vitro, demonstrated that a subgroup PTHrP+(positive) stem cell evolved predominantly into columnar chondrocytes11. Together with another yet unidentified PTHrP−(negative) subgroup located at the top of the RZ, adjacent to the SOC, PTHrP+ cells, located primarily at the bottom of the RZ, contributed to long-term GP growth11. In the present work, we prove that a population of FoxA2+col10−cells, located at the top of the RZ, are the PTHrP-(negative) stem cell population, and represent a subgroup of long-term skeletal stem cells (LTSSC) capable of dual osteo-chondro-progenitor activity during early postnatal development (P0-P28) and chondrogenic potential beyond P28, fundamental to GP turnover and regeneration following injury.Recently Newton et al., using clonal genetic tracing with a 12. FoxA1-3 are highly expressed in the hypertrophic zone (HZ) of newborn mice GP  are col.1+expressing bone cells   Fig. .AggrecanCreERT/+;Tomatofl/+ mice  dye, which labels efficiently the bone matrix but not the cartilage matrix in FoxA2CreERT/+;Tomatofl/+ mice. Tamoxifen pulses at P3-4 (during SOC prefiguration), at P7-8 (SOC hypertrophy), and P13-14 (SOC enlargement) to FoxA2CreERT/+;Tomatofl/+ mice gave rise to Tomato+ labeled cells which, 3 months later, were still present in the RZ either as single cells, clusters or columns of progeny  still persist in the GP cartilage 3 months later, we performed a long-term pulse chase of the eny Fig. , or becaeny Fig. . Altoget11, and that FoxA2+ cells are located in close proximity to the SOC, which is viewed as a signaling center of RZ stemness10, we asked whether FoxA2+ cells are different than the previously characterized PTHrP+ stem cell subset11. Using a PTHrPmcherry mouse line, Mizuhashi et al. have recently shown that PTHrP+ cells are restricted to the bottom of the RZ, and they are dedicated, at least to some degree, to giving rise to columns of progeny11. Here we show that FoxA2+ cells are located at the top of the RZ and they are geographically separated from the previously characterized PTHrP+ cells11, in both tibia and ulna of FoxA2CreERT/+;ZsGreenfl/+;PTHrPmcherry mice  cells, the PTHrP+(mCherry+) cells and the double positive (DP) cells. Consistent with the histological data, we observed no overlap between FoxA2+ cells and PTHrP+ cells  and low levels of bone markers  when compared with bone marrow mesenchymal cells (BMSC) , with that of PTHrP+ cells (isolated from PTHrPmcherry mice)  assay, RZ FoxA2+col.10-(green) cells formed distinct colonies  Fig. . At platce) Fig. . FoxA2+cce) Fig. . To inquce) Fig. . AltogetFoxA2CreERT/+;Tomatofl/+ mice, labeled 7.92 ± 0.9 FoxA2+ cells, which is 8% from the total number of FoxA2+ cells detected by immunohistochemistry at P14 (97.67 ± 9 cells)  labeled 22.6 ± 4 FoxA2+ cells, increasing the labeling efficiency relative to the number of FoxA2+ cells identified via immunohistochemistry, from 8 to 23% , labeled 36.9 ± 2.3 FoxA2+ cells, increasing the efficiency of labeling from 23%  to 38%    over time. Two tamoxifen injections, administered P14-P15 to le) Fig. .Fig. 4DuFoxA2CreERT2/+;Tomatofl/+ mice were pulsed with tamoxifen P14-P18 and chased for 1 day or 1, 3, 9 months we observed increasing numbers of doublets, clusters, and columns of progeny , but drops to 31% by 9 months, when FoxA2+ column formation is high , but this number decreases significantly (to 41 FoxA2+ clones/hindlimb) by 3 months after the initial labeling  the number of FoxA2+ clones is not significantly changed between 1-day (55 FoxA2+clones/hindlimb) and 3 months (57 FoxA2+ clones/hindlimb) after the initial labeling , with the clonality of FoxA2+ cells, labeled during SOC osteogenic maturation (P28-P37). Quantification of the percentage of FoxA2+ clones  to the total number of Tomato+units, shows a similar dynamic. Both FoxA2+ cells labeled at a younger age (tamoxifen P14-18), or at an older age (tamoxifen P28-37), exhibit the same LTSSC characteristics in vivo, as shown by an increased percentage of FoxA2+ columns over time Fig. . Howevering Fig. . In conting Fig. . These rFoxA2CreERT2/+;Tomatofl/+ mice treated with tamoxifen P14 –P18, and harvested at P19, a 1-day chase reveals that 137 cells of the FoxA2+ cells are in the GP and 43 in the SOC  post-transplantion  was 75 ± 1% prior to transplantation (D0), but it was reduced to 27.3 ± 1.1% in the cells derived from grafts at D30 post-transplantation  was significantly increased from 20.3 ± 0.6% at D0 to 42.3 ± 1.1% at D30  cells from tes Fig. . After 1tes Fig. . In addites Fig. . Peak Diion Fig. . The perion Fig. . SimultaD30 Fig. . This suhigh cell population (from 75.3 ± 1.5% on D0 to 29.7 ± 1.5% on D30) and a proportionate increase in DiDlow cell population (from 19.7 ± 0.6% on D0 to 46.7 ± 1.1% on D30) were consistent with the properties observed with cells derived from primary grafts cells from the primary graft were stained with DiD dye and transplanted into the tertiary recipient mice Fig. . A similfts Fig. . In addifts Fig. .The cells collected at the end of serial transplantation exhibited multi-lineage differentiation capabilities, as indicated by positive Alcian blue, LipidTox or Alizarin Red-S staining in cross-sections of tertiary grafts 18 , which involve separation across the entire physis, through the HZ, have the best prognosis for repair and are the least likely to cause growth arrest)18 Fig. . Quantit)18 Fig. . Color m)18 Fig. . The injt-op Fig.. Overallt-op Fig..Fig. 6A At 1-day post-op, the injured GP displays significant damage in the HZ Fig. F. WithinFoxA2CreERT/+;Tomatofl/+ mice, there was substantial expansion of Tomato+ cells in the injured GP  cells are significantly reduced in the GP tissue, and in parallel, we see a surge in Tomato+ cells in the MB (3-fold increase) Fig. . This suFoxA2Cre.ERT/+;ZsGreenf/+;Tg.col10mcherry mice demonstrates that FoxA2+(ZsGreen+) cells generate yellow hypertrophic cells (col.10mcherry+)  cells transit in the metaphyseal bone to become yellow bone cells (col.1GFP+)  or col.1+ cells (osteogenic lineage). SH1-like surgery on y+) Fig. , while SP+) Fig. .Fig. 8AbFoxA2CreERT/+;DTAf/f mice, in which FoxA2+ cells were ablated by production of diphtheria toxin (DTA). In FoxA2CreERT2/+;DTAf/+;Tomatofl/+ mice there was a 36% decrease in FoxA2+(Tomato+) cells compared with FoxA2CreERT2/+;Tomatofl/+ littermates  compared with control mice  is followed by osteogenesis (P9-P10) emerging in the center of the epiphysis and by SOC enlargement and maturation thereafter19. A rise in serum thyroid hormone (TH) level during the murine prepubertal growth period initiates SOC activity, by promoting conversion of Osterix-expressing chondrocytes into bone matrix producing osteoblasts via a hedgehog signaling pathway19.While POC (primary ossification centers) of the long bones form at E15.5, during mouse embryonic development, the SOCs appear sometime later, primarily after birth, around postnatal day P723. This FoxA factors-chromatin interaction facilitates binding of other transcription factors including C/EBP, GATA4, Nf1, androgen and glucocorticoid receptors to adjacent binding sites25. Thus, it is possible that FoxA factors provide competence for chondrocyte hypertrophy by promoting interaction with other transcription factors.Our findings provide experimental evidence that SOC formation starts earlier than previously thought. Lineage tracing shows that the cells fated to become SOC are marked by FoxA2 expression as early as P0, and are morphologically indistinguishable from the columnar GP cells before P5. Since the first col10+ hypertrophic chondrocytes appear by P7, FoxA2 expression prefigures SOC formation, before overt chondrocyte differentiation. These findings are consistent with the role of FoxA factors as pioneer transcription factors, able to bind nucleosomal covered DNA and to disrupt local chromatin structure prior to gene transcriptionAt the interface between SOC and GP, a population of FoxA2+ cells builds up in the GP cartilage during early postnatal skeletal development. These FoxA2+ cells are left behind from epiphyseal cartilage postnatal development , and give rise to the GP RZ top layer. At 3 months after the initial labeling, FoxA2+ cells are still present in the GP and give rise to columns of progeny. By 9 months, the number of columns significantly increases, indicating that the FoxA2+ cells increasingly contribute to GP cartilage over time. In addition, FoxA2+ cells can also give rise to col.1+bone cells in the SOC during early postnatal development. However, it is unclear whether the same FoxA2+ cell has a dual fate, or the FoxA2+population is heterogeneous, with some cells destined for osteogenic lineage, and others for the chondrogenic lineage.10. Newton et al. describe at least two sub-populations of stem cells, with different proliferation rates, similar to stem cell niches in hair follicles27, bone marrow28 and intestine29. In addition to the sub-population dedicated to making columnar chondrocytes, there seems to be another, more dormant, sub-population of LRC (label-retaining cells)10. Even after 1–2 months of tracing, some LRCs remained at the top of the RZ, either as single cells, or as small clusters, but not columns of progeny. This more quiescent group is primarily located next to the epiphyseal bone and the SOC, reinforcing the idea of the SOC as a center of signaling that guides, in some –not yet fully understood- way, the stemness of the RZ cells10. Our experiments support these previous findings and show that FoxA2+ cells constitute this more quiescent, LTSSC population, located at the border between GP and the SOC.After P28, FoxA2+ cells remain mostly in GP, and their contribution to the SOC slows down significantly. This shift in clonality coincides with the maturation of the SOC into epiphyseal bone, as previously reported by Newton et al. who reveal a significant role for the SOC in the renewal of GP chondroprogenitors by providing a stem cell niche11, and exhibit higher clonogenicity and longevity. Mizuhashi et al. noted higher longevity for PTHrPCreERT2/+;Tomatofl/+ clones, but the labeling involves an early-pulse  and a long-chase, which caused extensive marking of PTHrP+ descendants in the RZ11. Some labeled cells reach as high as the FoxA2+ expression domain, next to the newly formed SOC. Thus, this broad labeling may include some of the FoxA2+ cells, hence the LTSSC characteristics of some established clones. In contrast, we demonstrate that PTHrPmcherry cells exhaust within five passages in a cfu assay, unlike FoxA2+ cells, which maintain the colony forming ability beyond nine passages, demonstrating long term self-renewability.FoxA2+ cells are mutually exclusive with the previously characterized PTHrP+ stem cells30, and it is required for proper chromatin remodeling during human pluripotent stem cells differentiation to pancreatic progenitors31. It will be interesting to establish whether FoxA2-dependent transcription is required for FoxA2+ LTSSC self-renewability.In addition to demonstrating that FoxA2+ stem cells retain long-term self-renewability in vitro, we also found that FoxA2+ cells preserve self-renewal ability and multipotency in a serial transplantation assay. Published reports indicate that FoxA2 serves as a stemness regulator, controlling the self-renewal of ovarian cancer stem cells3. Salter–Harris fractures through the physis often result in the formation of a “bony bar” bridging the epiphysis to the metaphysis34. In contrast, Salter–Harris type1 fractures where the cleavage plane is along the GP, preserving the RZ, heal without significant growth disturbance4. Thus, we developed a surgical model that emulates SH1 fracture morphology by creating a discrete defect through the HZ of the physis. Within days, FoxA2+ cells expand in response to injury allowing for regeneration of physeal cartilage and maintenance of longitudinal bone growth. This is consistent with previous published reports where removal of PZ and HZ from the GP allowed for GP regeneration, as long as the RZ is preserved6. However, when the GP was progressively reduced to the RZ top compartment, regeneration of the GP took longer5. This supports our findings that FoxA2+ cells, located in the RZ top compartment, behave as LTSSC, first giving rise to progeny, which then produce columns of chondrocytes. These results are relevant for understanding the pathophysiology of GP injuries in children and deriving treatment strategies to prevent subsequent disturbances to symmetric, longitudinal bone growth that can cause disability in adulthood2.Salter–Harris classification system divides GP fractures into five groups predicated on fracture morphology involving the physis ± epiphysis and/or metaphysis and the deleterious effect on appendicular growth39: FoxA2Cre.ERT/+ mice12 (JAX stock#008464), AggCre.ERT/+ mice (JAX stock#019148), Tomatofl/fl mice36 (JAX stock#007914), ZsGreenfl/fl mice36 (JAX stock #007906), DTAf/f mice39 (JAX stock#009669), Tg.col1gfp mice35 (JAX stock #013134), PTHrPmcherry mice38 (JAX stock#032872) and Tg.col10a1mCherry mice37 (JAX stock#017465). All genetically modified mouse lines used in this study were backcrossed to a C57Bl/6J background. Mice with both sexes  were used up to 1 year of age. The animals were housed in Northeastern University accredited facilities with free access to standard rodent chow and water, with a controlled 12 h light-dark cycle. Intraperitoneal tamoxifen (100 mg/kg/day) injection was given daily to activate CRE expression as indicated to generate time-dependent and tissue-specific mutant mice. All studies were carried out using littermate cohorts. All procedures involving mice were performed with approved protocols from Northeastern Institutional Animal Care and Use Committee (IACUC). For BrdU labeling, mice were administered 3 injections  at 12 h intervals before harvest and sacrificed 2 h after the last injection.Mouse strainsFoxA2CreERT/+;ZsGreenfl/+;Tg.col10mCherry mice or PTHrPmcherry mice that received intraperitoneal tamoxifen, 100 mg/kg/day  at postnatal days P13-17. The surrounding tissues adhering to the growth plate were removed and the growth plate cartilage was subjected to predigestion with 0.2% Collagenase P  in DMEM high glucose  in a shaking waterbath at 37 °C, 150 rpm for 40 min. The predigested growth plates were cut into 1–2 mm pieces with a sterile scalpel and subjected to final digestion with enzymes, 0.1 % collagenase II  and 0.05 % trypsin  at 37 °C in a CO2 incubator for 4 h. After complete digestion, the cell suspension was passed through a 70 μm cell strainer, centrifuged at 1000 × g for 20 min and the pellet was resuspended in complete growth medium (DMEM containing high glucose (4.5 g/liter), 1% l-glutamine, 10% fetal bovine serum, 100 U/ml of penicillin, and 100 mg/ml of streptomycin).Isolation of GP cells. GP cartilage was dissected out of proximal tibia, distal radius and distal ulna of P18 Flow cytometry and cell sorting. For Fluorescence-activated cell sorting (FACS), on average 500,000 cells (from 5 to 6 mice) were washed 2x with 2% heat-inactivated fetal calf serum in PBS, and then resuspended in PBS. The analysis and sorting for ZsGreen or mcherry were performed with BD FACS Aria II equipped with six laser system 355, 405, 445, 488, 561, and 641 nm. FACS images show the representative contour plots derived from the analysis of three independent litters.TM Fast Advanced Master Mix . All quantitative gene expression data were normalized to the expression levels of PPIB. The TaqMan probe ID were Mm00545794 (Aggrecan), Mm01309565 (COL2A1), Mm00801666 (COL1A1), Mm00475834 (ALP) and Mm00478295 (PPIB) purchased from ThermoFisher.Real-time PCR analysis. Real-time PCR was performed using TaqMan3cells/10 cm culture dishes or 5–10 cells/well into 96-well plates. They were incubated for 21 days in a complete growth medium, with media change every 4–5 days. Cloning cylinders  were positioned around each single colony for trypsinization of cells. Progeny cells from each colony underwent serial passages after seeding at the same density for colony forming unit assay as mentioned above for the parent cells.Colony Forming Unit Assays. FoxA2+col.10- (green) cells, FoxA2+col.10+(yellow) cells, or PTHrP+ cells were seeded at a density of 1041. Cells were seeded in triplicates in 12-well plates (5 × 104 cells/well) and grown for 24 h in complete growth medium. To induce osteoblast differentiation, cells were cultured in osteogenic medium  supplemented with 1% L-glutamine, 10% fetal bovine serum, 10 mM β-glycerophosphate  and 50 μg/ml ascorbic acid) for 4 weeks. Osteogenic nodules formed after differentiation were stained by Alizarin Red-S  staining as described previously42 and visualized using an inverted light microscope. For adipogenesis, cells were treated with MEM α supplemented with 1% L-glutamine, 10% fetal bovine serum, 60 μM Indomethacin , 0.1 μM Dexamethasone  and 50 μg/ml ascorbic acid for 4 weeks as described before41. Neutral lipid deposits in differentiated adipocytes were stained by LipidTOX red neutral lipid fluorescent stain . Chondrogenesis was induced by culturing cell pellets in StemXVivo chondrogenic base media  supplemented with StemXVivo chondrogenic supplement  according to procedures described by manufacturer’s instructions. After 28 days, the proteoglycan deposits in the cartilage matrix were stained by Alcian-Blue 8GX  staining as described previously43 and visualized using an inverted light microscope.Trilineage differentiation. Osteogenesis was induced as described previouslyFoxA2Cre.ERT/+ZsGreenf/+Tg.col10mcherry mice were expanded in culture for 30 days. The cells were then collected, labeled with DiD lipophilic dye  and resuspended in Matrigel  at 4 °C resulting in a final cell concentration of 105 cells/200 µL that was injected subcutaneously into Zsgreen- littermates. Injected zsGreen+ cells developed into a graft after 30 days. The grafts were surgically removed for cell isolation and DiD analysis (by flow cytometry) to monitor cell proliferation by dye dilution. To determine if cells retained self-renewal ability after expansion in the recipient mice, the FoxA2+ cells isolated from each graft were examined for colony forming abilities under culture conditions. For serial transplantation, FoxA2+ stem cells isolated from the grafts of secondary recipients were re-stained with DiD, re-transplanted into a tertiary recipient, allowed to develop in vivo for 30 days, after which they were tested for cell proliferation  and for colony forming abilities in culture. To determine, if cells retained multi-lineage differentiation capabilities at the end of serial transplantation, the ZsGreen+ cells isolated at the end of serial transplantation assays were lineage-primed under chondrogenic, adipogenic or osteogenic conditions, for 3 days, prior to subcutaneous transplantation in ZsGreen− littermates. After 30 days, the grafts were surgically removed, fixed in 10% neutral buffered formalin, and cryoprotected in 30% sucrose solution before freezing in Optimal cutting temperature (OCT) compound blocks. Cryostat sections from the blocks were stained using Alcian blue, LipidTox or Alizarin Red-S staining procedures for examining differentiated cell types.In vivo serial transplantation of cells. FoxA2+(ZsGreen+) cells isolated from SH1-like surgery. A SH1-like injury model was established by inducing a transverse excisional biopsy in C57B6 mice, across the lower region (HZ) of proximal tibial GP, using a 30 g needle. Surgical manipulations in mice were performed under general anesthesia by isoflurane  inhalation at ~4% (v/v) for induction and at ~2.5% (v/v) for maintenance, which was delivered with oxygen through nose cone for 15–20 min. Prior to surgery, the animals’ knees were shaved, and treated with betadine and alcohol . The joint capsule immediately medial to the patellar tendon was incised and opened with micro Iris scissors. A 30 g needle (0.312 mm outer diameter) was passed, in the transverse plane, through the lowest part of murine proximal tibial physis. The needle advances anteromedial to posterolateral, creating a discrete cylinder-shaped hole through the HZ of the physis. Control mice are sham-operated mice. The joint capsule was closed with a continuous 5–0 Vicryl suture .44 , 5- Aminofluorescein (5-DTAF), Hoechst dye .Histology. For histological staining, hindlimbs were fixed in 10% neutral buffered formalin solution, decalcified in 10% EDTA, frozen-embedded and sectioned at 10 µm, then stained with Hematoxylin /Eosin , SafraninO/Fast Greenm. ImageJ quantification of the SH1-like injury involves measurement of Area A as SafraninO+ area of healthy cartilage, Area B as the injured area, characterized by Safranin O loss, and Area A + B as the total area. Percentage (%) regeneration is calculated by dividing the area of SO+healthy GP cartilage (A) to the total GP area (A + B) from n = 3 sections collected from the lesion area, per hindlimb. Quantification FoxA2+(Tomato+) cells at d1, d3 & d7 post-op in the metaphysis, SOC and GP was performed in tibial sections from control and operated limbs. The number of Tomato+ cells in the metaphyseal bone (MB) is counted in an area extending 100 µm away from the GP/MB interface, towards the MB. The number of Tomato+ cells in the SOC is counted in an area extending 100 µm away from the GP/SOC interface, towards the SOC. The number of Tomato+ cells in the GP is counted in an area extending 100 µm away from the GP/SOC interface, towards the GP. Each point represents an average of n = 3 sections collected from the lesion area, per hindlimb.Histomorphometry. Histomorphometry was performed using ImageJ image processing and analysis software . For SH1 surgery, we calibrated the images by using a global scale bar of 100 μn = 8 sections per mouse hindlimb. Percentage (%) cells in the SOC was calculated by dividing the number of Tomato+ cells in the SOC (inside the green 5-DTAF domain) by the total number of Tomato+ cells (located in both GP and SOC). To assess the ability of FoxA2+ cells to give rise to descendants over time, we quantified the number of FoxA2+ single cells, clusters and columns at 1 day, and 1, 3, 9 months after labeling. Each point represents the sum of n = 8 sections per mouse hindlimb. Percentage (%) single cells was calculated from the ratio of number of single Tomato+ cells in the GP (outside the green 5-DTAF domain) to the total number of Tomato+ units . One Tomato+ unit, is defined by 1 single cell, or 1 cluster comprised of two or more cells grouped together, or by a column of cells. Same calculations were performed for clusters and columns of progeny. Quantification of tamoxifen labeling efficiency was performed by calculating the number of FoxA2+ cells detected via IHC or by Tomato+ fluorescence per 10 µm thickness section, as an average of n = 4 sections. The number of FoxA2+ cells in the GP is counted in an area extending 100 µm away from the GP/SOC interface, towards the GP.Clone number quantification. To quantify the number of FoxA2+(ZsGreen+) cells present in the GP, during early postnatal development, we counted the number of FoxA2+ cells located in GP . To calculate the percentage (%) cells in the GP v. SOC, we divided the number of Tomato+ cells in the GP (outside the green 5-DTAF domain) by the total number of Tomato+ cells (located in both GP and SOC). The number of Tomato+ cells represents a sum of Immunohistochemistry. For immunohistochemistry, we used frozen-embedded sections. The following antibodies were used: FoxA1 , FoxA2 , FoxA3 , Ki67 , BrdU . The sections were incubated with Dual Endogenous Enzyme Block  for 30 min at room temperature in order to suppress endogenous alkaline phosphatase and peroxidase enzymes. Blocking was performed in 0.5% TNB buffer  for 1 h. The samples were washed with TBST ) and incubated with anti-rabbit IgG horseradish peroxidase , for 45 min at room temperature. After washing the samples in TBST, the slides were incubated for 5 min in a tyramide-biotin working solution, which was prepared in a 1:50 dilution using the TSA™ Biotin System (PerkinElmer cat#NEL700A001KT). For detection, the samples were incubated in strepdavidin conjugated with Alexa Fluor 568 dye, diluted 1:500 in 0.5% TNB buffer, for 1 h in the dark at room temperature. Lastly, the samples were mounted using ProLong™ Diamond Antifade Mountant with DAPI .CECT and X-ray Imaging and Analysis. Non-contrast-enhanced μCT cannot directly visualize unmineralized tissues such as cartilage, owing to the low x-ray attenuation coefficient of these tissues. To counteract this, we immersed the samples in CA4+, an iodinated, cationic contrast agent, that is electrostatically attracted to the anionic glycosaminoglycans (GAG) of the cartilage ECM. The X-ray attenuation induced by the CA4+ is highly correlated with the fixed negative charge density of the tissue and is thereby a reliable tool for amplifying attenuation of GAG-rich tissues such as cartilage. Contralateral and surgical mouse tibial explants from 1, 3, 7, and 21 days post-surgery were positioned in airtight CT vials. Baseline CT slices were acquired transaxially at 18 μm resolution, 70 kVp tube voltage, 114 mA, 300 ms integration time . Samples were then immersed in CA4 + solution  prior to CECT imaging using the same settings and μCT scanner. The μCT data was then converted to DICOM format. The GP cartilage was segmented from bone and surrounding soft tissues in a semi-automated process in order to obtain cartilage thickness, lesion dimensions, and lesion volume . Select images were converted to color maps to enhance visualization of the GP cartilage. For X-ray Imaging and Analysis, tibial explants from 1, 3, 7, and 21 days post-operation were X-rayed at 75kVp, 6 min exposure time . The film was developed, and calipers were used to measure tibial length for each specimen.t test. Comparisons between multiple groups were performed using a one‐way analysis of variance (ANOVA) with Tukey’s post hoc analysis. A p-value below less than 0.05 was considered statistically significant. Single asterisks denote p < 0.05 (*p  <  0.05); Double asterisks denote p < 0.01 (**p  < 0.01); Triple asterisks denote p < 0.001 (***P < 0.001).Statistics and Reproducibility. Data were expressed as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism. Experiments are performed in triplicate, unless otherwise noted in figure legends. Comparisons between two independent groups were performed using an unpaired Student’s Further information on research design is available in the Supplementary InformationReporting Summary


In vitro results indicated that hucMSC-Exos combined with the autophagy activator had positive effects in promoting the cell proliferation, migration capacity, and the cell cycle by upregulating the proportions of cells in the S phase and the expression of PCNA, Cyclin A, Cyclin E, and CDK2. Meanwhile, the combination treatment reduced the apoptotic rate of HCECs. In vivo results indicated that hucMSC-Exos especially combined them with the autophagy activator significantly alleviated corneal epithelial defects and stromal opacity, reduced the levels of the apoptotic markers Bax and cleaved Caspase-3, reduced the inflammatory response products TNF-α, IL-1β, IL-6, and CXCL-2, and increased the Bcl-2. This was achieved by upregulating pAMPK/AMPK and pULK1/ULK1 ratios, and Beclin-1 and LC3B II/I, and by downregulating the pmTOR/mTOR ratio and p62. In contrast, clinical indications, apoptosis, and inflammation were aggravated after the application of the autophagy inhibitor. HucMSC-Exos combined with an autophagy activator significantly enhanced HCECs functions and alleviated corneal defects, apoptosis, and inflammation by activating the autophagy signaling pathway, AMPK-mTOR-ULK1, providing a new biological therapy for corneal wound healing and ocular surface regeneration.Corneal injury (CI) affects corneal integrity and transparency, deteriorating the patient’s quality of life. This study aimed to explore the molecular mechanisms by which exosomes secreted from human umbilical cord mesenchymal stem cells (hucMSC-Exos) affect autophagy in human corneal epithelial cells (HCECs) and CI models. We isolated and identified hucMSC-Exos using nanoparticle tracking analysis, transmission electron microscopy, and western blotting. The effects of hucMSC-Exos combined with autophagy regulators on HCECs and CI mice were assessed using cell viability assays, scratch assay, cell cycle assay, apoptosis assay, corneal fluorescein staining, haze grades, pathological examinations, western blotting, and quantitative polymerase chain reaction (qPCR).  The cornea is situated on the most anterior segment of the eye and acts as the first refractive element . CornealTherapies involving mesenchymal stem cells (MSCs) obtained from adipose, bone marrow, or umbilical cord tissues promoted ocular surface healing . With thMultivesicular body-related secretory pathways and autophagy are interconnected at many levels . On the In all eukaryotic cells, autophagy is a self-degradation process that maintains homeostasis in the synthesis, degradation, and circulation of cellular components . Autophain vitro, corneal fluorescein staining, haze grades, pathological staining, terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay, western blotting, and quantitative polymerase chain reaction in vivo Furthermore, we studied the occurrence and development of autophagy in CI models.To the best of our knowledge, it isunknown whether the exosomes secreted from hucMSCs (hucMSC-Exos) and associated autophagy can exert a therapeutic effect in events of CI. The purpose of this research is to observe the mechanisms through which hucMSC-Exos and autophagy affect CI. For this, we performed cell viability assays, scratch wound assay,cell apoptosis assay, cell cycle assay g for 5 min, and analyzed by flow cytometry . Human corneal epithelial cells transformed with Simian virus 40 (HCECs) were cultivated in Dulbecco’s Modified Eagle Medium/F-12  supplemented with 10% fetal bovine serum , 10 ng/ml recombinant human epidermal growth factor , 5 μg/ml insulin , and 1% penicillin-streptomycin (Gibco). Cells were maintained at 37 C in a 5% CO2 incubator  until they reached 80–90% confluence.The isolation of hucMSCs was approved by the Medical Ethical Committee of the First Affiliated Hospital of Jinan University . The donors provided written informed consent in accordance with the Declaration of Helsinki. Cells were isolated and cultured as described by Gu et al. . Osteogeg for 10 min, 2,000 × g for 10 min, and 15,000 × g for 30 min. The supernatant was filtered, concentrated, and ultracentrifuged at 120,000 × g for 70 min . Exosomes were collected and resuspended in phosphate buffer solution . Their size, polydispersity, morphology, and surface marker antibodies CD9 , CD63 , CD81 , TSG101 , HSP70  and Calnexin  were determined by nanoparticle tracking analysis , transmission electron microscopy , and western blotting. As previously reported, the number of exosomes was quantified by the EXOCET exosomes quantification kit , according to the manufacturer’s instructions  with hucMSCs was collected and centrifuged at 300 × ructions . The colg for 90 min. HCECs and mouse corneas were treated with labelled hucMSC-Exos or PBS for 24 h, after which nuclei were stained with 4’,6-diamidino-2phenylindole , and photographed using a confocal microscope .The hucMSC-Exos were added into a mixture of PKH26 and Diluent C  for 5 min. After adding 0.5% FBS to terminate the reaction, exosomes were ultracentrifuged in the dark at 120,000 × in vitro and in vivo experiments are shown as follows (The autophagy activator (AA) Rapamycin  and the autophagy inhibitor (AI) dorsomorphin/Compound C  were dissolved and stored at 4 C. Group designs and schematic protocol of  follows .6/μl hucMSC-Exos; HCECs in the AA group were treated with 50 nM Rapamycin; HCECs in the AI group were treated with 5 μM Compound C; HCECs in the Exo + AA group were treated with 1 × 106/μl hucMSC-Exos and 50 nM Rapamycin; and HCECs in the Exo + AI group were treated with 1 × 106/μl hucMSC-Exos and 5 μM Compound C.HCECs in the control group were treated with PBS; HCECs in the Exo group were treated with 1 × 105/μl hucMSC-Exos; injured corneas in the CI + M-Exo group were treated with 1 × 106/μl hucMSC-Exos; injured corneas in the CI + H-Exo group were treated with 1 × 107/μl hucMSC-Exos; injured corneas in the CI + Exo + AA group were treated with 1 × 106/μl hucMSC-Exos and 10 μM Rapamycin; and injured corneas in the CI + Exo + AI group were treated with 1 × 106/μl hucMSC-Exos and 50 μM Compound C.Normal corneas in the control group were treated with PBS; injured corneas in the CI + PBS group were treated with PBS; injured corneas in the CI + L-Exo group were treated with 1 × 10HCECs were seeded into 96-well plates in triplicates and grown to 60% confluence. The cells were cultured in growth factor-starved medium with various treatments. The medium was replaced with 10 µl of Cell Counting Kit-8  solution after 24 h and incubated for 1 h at 37 C. Optical density was measured at 450 nm using a microplate reader .Fixed with a sterile ruler, confluent HCECs were scratched with a sterile standard 10 μl pipette tip , then treated for 24 h. Wound closure was recorded using a fluorescent microscope  and measured using ImageJ software . The wound closure ratio = [Cell-free area (0 h)- Cell-free area (24 h)]/Cell-free area (0 h) × 100 % .HCECs were mixed with 2.5 μl FITC Annexin V and with 2.5 μl propidium iodide staining solution  and incubated in darkness for 15 min. Pellets were resuspended in binding buffer and apoptosis rates were analyzed using flow cytometry.Harvested HCECs were incubated with cold 70% ethanol for 2 h at -20°C. The cells were washed with PBS to remove the ethanol then resuspended in PI/RNase Staining Buffer  for 15 min and immediately analyzed using a flow cytometer .The animal procedures were approved by the Laboratory Animal Ethics Committee of Jinan University . Experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Male C57BL/6 J mice aged 6–8 weeks  were anaesthetized using pentobarbital sodium solution (70 mg/kg) and proparacaine hydrochloride eye drops . The whole corneal epithelium and anterior stroma were scratched with AlgerBrush II corneal remover  after being marked with 2.5 mm trephine. Each group was treated three times daily.Fluorescein sodium  was applied to the corneal epithelium to observe the defects under cobalt blue light from a slit lamp . Corneal haze was observed and graded based on the haze grading method proposed by Zhou et al. .The mice were sacrificed and the eyeballs were fixed in 2.5% glutaraldehyde, dehydrated with ethanol and acetone, and embedded in epoxy resin. Ultrathin sagittal sections (60 nm) sliced using an Ultracut ultramicrotome  were placed on slot grids coated with polyvinyl formal, and counterstained with 1% uranium acetate.The eyeballs were fixed in 4% paraformaldehyde, dehydrated with a gradient of aqueous alcohol and xylene, and embedded in paraffin. The corneal structures were assessed by staining with hematoxylin-eosin . Tissue sections were incubated with Ki-67  overnight. Antigens were retrieved; non-specific antigen binding was blocked; then the sections were incubated with the secondary antibody goat anti-rabbit IgG H&L  and 3, 3′-diaminobenzidine (Servicebio).Label solution containing terminal deoxynucleotidyl transferase was incubated with paraffin-embedded sections in darkness for 2 h Nuclei were then stained with DAPI. Images were captured using a confocal microscope.Corneas were suspended in lysis buffer , homogenized , and centrifuged at 12,000 × g for 5 min at 4°C. Protein concentrations were determined using bicinchoninic acid standards (Leagene). Samples in loading buffer (Leagene), were denatured by boiling for 5 min, resolved by electrophoresis on 8–12% sodium dodecyl sulfate polyacrylamide gels (KeyGEN), then electroblotted onto polyvinylidene fluoride membranes . Non-specific antigen binding was blocked using 5% bovine serum albumin (Sigma Aldrich). The membranes were then incubated with primary antibodies Phospho-AMPKα , AMPKα , Phospho-mTOR , mTOR , Phospho-ULK1 , ULK1 , LC3B , SQSTM1/p62 , Beclin-1 , Bcl-2 , Bax , cleaved Caspase-3  and β-Actin  for 16–20 h at 4 C. The membranes were washed, incubated with secondary antibody goat anti-rabbit IgG H&L , and examined using a chemiluminescence apparatus . Images were analyzed using ImageJ.Total RNA was extracted using RNAiso Plus  and quantified using a spectrophotometer . Gene expression was measured by real-time quantitative PCR (qPCR) using designed primers , TB GreeMultiple comparisons were evaluated using the one-way analysis of variance (ANOVA) with Tukey or Dunnett’s post hoc tests using SPSS 24.0  and GraphPad Prism 9.0 . Results are presented as the means ± standard deviation (SD) of three independent experiments, and differences were considered statistically significant at p < 0.05.in vivo or in vitro.Typical spindle shapes were displayed by the hucMSCs . Flow cy6/μl (6/μl hucMSC-Exos and 50 nM Rapamycin) showed increased cell viability compared with that shown by Exo and AA (6/μl hucMSC-Exos and 5 μM Compound C) showed little effect on above cell functions. The number of HCECs in the S phase, but a distinct decline in these parameters was observed during in the G0/G1 phase by cell cycle assay  and applied to the following tests as the Exo group. Then, we systematically investigated the effects of hucMSC-Exos, autophagy activator, autophagy inhibitors, and hucMSC-Exos combined with those autophagy regulators on HCECs. Exosomes and Rapmycin both promoted cell proliferation and migration, but there was no significant difference between them. Interestingly, the combination treatment of hucMSC-Exos and Rapmycin could further contribute those positive effects. The optimal mechanism of tissue repair is that remaining live cells reenter the cell cycle (In the g et al. . As prevg et al. , differeg et al. . We idenll cycle . The proll cycle . Taking ll cycle . Our resin vivo experiments, we established and monitored a murine model with mechanical CI for 48 h. Our findings showed for the first time that the formation of numerous autophagosomes, elevated autophagic marker protein expression in the mechanically injured corneas. The occurrence and development of autophagy uncovered herein suggested that autophagy is directly related to corneal homeostasis. The level of autophagy is low in healthy corneas, and autophagic activation through the AMPK-mTOR-ULK1 signaling pathway was associated with mechanical ocular surface damage. Autophagy induction and the release of numerous pro-inflammatory cytokines were most likely owing to CI-induced stress responses (For the esponses . In the esponses . Our datesponses . This inesponses , we specin vivo was 1.0 × 106/μl, which was similar to the results in vitro. In addition to accelerating corneal re-epithelialization and reducing haze grades, exosomes exerted anti-apoptotic and anti-inflammatory effects on corneal wound healing. These were significantly reflected in the decreased levels of apoptotic cells, Bax, cleaved Caspase-3, and TNF-α, IL-1β, IL-6, and CXCL-2, and the increased level of Bcl-2. The mechanisms of apoptosis, inflammation and autophagy are interrelated. Physical wounds trigger the release of inflammatory cytokines from epithelial cells and the rapid apoptosis of keratocytes (Next, we compared the effects of various concentrations of hucMSC-Exos and combined them with autophagy regulators to treat corneal defects. The results showed that hucMSC-Exos exerted therapeutic effects, and that the optimal concentration for corneal epithelial defect healing atocytes . AMPK-statocytes  and workatocytes . In thisOur findings demonstrated that autophagy plays an essential role in corneal wound healing. HucMSC-Exos, when combined with autophagy activators, exerted protective effects on mechanical CI and accelerated cell proliferation and migration whilst attenuating apoptosis and inflammation via the activation of the AMPK-mTOR-ULK1 autophagy pathway. Therefore, exosomes and autophagy activators may represent a novel approach to corneal wound healing and ocular surface regeneration.