Small extracellular vesicles from hypoxia-preconditioned bone marrow mesenchymal stem cells attenuate spinal cord injury via miR-146a-5p-mediated regulation of macrophage polarization

Spinal cord injury is a disabling condition with limited treatment options.Multiple studies have provided evidence suggesting that small extracellular vesicles(SEVs)secreted by bone marrow mesenchymal stem cells(MSCs)help mediate the beneficial effects conferred by MSC transplantation following spinal cord injury.Strikingly,hypoxia-preconditioned bone marrow mesenchymal stem cell-derived SEVs(HSEVs)exhibit increased therapeutic potency.We thus explored the role of HSEVs in macrophage immune regulation after spinal cord injury in rats and their significance in spinal cord repair.SEVs or HSEVs were isolated from bone marrow MSC supernatants by density gradient ultracentrifugation.HSEV administration to rats via tail vein injection after spinal cord injury reduced the lesion area and attenuated spinal cord inflammation.HSEVs regulate macrophage polarization towards the M2 phenotype in vivo and in vitro.Micro RNA sequencing and bioinformatics analyses of SEVs and HSEVs revealed that mi R-146a-5p is a potent mediator of macrophage polarization that targets interleukin-1 receptor-associated kinase 1.Reducing mi R-146a-5p expression in HSEVs partially attenuated macrophage polarization.Our data suggest that HSEVs attenuate spinal cord inflammation and injury in rats by transporting mi R-146a-5p,which alters macrophage polarization.This study provides new insights into the application of HSEVs as a therapeutic tool for spinal cord injury.

Bone marrow-derived mesenchymal stem cell-derived exosomeloaded miR-129-5p targets high-mobility group box 1 attenuates neurological-impairment after diabetic cerebral hemorrhage

BACKGROUND Diabetic intracerebral hemorrhage(ICH)is a serious complication of diabetes.The role and mechanism of bone marrow mesenchymal stem cell(BMSC)-derived exosomes(BMSC-exo)in neuroinflammation post-ICH in patients with diabetes are unknown.In this study,we investigated the regulation of BMSC-exo on hyperglycemia-induced neuroinflammation.AIM To study the mechanism of BMSC-exo on nerve function damage after diabetes complicated with cerebral hemorrhage.METHODS BMSC-exo were isolated from mouse BMSC media.This was followed by transfection with microRNA-129-5p(miR-129-5p).BMSC-exo or miR-129-5poverexpressing BMSC-exo were intravitreally injected into a diabetes mouse model with ICH for in vivo analyses and were cocultured with high glucoseaffected BV2 cells for in vitro analyses.The dual luciferase test and RNA immunoprecipitation test verified the targeted binding relationship between miR-129-5p and high-mobility group box 1(HMGB1).Quantitative polymerase chain reaction,western blotting,and enzyme-linked immunosorbent assay were conducted to assess the levels of some inflammation factors,such as HMGB1,interleukin 6,interleukin 1β,toll-like receptor 4,and tumor necrosis factorα.Brain water content,neural function deficit score,and Evans blue were used to measure the neural function of mice.RESULTS Our findings indicated that BMSC-exo can promote neuroinflammation and functional recovery.MicroRNA chip analysis of BMSC-exo identified miR-129-5p as the specific microRNA with a protective role in neuroinflammation.Overexpression of miR-129-5p in BMSC-exo reduced the inflammatory response and neurological impairment in comorbid diabetes and ICH cases.Furthermore,we found that miR-129-5p had a targeted binding relationship with HMGB1 mRNA.CONCLUSION We demonstrated that BMSC-exo can reduce the inflammatory response after ICH with diabetes,thereby improving the neurological function of the brain.

Neural stem cell-derived exosomes regulate cell proliferation,migration,and cell death of brain microvascular endothelial cells via the miR-9/Hes1 axis under hypoxia

Background:Our previous study found that mouse embryonic neural stem cell(NSC)-derived exosomes(EXOs)regulated NSC differentiation via the miR-9/Hes1 axis.However,the effects of EXOs on brain microvascular endothelial cell(BMEC)dysfunction via the miR-9/Hes1 axis remain unknown.Therefore,the current study aimed to determine the effects of EXOs on BMEC proliferation,migration,and death via the miR-9/Hes1 axis.Methods:Immunofluorescence,quantitative real-time polymerase chain reaction,cell counting kit-8 assay,wound healing assay,calcein-acetoxymethyl/propidium iodide staining,and hematoxylin and eosin staining were used to determine the role and mechanism of EXOs on BMECs.Results:EXOs promoted BMEC proliferation and migration and reduced cell death under hypoxic conditions.The overexpression of miR-9 promoted BMEC prolifera-tion and migration and reduced cell death under hypoxic conditions.Moreover,miR-9 downregulation inhibited BMEC proliferation and migration and also promoted cell death.Hes1 silencing ameliorated the effect of amtagomiR-9 on BMEC proliferation and migration and cell death.Hyperemic structures were observed in the regions of the hippocampus and cortex in hypoxia-induced mice.Meanwhile,EXO treatment improved cerebrovascular alterations.Conclusion:NSC-derived EXOs can promote BMEC proliferation and migra-tion and reduce cell death via the miR-9/Hes1 axis under hypoxic conditions.Therefore,EXO therapeutic strategies could be considered for hypoxia-induced vascular injury.

TAX1BP1 and FIP200 orchestrate non-canonical autophagy of p62 aggregates for mouse neural stem cell maintenance

Autophagy plays a pivotal role in diverse biological processes,including the maintenance and differentiation of neural stem cells(NSCs).Interestingly,while complete deletion of Fip200 severely impairs NSC maintenance and differentiation,inhibiting canonical autophagy via deletion of core genes,such as Atg5,Atg16l1,and Atg7,or blockade of canonical interactions between FIP200 and ATG13(designated as FIP200-4A mutant or FIP200 KI)does not produce comparable detrimental effects.This highlights the likely critical involvement of the non-canonical functions of FIP200,the mechanisms of which have remained elusive.Here,utilizing genetic mouse models,we demonstrated that FIP200 mediates non-canonical autophagic degradation of p62/sequestome1,primarily via TAX1BP1 in NSCs.Conditional deletion of Tax1bp1 in fip200hGFAP conditional knock-in(cKI)mice led to NSC deficiency,resembling the fip200hGFAP conditional knockout(cKO)mouse phenotype.Notably,reintroducing wild-type TAX1BP1 not only restored the maintenance of NSCs derived from tax1bp1-knockout fip200hGFAP cKI mice but also led to a marked reduction in p62 aggregate accumulation.Conversely,a TAX1BP1 mutant incapable of binding to FIP200 or NBR1/p62 failed to achieve this restoration.Furthermore,conditional deletion of Tax1bp1 in fip200hGFAP cKO mice exacerbated NSC deficiency and p62 aggregate accumulation compared to fip200hGFAP cKO mice.Collectively,these findings illustrate the essential role of the FIP200-TAX1BP1 axis in mediating the non-canonical autophagic degradation of p62 aggregates towards NSC maintenance and function,presenting novel therapeutic targets for neurodegenerative diseases.

Effect of ginsenoside Rg1 on hematopoietic stem cells in treating aplastic anemia in mice via MAPK pathway

BACKGROUND Aplastic anemia(AA)presents a significant clinical challenge as a life-threatening condition due to failure to produce essential blood cells,with the current the-rapeutic options being notably limited.AIM To assess the therapeutic potential of ginsenoside Rg1 on AA,specifically its protective effects,while elucidating the mechanism at play.METHODS We employed a model of myelosuppression induced by cyclophosphamide(CTX)in C57 mice,followed by administration of ginsenoside Rg1 over 13 d.The invest-igation included examining the bone marrow,thymus and spleen for pathological changes via hematoxylin-eosin staining.Moreover,orbital blood of mice was collected for blood routine examinations.Flow cytometry was employed to identify the impact of ginsenoside Rg1 on cell apoptosis and cycle in the bone marrow of AA mice.Additionally,the study further evaluated cytokine levels with enzyme-linked immunosorbent assay and analyzed the expression of key proteins in the MAPK signaling pathway via western blot.RESULTS Administration of CTX led to significant damage to the bone marrow’s structural integrity and a reduction in hematopoietic cells,establishing a model of AA.Ginsenoside Rg1 successfully reversed hematopoietic dysfunction in AA mice.In comparison to the AA group,ginsenoside Rg1 provided relief by reducing the induction of cell apoptosis and inflammation factors caused by CTX.Furthermore,it helped alleviate the blockade in the cell cycle.Treatment with ginsenoside Rg1 significantly alleviated myelosuppression in mice by inhibiting the MAPK signaling pathway.CONCLUSION This study suggested that ginsenoside Rg1 addresses AA by alleviating myelosuppression,primarily through modulating the MAPK signaling pathway,which paves the way for a novel therapeutic strategy in treating AA,highlighting the potential of ginsenoside Rg1 as a beneficial intervention.

Silencing of Jumonji domain-containing 1C inhibits the osteogenic differentiation of bone marrow mesenchymal stem cells via nuclear factor-κB signaling

BACKGROUND Osteoporosis is a common metabolic bone disorder induced by an imbalance between osteoclastic activity and osteogenic activity.During osteoporosis,bone mesenchymal stem cells(BMSCs)exhibit an increased ability to differentiate into adipocytes and a decreased ability to differentiate into osteoblasts,resulting in bone loss.Jumonji domain-containing 1C(JMJD1C)has been demonstrated to suppress osteoclastogenesis.AIM To examine the effect of JMJD1C on the osteogenesis of BMSCs and the potential underlying mechanism.METHODS BMSCs were isolated from mouse bone marrow tissues.Oil Red O staining,Alizarin red staining,alkaline phosphatase staining and the expression of adipo-genic and osteogenic-associated genes were assessed to determine the differen-tiation of BMSCs.Bone marrow-derived macrophages(BMMs)were incubated with receptor activator of nuclear factor-kappaΒligand to induce osteoclast differentiation,and osteoclast differen-tiation was confirmed by tartrate-resistant acid phosphatase staining.Other related genes were measured via reverse transcription coupled to the quantitative polymerase chain reaction and western blotting.Enzyme-linked immunosorbent assays were used to measure the levels of inflammatory cytokines,including tumor necrosis factor alpha,interleukin-6 and interleukin-1 beta.RESULTS The osteogenic and adipogenic differentiation potential of BMSCs isolated from mouse bone marrow samples was evaluated.JMJD1C mRNA and protein expression was upregulated in BMSCs after osteoblast induction,while p-nuclear factor-κB(NF-κB)and inflammatory cytokines were not significantly altered.Knockdown of JMJD1C repressed osteogenic differentiation and enhanced NF-κB activation and inflammatory cytokine release in BMSCs.Moreover,JMJD1C expression decreased during BMM osteoclast differentiation.CONCLUSION The JMJD1C/NF-κB signaling pathway is potentially involved in BMSC osteogenic differentiation and may play vital roles in the pathogenesis of osteoporosis.

Gossypol acetic acid regulates leukemia stem cells by degrading LRPPRC via inhibiting IL-6/JAK1/STAT3 signaling or resulting mitochondrial dysfunction

BACKGROUND Leukemia stem cells(LSCs)are found to be one of the main factors contributing to poor therapeutic effects in acute myeloid leukemia(AML),as they are protected by the bone marrow microenvironment(BMM)against conventional therapies.Gossypol acetic acid(GAA),which is extracted from the seeds of cotton plants,exerts anti-tumor roles in several types of cancer and has been reported to induce apoptosis of LSCs by inhibiting Bcl2.AIM To investigate the exact roles of GAA in regulating LSCs under different microenvironments and the exact mechanism.METHODS In this study,LSCs were magnetically sorted from AML cell lines and the CD34+CD38-population was obtained.The expression of leucine-rich pentatricopeptide repeat-containing protein(LRPPRC)and forkhead box M1(FOXM1)was evaluated in LSCs,and the effects of GAA on malignancies and mitochondrial RESULTS LRPPRC was found to be upregulated,and GAA inhibited cell proliferation by degrading LRPPRC.GAA induced LRPPRC degradation and inhibited the activation of interleukin 6(IL-6)/janus kinase(JAK)1/signal transducer and activator of transcription(STAT)3 signaling,enhancing chemosensitivity in LSCs against conventional chemotherapies,including L-Asparaginase,Dexamethasone,and cytarabine.GAA was also found to downregulate FOXM1 indirectly by regulating LRPPRC.Furthermore,GAA induced reactive oxygen species accumulation,disturbed mitochondrial homeostasis,and caused mitochondrial dysfunction.By inhibiting IL-6/JAK1/STAT3 signaling via degrading LRPPRC,GAA resulted in the elimination of LSCs.Meanwhile,GAA induced oxidative stress and subsequent cell damage by causing mitochondrial damage.CONCLUSION Taken together,the results indicate that GAA might overcome the BMM protective effect and be considered as a novel and effective combination therapy for AML.

Neuroprotective effects of neural stem cells pretreated with neuregulin1β on PC12 cells exposed to oxygen-glucose deprivation/reoxygenation

Studies on ischemia/reperfusion(I/R)injury suggest that exogenous neural stem cells(NSCs)are ideal candidates for stem cell therapy reperfusion injury.However,NSCs are difficult to obtain owing to ethical limitations.In addition,the survival,differentiation,and proliferation rates of transplanted exogenous NSCs are low,which limit their clinical application.Our previous study showed that neuregulin1β(NRG1β)alleviated cerebral I/R injury in rats.In this study,we aimed to induce human umbilical cord mesenchymal stem cells into NSCs and investigate the improvement effect and mechanism of NSCs pretreated with 10 nM NRG1βon PC12 cells injured by oxygen-glucose deprivation/reoxygenation(OGD/R).Our results found that 5 and 10 nM NRG1βpromoted the generation and proliferation of NSCs.Co-culture of NSCs and PC12 cells under condition of OGD/R showed that pretreatment of NSCs with NRG1βimproved the level of reactive oxygen species,malondialdehyde,glutathione,superoxide dismutase,nicotinamide adenine dinucleotide phosphate,and nuclear factor erythroid 2-related factor 2(Nrf2)and mitochondrial damage in injured PC12 cells;these indexes are related to ferroptosis.Research has reported that p53 and solute carrier family 7 member 11(SLC7A11)play vital roles in ferroptosis caused by cerebral I/R injury.Our data show that the expression of p53 was increased and the level of glutathione peroxidase 4(GPX4)was decreased after RNA interference-mediated knockdown of SLC7A11 in PC12 cells,but this change was alleviated after co-culturing NSCs with damaged PC12 cells.These findings suggest that NSCs pretreated with NRG1βexhibited neuroprotective effects on PC12 cells subjected to OGD/R through influencing the level of ferroptosis regulated by p53/SLC7A11/GPX4 pathway.

Effects of different concentrations of nicotinamide on hematopoietic stem cells cultured in vitro

BACKGROUND In vitro expansion to increase numbers of hematopoietic stem cells(HSCs)in cord blood could improve clinical efficacy of this vital resource.Nicotinamide(NAM)can promote HSC expansion ex vivo,but its effect on hematopoietic stem and progenitor cells(HSPCs,CD34^(+)CD38)and functional subtypes of HSCs-shortterm repopulating HSCs(ST-HSCs,CD34^(+)CD38CD45RACD49f^(+))and long-term repopulating HSCs(LT-HSCs,CD34^(+)CD38CD45RACD49f^(+)CD90^(+))is not yet known.As a sirtuin 1(SIRT1)inhibitor,NAM participates in regulating cell adhesion,polarity,migration,proliferation,and differentiation.However,SIRT1 exhibits dual effects by promoting or inhibiting differentiation in different tissues or cells.We propose that the concentration of NAM may influence proliferation,differentiation,and SIRT1 signaling of HSCs.AIM To evaluate the effects and underlying mechanisms of action of different concentrations of NAM on HSC proliferation and differentiation.METHODS CD34^(+)cells were purified from umbilical cord blood using MacsCD34 beads,and cultured for 10-12 d in a serum-free medium supplemented with cytokines,with different concentrations of NAM added according to experimental requirements.Flow cytometry was used to detect phenotype,cell cycle distribution,and apoptosis of the cultured cells.Real-time polymerase chain reaction was used to detect the transcription levels of target genes encoding stemness-related factors,che mokines,components of hypoxia pathways,and antioxidant enzymes.Dichloro-dihydro-fluorescein diacetate probes were used to evaluate intracellular production of reactive oxygen species(ROS).Determination of the effect of different culture conditions on the balance of cytokine by cytometric bead array.RESULTS Compared with the control group,the proportion and expansion folds of HSPCs(CD34^(+)CD38)incubated with 5 mmol/L or 10 mmol/L NAM were significantly increased(all P<0.05).The ST-HSCs ratio and fold expansion of the 5 mmol/L NAM group were significantly higher than those of the control and 10 mmol/L NAM groups(all P<0.001),whereas the LT-HSCs ratio and fold expansion of the 10 mmol/L NAM group were significantly higher than those of the other two groups(all P<0.05).When the NAM concentration was>10 mmol/L,cell viability significantly decreased.In addition,compared with the 5 mmol/L NAM group,the proportion of apoptotic cells in the 10 mmol/L NAM group increased and the proportion of cells in S and G2 phase decreased.Compared with the 5 mmol/L NAM group,the HSCs incubated with 10 mmol/L NAM exhibited significantly inhibited SIRT1 expression,increased intracellular ROS content,and downregulated expression of genes encoding antioxidant enzymes(superoxide dismutase 1,peroxiredoxin 1).CONCLUSION Low concentrations(5 mmol/L)of NAM can better regulate the balance between proliferation and differentiation,thereby promoting expansion of HSCs.These findings allow adjustment of NAM concentrations according to expansion needs.

Reveal more mechanisms of precondition mesenchymal stem cells inhibiting inflammation

Hypoxia can get more ability to inhibit inflammation.But how it impact on survival time of mesenchymal stem cells(MSCs)is confusing and how preconditioned MSCs inhibiting inflammation are partially known.Those issues decided the value of preconditioned MSCs by hypoxia.

High mobility group box 1 in the central nervous system:regeneration hidden beneath inflammation

High-mobility group box 1 was first discovered in the calf thymus as a DNA-binding nuclear protein and has been widely studied in diverse fields,including neurology and neuroscience.High-mobility group box 1 in the extracellular space functions as a pro-inflammatory damage-associated molecular pattern,which has been proven to play an important role in a wide variety of central nervous system disorders such as ischemic stroke,Alzheimer’s disease,frontotemporal dementia,Parkinson’s disease,multiple sclerosis,epilepsy,and traumatic brain injury.Several drugs that inhibit high-mobility group box 1 as a damage-associated molecular pattern,such as glycyrrhizin,ethyl pyruvate,and neutralizing anti-high-mobility group box 1 antibodies,are commonly used to target high-mobility group box 1 activity in central nervous system disorders.Although it is commonly known for its detrimental inflammatory effect,high-mobility group box 1 has also been shown to have beneficial pro-regenerative roles in central nervous system disorders.In this narrative review,we provide a brief summary of the history of high-mobility group box 1 research and its characterization as a damage-associated molecular pattern,its downstream receptors,and intracellular signaling pathways,how high-mobility group box 1 exerts the repair-favoring roles in general and in the central nervous system,and clues on how to differentiate the pro-regenerative from the pro-inflammatory role.Research targeting high-mobility group box 1 in the central nervous system may benefit from differentiating between the two functions rather than overall suppression of high-mobility group box 1.

Exosomes from circ-Astn1-modified adipose-derived mesenchymal stem cells enhance wound healing through miR-138-5p/SIRT1/FOXO1 axis regulation

BACKGROUND Wound healing impairment is a dysfunction induced by hyperglycemia and its effect on endothelial precursor cells(EPCs)in type 2 diabetes mellitus.There is increasing evidence showing that exosomes(Exos)derived from adipose-derived mesenchymal stem cells(ADSCs)exhibit the potential to improve endothelial cell function along with wound healing.However,the potential therapeutic mechanism by which ADSC Exos contribute to wound healing in diabetic mice remains unclear.AIM To reveal the potential therapeutic mechanism of ADSC Exos in wound healing in diabetic mice.METHODS Exos from ADSCs and fibroblasts were used for high-throughput RNA sequencing(RNA-Seq).ADSC-Exo-mediated healing of full-thickness skin wounds in a diabetic mouse model was investigated.We employed EPCs to investigate the therapeutic function of Exos in cell damage and dysfunction caused by high glucose(HG).We utilized a luciferase reporter(LR)assay to analyze interactions among circular RNA astrotactin 1(circ-Astn1),sirtuin(SIRT)and miR-138-5p.A diabetic mouse model was used to verify the therapeutic effect of circ-Astn1 on Exo-mediated wound healing.RESULTS High-throughput RNA-Seq analysis showed that circ-Astn1 expression was increased in ADSC Exos compared with Exos from fibroblasts.Exos containing high concentrations of circ-Astn1 had enhanced therapeutic effects in restoring EPC function under HG conditions by promoting SIRT1 expression.Circ-Astn1 expression enhanced SIRT1 expression through miR-138-5p adsorption,which was validated by the LR assay along with bioinformatics analyses.Exos containing high concentrations of circ-Astn1 had better therapeutic effects on wound healing in vivo compared to wild-type ADSC Exos.Immunofluorescence and immunohistochemical investigations suggested that circ-Astn1 enhanced angiopoiesis through Exo treatment of wounded skin as well as by suppressing apoptosis through promotion of SIRT1 and decreased forkhead box O1 expression.CONCLUSION Circ-Astn1 promotes the therapeutic effect of ADSC-Exos and thus improves wound healing in diabetes via miR-138-5p absorption and SIRT1 upregulation.Based on our data,we advocate targeting the circ-Astn1/miR-138-5p/SIRT1 axis as a potential therapeutic option for the treatment of diabetic ulcers.

Low-temperature 3D-printed collagen/chitosan scaffolds loaded with exosomes derived from neural stem cells pretreated with insulin growth factor-1 enhance neural regeneration after traumatic brain injury

There are various clinical treatments for traumatic brain injury,including surgery,drug therapy,and rehabilitation therapy;howeve r,the therapeutic effects are limited.Scaffolds combined with exosomes represent a promising but challenging method for improving the repair of traumatic brain injury.In this study,we determined the ability of a novel 3D-printed collagen/chitosan scaffold loaded with exosomes derived from neural stem cells pretreated with insulin-like growth factor-1(3D-CC-INEXOS) to improve traumatic brain injury repair and functional recove ry after traumatic brain injury in rats.Composite scaffolds comprising collagen,chitosan,and exosomes derived from neural stem cells pretreated with insulin-like growth fa ctor-1(INEXOS) continuously released exosomes for 2weeks.Transplantation of 3D-CC-INExos scaffolds significantly improved motor and cognitive functions in a rat traumatic brain injury model,as assessed by the Morris water maze test and modified neurological seve rity scores.In addition,immunofluorescence staining and transmission electron microscopy showed that3D-CC-INExos implantation significantly improved the recove ry of damaged nerve tissue in the injured area.In conclusion,this study suggests that transplanted3D-CC-INExos scaffolds might provide a potential strategy for the treatment of traumatic brain injury and lay a solid foundation for clinical translation.

Cardiac differentiation is modulated by anti-apoptotic signals in murine embryonic stem cells

BACKGROUND Embryonic stem cells(ESCs)serve as a crucial ex vivo model,representing epiblast cells derived from the inner cell mass of blastocyst-stage embryos.ESCs exhibit a unique combination of self-renewal potency,unlimited proliferation,and pluripotency.The latter is evident by the ability of the isolated cells to differ-entiate spontaneously into multiple cell lineages,representing the three primary embryonic germ layers.Multiple regulatory networks guide ESCs,directing their self-renewal and lineage-specific differentiation.Apoptosis,or programmed cell death,emerges as a key event involved in sculpting and forming various organs and structures ensuring proper embryonic development.How-ever,the molecular mechanisms underlying the dynamic interplay between diffe-rentiation and apoptosis remain poorly understood.AIM To investigate the regulatory impact of apoptosis on the early differentiation of ESCs into cardiac cells,using mouse ESC(mESC)models-mESC-B-cell lym-phoma 2(BCL-2),mESC-PIM-2,and mESC-metallothionein-1(MET-1)-which overexpress the anti-apoptotic genes Bcl-2,Pim-2,and Met-1,respectively.METHODS mESC-T2(wild-type),mESC-BCL-2,mESC-PIM-2,and mESC-MET-1 have been used to assess the effect of potentiated apoptotic signals on cardiac differentiation.The hanging drop method was adopted to generate embryoid bodies(EBs)and induce terminal differentiation of mESCs.The size of the generated EBs was measured in each condition compared to the wild type.At the functional level,the percentage of cardiac differentiation was measured by calculating the number of beating cardiomyocytes in the manipulated mESCs compared to the control.At the molecular level,quantitative reverse transcription-polymerase chain reaction was used to assess the mRNA expression of three cardiac markers:Troponin T,GATA4,and NKX2.5.Additionally,troponin T protein expression was evaluated through immunofluorescence and western blot assays.RESULTS Our findings showed that the upregulation of Bcl-2,Pim-2,and Met-1 genes led to a reduction in the size of the EBs derived from the manipulated mESCs,in comparison with their wild-type counterpart.Additionally,a decrease in the count of beating cardiomyocytes among differentiated cells was observed.Furthermore,the mRNA expression of three cardiac markers-troponin T,GATA4,and NKX2.5-was diminished in mESCs overexpressing the three anti-apoptotic genes compared to the control cell line.Moreover,the overexpression of the anti-apoptotic genes resulted in a reduction in troponin T protein expression.CONCLUSION Our findings revealed that the upregulation of Bcl-2,Pim-2,and Met-1 genes altered cardiac differentiation,providing insight into the intricate interplay between apoptosis and ESC fate determination.

Self-assembly of differentiated dental pulp stem cells facilitates spheroid human dental organoid formation and prevascularization

BACKGROUND The self-assembly of solid organs from stem cells has the potential to greatly expand the applicability of regenerative medicine.Stem cells can self-organise into microsized organ units,partially modelling tissue function and regeneration.Dental pulp organoids have been used to recapitulate the processes of tooth development and related diseases.However,the lack of vasculature limits the utility of dental pulp organoids.AIM To improve survival and aid in recovery after stem cell transplantation,we demonstrated the three-dimensional(3D)self-assembly of adult stem cell-human dental pulp stem cells(hDPSCs)and endothelial cells(ECs)into a novel type of spheroid-shaped dental pulp organoid in vitro under hypoxia and conditioned medium(CM).METHODS During culture,primary hDPSCs were induced to differentiate into ECs by exposing them to a hypoxic environment and CM.The hypoxic pretreated hDPSCs were then mixed with ECs at specific ratios and conditioned in a 3D environment to produce prevascularized dental pulp organoids.The biological characteristics of the organoids were analysed,and the regulatory pathways associated with angiogenesis were studied.RESULTS The combination of these two agents resulted in prevascularized human dental pulp organoids(Vorganoids)that more closely resembled dental pulp tissue in terms of morphology and function.Single-cell RNA sequencing of dental pulp tissue and RNA sequencing of Vorganoids were integrated to analyse key regulatory pathways associated with angiogenesis.The biomarkers forkhead box protein O1 and fibroblast growth factor 2 were identified to be involved in the regulation of Vorganoids.CONCLUSION In this innovative study,we effectively established an in vitro model of Vorganoids and used it to elucidate new mechanisms of angiogenesis during regeneration,facilitating the development of clinical treatment strategies.

Stromal cell-derived factor-1α regulates chondrogenic differentiation via activation of the Wnt/β-catenin pathway in mesenchymal stem cells

BACKGROUND Mesenchymal stem cells(MSCs)have been applied to treat degenerative articular diseases,and stromal cell-derived factor-1α(SDF-1α)may enhance their therapeutic efficacy.However,the regulatory effects of SDF-1αon cartilage differentiation remain largely unknown.Identifying the specific regulatory effects of SDF-1αon MSCs will provide a useful target for the treatment of degenerative articular diseases.AIM To explore the role and mechanism of SDF-1αin cartilage differentiation of MSCs and primary chondrocytes.METHODS The expression level of C-X-C chemokine receptor 4(CXCR4)in MSCs was assessed by immunofluorescence.MSCs treated with SDF-1αwere stained for alkaline phosphatase(ALP)and with Alcian blue to observe differentiation.Western blot analysis was used to examine the expression of SRY-box transcription factor 9,aggrecan,collagen II,runt-related transcription factor 2,collagen X,and matrix metalloproteinase(MMP)13 in untreated MSCs,of aggrecan,collagen II,collagen X,and MMP13 in SDF-1α-treated primary chondrocytes,of glycogen synthase kinase 3β(GSK3β)p-GSK3βandβ-catenin expression in SDF-1α-treated MSCs,and of aggrecan,collagen X,and MMP13 in SDF-1α-treated MSCs in the presence or absence of ICG-001(SDF-1αinhibitor).RESULTS Immunofluorescence showed CXCR4 expression in the membranes of MSCs.ALP stain was intensified in MSCs treated with SDF-1αfor 14 d.The SDF-1αtreatment promoted expression of collagen X and MMP13 during cartilage differentiation,whereas it had no effect on the expression of collagen II or aggrecan nor on the formation of cartilage matrix in MSCs.Further,those SDF-1α-mediated effects on MSCs were validated in primary chondrocytes.SDF-1αpromoted the expression of p-GSK3βandβ-catenin in MSCs.And,finally,inhibition of this pathway by ICG-001(5μmol/L)neutralized the SDF-1α-mediated up-regulation of collagen X and MMP13 expression in MSCs.CONCLUSION SDF-1αmay promote hypertrophic cartilage differentiation in MSCs by activating the Wnt/β-catenin pathway.These findings provide further evidence for the use of MSCs and SDF-1αin the treatment of cartilage degeneration and osteoarthritis.

Interferon-gamma and tumor necrosis factor-alpha synergistically enhance the immunosuppressive capacity of human umbilical-cordderived mesenchymal stem cells by increasing PD-L1 expression

BACKGROUND The immunosuppressive capacity of mesenchymal stem cells(MSCs)is dependent on the“license”of several proinflammatory factors to express immunosuppressive factors such as programmed cell death 1 ligand 1(PD-L1),which determines the clinical therapeutic efficacy of MSCs for inflammatory or immune diseases.In MSCs,interferon-gamma(IFN-γ)is a key inducer of PD-L1 expression,which is synergistically enhanced by tumor necrosis factor-alpha(TNF-α);however,the underlying mechanism is unclear.AIM To reveal the mechanism of pretreated MSCs express high PD-L1 and explore the application of pretreated MSCs in ulcerative colitis.METHODS We assessed PD-L1 expression in human umbilical-cord-derived MSCs(hUC-MSCs)induced by IFN-γand TNF-α,alone or in combination.Additionally,we performed signal pathway inhibitor experiments as well as RNA interference experiments to elucidate the molecular mechanism by which IFN-γalone or in combination with TNF-αinduces PD-L1 expression.Moreover,we used luciferase reporter gene experiments to verify the binding sites of the transcription factors of each signal transduction pathway to the targeted gene promoters.Finally,we evaluated the immunosuppressive capacity of hUC-MSCs treated with IFN-γand TNF-αin both an in vitro mixed lymphocyte culture assay,and in vivo in mice with dextran sulfate sodium-induced acute colitis.RESULTS Our results suggest that IFN-γinduction alone upregulates PD-L1 expression in hUC-MSCs while TNF-αalone does not,and that the co-induction of IFN-γand TNF-αpromotes higher expression of PD-L1.IFN-γinduces hUCMSCs to express PD-L1,in which IFN-γactivates the JAK/STAT1 signaling pathway,up-regulates the expression of the interferon regulatory factor 1(IRF1)transcription factor,promotes the binding of IRF1 and the PD-L1 gene promoter,and finally promotes PD-L1 mRNA.Although TNF-αalone did not induce PD-L1 expression in hUCMSCs,the addition of TNF-αsignificantly enhanced IFN-γ-induced JAK/STAT1/IRF1 activation.TNF-αupregulated IFN-γreceptor expression through activation of the nuclear factor kappa-B signaling pathway,which significantly enhanced IFN-γsignaling.Finally,co-induced hUC-MSCs have a stronger inhibitory effect on lymphocyte proliferation,and significantly ameliorate weight loss,mucosal damage,inflammatory cell infiltration,and up-regulation of inflammatory factors in colitis mice.CONCLUSION Overall,our results suggest that IFN-γand TNF-αenhance both the immunosuppressive ability of hUC-MSCs and their efficacy in ulcerative colitis by synergistically inducing high expression of PD-L1.

Magnetic resonance imaging focused on the ferritin heavy chain 1 reporter gene detects neuronal differentiation in stem cells

The neuronal differentiation of mesenchymal stem cells offers a new strategy for the treatment of neurological disorders.Thus,there is a need to identify a noninvasive and sensitive in vivo imaging approach for real-time monitoring of transplanted stem cells.Our previous study confirmed that magnetic resonance imaging,with a focus on the ferritin heavy chain 1 reporter gene,could track the proliferation and differentiation of bone marrow mesenchymal stem cells that had been transduced with lentivirus carrying the ferritin heavy chain 1 reporter gene.However,we could not determine whether or when bone marrow mesenchymal stem cells had undergone neuronal differentiation based on changes in the magnetic resonance imaging signal.To solve this problem,we identified a neuron-specific enolase that can be differentially expressed before and after neuronal differentiation in stem cells.In this study,we successfully constructed a lentivirus carrying the neuron-specific enolase promoter and expressing the ferritin heavy chain 1 reporter gene;we used this lentivirus to transduce bone marrow mesenchymal stem cells.Cellular and animal studies showed that the neuron-specific enolase promoter effectively drove the expression of ferritin heavy chain 1 after neuronal differentiation of bone marrow mesenchymal stem cells;this led to intracellular accumulation of iron and corresponding changes in the magnetic resonance imaging signal.In summary,we established an innovative magnetic resonance imaging approach focused on the induction of reporter gene expression by a neuron-specific promoter.This imaging method can be used to noninvasively and sensitively detect neuronal differentiation in stem cells,which may be useful in stem cell-based therapies.

Oscillating field stimulation promotes neurogenesis of neural stem cells through miR-124/Tal1 axis to repair spinal cord injury in rats

Spinal cord injury often leads to severe motor and sensory deficits,and prognosis using the currently available therapies remains poor.Therefore,we aimed to explore a novel therapeutic approach for improving the prognosis of spinal cord injury.In this study,we implanted oscillating field stimulation devices and transplanted neural stem cells into the thoracic region(T9–T10)of rats with a spinal cord contusion.Basso-Beattie-Bresnahan scoring revealed that oscillating field stimulation combined with neural stem cells transplantation promoted motor function recovery following spinal cord injury.In addition,we investigated the regulation of oscillating field stimulation on the miR-124/Tal1 axis in neural stem cells.Transfection of lentivirus was performed to investigate the role of Tal1 in neurogenesis of neural stem cells induced by oscillating field stimulation.Quantitative reverse transcription-polymerase chain reaction,immunofluorescence and western blotting showed that oscillating field stimulation promoted neurogenesis of neural stem cells in vitro and in vivo.Hematoxylin and eosin staining showed that oscillating field stimulation combined with neural stem cells transplantation alleviated cavities formation after spinal cord injury.Taking the results together,we concluded that oscillating field stimulation decreased miR-124 expression and increased Tal1 content,thereby promoting the neurogenesis of neural stem cells.The combination of oscillating field stimulation and neural stem cells transplantation improved neurogenesis,and thereby promoted structural and functional recovery after spinal cord injury.

Chitosan conduits enriched with fibrin-collagen hydrogel with or without adipose-derived mesenchymal stem cells for the repair of 15-mm-long sciatic nerve defect

Hollow conduits of natural or synthetic origins have shown acceptable regeneration results in short nerve gap repair;however,results are still not comparable with the current gold standard technique“autografts”.Hollow conduits do not provide a successful regeneration outcome when it comes to critical nerve gap repair.Enriching the lumen of conduits with different extracellular materials and cells could provide a better biomimicry of the natural nerve regenerating environment and is expected to ameliorate the conduit performance.In this study,we evaluated nerve regeneration in vivo using hollow chitosan conduits or conduits enriched with fibrin-collagen hydrogels alone or with the further addition of adipose-derived mesenchymal stem cells in a 15 mm rat sciatic nerve transection model.Unexpected changes in the hydrogel consistency and structural stability in vivo led to a failure of nerve regeneration after 15 weeks.Nevertheless,the molecular assessment in the early regeneration phase(7,14,and 28 days)has shown an upregulation of useful regenerative genes in hydrogel enriched conduits compared with the hollow ones.Hydrogels composed of fibrin-collagen were able to upregulate the expression of soluble NRG1,a growth factor that plays an important role in Schwann cell transdifferentiation.The further enrichment with adipose-derived mesenchymal stem cells has led to the upregulation of other important genes such as ErbB2,VEGF-A,BDNF,c-Jun,and ATF3.