Conditioned medium from human dental pulp stem cells treats spinal cord injury by inhibiting microglial pyroptosis

Human dental pulp stem cell transplantation has been shown to be an effective therapeutic strategy for spinal cord injury.However,whether the human dental pulp stem cell secretome can contribute to functional recovery after spinal cord injury remains unclear.In the present study,we established a rat model of spinal cord injury based on impact injury from a dropped weight and then intraperitoneally injected the rats with conditioned medium from human dental pulp stem cells.We found that the conditioned medium effectively promoted the recovery of sensory and motor functions in rats with spinal cord injury,decreased expression of the microglial pyroptosis markers NLRP3,GSDMD,caspase-1,and interleukin-1β,promoted axonal and myelin regeneration,and inhibited the formation of glial scars.In addition,in a lipopolysaccharide-induced BV2 microglia model,conditioned medium from human dental pulp stem cells protected cells from pyroptosis by inhibiting the NLRP3/caspase-1/interleukin-1βpathway.These results indicate that conditioned medium from human dental pulp stem cells can reduce microglial pyroptosis by inhibiting the NLRP3/caspase-1/interleukin-1βpathway,thereby promoting the recovery of neurological function after spinal cord injury.Therefore,conditioned medium from human dental pulp stem cells may become an alternative therapy for spinal cord injury.

Enhancing Erectile Function and Alleviating Andropause Symptoms: Clinical Efficacy of a Human Stem Cell Conditioned Medium Cream

Erectile dysfunction (ED) is increasingly prevalent in Japan, exceeding 30%, and increasing with age. Unhealthy lifestyle habits, obesity, insufficient exercise, and smoking have been implicated in its pathogenesis, along with endothelial dysfunction of the corpora cavernosa and impaired blood flow to the penis considered underlying factors. However, the current treatments are limited to Phosphodiesterase-5 (PDE5) inhibitors. ED is the primary symptom of andropathy. This study reports the clinical efficacy of human stem cell-conditioned medium cream for ED treatment. Ten men without underlying diseases suspected of andropause with ED (mean age 43.2 ± 4.4 y, Hb 15.2 ± 0.6 gm/dL, AST/ALT 30.2/37.9 ± 12.4/14.0, eGFR 82.7 ± 12.4 mL/min/1.73 m2) were targeted. The cream was applied twice daily to the genital and scrotal areas. The erectile hardness score (EHS), International Index of Erectile Function-5 (IIEF-5), and Aging Male Symptoms (AMS) scale were used to evaluate the participants before and 30 days after use, and the results were compared using paired t-tests. The post-use qualitative opinions were collected through interviews. Significant improvements were observed compared to baseline in the IIEF-5 (11.8 ± 4.6→17.2 ± 5.1, P < 0.001), and AMS (46.3 ± 6.7→37.6 ± 5.3, P < 0.001) scores post cream use. EHS did not show a statistically significant difference, but a trend towards improvement was observed. Qualitative feedback included increased morning erection, improved maintenance of erection during intercourse, and reduced post work fatigue. Human stem cell-conditioned medium contains endothelial growth factors that potentially contribute to the improvement of ED and andropause by enhancing corporal endothelial function. Future studies should include control groups to further investigate the efficacy of these treatments.

Conditioned secretome of adipose-derived stem cells improves dextran sulfate sodium-induced colitis in mice

BACKGROUND Inflammatory bowel diseases(IBD)is related to uncontrolled immune response.Currently,there is no successful treatment for significant improvement in IBD.Stem cells display their therapeutic effects through their repopulating capacity or secreting factors.AIM To investigate the effects of conditioned mouse adipose-derived stem cells(mADSCs)secretome on colitis-induced mice.METHODS mADSCs were isolated from adipose tissue of C57BL/6 mice.Conditioned mADSCs secrectome was obtained by culturing of mADSCs with lipopolysaccharides(LPS,1μg/mL)for 24 h.Acute colitis was induced by 2%dextran sulfate sodium(DSS)drinking water for 7 d and then normal drinking water for 4 d.The mice were treated with normal culture medium(NM group),conditioned mADSCs secretome(CM group)or mADSCs(SC group).The length of colon and histopatholgy of colon tissues were evaluated.The mRNA expression levels of inflammatory cytokines in colon tissue and the serum interleukin(IL)-6 levels were determined.RESULTS The isolated mADSCs maintained the mADSCs specific gene expression profiles during experiment.The conditioned mADSCs secretome released by the treatment of mADSCs with LPS contained mainly inflammatory chemokines,colony-stimulating factors and inflammatory cytokines.The loss of body weight and reduction in colon length were ameliorated in the CM group.The conditioned mADSCs secretome reduced the histological score in colon tissue.The expression of IL-1b and IL-6 mRNAs in colon tissues significantly inhibited in the CM group compared to SC group and NM group,respectively.The elevation of serum IL-6 levels was also ameliorated in the CM group.These results indicate that the conditioned mADSCs secretome suppressed the synthesis of inflammatory cytokines in damaged colon tissue and the elevation of serum IL-6 concentration in DSS-induced mice CONCLUSION Conditioned mADSCs secretome might play regenerative roles by the suppression of IL-6 in serum and tissue during acute colitis,and may be more effective than stem cells themselves in the regeneration of colon tissue.

Effects of feeder layer and BRL conditioned medium on mouse embryonic stem cells

In vitro growth and maintenance of embryonic stem (ES) cell lines derived from ICM cells of various blastocysts of 129 strain mice,the sustenance of their pluripotency and normal karyotype depend on the feeder layer of mouse embryonic fibroblasts (MEF).Compared with the feeder layer of MEF cells,medium conditioned by Buffalo rat liver cells (BRL-CM) is able to maintain pluripotency and karyotypic normality of ES cells only in short term cell propagation.Besides,ES cells grown in BRL-CM are also capable of aggregation with 8-cell embryos of Swiss strain and develop into germ line chimaeras.Modification to the method of aggregating ES cells with early embryos by making a hole in agar layer on the top of MEF feeder cells was shown to be more converient and efficient than the conventional microdrop method.

Effect of conditioned medium from neural stem cells on glioma progression and its protein expression profile analysis

BACKGROUND Emerging evidence suggests that the spread of glioma to the subventricular zone(SVZ)is closely related to glioma recurrence and patient survival.Neural stem cells(NSCs)are the main cell type in the SVZ region and exhibit tumor-homing ability.AIM To evaluate the effects of conditioned medium(CM)derived from SVZ NSCs on the cancer-related behaviors of glioma cells.METHODS The characteristics of SVZ hNSCs were identified by immunofluorescence.The normoxic-hNSC-CM and hypoxic-hNSC-CM(3%O2,oxygen-glucose deprived[OGD]culturing)were collected from 80%-90%confluent SVZ NSCs in sterile conditions.The CCK8 and Transwell assays were used to compare and evaluate the effects of normoxic-CM and hypoxic-CM on glioma proliferation and invasion.Then proteins secreted from SVZ NSCs into the CM were investigated by mass spectrometry,and the potential effects of candidate protein NCAN in the regulation of glioma progression were examined by CCK8 and Transwell assays.RESULTS The CM from SVZ NSCs significantly increased the proliferation and invasion of glioma cells,particularly the CM from OGD NSCs induced under hypoxic conditions.Furthermore,the secreted protein neurocan(NCAN)in CM from OGD NSCs was identified by proteomic analysis.NCAN was expressed in glioma cells and played regulatory roles in mediating the progression of glioma cells mainly via the Rho/Rho-associated protein kinase pathway.CONCLUSION Our study identified a potential interactive mechanism between SVZ NSCs and glioma cells,in which SVZ NSCs promote glioma progression via the secreted protein NCAN.These findings suggested that exploring the CM derived from cells could be a novel strategy for optimizing treatments and that NCAN derived from SVZ NSCs may be a potential new target in glioma progression.

Dental stem cell-conditioned medium for tissue regeneration: Optimization of production and storage

BACKGROUND Mesenchymal stem cells(MSC)effects on tissue regeneration are mainly mediated by their secreted substances(secretome),inducing their paracrine activity.This Conditioned medium(CM),including soluble factors(proteins,nucleic acids,lipids)and extracellular vesicles is emerging as a potential alternative to cell therapy.However,the manufacturing of CM suffers from variable procedures and protocols leading to varying results between studies.Besides,there is no welldefined optimized procedure targeting specific applications in regenerative medicine.AIM To focus on conditioned medium produced from dental MSC(DMSC-CM),we reviewed the current parameters and manufacturing protocols,in order to propose a standardization and optimization of these manufacturing procedures.METHODS We have selected all publications investigating the effects of dental MSC secretome in in vitro and in vivo models of tissue regeneration,in accordance with the PRISMA guidelines.RESULTS A total of 351 results were identified.And based on the inclusion criteria described above,118 unique articles were included in the systematic review.DMSC-CM production was considered at three stages:before CM recovery(cell sources for CM),during CM production(culture conditions)and after production(CM treatment).CONCLUSION No clear consensus could be recovered as evidence-based methods,but we were able to describe the most commonly used protocols:donors under 30 years of age,dental pulp stem cells and exfoliated deciduous tooth stem cells with cell passage between 1 and 5,at a confluence of 70%to 80%.CM were often collected during 48 h,and stored at-80°C.It is important to point out that the preconditioning environment had a significant impact on DMSCCM content and efficiency.

Intranasal Administration of Conditioned Medium from Cultured Mesenchymal Stem Cells Improves Cognitive Impairment in Olfactory Bulbectomized Mice

Alzheimer’s disease (AD) is the common cause of dementia which shows the neuro-pathologies like an accumulation of amyloid-β (Aβ) and degeneration of cholinergic neuron. Olfactory bulbectomized (OBX) mice show some of AD features, so they have been used to research as AD model. Mesenchymal stem cells (MSCs) can differentiate into many kinds of cells, including neuronal cells. In this study, we intranasally administrated the conditioned medium derived from cultured umbilical cord (UC) MSCs. The intranasal administration of the MSCs medium restored the cognitive impairment observed in OBX mice. In addition, the decreased number of choline acetyltransferase-positive cells in the medial septum was restored by the conditioned medium administration. In conclusion, MSCs-derived conditioned medium may have protective effects of cholinergic neurons in the medial septum, thereby rescuing the cognitive impairment of OBX.

Neural stem cell-conditioned medium upregulated the PCMT1 expression and inhibited the phosphorylation of MST1 in SHSY5Y cells induced by Aβ_(25-35)

A progressive neurodegenerative disease,Alzheimer’s disease(AD).Studies suggest that highly expressed protein isoaspartate methyltransferase 1(PCMT1)in brain tissue.In the current study,we explored the effects of neural stem cell-conditioned medium(NSC-CDM)on the PCMT1/MST1 pathway to alleviate Aβ_(25-35)-induced damage in SH-SY5Y cells.Our data suggested that Aβ_(25-35) markedly inhibited cell viability.NSC-CDM or Neural stem cell-complete medium(NSC-CPM)had a suppression effect on toxicity when treatment with Aβ_(25-35),with a greater effect observed with NSC-CDM.Aβ_(25-35)+NSC-CDM group exhibited an increase in PCMT1 expression.sh-PCMT1 markedly decreased cell proliferation and suppressed the protective role of NSC-CDM through the induction of apoptosis and improved p-MST1 expression.Overexpression of PCMT1 reversed the Aβ_(25-35)-induced decrease in cell proliferation and apoptosis.In summary,our findings suggest that NSC-CDM corrects the Aβ_(25-35)-induced damage to cells by improving PCMT1 expressions,which in turn reduces phosphorylation of MST1.

The Skin Whitening Effect of Co-Cultured Conditioned Medium: Involvement of Synergy between Stem Cells and Immune Cells

Many researchers have described that mesenchymal stem cells conditioned medium and immune cells conditioned medium have a clear whitening effect when they are used as cosmetic ingredients. In this study, we confirmed the whitening efficacy of various concentrations of immune cells and stem cell conditioned media. The author tried to study a conditioned medium that has a strong whitening effect even with a composition of less than 20% (the most used concentration in cosmetics). Because of the fact that the conditioned medium contains various cytokines and growth factors secreted by stem cells or immune cells, it is known to have effects such as wound healing, antioxidant, and whitening effect. Recently, stem cells have been used not only in the development of cosmetic raw materials but also in skincare procedures, and there are reports being released of cosmetics using immune cells conditioned medium. The concentration-dependent whitening effect equivalently increased as the concentration of the mono-cultured conditioned medium was obtained through the stem cells or immune cells culture. In the case of co-culture, whitening results are like the effect of positive control such as arbutin in the medium carrying only 10% of the co-cultured conditioned medium. It is possible that enhanced whitening efficiency in co-cultured conditioned medium leads to a major innovation in the global cosmetic market.

Effect of adipose-derived mesenchymal stem cells on hepatocellular carcinoma: In vitro inhibition of carcinogenesis

AIM To investigate the effect of adipose-derived mesenchymal stem cells(ADMSCs)and their conditioned media(CM) on hepatocellular carcinoma(HCC) cell tumorigenesis.METHODS The proliferation rate of HepG2 and PLC-PRF-5 HCC cancer cells was measured using the trypan blue exclusion method and confirmed using the cell-counting kit8(commonly known as CCK-8) assay. Apoptosis was detected by flow cytometry using annexin V-FITC. Protein and mRNA expression was quantified by ELISA and real time PCR, respectively. Migration and invasion rates were performed by Transwell migration and invasion assays. Wound healing was examined to confirm the data obtained from the migration assays.RESULTS Our data demonstrated that when co-culturing HCC cell lines with ADMSCs or treating them with ADMSC CM, the HCC cell proliferation rate was significantly inhibited and the apoptosis rate increased. The decreased proliferation rate was accompanied by an upregulation of P53 and Retinoblastoma mRNA and a downregulation of c-Myc and hTERT mRNA levels. More notably, ADMSCs and their CM suppressed the expression of the two important markers of HCC carcinogenicity, alpha-fetoprotein and Des-gamma-carboxyprothrombin. In addition, the migration and invasion levels of HepG2 and PLC-PRF-5 cells significantly decreased, potentially through increased expression of the tissue inhibitor metalloproteinases TIMP-1, TIMP-2 and TIMP-3.CONCLUSION These findings shed new light on a protective and therapeutic role for ADMSCs and their CM in controlling HCC invasiveness and carcinogenesis.

The Influence of Culture Medium Type on Cellular Phenotype of Canine Adipose Derived Stem Cells

Canine adipose derived stem cells (ASCs) hold a great promise for the therapy of osteoarthritis in veterinary medicine. Current therapy is an autologous, stromal vascular fraction. Allogeneic ASCs provide many advantages, including efficient, cost-effective treatments while eliminating a surgical procedure in a diseased animal. Cultured ASCs can be expanded and characterized, allowing selection of desirable qualities. Use of allogeneic ASCs requires selection of a culture medium that provides consistent, desirable cellular products. The supplements within a medium can greatly influence cellular phenotypes. We hypothesized that medium type influenced cellular phenotype, allowing selection of a specified cellular product for clinical applications. We evaluated ASCs derived from adipose tissue of six dogs, assessing mRNA expression of proinflammatory: interleukin-1b, cyclooxygenase-2, and anti-inflammatory mediators: tissue inhibitor metalloproteinase-2 and interleukin -1 receptor antagonist, via quantitative RT-PCR prior to, and following culture in five cell culture media: basic cell growth medium (BGM), Keratinocyte N acetyl-L-cysteine supplemented (KNAC) medium, Multipotent Adult Progenitor Cell (MAPC) medium, serum free medium (SFM) and xeno-free medium. Major histocompatability complex I (MHCI), major histocompatability complex II (MHCII), CD44 and CD90 immunophenotypes were assessed via flow cytometry analysis. Tri-lineage differentiation (bone, adipose and cartilage tissue) was utilized to verify multipotency. SFM and xeno-free culture conditions did not produce cell expansion sufficient to assess phenotype. ASCs prior to culture had wide variability in all mediator levels, while culturing in the remaining conditions resulted in more predictable expression levels of inflammatory mediators, with a decrease in all levels. Cultured ASCs retained expression of cell surface markers MHCI, CD44 and CD90, while decreasing MHCII expression levels. KNAC and MAPC medium conditions consistently produced tri-lineage differentation;BGM, SFM and xeno-free medium did not. Culture condition will influence phenotype of ASCs, and should be selected according to the intended therapeutic effect.

Human umbilical cord mesenchymal stem cells promote peripheral nerve repair via paracrine mechanisms

Human umbilical cord-derived mesenchymal stem cells （hUCMSCs） represent a promising young-state stem cell source for cell-based therapy. hUCMSC transplantation into the transected sciatic nerve promotes axonal regeneration and functional recovery. To further clarify the para-crine effects of hUCMSCs on nerve regeneration, we performed human cytokine antibody array analysis, which revealed that hUCMSCs express 14 important neurotrophic factors. Enzyme-linked immunosorbent assay and immunohistochemistry showed that brain-derived neurotrophic factor, glial-derived neurotrophic factor, hepatocyte growth factor, neurotrophin-3, basic fibroblast growth factor, type I collagen, fibronectin and laminin were highly expressed. Treatment with hUCMSC-conditioned medium enhanced Schwann cell viability and proliferation, increased nerve growth factor and brain-derived neurotrophic factor expression in Schwann cells, and enhanced neurite growth from dorsal root ganglion explants. These ifndings suggest that paracrine action may be a key mechanism underlying the effects of hUCMSCs in peripheral nerve repair.

Dental pulp stem cells stimulate neuronal differentiation of PC12 cells

Dental pulp stem cells(DPSCs) secrete neurotrophic factors which may play an important therapeutic role in neural development, maintenance and repair. To test this hypothesis, DPSCs-conditioned medium(DPSCs-CM) was collected from 72 hours serum-free DPSCs cultures. The impact of DPSCs-derived factors on PC12 survival, growth, migration and differentiation was investigated. PC12 cells were treated with nerve growth factor(NGF), DPSCs-CM or co-cultured with DPSCs using Transwell inserts for 8 days. The number of surviving cells with neurite outgrowths and the length of neurites were measured by image analysis. Immunocytochemical staining was used to evaluate the expression of neuronal markers NeuN, microtubule associated protein 2(MAP-2) and cytoskeletal marker βIII-tubulin. Gene expression levels of axonal growth-associated protein 43 and synaptic protein Synapsin-I, NeuN, MAP-2 and βIII-tubulin were analysed by quantitative polymerase chain reaction(qRT-PCR). DPSCs-CM was analysed for the neurotrophic factors(NGF, brain-derived neurotrophic factor [BDNF], neurotrophin-3, and glial cell-derived neurotrophic factor [GDNF]) by specific ELISAs. Specific neutralizing antibodies against the detected neurotrophic factors were used to study their exact role on PC12 neuronal survival and neurite outgrowth extension. DPSCs-CM significantly promoted cell survival and induced the neurite outgrowth confirmed by NeuN, MAP-2 and βIII-tubulin immunostaining. Furthermore, DPSCsCM was significantly more effective in stimulating PC12 neurite outgrowths than live DPSCs/PC12 co-cultures over the time studied. The morphology of induced PC12 cells in DPSCs-CM was similar to NGF positive controls;however, DPSCs-CM stimulation of cell survival was significantly higher than what was seen in NGF-treated cultures. The number of surviving PC12 cells treated with DPSCs-CM was markedly reduced by the addition of anti-GDNF, whilst PC12 neurite outgrowth was significantly attenuated by anti-NGF, anti-GDNF and anti-BDNF antibodies. These findings demonstrated that DPSCs were able to promote PC12 survival and differentiation. DPSCs-derived NGF, BDNF and GDNF were involved in the stimulatory action on neurite outgrowth, whereas GDNF also had a significant role in promoting PC12 survival. DPSCs-derived factors may be harnessed as a cell-free therapy for peripheral nerve repair. All experiments were conducted on dead animals that were not sacrificed for the purpose of the study. All the methods were carried out in accordance with Birmingham University guidelines and regulations and the ethical approval is not needed.

Mesenchymal stem cells and cell-free preparations for treating atopic dermatitis

Atopic dermatitis(AD)is a chronic cutaneous inflammatory disease caused by an interaction between genetic,immune and epidermal barrier factors.Several treatments can be used to treat this disease but there are patients that do not respond to actual drugs.So,there is a need to develop effective therapies for AD.Mesenchymal stem cells(MSCs)are non-hematopoietic multipotent adult progenitor cells with immunomodulatory power and self-regenerating capacity to repair tissue damage,so they could be a potential effective treatment for AD.MSCs-Conditioned Medium(CM)and MSCs-exosomes are cell-free preparation with molecules secreted by stem cells that could be also beneficial for AD.This viewpoint reviews the actual development of MSCs,MSCs-CM and MSCs-exosomes for treating patients with AD.

Induced pluripotent stem cells as suitable sensors for fibromyalgia and myalgic encephalomyelitis/chronic fatigue syndrome

BACKGROUND Fibromyalgia(FM)and myalgic encephalomyelitis/chronic fatigue syndrome(ME/CFS)are devastating metabolic neuroimmune diseases that are difficult to diagnose because of the presence of numerous symptoms and a lack of specific biomarkers.Despite patient heterogeneity linked to patient subgroups and variation in disease severity,anomalies are found in the blood and plasma of these patients when compared with healthy control groups.The seeming specificity of these“plasma factors”,as recently reported by Ron Davis and his group at Stanford University,CA,United States,and observations by our group,have led to the proposal that induced pluripotent stem cells(iPSCs)may be used as metabolic sensors for FM and ME/CFS,a hypothesis that is the basis for this indepth review.AIM To identify metabolic signatures in FM and/or ME/CFS supporting the existence of disease-associated plasma factors to be sensed by iPSCs.METHODS A PRISMA(Preferred Reported Items for Systematic Reviews and Meta-analysis)-based systematic review of the literature was used to select original studies evaluating the metabolite profiles of FM and ME/CFS body fluids.The MeSH terms“metabolomic”or“metabolites”in combination with FM and ME/CFS disease terms were screened against the PubMed database.Only original studies applying omics technologies,published in English,were included.The data obtained were tabulated according to the disease and type of body fluid analyzed.Coincidences across studies were searched and P-values reported by the original studies were gathered to document significant differences found in the disease groups.RESULTS Eighteen previous studies show that some metabolites are commonly altered in ME/CFS and FM body fluids.In vitro cell-based assays have the potential to be developed as screening platforms,providing evidence for the existence of factors in patient body fluids capable of altering morphology,differentiation state and/or growth patterns.Moreover,they can be further developed using approaches aimed at blocking or reversing the effects of specific plasma/serum factors seen in patients.The documented high sensitivity and effective responses of iPSCs to environmental cues suggests that these pluripotent cells could form robust,reproducible reporter systems of metabolic diseases,including ME/CFS and FM.Furthermore,culturing iPSCs,or their mesenchymal stem cell counterparts,in patient-conditioned medium may provide valuable information to predict individual outcomes to stem-cell therapy in the context of precision medicine studies.CONCLUSION This opinion review explains our hypothesis that iPSCs could be developed as a screening platform to provide evidence of a metabolic imbalance in FM and ME/CFS.

Low level of activin A secreted by fibroblast feeder cells accelerates early stage differentiation of retinal pigment epithelial cells from human pluripotent stem cells

Human pluripotent stem cells (hPSC) differentiated to retinal pigment epithelial cells (RPE) provide a promising tool for cell replacement therapies of retinal degenerative diseases. The in vitro differentiation of hPSC-RPE is still poorly understood and current differentiation protocols rely on spontaneous differentiation on fibroblast feeder cells or as floating cell aggregates in suspension. The fibroblast feeder cells may have an inductive effect on the hPSC-RPE differentiation, providing variable signals mimicking the extraocular mesenchyme that directs the differentiation in vivo. The effect of the commonly used fibroblast feeder cells on the hPSCRPE differentiation was studied by comparing suspension differentiation in standard RPEbasic (no bFGF) medium to RPEbasic medium conditioned with mouse embryonic (mEF-CM) and human foreskin (hFF-CM) fibroblast feeder cells. The fibroblast secreted factors were found to enhance early hPSC-RPE differentiation. The onset of pigmentation was faster in the conditioned media (CM) compared to RPEbasic for both human embryonic (hESC) and induced pluripotent (iPSC) stem cells, with the first pigments appearing around two weeks of differentiation. After four weeks of differentiation, CM conditions consistently contained higher number of pigmented cell aggregates. The ratio of PAX6 and MITF positive cells was quantified to be clearly higher in the CM conditions, with mEFCM containing most positive cells. The mEF cells were found to secrete low levels of activin A growth factor that is known to regulate eye field differentiation. As RPEbasic was supplemented with corresponding, low level (10 ng/ml) of recombinant human activin A, a clear increase in the hPSC-RPE differentiation was achieved. Thus, inductive effect provided by feeder cells was at least partially driven by activin A and could be substituted with a low level of recombinant growth factor in contrasts to previously reported much higher concentrations.

Ginsenoside-Rg1 combined with a conditioned medium from induced neuron-like hUCMSCs alleviated the apoptosis in a cell model of ALS through regulating the NF-κB/Bcl-2 pathway

Amyotrophic lateral sclerosis(ALS)is a fatal neurodegenerative disease affecting both upper and lower motor neurons in the brain and spinal cord.One important aspect of ALS pathogenesis is superoxide dismutase 1(SOD1)mutant-mediated mitochondrial toxicity,leading to apoptosis in neurons.This study aimed to evaluate the neural protective synergistic effects of ginsenosides Rg1(G-Rg1)and conditioned medium(CM)on a mutational SOD1 cell model,and to explore the underlying mechanisms.We found that the contents of nerve growth factor,glial cell line-derived neurotrophic factor,and brain-derived neurotrophic factor significantly increased in CM after human umbilical cord mesenchymal stem cells(hUCMSCs)were exposed to neuron differentiation reagents for seven days.CM or G-Rg1 decreased the apoptotic rate of SOD1^(G93A)-NSC34 cell to a certain extent,but their combination brought about the least apoptosis,compared with CM or G-Rg1 alone.Further research showed that the anti-apoptotic protein Bcl-2 was upregulated in all the treatment groups.Proteins associated with mitochondrial apoptotic pathways,such as Bax,caspase 9(Cas-9),and cytochrome c(Cyt c),were downregulated.Furthermore,CM or G-Rg1 also inhibited the activation of the nuclear factor-kappa B(NF-κB)signaling pathway by reducing the phosphorylation of p65 and IκBa.CM/G-Rg1 or their combination also reduced the apoptotic rate induced by betulinic acid(BetA),an agonist of the NF-κB signaling pathway.In summary,the combination of CM and G-Rg1 effectively reduced the apoptosis of SOD1^(G93A)-NSC34 cells through suppresing the NF-κB/Bcl-2 sgaling pathway(Fig.1 is a graphcal representation of the abstract).

人骨髓间充质干细胞通过YAP影响人脂肪肉瘤SW872细胞的生物学行为

目的:观察人骨髓间充质干细胞(hMSCs)条件培养基(CM)与人脂肪肉瘤SW872细胞共培养后对肿瘤细胞增殖和迁移能力的影响,探讨hMSCs CM对脂肪肉瘤细胞的作用及可能的作用机制。方法:体外培养hMSCs,采用慢病毒方法分别转染慢病毒空载体shNS(对照组)和慢病毒shRNA Yes相关蛋白(YAP)(shYAP-hMSCs组),采用实时荧光定量PCR(RT-qPCR)法和Western blotting法检测各组hMSCs中YAP mRNA和蛋白表达水平,提取CM。体外培养SW872细胞,分为对照组(正常培养)、hMSCs CM组和shYAP-hMSCs CM组。采用CCK-8法检测各组细胞增殖活性,流式细胞术检测各组细胞凋亡率,细胞划痕实验检测各组细胞划痕愈合率,Western blotting法检测各组细胞中YAP、基质金属蛋白酶9(MMP-9)和细胞周期蛋白D1(cyclin D1)蛋白表达水平。结果:与对照组比较,shYAP-hMSCs组hMSCs中YAP mRNA和蛋白表达水平降低(P<0.01),表明成功构建了shYAP-hMSCs稳定转染细胞株。CCK-8法,与对照组比较,hMSCs CM组SW872细胞增殖活性升高(P<0.05),shYAP-hMSCs CM组SW872细胞增殖活性降低(P<0.01);流式细胞术,与对照组比较,hMSCs CM组SW872细胞凋亡率无明显变化(P>0.05),shYAP-hMSCs CM组SW872细胞凋亡率升高(P<0.01);细胞划痕实验,与对照组比较,hMSCs CM组SW872细胞划痕愈合率升高(P<0.05),shYAP-hMSCs CM组SW872细胞划痕愈合率降低(P<0.01);Western blotting法,与对照组比较,hMSCs CM组SW872细胞中YAP、MMP-9和cyclin D1蛋白表达水平差异无统计学意义(P>0.05),shYAP-hMSCs组SW872细胞中YAP、MMP-9和cyclin D1蛋白表达水平降低(P<0.05或P<0.01)。结论:hMSCs参与调控人脂肪肉瘤SW872细胞增殖和迁移,其机制可能与YAP表达有关。

Effectiveness of mesenchymal stem cellconditioned medium in bone regeneration in animal and human models:a systematic review and meta-analysis

Background:Given the limitations of current therapies for the reconstruction of bone defects,regenerative medicine has arisen as a new therapeutic strategy along with mesenchymal stem cells(MSCs),which,because of their osteogenic potential and immunomodulatory properties,have emerged as a promising alternative for the treatment of bone injuries.In vivo studies have demonstrated that MSCs have a positive effect on regeneration due to their secretion of cytokines and growth factors that,when collected in conditioned medium(MSC-CM)and applied to an injured tissue,can modulate and promote the formation of new tissue.Objective:To evaluate the effectiveness of application of conditioned medium derived from mesenchymal stem cells in bone regeneration in animal and human models.Methods:We conducted a systematic review with a comprehensive search through February of 2018 using several electronic databases(MEDLINE,EMBASE,SCOPUS,CENTRAL(Ovid),and LILACS),and we also used the“snowballing technique”.Articles that met the inclusion criteria were selected through abstract review and subsequent assessment of the full text.We assessed the risk of bias with the SYRCLE and Cochrane tools,and three metaanalyses were performed.Results:We included 21 articles,19 of which used animal models and 2 of which used human models.In animal models,the application of MSC-CM significantly increased the regeneration of bone defects in comparison with control groups.Human studies reported early mineralization in regenerated bones,and no bone resorption,inflammation,nor local or systemic alterations were observed in any case.The meta-analysis showed an overall favorable effect of the application of MSC-CM.Conclusions:The application of MSC-CM to bone defects has a positive and favorable effect on the repair and regeneration of bone tissue,particularly in animal models.It is necessary to perform additional studies to support the application of MSC-CM in clinical practice.

间充质干细胞治疗皮瓣缺血再灌注损伤的作用机制及优势

背景:间充质干细胞因具有抗氧化、抑制炎症反应和诱导血管新生等作用而被用于皮瓣缺血再灌注损伤中。目的:综述间充质干细胞在治疗皮瓣缺血再灌注损伤方面的作用机制和最新进展,为其进一步理论研究和临床合理应用提供依据。方法:检索CNKI中国期刊全文数据库、万方数据库和PubMed数据库收录的相关文献。中文检索词为“间充质干细胞;皮瓣缺血再灌注损伤;条件培养基;外泌体;氧化应激;炎症反应;血管新生”。英文检索词为“mesenchymal stem cells;flap ischemia reperfusion injury;Conditioned medium;exosomes;Oxidative stress;Inflammatory reaction;Angiogenesis”。检索2010年以来相关文献,通过阅读文章剔除研究内容与文章主题关系不大、质量较差及内容陈旧文献,最终纳入74篇文献进行归纳总结。结果与结论:①间充质干细胞在抗氧化、抑制炎性反应及诱导血管新生等方面作用显著,在治疗皮瓣缺血再灌注损伤中蕴含着巨大潜力。②然而,间充质干细胞自身的缺陷及近年来治疗效果的下降使间充质干细胞的发展和应用陷入了瓶颈期,间充质干细胞条件培养基及其外泌体和间充质干细胞可塑性研究应运而生,且治疗效果明显优于单纯使用间充质干细胞。③因此,更全面地了解间充质干细胞治疗皮瓣缺血再灌注损伤的作用机制和最新治疗进展,对于间充质干细胞的研究和皮瓣缺血再灌注损伤的治疗都具有非常重要的意义。