Advances in the differentiation of pluripotent stem cells into vascular cells

Blood vessels constitute a closed pipe system distributed throughout the body,transporting blood from the heart to other organs and delivering metabolic waste products back to the lungs and kidneys.Changes in blood vessels are related to many disorders like stroke,myocardial infarction,aneurysm,and diabetes,which are important causes of death worldwide.Translational research for new appro-aches to disease modeling and effective treatment is needed due to the huge socio-economic burden on healthcare systems.Although mice or rats have been widely used,applying data from animal studies to human-specific vascular physiology and pathology is difficult.The rise of induced pluripotent stem cells(iPSCs)provides a reliable in vitro resource for disease modeling,regenerative medicine,and drug discovery because they carry all human genetic information and have the ability to directionally differentiate into any type of human cells.This review summarizes the latest progress from the establishment of iPSCs,the strategies for differentiating iPSCs into vascular cells,and the in vivo trans-plantation of these vascular derivatives.It also introduces the application of these technologies in disease modeling,drug screening,and regenerative medicine.Additionally,the application of high-tech tools,such as omics analysis and high-throughput sequencing,in this field is reviewed.

Non-enzymatic methods for isolation of stromal vascular fraction and adipose-derived stem cells:A systematic review

BACKGROUND Adipose-derived stem cells(ADSCs)and the stromal vascular fraction(SVF)have garnered substantial interest in regenerative medicine due to their potential to treat a wide range of conditions.Traditional enzymatic methods for isolating these cells face challenges such as high costs,lengthy processing time,and regulatory complexities.AIM This systematic review aimed to assess the efficacy and practicality of nonenzymatic,mechanical methods for isolating SVF and ADSCs,comparing these to conventional enzymatic approaches.METHODS Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines,a comprehensive literature search was conducted across multiple databases.Studies were selected based on inclusion criteria focused on non-enzymatic isolation methods for SVF and ADSCs from adipose tissue.The risk of bias was assessed,and a qualitative synthesis of findings was performed due to the methodological heterogeneity of the included studies.RESULTS Nineteen studies met the inclusion criteria,highlighting various mechanical techniques such as centrifugation,vortexing,and ultrasonic cavitation.The review identified significant variability in cell yield and viability,and the integrity of isolated cells across different non-enzymatic methods compared to enzymatic procedures.Despite some advantages of mechanical methods,including reduced processing time and avoidance of enzymatic reagents,the evidence suggests a need for optimization to match the cell quality and therapeutic efficacy achievable with enzymatic isolation.CONCLUSION Non-enzymatic,mechanical methods offer a promising alternative to enzymatic isolation of SVF and ADSCs,potentially simplifying the isolation process and reducing regulatory hurdles.However,further research is necessary to standardize these techniques and ensure consistent,high-quality cell yields for clinical applications.The development of efficient,safe,and reproducible non-enzymatic isolation methods could significantly advance the field of regenerative medicine.

Human umbilical cord mesenchymal stem cells derivedexosomes on VEGF-A in hypoxic-induced mice retinal astrocytes and mice model of retinopathy of prematurity

AIM:To observe the effect of human umbilical cord mesenchymal stem cells(hUCMSCs)secretions on the relevant factors in mouse retinal astrocytes,and to investigate the effect of hUCMSCs on the expression of vascular endothelial growth factor-A(VEGF-A)and to observe the therapeutic effect on the mouse model of retinopathy of prematurity(ROP).METHODS:Cultured hUCMSCs and extracted exosomes from them and then retinal astrocytes were divided into control group and hypoxia group.MTT assay,flow cytometry,reverse transcription-polymerase chain reaction(RT-PCR)and Western blot were used to detect related indicators.Possible mechanisms by which hUCMSCs exosomes affect VEGF-A expression in hypoxia-induced mouse retinal astrocytes were explored.At last,the efficacy of exosomes of UCMSCs in a mouse ROP model was explored.Graphpad6 was used to comprehensively process data information.RESULTS:The secretion was successfully extracted from the culture supernatant of hUCMSCs by gradient ultracentrifugation.Reactive oxygen species(ROS)and hypoxia inducible factor-1α(HIF-1α)of mice retinal astrocytes under different hypoxia time and the expression level of VEGF-A protein and VEGF-A mRNA increased,and the ROP cell model was established after 6h of hypoxia.The secretions of medium and high concentrations of hUCMSCs can reduce ROS and HIF-1α,the expression levels of VEGF-A protein and VEGF-A mRNA are statistically significant and concentration dependent.Compared with the ROP cell model group,the expression of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)signal pathway related factors in the hUCMSCs exocrine group is significantly decreased.The intravitreal injection of the secretions of medium and high concentrations of hUCMSCs can reduce VEGF-A and HIF-1αin ROP model tissues.HE staining shows that the number of retinal neovascularization in ROP mice decreases with the increase of the dose of hUCMSCs secretion.CONCLUSION:In a hypoxia induced mouse retinal astrocyte model,hUCMSCs exosomes are found to effectively reduce the expression of HIF-1αand VEGF-A,which are positively correlated with the concentration of hUCMSCs exosomes.HUCMSCs exosomes can effectively reduce the number of retinal neovascularization and the expression of HIF-1αand VEGF-A proteins in ROP mice,and are positively correlated with drug dosage.Besides,they can reduce the related factors on the PI3K/AKT/mTOR signaling pathway.

Potential plausible role of Wharton’s jelly mesenchymal stem cells for diabetic bone regeneration

This letter addresses the review titled“Wharton’s jelly mesenchymal stem cells:Future regenerative medicine for clinical applications in mitigation of radiation injury”.The review highlights the regenerative potential of Wharton’s jelly mesenchymal stem cells(WJ-MSCs)and describes why WJ-MSCs will become one of the most probable stem cells for future regenerative medicine.The potential plausible role of WJ-MSCs for diabetic bone regeneration should be noticeable,which will provide a new strategy for improving bone regeneration under diabetic conditions.

Adipose-derived stem cells and knee osteoarthritis:New perspectives,old concerns

In this editorial,we comment on the paper by Muthu et al published in the recent issue of the journal.This editorial review focusses on the use of adipose-derived stem cells(ADSCs)in knee osteoarthritis treatment.We discuss the differences between the stromal vascular fraction and microfragmented adipose tissue and highlight the results of clinical studies comparing both treatments and the use of hyaluronic acid,platelet-rich plasma,and bone marrow aspirate concentrate.The use of expanded ADSCs is also discussed;moreover,concerns regarding treatment with ADSCs,particularly the heterogeneity of published studies and the need to standardize protocols to explore clinical potential is explored.

Early expressions of hypoxia-inducible factor 1alpha and vascular endothelial growth factor increase the neuronal plasticity of activated endogenous neural stem cells after focal cerebral ischemia

Endogenous neural stem cells become ＂activated＂ after neuronal injury, but the activation sequence and fate of endogenous neural stem cells in focal cerebral ischemia model are little known. We evaluated the relationships between neural stem cells and hypoxia-inducible factor-1α and vascular endothelial growth factor expression in a photothromobotic rat stroke model using immunohistochemistry and western blot analysis. We also evaluated the chronological changes of neural stem cells by 5-bromo-2′-deoxyuridine（BrdU） incorporation. Hypoxia-inducible factor-1α expression was initially increased from 1 hour after ischemic injury, followed by vascular endothelial growth factor expression. Hypoxia-inducible factor-1α immunoreactivity was detected in the ipsilateral cortical neurons of the infarct core and peri-infarct area. Vascular endothelial growth factor immunoreactivity was detected in bilateral cortex, but ipsilateral cortex staining intensity and numbers were greater than the contralateral cortex. Vascular endothelial growth factor immunoreactive cells were easily found along the peri-infarct area 12 hours after focal cerebral ischemia. The expression of nestin increased throughout the microvasculature in the ischemic core and the peri-infarct area in all experimental rats after 24 hours of ischemic injury. Nestin immunoreactivity increased in the subventricular zone during 12 hours to 3 days, and prominently increased in the ipsilateral cortex between 3–7 days. Nestin-labeled cells showed dual differentiation with microvessels near the infarct core and reactive astrocytes in the peri-infarct area. BrdU-labeled cells were increased gradually from day 1 in the ipsilateral subventricular zone and cortex, and numerous BrdU-labeled cells were observed in the peri-infarct area and non-lesioned cortex at 3 days. BrdU-labeled cells rather than neurons, were mainly co-labeled with nestin and GFAP. Early expressions of hypoxia-inducible factor-1α and vascular endothelial growth factor after ischemia made up the microenvironment to increase the neuronal plasticity of activated endogenous neural stem cells. Moreover, neural precursor cells after large-scale cortical injury could be recruited from the cortex nearby infarct core and subventricular zone.

Transplantation of vascular endothelial growth factor-modified neural stem/progenitor cells promotes the recovery of neurological function following hypoxic-ischemic brain damage

Neural stem/progenitor cell （NSC） transplantation has been shown to effectively improve neurological function in rats with hypoxic-isch- emic brain damage. Vascular endothelial growth factor （VEGF） is a signaling protein that stimulates angiogenesis and improves neural regeneration. We hypothesized that transplantation of VEGF-transfected NSCs would alleviate hypoxic-ischemic brain damage in neo- natal rats. We produced and transfected a recombinant lentiviral vector containing the VEGF165gene into cultured NSCs. The transfected NSCs were transplanted into the left sensorimotor cortex of rats 3 days after hypoxic-ischemic brain damage. Compared with the NSCs group, VEGF mRNA and protein expression levels were increased in the transgene NSCs group, and learning and memory abilities were significantly improved at 30 days. Furthermore, histopathological changes were alleviated in these animals. Our findings indicate that transplantation of VEGF-transfected NSCs may facilitate the recovery of neurological function, and that its therapeutic effectiveness is better than that of unmodified NSCs.

Response of the sensorimotor cortex of cerebral palsy rats receiving transplantation of vascular endothelial growth factor 165-transfected neural stem cells

Neural stem cells are characterized by the ability to differentiate and stably express exogenous ge- nes. Vascular endothelial growth factor plays a role in protecting local blood vessels and neurons of newborn rats with hypoxic-ischemic encephalopathy. Transplantation of vascular endothelial growth factor-transfected neural stem cells may be neuroprotective in rats with cerebral palsy. In this study, 7-day-old Sprague-Dawley rats were divided into five groups： （1） sham operation （control）, （2） cerebral palsy model alone or with （3） phosphate-buffered saline, （4） vascular en- dothelial growth factor 165 ＋ neural stem cells, or （5） neural stem cells alone. The cerebral palsy model was established by ligating the left common carotid artery followed by exposure to hypox- ia. Phosphate-buffered saline, vascular endothelial growth factor ＋ neural stem cells, and neural stem cells alone were administered into the sensorimotor cortex using the stereotaxic instrument and microsyringe. After transplantation, the radial-arm water maze test and holding test were performed. Immunohistochemistry for vascular endothelial growth factor and histology using hematoxylin-eosin were performed on cerebral cortex. Results revealed that the number of vas- cular endothelial growth factor-positive cells in cerebral palsy rats transplanted with vascular endothelial growth factor-transfected neural stem cells was increased, the time for finding water and the finding repetitions were reduced, the holding time was prolonged, and the degree of cell degeneration or necrosis was reduced. These findings indicate that the transplantation of vascu- lar endothelial growth factor-transfected neural stem cells alleviates brain damage and cognitive deficits, and is neuroprotective in neonatal rats with hypoxia ischemic-mediated cerebral palsy.

The interaction of stem cells and vascularity in peripheral nerve regeneration

The degree of nerve regeneration after peripheral nerve injury can be altered by the microenvironment at the site of injury. Stem cells and vascularity are postulated to be a part of a complex pathway that enhances peripheral nerve regeneration;however, their interaction remains unexplored. This review aims to summarize current knowledge on this interaction, including various mechanisms through which trophic factors are promoted by stem cells and angiogenesis. Angiogenesis after nerve injury is stimulated by hypoxia, mediated by vascular endothelial growth factor, resulting in the growth of preexisting vessels into new areas. Modulation of distinct signaling pathways in stem cells can promote angiogenesis by the secretion of various angiogenic factors. Simultaneously, the importance of stem cells in peripheral nerve regeneration relies on their ability to promote myelin formation and their capacity to be influenced by the microenvironment to differentiate into Schwann-like cells. Stem cells can be acquired through various sources that correlate to their differentiation potential, including embryonic stem cells, neural stem cells, and mesenchymal stem cells. Each source of stem cells serves its particular differentiation potential and properties associated with the promotion of revascularization and nerve regeneration. Exosomes are a subtype of extracellular vesicles released from cell types and play an important role in cell-to-cell communication. Exosomes hold promise for future transplantation applications, as these vesicles contain fewer membrane-bound proteins, resulting in lower immunogenicity. This review presents pre-clinical and clinical studies that focus on selecting the ideal type of stem cell and optimizing stem cell delivery methods for potential translation to clinical practice. Future studies integrating stem cell-based therapies with the promotion of angiogenesis may elucidate the synergistic pathways and ultimately enhance nerve regeneration.

Therapeutic effects of dental pulp stem cells on vascular dementia in rat models

Dental pulp stem cells are a type of adult stem cells with strong proliferative ability and multi-differentiation potential. There are no studies on treatment of vascular dementia with dental pulp stem cells. In the present study, rat models of vascular dementia were established by two-vessel occlusion, and 30 days later, rats were injected with 2 × 10^(7) dental pulp stem cells via the tail vein. At 70 days after vascular dementia induction, dental pulp stem cells had migrated to the brain tissue of rat vascular dementia models and differentiated into neuronlike cells. At the same time, doublecortin, neurofilament 200, and Neu N m RNA and protein expression levels in the brain tissue were increased, and glial fibrillary acidic protein m RNA and protein expression levels were decreased. Behavioral testing also revealed that dental pulp stem cell transplantation improved the cognitive function of rat vascular dementia models. These findings suggest that dental pulp stem cell transplantation is effective in treating vascular dementia possibly through a paracrine mechanism. The study was approved by the Animal Ethics Committee of Harbin Medical University(approval No. KY2017-132) in 2017.

Effects of bone marrow-derived mesenchymal stemcells engraftment on vascular endothelial cell growthfactor in lung tissue and plasma at early stage of smoke inhalation injury

BACKGROUND： This study was undertaken to determine the effect of mesenchymal stem cells （MSCs） engraftment on vascular endothelial cell growth factor （VEGF） in lung tissue, plasma and extravascular lung water at early stage of smoke inhalation injury.METHODS： A rabbit smoke inhalation injury model was established using a home-made smoke inhalation injury generator, and rabbits were divided into two groups randomly： a control group （S group, n=32） and a MSCs treatment group （M group, n=32）. 10 ml PBS was injected via the ear marginal vein immediately at injury into the S group. Third generation MSCs with a concentration of 1×107/10 ml PBS were injected via the ear marginal vein immediately at injury into the M group. VEGF in peripheral blood and lung tissue were measured at 0 （baseline）, 2, 4 and 6 hours after injection respectively and analyzed. The right lungs of rabbits were taken to measure lung water mass fraction.RESULTS： In the lung tissue, VEGF decreased gradually in the S group （P〈0.05） and signi？ cantly decreased in the M group （P〈0.05）, but it increased more signi？ cantly than the values at the corresponding time points （P〈0.05）. In peripheral blood, VEGF increased gradually in the S group （P〈0.05） and markedly increased in the M group （P〈0.05）, but it decreased more signi？ cantly than the values at corresponding time points （P〈0.05）.CONCLUSION： MSCs engraftment to smoke inhalation injury could increase VEGF in lung tissue, decrease VEGF in plasma and reduce extravascular lung water, indicating its protective effect on smoke inhalation injury.

Mechanoresponse of stem cells for vascular repair

Stem cells have shown great potential in vascular repair.Numerous evidence indicates that mechanical forces such as shear stress and cyclic strain can regulate the adhesion,proliferation,migration,and differentiation of stem cells via serious signaling pathways.The enrichment and differentiation of stem cells play an important role in the angiogenesis and maintenance of vascular homeostasis.In normal tissues,blood flow directly affects the microenvironment of vascular endothelial cells(ECs);in pathological status,the abnormal interactions between blood flow and vessels contribute to the injury of vessels.Next,the altered mechanical forces are transduced into cells by mechanosensors to trigger the reformation of vessels.This process occurs when signaling pathways related to EC differentiation are initiated.Hence,a deep understanding of the responses of stem cells to mechanical stresses and the underlying mechanisms involved in this process is essential for clinical translation.In this the review,we provide an overview of the role of stem cells in vascular repair,outline the performance of stem cells under the mechanical stress stimulation,and describe the related signaling pathways.

Vascularization and osteogenesis in ectopically implanted bone tissue-engineered constructs with endothelial and osteogenic differentiated adipose-derived stem cells

BACKGROUND A major problem in the healing of bone defects is insufficient or absent blood supply within the defect.To overcome this challenging problem,a plethora of approaches within bone tissue engineering have been developed recently.Bearing in mind that the interplay of various diffusible factors released by endothelial cells(ECs)and osteoblasts(OBs)have a pivotal role in bone growth and regeneration and that adjacent ECs and OBs also communicate directly through gap junctions,we set the focus on the simultaneous application of these cell types together with platelet-rich plasma(PRP)as a growth factor reservoir within ectopic bone tissue engineering constructs.AIM To vascularize and examine osteogenesis in bone tissue engineering constructs enriched with PRP and adipose-derived stem cells(ASCs)induced into ECs and OBs.METHODS ASCs isolated from adipose tissue,induced in vitro into ECs,OBs or just expanded were used for implant construction as followed:BPEO,endothelial and osteogenic differentiated ASCs with PRP and bone mineral matrix;BPUI,uninduced ASCs with PRP and bone mineral matrix;BC(control),only bone mineral matrix.At 1,2,4 and 8 wk after subcutaneous implantation in mice,implants were extracted and endothelial-related and bone-related gene expression were analyzed,while histological analyses were performed after 2 and 8 wk.RESULTS The percentage of vascularization was significantly higher in BC compared to BPUI and BPEO constructs 2 and 8 wk after implantation.BC had the lowest endothelial-related gene expression,weaker osteocalcin immunoexpression and Spp1 expression compared to BPUI and BPEO.Endothelial-related gene expression and osteocalcin immunoexpression were higher in BPUI compared to BC and BPEO.BPEO had a higher percentage of vascularization compared to BPUI and the highest CD31 immunoexpression among examined constructs.Except Vwf,endothelial-related gene expression in BPEO had a later onset and was upregulated and well-balanced during in vivo incubation that induced late onset of Spp1 expression and pronounced osteocalcin immunoexpression at 2 and 8 wk.Tissue regression was noticed in BPEO constructs after 8 wk.CONCLUSION Ectopically implanted BPEO constructs had a favorable impact on vascularization and osteogenesis,but tissue regression imposed the need for discovering a more optimal EC/OB ratio prior to considerations for clinical applications.

Mesenchymal stem cell-derived exosomes inhibit the VEGF-A expression in human retinal vascular endothelial cells induced by high glucose

AIM:To determine the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells(hUCMSCs)on the expression of vascular endothelial growth factor A(VEGF-A)in human retinal vascular endothelial cells(HRECs).METHODS:Exosomes were isolated from hUCMSCs using cryogenic ultracentrifugation and characterized by transmission electron microscopy,Western blotting and nanoparticle tracking analysis.HRECs were randomly divided into a normal control group(group A),a high glucose model group(group B),a high glucose group with 25μg/mL(group C),50μg/mL(group D),and 100μg/mL exosomes(group E).Twenty-four hours after coculture,the cell proliferation rate was detected using flow cytometry,and the VEGF-A level was detected using immunofluorescence.After coculture 8,16,and 24h,the expression levels of VEGF-A in each group were detected using PCR and Western blots.RESULTS:The characteristic morphology(membrane structured vesicles)and size(diameter between 50 and 200 nm)were observed under transmission electron microscopy.The average diameter of 122.7 nm was discovered by nanoparticle tracking analysis(NTA).The exosomal markers CD9,CD63,and HSP70 were strongly detected.The proliferation rate of the cells in group B increased after 24h of coculture.Immunofluorescence analyses revealed that the upregulation of VEGF-A expression in HRECs stimulated by high glucose could be downregulated by cocultured hUCMSC-derived exosomes(F=39.03,P<0.01).The upregulation of VEGF-A protein(group C:F=7.96;group D:F=17.29;group E:F=11.89;8h:F=9.45;16h:F=12.86;24h:F=42.28,P<0.05)and mRNA(group C:F=4.137;group D:F=13.64;group E:F=22.19;8h:F=7.253;16h:F=16.98;24h:F=22.62,P<0.05)in HRECs stimulated by high glucose was downregulated by cocultured hUCMSC-derived exosomes(P<0.05).CONCLUSION:hUCMSC-derived exosomes downregulate VEGF-A expression in HRECs stimulated by high glucose in time and concentration dependent manner.

MicroRNA-146a Promotes Embryonic Stem Cell Differentiation towards Vascular Smooth Muscle Cells through Regulation of Kruppel-like Factor 4

Objective Vascular smooth muscle cell(VSMC)differentiation from stem cells is one source of the increasing number of VSMCs that are involved in vascular remodeling-related diseases such as hypertension,atherosclerosis,and restenosis.MicroRNA-146a(miR-146a)has been proven to be involved in cell proliferation,migration,and tumor metabolism.However,little is known about the functional role of miR-146a in VSMC differentiation from embryonic stem cells(ESCs).This study aimed to determine the role of miR-146a in VSMC differentiation from ESCs.Methods Mouse ESCs were differentiated into VSMCs,and the cell extracts were analyzed by Western blotting and RT-qPCR.In addition,luciferase reporter assays using ESCs transfected with miR-146a/mimic and plasmids were performed.Finally,C57BL/6J female mice were injected with mimic or miR-146a-overexpressing ESCs,and immunohistochemistry,Western blotting,and RT-qPCR assays were carried out on tissue samples from these mice.Results miR-146a was significantly upregulated during VSMC differentiation,accompanied with the VSMC-specific marker genes smooth muscle-alpha-actin(SMαA),smooth muscle 22(SM22),smooth muscle myosin heavy chain(SMMHC),and h1-calponin.Furthermore,overexpression of miR-146a enhanced the differentiation process in vitro and in vivo.Concurrently,the expression of Kruppel-like factor 4(KLF4),predicted as one of the top targets of miR-146a,was sharply decreased in miR-146a-overexpressing ESCs.Importantly,inhibiting KLF4 expression enhanced the VSMC-specific gene expression induced by miR-146a overexpression in differentiating ESCs.In addition,miR-146a upregulated the mRNA expression levels and transcriptional activity of VSMC differentiation-related transcription factors,including serum response factor(SRF)and myocyte enhancer factor 2c(MEF-2c).Conclusion Our data support that miR-146a promotes ESC-VSMC differentiation through regulating KLF4 and modulating the transcription factor activity of VSMCs.

Lentiviral-mediated vascular endothelial growth factor 165 gene transfer into neural stem cells promotes proliferation

We constructed a lentiviral vector carrying vascular endothelial growth factor 165, which was used to transfect neural stem cells. The transfection rate was approximately 50%, as determined by flow cytometry. Vascular endothelial growth factor protein was detected in neural stem cells and promoted proliferation.

Adipose tissue-derived stem cells as a therapeutic tool for cardiovascular disease

Adipose tissue-deried stem cells( ADSCs) are adult stem cells that can be easily harvested from subcutaneous adipose tissue. Many studies have demonstrated that ADSCs differentiate into vascular endothelial cells(VECs), vascular smooth muscle cells(VSMCs), and cardiomyocytes in vitro and in vivo. However, ADSCs may fuse with tissue-resident cells and obtain the corresponding characteristics of those cells. If fusion occurs, ADSCs may express markers of VECs, VSMCs, and cardiomyocytes without direct differentiation into these cell types. ADSCs also produce a variety of paracrine factors such as vascular endothelial growth factor, hepatocyte growth factor, and insulin-like growth factor-1 that have proangiogenic and/or antiapoptotic activities. Thus, ADSCs have the potential to regenerate the cardiovascular system via direct differentiation into VECs, VSMCs, and cardiomyocytes, fusion with tissueresident cells, and the production of paracrine factors. Numerous animal studies have demonstrated the efficacy of ADSC implantation in the treatment of acute myocardial infarction(AMI), ischemic cardiomyopathy(ICM), dilated cardiomyopathy, hindlimb ischemia, and stroke. Clinical studies regarding the use of autologous ADSCs for treating patients with AMI and ICM have recently been initiated. ADSC implantation has been reported as safe and effective so far. Therefore, ADSCs appear to be useful for the treatment of cardiovascular disease. However, the tumorigenic potential of ADSCs requires careful evaluation before their safe clinical application.

Inhibition of VEGF-A expression in hypoxia-exposed fetal retinal microvascular endothelial cells by exosomes derived from human umbilical cord mesenchymal stem cells

Objective:This study aimed to investigate the potential of human umbilical cord mesenchymal stem cell(hucMSC)-derived exosomes(hucMSC-Exos)in inhibiting hypoxia-induced cell hyper proliferation and overexpression of vascular endothelial growth factor A(VEGF-A)in immature human fetal retinal microvascular endothelial cells(hfRMECs).Methods:Exosomes were isolated from hucMSCs using cryogenic ultracentrifugation and characterized through various techniques,including transmission electron microscopy,nanoparticle tracking analysis,bicinchoninic acid assays,and western blotting.The hfRMECs were identified using von Willebrand factor(vWF)co-staining and divided into four groups:a control group cultured under normoxic condition,a hypoxic model group,a hypoxic group treated with low-concentration hucMSC-Exos(75μg/mL)and a hypoxic group treated with high-concentration hucMSC-Exos(100μg/mL).Cell viability and proliferation were assessed using Cell Counting Kit-8(CCK-8)assay and EdU(5-ethynyl-2′-deoxyuridine)assay respectively.Expression levels of VEGF-A were evaluated using RT-PCR,western blotting and immunofluorescence.Results:Hypoxia significantly increased hfRMECs’viability and proliferation by upregulating VEGF-A levels.The administration of hucMSC-Exos effectively reversed this response,with the high-concentration group exhibiting greater efficacy compared to the lowconcentration group.Conclusion:In conclusion,hucMSC-Exos can dose-dependently inhibit hypoxia-induced hyperproliferation and VEGF-A overexpression in immature fetal retinal microvascular endothelial cells.

Effects of Hydrocortisone, Glycerophosphate and Retinol on the Differentiation of Mesenchymal Stem Cells and Vascular Endothelial Cells to Osteoblasts

Vascular calcification, which causes occlusion and rupture of the vascular, is often observed in patients in the advanced stages of arteriosclerosis. One of the best procedures for inhibiting the accumulation of vascular calcification is to obstruct the differentiation of mesenchymal stem cells (MSCs) and/or vascular endothelial cells (VECs) in the vascular to osteoblasts. In this study, we evaluated the biochemical and genetic characteristics of the process of differentiation of MSCs and VECs to osteoblasts. C3H10T1/2 MSCs, TKD2 VECs and MC3T3-E1 preosteoblasts (POBs) were cultured in medium containing both hydrocortisone and glycerophosphate. These compounds showed strong effects promoting the differentiation of VECs as well as POBs, although the effect was weak in the MSCs. Moreover, C3H10T1/2 MSCs and TKD2 VECs were cultured in medium containing 10 mM retinol, after which the alkali phosphatase (ALP) activity of the MSCs and production of calcified nodules of TKD2 were significantly increased, whereas the marker genes for the osteoblasts were not. These results suggest that retinol does not have an effect in inducing the differentiation of VECs to osteoblasts, but rather exhibits a strong promoting effect on differentiation.

Application of vascular endothelial cells in stem cell medicine

Stem cell medicine is gaining momentum in the development of therapy for various end-stage diseases.The search for new seed cells and exploration of their application prospects are topics of interest in stem cell medicine.In recent years,vascular endothelial cells(VECs)have attracted wide attention from scholars.VECs,which form the inner lining of blood vessels,are critically involved in many physiological functions,including permeability,angiogenesis,blood pressure regulation,immunity,and pathological development,such as atherosclerosis and malignant tumors.VECs have significant therapeutic effects and broad application prospects in stem cell medicine for the treatment of various refractory diseases,including atherosclerosis,myocardial infarction,diabetic complications,hypertension,coronavirus disease 2019,and malignant tumors.On the one hand,VECs and their extracellular vesicles can be directly used for the treatment of these diseases.On the other hand,VECs can be used as therapeutic targets for some diseases.However,there are still some obstacles to the use of VECs in stem cell medicine.In this review,advances in the applications and challenges that come with the use of these cells are discussed.