Expansion of human umbilical cord derived mesenchymal stem cells in regenerative medicine

BACKGROUND Stem cells are undifferentiated cells that possess the potential for self-renewal with the capacity to differentiate into multiple lineages.In humans,their limited numbers pose a challenge in fulfilling the necessary demands for the regeneration and repair of damaged tissues or organs.Studies suggested that mesenchymal stem cells(MSCs),necessary for repair and regeneration via transplantation,require doses ranging from 10 to 400 million cells.Furthermore,the limited expansion of MSCs restricts their therapeutic application.AIM To optimize a novel protocol to achieve qualitative and quantitative expansion of MSCs to reach the targeted number of cells for cellular transplantation and minimize the limitations in stem cell therapy protocols.METHODS Human umbilical cord(hUC)tissue derived MSCs were obtained and re-cultured.These cultured cells were subjected to the following evaluation pro-cedures:Immunophenotyping,immunocytochemical staining,trilineage differentiation,population doubling time and number,gene expression markers for proliferation,cell cycle progression,senescence-associatedβ-galactosidase assay,human telomerase reverse transcriptase(hTERT)expression,mycoplasma,cytomegalovirus and endotoxin detection.RESULTS Analysis of pluripotent gene markers Oct4,Sox2,and Nanog in recultured hUC-MSC revealed no significant differences.The immunophenotypic markers CD90,CD73,CD105,CD44,vimentin,CD29,Stro-1,and Lin28 were positively expressed by these recultured expanded MSCs,and were found negative for CD34,CD11b,CD19,CD45,and HLA-DR.The recultured hUC-MSC population continued to expand through passage 15.Proliferative gene expression of Pax6,BMP2,and TGFb1 showed no significant variation between recultured hUC-MSC groups.Nevertheless,a significant increase(P<0.001)in the mitotic phase of the cell cycle was observed in recultured hUC-MSCs.Cellular senescence markers(hTERT expression andβ-galactosidase activity)did not show any negative effect on recultured hUC-MSCs.Additionally,quality control assessments consistently confirmed the absence of mycoplasma,cytomegalovirus,and endotoxin contamination.CONCLUSION This study proposes the development of a novel protocol for efficiently expanding stem cell population.This would address the growing demand for larger stem cell doses needed for cellular transplantation and will significantly improve the feasibility of stem cell based therapies.

Zinc enhances the cell adhesion,migration,and self-renewal potential of human umbilical cord derived mesenchymal stem cells

BACKGROUND Zinc(Zn)is the second most abundant trace element after Fe,present in the human body.It is frequently reported in association with cell growth and proliferation,and its deficiency is considered to be a major disease contributing factor.AIM To determine the effect of Zn on in vitro growth and proliferation of human umbilical cord(hUC)-derived mesenchymal stem cells(MSCs).METHODS hUC-MSCs were isolated from human umbilical cord tissue and characterized based on immunocytochemistry,immunophenotyping,and tri-lineage differentiation.The impact of Zn on cytotoxicity and proliferation was determined by MTT and Alamar blue assay.To determine the effect of Zn on population doubling time(PDT),hUC-MSCs were cultured in media with and without Zn for several passages.An in vitro scratch assay was performed to analyze the effect of Zn on the wound healing and migration capability of hUC-MSCs.A cell adhesion assay was used to test the surface adhesiveness of hUC-MSCs.Transcriptional analysis of genes involved in the cell cycle,proliferation,migration,and selfrenewal of hUC-MSCs was performed by quantitative real-time polymerase chain reaction.The protein expression of Lin28,a pluripotency marker,was analyzed by immunocytochemistry.RESULTS Zn at lower concentrations enhanced the rate of proliferation but at higher concentrations(>100μM),showed concentration dependent cytotoxicity in hUC-MSCs.hUC-MSCs treated with Zn exhibited a significantly greater healing and migration rate compared to untreated cells.Zn also increased the cell adhesion rate,and colony forming efficiency(CFE).In addition,Zn upregulated the expression of genes involved in the cell cycle(CDC20,CDK1,CCNA2,CDCA2),proliferation(transforming growth factorβ1,GDF5,hypoxia-inducible factor 1α),migration(CXCR4,VCAM1,VEGF-A),and self-renewal(OCT4,SOX2,NANOG)of hUC-MSCs.Expression of Lin28 protein was significantly increased in cells treated with Zn.CONCLUSION Our findings suggest that zinc enhances the proliferation rate of hUC-MSCs decreasing the PDT,and maintaining the CFE.Zn also enhances the cell adhesion,migration,and self-renewal of hUC-MSCs.These results highlight the essential role of Zn in cell growth and development.

Towards Cache-Assisted Hierarchical Detection for Real-Time Health Data Monitoring in IoHT

Real-time health data monitoring is pivotal for bolstering road services’safety,intelligence,and efficiency within the Internet of Health Things(IoHT)framework.Yet,delays in data retrieval can markedly hinder the efficacy of big data awareness detection systems.We advocate for a collaborative caching approach involving edge devices and cloud networks to combat this.This strategy is devised to streamline the data retrieval path,subsequently diminishing network strain.Crafting an adept cache processing scheme poses its own set of challenges,especially given the transient nature of monitoring data and the imperative for swift data transmission,intertwined with resource allocation tactics.This paper unveils a novel mobile healthcare solution that harnesses the power of our collaborative caching approach,facilitating nuanced health monitoring via edge devices.The system capitalizes on cloud computing for intricate health data analytics,especially in pinpointing health anomalies.Given the dynamic locational shifts and possible connection disruptions,we have architected a hierarchical detection system,particularly during crises.This system caches data efficiently and incorporates a detection utility to assess data freshness and potential lag in response times.Furthermore,we introduce the Cache-Assisted Real-Time Detection(CARD)model,crafted to optimize utility.Addressing the inherent complexity of the NP-hard CARD model,we have championed a greedy algorithm as a solution.Simulations reveal that our collaborative caching technique markedly elevates the Cache Hit Ratio(CHR)and data freshness,outshining its contemporaneous benchmark algorithms.The empirical results underscore the strength and efficiency of our innovative IoHT-based health monitoring solution.To encapsulate,this paper tackles the nuances of real-time health data monitoring in the IoHT landscape,presenting a joint edge-cloud caching strategy paired with a hierarchical detection system.Our methodology yields enhanced cache efficiency and data freshness.The corroborative numerical data accentuates the feasibility and relevance of our model,casting a beacon for the future trajectory of real-time health data monitoring systems.

Wnt signaling pathway inhibitor promotes mesenchymal stem cells differentiation into cardiac progenitor cells in vitro and improves cardiomyopathy in vivo

BACKGROUND Cardiovascular diseases particularly myocardial infarction(MI)are the leading cause of mortality and morbidity around the globe.As cardiac tissue possesses very limited regeneration potential,therefore use of a potent small molecule,inhibitor Wnt production-4(IWP-4)for stem cell differentiation into cardiomyocytes could be a promising approach for cardiac regeneration.Wnt pathway inhibitors may help stem cells in their fate determination towards cardiomyogenic lineage and provide better homing and survival of cells in vivo.Mesenchymal stem cells(MSCs)derived from the human umbilical cord have the potential to regenerate cardiac tissue,as they are easy to isolate and possess multilineage differentiation capability.IWP-4 may promote the differentiation of MSCs into the cardiac lineage.AIM To evaluate the cardiac differentiation ability of IWP-4 and its subsequent in vivo effects.METHODS Umbilical cord tissue of human origin was utilized to isolate the MSCs which were characterized by their morphology,immunophenotyping of surface markers specific to MSCs,as well as by tri-lineage differentiation capability.Cytotoxicity analysis was performed to identify the optimal concentration of IWP-4.MSCs were treated with 5μM IWP-4 at two different time intervals.Differentiation of MSCs into cardiomyocytes was evaluated at DNA and protein levels.The MI ratmodel was developed.IWP-4 treated as well as untreated MSCs were implanted in the MI model,then the cardiac function was analyzed via echocardiography.MSCs were labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate(DiI)dye for tracking,while the regeneration of infarcted myocardium was examined by histology and immunohistochemistry.RESULTS MSCs were isolated and characterized.Cytotoxicity analysis showed that IWP-4 was non-cytotoxic at 5μM concentration.Cardiac specific gene and protein expression analyses exhibited more remarkable results in fourteen days treated group that was eventually selected for in vivo transplantation.Cardiac function was restored in the IWP-4 treated group in comparison to the MI group.Immunohistochemical analysis confirmed the homing of pre-differentiated MSCs that were labeled with DiI cell labeling dye.Histological analysis confirmed the significant reduction in fibrotic area,and improved left ventricular wall thickness in IWP-4 treated MSC group.CONCLUSION Treatment of MSCs with IWP-4 inhibits Wnt pathway and promotes cardiac differentiation.These pre-conditioned MSCs transplanted in vivo improved cardiac function by cell homing,survival,and differentiation at the infarcted region,increased left ventricular wall thickness,and reduced infarct size.

Effect of glycyrrhizic acid and 18β-glycyrrhetinic acid on the differentiation of human umbilical cord-mesenchymal stem cells into hepatocytes

BACKGROUND End-stage liver disease is a global health complication with high prevalence and limited treatment options.Cell-based therapies using mesenchymal stem cells(MSCs)emerged as an alternative approach to support hepatic regeneration.In vitro preconditioning strategies have been employed to strengthen the regenerative and differentiation potential of MSCs towards hepatic lineage.Chemical compounds of the triterpene class;glycyrrhizic acid(GA)and 18β-glycyrrhetinic acid(GT)possess diverse therapeutic properties including hepatoprotection and anti-fibrosis characteristics.They are capable of modulating several signaling pathways that are crucial in hepatic regeneration.Preconditioning with hepato-protective triterpenes may stimulate MSC fate transition towards hepatocytes.AIM To explore the effect of GA and GT on hepatic differentiation of human umbilical cord-MSCs(hUC-MSCs).METHODS hUC-MSCs were isolated and characterized phenotypically by flow cytometry and immunocytochemistry for the expression of MSC-associated surface molecules.Isolated cells were treated with GA,GT,and their combination for 24 h and then analyzed at three time points;day 7,14,and 21.qRT-PCR was performed for the expression of hepatic genes.Expression of hepatic proteins was analyzed by immunocytochemistry at day 21.Periodic acid Schiff staining was performed to determine the functional ability of treated cells.RESULTS The fusiform-shaped morphology of MSCs in the treatment groups in comparison with the untreated control,eventually progressed towards the polygonal morphology of hepatocytes with the passage of time.The temporal transcriptional profile of preconditioned MSCs displayed significant expression of hepatic genes with increasing time of differentiation.Preconditioned cells showed positive expression of hepatocyte-specific proteins.The results were further corroborated by positive periodic acid Schiff staining,indicating the presence of glycogen in their cytoplasm.Moreover,bi-nucleated cells,which is the typical feature of hepatocytes,were also seen in the preconditioned cells.CONCLUSION Preconditioning with glycyrrhizic acid,18β-glycyrrhetinic acid and their combination,successfully differentiates hUC-MSCs into hepatic-like cells.These MSCs may serve as a better therapeutic option for degenerative liver diseases in future.

Umbilical cord-derived mesenchymal stem cells preconditioned with isorhamnetin:potential therapy for burn wounds

BACKGROUND Impaired wound healing can be associated with different pathological states.Burn wounds are the most common and detrimental injuries and remain a major health issue worldwide.Mesenchymal stem cells(MSCs)possess the ability to regenerate tissues by secreting factors involved in promoting cell migration,proliferation and differentiation,while suppressing immune reactions.Preconditioning of MSCs with small molecules having cytoprotective properties can enhance the potential of these cells for their use in cell-based therapeutics.AIM To enhance the therapeutic potential of MSCs by preconditioning them with isorhamnetin for second degree burn wounds in rats.METHODS Human umbilical cord MSCs(hU-MSCs)were isolated and characterized by surface markers,CD105,vimentin and CD90.For preconditioning,hU-MSCs were treated with isorhamnetin after selection of the optimized concentration(5μmol/L)by cytotoxicity analysis.The migration potential of these MSCs was analyzed by the in vitro scratch assay.The healing potential of normal,and preconditioned hU-MSCs was compared by transplanting these MSCs in a rat model of a second degree burn wound.Normal,and preconditioned MSCs(IH+MSCs)were transplanted after 72 h of burn injury and observed for 2 wk.Histological and gene expression analyses were performed on day 7 and 14 after cell transplantation to determine complete wound healing.RESULTS The scratch assay analysis showed a significant reduction in the scratch area in the case of IH+MSCs compared to the normal untreated MSCs at 24 h,while complete closure of the scratch area was observed at 48 h.Histological analysis showed reduced inflammation,completely remodeled epidermis and dermis without scar formation and regeneration of hair follicles in the group that received IH+MSCs.Gene expression analysis was time dependent and more pronounced in the case of IH+MSCs.Interleukin(IL)-1β,IL-6 and Bcl-2 associated X genes showed significant downregulation,while transforming growth factorβ,vascular endothelial growth factor,Bcl-2 and matrix metallopeptidase 9 showed significant upregulation compared to the burn wound,showing increased angiogenesis and reduced inflammation and apoptosis.CONCLUSION Preconditioning of hU-MSCs with isorhamnetin decreases wound progression by reducing inflammation,and improving tissue architecture and wound healing.The study outcome is expected to lead to an improved cell-based therapeutic approach for burn wounds.

Regulating the fate of stem cells for regenerating the intervertebral disc degeneration

Lower back pain is a leading cause of disability and is one of the reasons for the substantial socioeconomic burden.The etiology of intervertebral disc(IVD)degeneration is complicated,and its mechanism is still not completely understood.Factors such as aging,systemic inflammation,biochemical mediators,toxic environmental factors,physical injuries,and genetic factors are involved in the progression of its pathophysiology.Currently,no therapy for restoring degenerated IVD is available except pain management,reduced physical activities,and surgical intervention.Therefore,it is imperative to establish regenerative medicine-based approaches to heal and repair the injured disc,repopulate the cell types to retain water content,synthesize extracellular matrix,and strengthen the disc to restore normal spine flexion.Cellular therapy has gained attention for IVD management as an alternative therapeutic option.In this review,we present an overview of the anatomical and molecular structure and the surrounding pathophysiology of the IVD.Modern therapeutic approaches,including proteins and growth factors,cellular and gene therapy,and cell fate regulators are reviewed.Similarly,small molecules that modulate the fate of stem cells for their differentiation into chondrocytes and notochordal cell types are highlighted.

Transcription regulators differentiate mesenchymal stem cells into chondroprogenitors,and their in vivo implantation regenerated the intervertebral disc degeneration

BACKGROUND Intervertebral disc degeneration(IVDD)is the leading cause of lower back pain.Disc degeneration is characterized by reduced cellularity and decreased production of extracellular matrix(ECM).Mesenchymal stem cells(MSCs)have been envisioned as a promising treatment for degenerative illnesses.Cell-based therapy using ECM-producing chondrogenic derivatives of MSCs has the potential to restore the functionality of the intervertebral disc(IVD).AIM To investigate the potential of chondrogenic transcription factors to promote differentiation of human umbilical cord MSCs into chondrocytes,and to assess their therapeutic potential in IVD regeneration.METHODS MSCs were isolated and characterized morphologically and immunologically by the expression of specific markers.MSCs were then transfected with Sox-9 and Six-1 transcription factors to direct differentiation and were assessed for chondrogenic lineage based on the expression of specific markers.These differentiated MSCs were implanted in the rat model of IVDD.The regenerative potential of transplanted cells was investigated using histochemical and molecular analyses of IVDs.RESULTS Isolated cells showed fibroblast-like morphology and expressed CD105,CD90,CD73,CD29,and Vimentin but not CD45 antigens.Overexpression of Sox-9 and Six-1 greatly enhanced the gene expression of transforming growth factor beta-1 gene,BMP,Sox-9,Six-1,and Aggrecan,and protein expression of Sox-9 and Six-1.The implanted cells integrated,survived,and homed in the degenerated intervertebral disc.Histological grading showed that the transfected MSCs regenerated the IVD and restored normal architecture.CONCLUSION Genetically modified MSCs accelerate cartilage regeneration,providing a unique opportunity and impetus for stem cell-based therapeutic approach for degenerative disc diseases.

Combination of mesenchymal stem cells and three-dimensional collagen scaffold preserves ventricular remodeling in rat myocardial infarction model

BACKGROUND Cardiovascular diseases are the major cause of mortality worldwide.Regeneration of the damaged myocardium remains a challenge due to mechanical constraints and limited healing ability of the adult heart tissue.Cardiac tissue engineering using biomaterial scaffolds combined with stem cells and bioactive molecules could be a highly promising approach for cardiac repair.Use of biomaterials can provide suitable microenvironment to the cells and can solve cell engraftment problems associated with cell transplantation alone.Mesenchymal stem cells(MSCs)are potential candidates in cardiac tissue engineering because of their multilineage differentiation potential and ease of isolation.Use of DNA methyl transferase inhibitor,such as zebularine,in combination with three-dimensional(3D)scaffold can promote efficient MSC differentiation into cardiac lineage,as epigenetic modifications play a fundamental role in determining cell fate and lineage specific gene expression.AIM To investigate the role of collagen scaffold and zebularine in the differentiation of rat bone marrow(BM)-MSCs and their subsequent in vivo effects.METHODS MSCs were isolated from rat BM and characterized morphologically,immunophenotypically and by multilineage differentiation potential.MSCs were seeded in collagen scaffold and treated with 3μmol/L zebularine in three different ways.Cytotoxicity analysis was done and cardiac differentiation was analyzed at the gene and protein levels.Treated and untreated MSC-seeded scaffolds were transplanted in the rat myocardial infarction(MI)model and cardiac function was assessed by echocardiography.Cell tracking was performed by DiI dye labeling,while regeneration and neovascularization were evaluated by histological and immunohistochemical analysis,respectively.RESULTS MSCs were successfully isolated and seeded in collagen scaffold.Cytotoxicity analysis revealed that zebularine was not cytotoxic in any of the treatment groups.Cardiac differentiation analysis showed more pronounced results in the type 3 treatment group which was subsequently chosen for the transplantation in the in vivo MI model.Significant improvement in cardiac function was observed in the zebularine treated MSC-seeded scaffold group as compared to the MI control.Histological analysis also showed reduction in fibrotic scar,improvement in left ventricular wall thickness and preservation of ventricular remodeling in the zebularine treated MSC-seeded scaffold group.Immunohistochemical analysis revealed significant expression of cardiac proteins in DiI labeled transplanted cells and a significant increase in the number of blood vessels in the zebularine treated MSC-seeded collagen scaffold transplanted group.CONCLUSION Combination of 3D collagen scaffold and zebularine treatment enhances cardiac differentiation potential of MSCs,improves cell engraftment at the infarcted region,reduces infarct size and improves cardiac function.

Overexpression of GATA binding protein 4 and myocyte enhancer factor 2C induces differentiation of mesenchymal stem cells into cardiac-like cells

BACKGROUND Heart diseases are the primary cause of death all over the world.Following myocardial infarction,billions of cells die,resulting in a huge loss of cardiac function.Stem cell-based therapies have appeared as a new area to support heart regeneration.The transcription factors GATA binding protein 4(GATA-4)and myocyte enhancer factor 2C(MEF2C)are considered prominent factors in the development of the cardiovascular system.AIM To explore the potential of GATA-4 and MEF2C for the cardiac differentiation of human umbilical cord mesenchymal stem cells(hUC-MSCs).METHODS hUC-MSCs were characterized morphologically and immunologically by the presence of specific markers of MSCs via immunocytochemistry and flow cytometry,and by their potential to differentiate into osteocytes and adipocytes.hUC-MSCs were transfected with GATA-4,MEF2C,and their combination to direct the differentiation.Cardiac differentiation was confirmed by semiquant itative real-time polymerase chain reaction and immunocytochemistry.RESULTS hUC-MSCs expressed specific cell surface markers CD105,CD90,CD44,and vimentin but lack the expression of CD45.The transcription factors GATA-4 and MEF2C,and their combination induced differentiation in hUC-MSCs with significant expression of cardiac genes i.e.,GATA-4,MEF2C,NK2 homeobox 5(NKX2.5),MHC,and connexin-43,and cardiac proteins GATA-4,NKX2.5,cardiac troponin T,and connexin-43.CONCLUSION Transfection with GATA-4,MEF2C,and their combination effectively induces cardiac differentiation in hUC-MSCs.These genetically modified MSCs could be a promising treatment option for heart diseases in the future.

大数据是个大谎言!

天又一次塌下来了。这一次是“大数据”让IT部门如临大敌。但是就像“世界末日”和“外星人”一样，“大数据”是虚构的，是一个“大谎言”。正如街谈巷议的传闻一样，不管你走到哪里，关于“大数据”的讨论无处不在。