Use Of Limiting Dilution Method For Isolation Of Rat Nucleuspulposus Mesenchymal Stem Cells And Effects On Biological Characteristics

BackgroundLow back pain(LBP)is a public health problem,with estimated annual health care system costs exceeding $100 billion in the United States.And degenerative disc disease(DDD)is one of the main causes of LBP.Despite the numbers of affected individuals,the pathogenic mechanisms contributing to DDD remain poorly understood.In recent years,endogenous stem cells have been isolated from almost all tissues and organs of the body.Stem cells play a key role in maintaining homeostasis and are now believed to serve as important disease-causing determinants.Hence,mesenchymal stem cells(MSCs)implantation has become a promising method for the treatment of DDD.Recently,mesenchymal stem cells were found in the nucleus pulposus(NP)and dubbed nucleus pulposus mesenchymal stem cells(NPMSCs).NPMSCs showed similar biological characteristics to MSCs.NPMSCs cultured in an intervertebral disc(IVD)mimicking microenvironments differentiate along a chondrogenic lineage,with a characteristic extracellular matrix secreted.Furthermore,under these conditions,NPMSCs showed a greater bioactivity when compared to adipose-derived MSCs,thus making NPMSCs a potential IVD regeneration therapy.While stem cells possess therapeutic potential,one of the largest limiting factors is isolating seed cells.Presently,there are several ways to select and purify stem cells in vitro,such as an attachment-based culturing method,fluorescent-based or magnetic-based cell sorting or density gradient centrifugation method.Among these methods,the best way to isolate NPMSCs remains unclear.ObjectivesThe aim of this study was to investigate the effect of cell densities on the biological characteristics and plasticity of NPMSCs,and to isolate and culture NPMSCs on high,medium and low cell seeding densities(low cell density group was also called limiting dilution method in other research).Thus,this study examined rat NPMSCs cultured in three different seeding densities and characterized differences in morphology,proliferative ability,migrative ability,stem cell biomarker expression(Sox2,Oct4 and Nanog),immunophenotyping and multi-lineage differentiation potential(osteogenic,chondrogenic and adipogenic).MethodsNPMSCs were isolated from Nucleus pulposus tissues of 12-week-old male Sprague-Dawley rats.The obtained cells were then classified into 3 groups according to plating density as follows:low densitygroup(LD,5 cells/cm2),medium densitygroup(MD,100 cells/cm2)and high density group(HD,10000 cells/cm2).NPMSC morphologies were observed and colony-forming units determined.The migration abilities,proliferative capacities,cell cycle percentages,multi-lineage differentiation capacities,stem cell biomarker expression levels and immunophenotyping were also examined in each group.ResultsNPMSCs from LD group had higher morphological homogeneity,a stronger colony-forming ability,a higher cell proliferation capacity and enhanced cell migration ability relative to the other two groups(p<0.05).Moreover,NPMSCs from LD group had more cells entering S phase,with fewer cells arrested in G0/G1 phase(p<0.05).While all three density groups showed a multi-lineage differentiation potential,the LD group showed a higher degree of observed and semi-quantified lineage specific staining(p<0.05).Furthermore,NPMSCs from LD group displayed higher expression levels of stem cell biomarkers(Nanog,Oct4 and Sox2)and showed higher percentages of CD44+ and CD90+ cells(p<0.05)following flow cytometry analysis.ConclusionsThe limiting dilution method and a cell plating densities of 5 cells/cm2 are suggested when isolating NPMSCs as a means of improving cell activity and plasticity.Therefore,this method can be chosen to isolate the NPMSCs and apply in tissue-engineering and regenerative medicine.