Comparison Of Multi-source Derived Mesenchymal Stem Cells And Screening Of Long Non-coding RNAs Relate To Osteogenic Differentiation For Bone Regeneration

Bone is a unique organ with self-repairing and regenerative capacity.Post-natal bone retains its inherent ability to grow and remodel in response to mechanical stimuli and regenerate after injury.However,if severe trauma or systemic disease,pathological fractures,infection of the bone and/or surrounding tissue,or impaired blood supply,the ability to adequately self-heal may be lost,which may result in permanent bone defects.Thereby the function of the relevant part of the affected individual is lost.In addition,this ability to regenerate will decline with aging,as reflected in osteoporosis.The advancement of medicine in the past century has led to an increase in population aging which makes this situation more and more serious.In the oral and maxillofacial region,the defect of the bone caused by congenital malformation,trauma,tumor,inflammation,etc.not only causes facial deformity,but also causes disorders of oral function such as language,chewing,and breathing,and gives the patient a lot of adverse effects on physiological and psychological aspects.Therefore,the treatment of patients with musculoskeletal diseases has been highly valued by clinicians,and the pursuit of new treatment methods is also the direction of clinical researchers.Tissue engineering is a new subject emerging from the end of the 20th century.It integrates bioengineering,cell biology,molecular biology and materials science together and proposes a new feature for the functional repair and reconstruction of bone defects.Moreover,with the deepening of the larger concept of tissue engineering and regenerative medicine,it has become an irresistible trend to restore the missing structure and function of patients by regenerative treatment in vivo or in vitro.May be the most important component in this field is the seed cells with multipotential differentiation potential.As candidates for seed cells,various stem cells have been included in the research of scholars.To date,stem cells have been identified from three main sources:embryonic stem cells,adult stem cells,and pluripotent stem cells induced by genetic manipulation.Among them,adult stem cells have become the mainstream cells in the application of regenerative medicine because of their safety,low ethical controversy and the possibility to be autologous transplanted.Mesenchymal stem cells(MSCs)are part of adult stem cells and hold great promise for tissue regeneration in clinical applications owing to their availability,high proliferative capacity and multipotency.MSCs can be obtained from a variety of tissues and organs of the body and have been shown to existing differences in their properties.However,the developmental relationships between these different MSCs populations have not yet been determined.Therefore,which kind of MSCs should be selected from the large number of stem cells as the seed cells for bone regeneration are inconclusive.On the other hand,the regulation mechanism of cell-directed osteogenic differentiation is also a fundamental problem in the process of bone regeneration.Many factors regulate the expression of genes and proteins in cells through classical genetic or epigenetic mechanisms and thus affecting the various biological behaviors of the cells.With the continuous research on gene regulation,it has been found that various regulatory methods are emerging in the cells.One of the new aspects is the regulation of gene expression by non-coding RNAs which accounts for more than 95%of the transcriptome.Long non-coding RNA(IncRNA)is a type of RNA that does not encode protein with a transcript of more than 200 nucleotides in length,and is the least well-understood species.Recent studies have shown that IncRNA has spatio-temporal specific expression at different stages of ontogenesis and owns a regulatory role at various levels of gene expression in a variety of mechanisms.Many studies have also shown that it is involved in the regulation of osteogenic differentiation of MSCs.But the researches are still in their infancy.In the present study,we isolated and cultured MSCs from various tissue origins and compared the proliferation,apoptosis resistance and osteogenic differentiation ability of them to providing reference for selecting suitable seed cells for bone tissue regeneration.Secondly,the expression profiles of mRNAs and lncRNAs of periodontal ligament Stem cells(PDLSCs)and gingival stem cells(GMSCs)during osteogenic induction were detected respectively through high throughput microarray technology to explore the role of lncRNA in stem cell differentiation.Then,the differences in the expression profiles of mRNAs and IncRNAs between uninduced PDLSCs and GMSCs were compared to explore the inherent differences between the two MSCs at gene levels.Finally,the differentially expressed mRNAs(DEGs)and lncRNAs(DELs)during the osteogenic induction of PDLSCs and GMSCs were compared to screen possible IncRNAs which play an important role in stem cell osteogenic differentiation,and also provide potential targets for seed cell performance optimization.Methods1.Isolation and culture of MSCs from dental pulp,periodontal ligament,gingiva,dental follicle,bone marrow,umbilical cord(Wharton's jelly)and adipose tissue,followed by purification.And then mesenchymal immunophenotypic and multi-directional differentiation(osteogenic,adipogenic and chondrogenic)ability were identificated.The proliferative ability were detected and compared using Cell Counting Kit-8(CCK-8)and 5-ethynyl-2'-deoxyuridine kit(EdU).Annexin V staining were used to analyze the apoptosis of cells under the condition of nutrient deprivation combined with oxidative stress.And the expression levels of major pro/anti-apoptosis proteins were detected using western blot.The activities of alkaline phosphatase(ALP)were detected by staining and quantitative assays and the expression of osteogenic differentiation-related marker proteins were examined at day 14.Alizarin red staining and quantitative analysis was performed on the 28th day of osteogenic differentiation to detect the formation of mineralized nodules.The expression levels of osteogenic proteins were also detected.Western blot analysis was used to detect changes in several canonical cascade kinase signaling pathways in each cells at day 7 of osteogenic induction.2.The osteogenic differentiation of PDLSCs and GMSCs was induced,and the uninduced cells were simultaneously cultured as the respective control groups.Total RNA was extracted from cells on day 7 and the expression profiles of mRNAs and lncRNAs during PDLSCs and GMSCs osteogenic differentiation were detected using Affymetrix GeneChip(?)Human Transcriptome Array 2.0.Significant differences and cluster analysis were performed.Expression verification of representative DEGs and DELs was performed by quantitative real-time PCR(qRT-PCR).Bioinformatics analysis of gene ontology(GO)and signal pathway was perform on DEGs to observe their main functions.A coding-non-coding co-expression(CNC)network was constructed to predict functional lncRNAs associated with osteogenic differentiation.3.The microarray results of undifferentiated PDLSCs and GMSCs were compared and clustered and verified by qRT-PCR.GO analysis and signal pathway analysis was performed on DEGs.CNC network was constructed to predict specific IncRNAs associated with cell types.4.The DEGs and DELs during osteogenic differentiation of PDLSCs and GMSCs were compared to acquire common and cell-specific DEGs and DELs.Compare them with previous GO and signal pathway analysis results to gain the function of cell common and specific DEGs.Key mRNAs and lncRNAs that regulate osteogenic differentiation were predicted based on cellular characteristics.Results1.MSCs can be obtained from different tissues of the body by similar methods.They all possess self-renewal and multi-directional differentiation ability under the same culture conditions and existing similar immunophenotypes,in line with the standards of MSCs.PDLSCs own stronger proliferative capacity than BM-MSCs and ADSCs,and more resistant to apoptosis than GMSCs,DFSCs,BM-MSCs and UCMSCs.They also have stronger osteogenic differentiation ability than DPSCs,GMSCs and UCMSCs.GMSCs and UCMSCs own the lowest capabilities of ossification.Different kinase cascade signaling pathways were activated differently in different cells at day 7 of induction.2.PDLSCs differentially expressed 487 mRNAs and 119 lncRNAs during osteogenic induction.Through bioinformatics analysis,we noticed that DEGs are mainly enriched in biological processes related to various amino acid and protein metabolism in cells.At the same time,we also found that there are DEGs enrichment in several biological processes and signaling pathways directly related to stem cell osteogenic differentiation.Based on these mRNAs and CNC networks,39 IncRNAs that may be involved in the regulation of osteogenic differentiation of PDLSCs were predicted.3.GMSCs were one of the MSCs with the lowest ability to differentiate into osteoblasts.The expression of 112 mRNAs and 66 lncRNAs was changed during induction of osteogenic differentiation,which is significantly less than that of PDLSCs.Bioinformatics analysis found that these DEGs enriched to less biological processes and signaling pathways directly related to osteogenesis,on the contrary,more biological processes related to nervous system function.29 lncRNAs that may be involved in the differentiation of GMSCs were predicted based on mRNAs function.4.With the comparation of mRNAs and IncRNAs expression profiles of undifferentiated cells of PDLSCs and GMSCs,we obtained 133 mRNAs and 99 lncRNAs specifically expressed in PDLSCs,76 mRNAs and 437 IncRNAs were highly expressed in GMSCs.The intercellular differences in IncRNAs were significantly greater than mRNAs and those in cell differentiation.The expression of different mRNAs in the two MSCs is related to their functional properties according to GO analysis,and the CNC network also predicted 37 IncRNAs that may be involved in controlling cell specificity.5.When comparing the DEGs and DELs between PDLSCs and GMSCs and further comparing the results with previous bioinformatics analysis,we screened a series of mRNAs and IncRNAs with specific changes during osteogenic differentiation of PDLSCs which own better osteogensis.Conclusions1.PDLSCs could be a good alternative to BM-MSCs in bone regeneration therapy.The canonical intracellular protein kinase cascade signaling pathways function in cell type specific ways.2.The early stage of PDLSCs differentiation is a process of amino acid and protein metabolism changes.The interaction between DELs and DEGs suggests that IncRNAs may be involved in the regulation of cell differentiation.3.The weaker osteogenic differentiation of GMSCs may be due to its insensitivity to conventional osteogenic differentiation-inducing stimuli,and they can maintain a certain degree of stemness under induction conditions.Searching for more suitable induction means or performing gene editing may enhance their ability to di0fferentiate into osteoblasts.4.The difference in mRNA expression between cells leads to different characteristics.LncRNAs may be extensively involved in the regulation of cell specificity,and its role in this aspect maybe more important and complicated than in regulating cell differentiation.5.There are several specific DEGs related to osteogenic differentiation in PDLSCs,which may be the key factors for the stronger osteogenic differentiation of PDLSCs compared to GMSCs.The related IncRNAs may also become important switches for osteogenic differentiation of cells.They can also serve as genetic editing targets for enhancing osteogenic differentiation of GMSCs.