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.

RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation

BACKGROUND Validation of the reference gene(RG)stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction(RT-qPCR)data normalisation.Commonly,in an unreliable way,several studies use genes involved in essential cellular functions[glyceraldehyde-3-phosphate dehydro-genase(GAPDH),18S rRNA,andβ-actin]without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes.Furthermore,such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recom-mend two or more genes.It impacts the credibility of these studies and causes dis-tortions in the gene expression findings.For tissue engineering,the accuracy of gene expression drives the best experimental or therapeutical approaches.We cultivated DPSCs under two conditions:Undifferentiated and osteogenic dif-ferentiation,both for 35 d.We evaluated the gene expression of 10 candidates for RGs[ribosomal protein,large,P0(RPLP0),TATA-binding protein(TBP),GAPDH,actin beta(ACTB),tubulin(TUB),aminolevulinic acid synthase 1(ALAS1),tyro-sine 3-monooxygenase/tryptophan 5-monooxygenase activation protein,zeta(YWHAZ),eukaryotic translational elongation factor 1 alpha(EF1a),succinate dehydrogenase complex,subunit A,flavoprotein(SDHA),and beta-2-micro-globulin(B2M)]every 7 d(1,7,14,21,28,and 35 d)by RT-qPCR.The data were analysed by the four main algorithms,ΔCt method,geNorm,NormFinder,and BestKeeper and ranked by the RefFinder method.We subdivided the samples into eight subgroups.RESULTS All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm.The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs.Either theΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes.However,geNorm analysis showed RPLP0/EF1αin the first place.These algorithms’two least stable RGs were B2M/GAPDH.For BestKeeper,ALAS1 was ranked as the most stable RG,and SDHA as the least stable RG.The pair RPLP0/TBP was detected in most subgroups as the most stable RGs,following the RefFinfer ranking.CONCLUSION For the first time,we show that RPLP0/TBP are the most stable RGs,whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.

Hepatocyte growth factor enhances the ability of dental pulp stem cells to ameliorate atherosclerosis in apolipoprotein E-knockout mice

BACKGROUND Atherosclerosis(AS),a chronic inflammatory disease of blood vessels,is a major contributor to cardiovascular disease.Dental pulp stem cells(DPSCs)are capable of exerting immunomodulatory and anti-inflammatory effects by secreting cytokines and exosomes and are widely used to treat autoimmune and inflam-mation-related diseases.Hepatocyte growth factor(HGF)is a pleiotropic cytokine that plays a key role in many inflammatory and autoimmune diseases.AIM To modify DPSCs with HGF(DPSC-HGF)and evaluate the therapeutic effect of DPSC-HGF on AS using an apolipoprotein E-knockout(ApoE-/-)mouse model and an in vitro cellular model.METHODS ApoE-/-mice were fed with a high-fat diet(HFD)for 12 wk and injected with DPSC-HGF or Ad-Null modified DPSCs(DPSC-Null)through tail vein at weeks 4,7,and 11,respectively,and the therapeutic efficacy and mechanisms were analyzed by histopathology,flow cytometry,lipid and glucose measurements,real-time reverse transcription polymerase chain reaction(RT-PCR),and enzyme-linked immunosorbent assay at the different time points of the experiment.An in vitro inflammatory cell model was established by using RAW264.7 cells and human aortic endothelial cells(HAOECs),and indirect co-cultured with supernatant of DPSC-Null(DPSC-Null-CM)or DPSC-HGF-CM,and the effect and mechanisms were analyzed by flow cytometry,RT-PCR and western blot.Nuclear factor-κB(NF-κB)activators and inhibitors were also used to validate the related signaling pathways.RESULTS DPSC-Null and DPSC-HGF treatments decreased the area of atherosclerotic plaques and reduced the expression of inflammatory factors,and the percentage of macrophages in the aorta,and DPSC-HGF treatment had more pronounced effects.DPSCs treatment had no effect on serum lipoprotein levels.The FACS results showed that DPSCs treatment reduced the percentages of monocytes,neutrophils,and M1 macrophages in the peripheral blood and spleen.DPSC-Null-CM and DPSC-HGF-CM reduced adhesion molecule expression in tumor necrosis factor-αstimulated HAOECs and regulated M1 polarization and inflammatory factor expression in lipopolysaccharide-induced RAW264.7 cells by inhibiting the NF-κB signaling pathway.CONCLUSION This study suggested that DPSC-HGF could more effectively ameliorate AS in ApoE-/-mice on a HFD,and could be of greater value in stem cell-based treatments for AS.

Human dental pulp stem/stromal cells in clinical practice

Dental pulp stem/stromal cells(DPSCs)are fibroblast-like,neural crest-derived,and multipotent cells that can differentiate into several lineages.They are relatively easy to isolate from healthy and inflamed pulps,with little ethical concerns and can be successfully cryopreserved and thawed.The therapeutic effects of DPSCs derived from animal or human sources have been extensively studied through in-vitro and in-vivo animal experiments and the findings indicated that DPSCs are effective not only for dental diseases but also for systemic diseases.Understanding that translational research is a critical step through which the fundamental scientific discoveries could be translated into applicable diagnostics and therapeutics that directly benefit humans,several clinical studies were carried out to generate evidence for the efficacy and safety of autogenous or allogeneic human DPSCs(hDPSCs)as a treatment modality for use in cell-based therapy,regenerative medicine/dentistry and tissue engineering.In clinical medicine,hDPSCs were effective for treating acute ischemic stroke and human exfoliated deciduous teeth-conditioned medium(SHED-CM)repaired vascular damage of the corpus cavernous,which is the main cause of erectile dysfunction.Whereas in clinical dentistry,autologous SHED was able to rege-nerate necrotic dental pulp after implantation into injured teeth,and micrografts enriched with autologous hDPSCs and collagen sponge were considered a treatment option for human intrabony defects.In contrast,hDPSCs did not add a significant regenerative effect when they were used for the treatment of post-extraction sockets.Large-scale clinical studies across diverse populations are still lacking to provide robust evidence on the safety and efficacy of hDPSCs as a new treatment option for various human diseases including dental-related problems.

Self-assembly of differentiated dental pulp stem cells facilitates spheroid human dental organoid formation and prevascularization

BACKGROUND The self-assembly of solid organs from stem cells has the potential to greatly expand the applicability of regenerative medicine.Stem cells can self-organise into microsized organ units,partially modelling tissue function and regeneration.Dental pulp organoids have been used to recapitulate the processes of tooth development and related diseases.However,the lack of vasculature limits the utility of dental pulp organoids.AIM To improve survival and aid in recovery after stem cell transplantation,we demonstrated the three-dimensional(3D)self-assembly of adult stem cell-human dental pulp stem cells(hDPSCs)and endothelial cells(ECs)into a novel type of spheroid-shaped dental pulp organoid in vitro under hypoxia and conditioned medium(CM).METHODS During culture,primary hDPSCs were induced to differentiate into ECs by exposing them to a hypoxic environment and CM.The hypoxic pretreated hDPSCs were then mixed with ECs at specific ratios and conditioned in a 3D environment to produce prevascularized dental pulp organoids.The biological characteristics of the organoids were analysed,and the regulatory pathways associated with angiogenesis were studied.RESULTS The combination of these two agents resulted in prevascularized human dental pulp organoids(Vorganoids)that more closely resembled dental pulp tissue in terms of morphology and function.Single-cell RNA sequencing of dental pulp tissue and RNA sequencing of Vorganoids were integrated to analyse key regulatory pathways associated with angiogenesis.The biomarkers forkhead box protein O1 and fibroblast growth factor 2 were identified to be involved in the regulation of Vorganoids.CONCLUSION In this innovative study,we effectively established an in vitro model of Vorganoids and used it to elucidate new mechanisms of angiogenesis during regeneration,facilitating the development of clinical treatment strategies.

Genetic modification of miR-34a enhances efficacy of transplanted human dental pulp stem cells after ischemic stroke

Human dental pulp stem cells(hDPSCs) promote recovery after ischemic stro ke;however,the therapeutic efficacy is limited by the poor survival of transplanted cells.For in vitro expe riments in the present study,we used oxygen-glucose deprivation/reoxygenation in hDPSCs to mimic cell damage induced by ischemia/reperfusion.We found that miRNA-34a-5p(miR-34a) was elevated under oxygen-glucose deprivation/reoxygenation conditions in hDPSCs.Inhibition of miR-34a facilitated the prolife ration and antioxidant capacity and reduced the apoptosis of hDPSCs.Moreove r,dual-luciferase reporter gene assay showed WNT1and SIRT1 as the targets of miR-34a.In miR-34a knockdown cell lines,WNT1 suppression reduced cell prolife ration,and SIRT1 suppression decreased the antioxidant capacity.Togethe r,these results indicated that miR-34a regulates cell prolife ration and antioxidant stress via targeting WNT1 and SIRT1,respectively.For in vivo expe riments,we injected genetically modified hDPSCs(anti34a-hDPSCs) into the brains of mice.We found that anti34a-hDPSCs significantly inhibited apoptosis,reduced cerebral edema and cerebral infarct volume,and improved motor function in mice.This study provides new insights into the molecular mechanism of the cell prolife ration and antioxidant capacity of hDPSCs,and suggests a potential gene that can be targeted to improve the survival rate and efficacy of transplanted hDPSCs in brain after ischemic stroke.

Potential of dental pulp stem cells and their products in promoting peripheral nerve regeneration and their future applications

Peripheral nerve injury(PNI)seriously affects people’s quality of life.Stem cell therapy is considered a promising new option for the clinical treatment of PNI.Dental stem cells,particularly dental pulp stem cells(DPSCs),are adult pluripotent stem cells derived from the neuroectoderm.DPSCs have significant potential in the field of neural tissue engineering due to their numerous advantages,such as easy isolation,multidifferentiation potential,low immunogenicity,and low transplant rejection rate.DPSCs are extensively used in tissue engineering and regenerative medicine,including for the treatment of sciatic nerve injury,facial nerve injury,spinal cord injury,and other neurodegenerative diseases.This article reviews research related to DPSCs and their advantages in treating PNI,aiming to summarize the therapeutic potential of DPSCs for PNI and the underlying mechanisms and providing valuable guidance and a foundation for future research.

Therapeutic and regenerative potential of different sources of mesenchymal stem cells for cardiovascular diseases

Mesenchymalstemcells(MSCs)areidealcandidatesfortreatingmanycardiovasculardiseases.MSCscanmodify the internal cardiac microenvironment to facilitate their immunomodulatory and differentiation abilities,which are essential to restore heart function.MSCs can be easily isolated from different sources,including bone marrow,adipose tissues,umbilical cord,and dental pulp.MSCs from various sources differ in their regenerative and therapeutic abilities for cardiovascular disorders.In this review,we will summarize the therapeutic potential of each MSC source for heart diseases and highlight the possible molecular mechanisms of each source to restore cardiac function.

Clinical trials using dental stem cells:2022 update

For nearly 20 years,dental stem cells(DSCs)have been successfully isolated from mature/immature teeth and surrounding tissue,including dental pulp of permanent teeth and exfoliated deciduous teeth,periodontal ligaments,dental follicles,and gingival and apical papilla.They have several properties(such as self-renewal,multidirectional differentiation,and immunomodulation)and exhibit enormous potential for clinical applications.To date,many clinical articles and clinical trials using DSCs have reported the treatment of pulpitis,periapical lesions,periodontitis,cleft lip and palate,acute ischemic stroke,and so on,and DSC-based therapies obtained satisfactory effects in most clinical trials.In these studies,no adverse events were reported,which suggested the safety of DSC-based therapy.In this review,we outline the characteristics of DSCs and summ-arize clinical trials and their safety as DSC-based therapies.Meanwhile,we also present the current limitations and perspectives of DSC-based therapy(such as harvesting DSCs from inflamed tissue,applying DSC-conditioned medi-um/DSC-derived extracellular vesicles,and expanding-free strategies)to provide a theoretical basis for their clinical applications.

Human dental pulp stem cells: Applications in future regenerative medicine

Stem cells are pluripotent cells, having a property of differentiating into various types of cells of human body. Several studies have developed mesenchymal stem cells(MSCs) from various human tissues,peripheral blood and body fluids. These cells are then characterized by cellular and molecular markers to understand their specific phenotypes. Dental pulp stem cells(DPSCs) are having a MSCs phenotype and they are differentiated into neuron, cardiomyocytes, chondrocytes, osteoblasts, liver cells and β cells of islet of pancreas. Thus, DPSCs have shown great potentiality to use in regenerative medicine for treatment of various human diseases including dental related problems. These cells can also be developed into induced pluripotent stem cells by incorporation of pluripotency markers and use for regenerative therapies of various diseases. The DPSCs are derived from various dental tissues such as human exfoliated deciduous teeth, apical papilla, periodontal ligament and dental follicle tissue. This review will overview the information about isolation, cellular and molecular characterization and differentiation of DPSCs into various types of human cells and thus these cells have important applications in regenerative therapies for various diseases. This review will be most useful for postgraduate dental students as well as scientists working in the field of oral pathology and oral medicine.

Neural crest derived stem cells from dental pulp and tooth-associated stem cells for peripheral nerve regeneration

The peripheral nerve injuries,representing some of the most common types of traumatic lesions affecting the nervous system,are highly invalidating for the patients besides being a huge social burden.Although peripheral nervous system owns a higher regenerative capacity than does central nervous system,mostly depending on Schwann cells intervention in injury repair,several factors determine the extent of functional outcome after healing.Based on the injury type,different therapeutic approaches have been investigated so far.Nerve grafting and Schwann cell transplantation have represented the gold standard treatment for peripheral nerve injuries,however these approaches own limitations,such as scarce donor nerve availability and donor site morbidity.Cell based therapies might provide a suitable tool for peripheral nerve regeneration,in fact,the ability of different stem cell types to differentiate towards Schwann cells in combination with the use of different scaffolds have been widely investigated in animal models of peripheral nerve injuries in the last decade.Dental pulp is a promising cell source for regenerative medicine,because of the ease of isolation procedures,stem cell proliferation and multipotency abilities,which are due to the embryological origin from neural crest.In this article we review the literature concerning the application of tooth derived stem cell populations combined with different conduits to peripheral nerve injuries animal models,highlighting their regenerative contribution exerted through either glial differentiation and neuroprotective/neurotrophic effects on the host tissue.

Dental pulp stem cells express tendon markers under mechanical loading and are a potential cell source for tissue engineering of tendon-like tissue

Postnatal mesenchymal stem cells have the capacity to differentiate into multiple cell lineages. This study explored the possibility of dental pulp stem cells （DPSCs） for potential application in tendon tissue engineering. The expression of tendon- related markers such as scleraxis, tenascin-C, tenomodulin, eye absent homologue 2, collagens I and VI was detected in dental pulp tissue. Interestingly, under mechanical stimulation, these tendon-related markers were significantly enhanced when DPSCs were seeded in aligned polyglycolic acid （PGA） fibre scaffolds. Furthermore, mature tendon-like tissue was formed after transplantation of DPSC-PGA constructs under mechanical loading conditions in a mouse model. This study demonstrates that DPSCs could be a ootential stem cell source for tissue enEineerin~ of tendon-like tissue.

Neurotrophic effects of dental pulp stem cells in repair of peripheral nerve after crush injury

BACKGROUND Nerve diseases and injuries,which are usually accompanied by motor or sensory dysfunction and disorder,impose a heavy burden upon patients and greatly reduce their quality of life.Dental pulp stem cells(DPSCs),derived from the neural crest,have many characteristics that are similar to those of neural cells,indicating that they can be an ideal source for neural repair.AIM To explore the potential roles and molecular mechanisms of DPSCs in crushed nerve recovery.METHODS DPSCs were isolated,cultured,and identified by multilineage differentiation and flow cytometry.Western blot and immunofluorescent staining were applied to analyze the expression levels of neurotrophic proteins in DPSCs after neural induction.Then,we collected the secretions of DPSCs.We analyzed their effects on RSC96 cell proliferation and migration by CCK8 and transwell assays.Finally,we generated a sciatic nerve crush injury model in vivo and used the sciatic function index,walking track analysis,muscle weight,and hematoxylin&eosin(H&E)staining to further evaluate the nerve repair ability of DPSCs.RESULTS DPSCs highly expressed several specific neural markers,including GFAP,S100,Nestin,P75,and NF200,and were inclined toward neural differentiation.Furthermore,neural-induced DPSCs(N-DPSCs)could express neurotrophic factors,including NGF,BDNF,and GDNF.The secretions of N-DPSCs could enhance the proliferation and migration of Schwann cells.In vivo,both DPSC and N-DPSC implants alleviated gastrocnemius muscle atrophy.However,in terms of anatomy and motor function,as shown by H&E staining,immunofluorescent staining,and walking track analyses,the repair effects of N-DPSCs were more sustained,potent,and effective than those of DPSCs and the controls.CONCLUSION In summary,this study demonstrated that DPSCs are inclined to differentiate into neural cells.N-DPSCs express neurotrophic proteins that could enhance the proliferation and migration of SCs.Furthermore,our results suggested that NDPSCs could help crushed nerves with functional recovery and anatomical repair in vivo.Thus,DPSCs or N-DPSCs could be a promising therapeutic cell source for peripheral nerve repair and regeneration.

Multilineage Differentiation of Dental Pulp Stem Cells from Green Fluorescent Protein Transgenic Mice

Aim The aim of this study was to confirm the multilineage differentiation ability of dental pulp stem cells （DPSCs） from green fluorescent protein （GFP） transgenic mice. The expression of GFP in DPSCs was also observed during differentiation. Methodology DPSCs were harvested from the dental pulp tissue of transgenic nude mice, and then transferred to osteogenic, adipogenic, and chondrogenic media. The morphological characterization of induced cells was observed by microscopy and histological staining. The expression of marker genes was measured by RT-PCR. Results The endogenous GFP and multilineage potential of transgenic DPSCs had no influence on each other. Moreover, the results of fluorescence microscopic imaging suggest that there was no significant decline of GFP expression during DPSCs differentiation. Conclusion As the population of GFP labeled DPSCs can be easily identified, this will be a promising method for tracking DPSCs in vivo.

Epigenetic regulation of dental pulp stem cells and its potential in regenerative endodontics

Regenerative endodontics(RE)therapy means physiologically replacing damaged pulp tissue and regaining functional dentin–pulp complex.Current clinical RE procedures recruit endogenous stem cells from the apical papilla,periodontal tissue,bone marrow and peripheral blood,with or without application of scaffolds and growth factors in the root canal space,resulting in cementum-like and bone-like tissue formation.Without the involvement of dental pulp stem cells(DPSCs),it is unlikely that functional pulp regeneration can be achieved,even though acceptable repair can be acquired.DPSCs,due to their specific odontogenic potential,high proliferation,neurovascular property,and easy accessibility,are considered as the most eligible cell source for dentin–pulp regeneration.The regenerative potential of DPSCs has been demonstrated by recent clinical progress.DPSC transplantation following pulpectomy has successfully reconstructed neurovascularized pulp that simulates the physiological structure of natural pulp.The self-renewal,proliferation,and odontogenic differentiation of DPSCs are under the control of a cascade of transcription factors.Over recent decades,epigenetic modulations implicating histone modifications,DNA methylation,and noncoding(nc)RNAs have manifested as a new layer of gene regulation.These modulations exhibit a profound effect on the cellular activities of DPSCs.In this review,we offer an overview about epigenetic regulation of the fate of DPSCs;in particular,on the proliferation,odontogenic differentiation,angiogenesis,and neurogenesis.We emphasize recent discoveries of epigenetic molecules that can alter DPSC status and promote pulp regeneration through manipulation over epigenetic profiles.

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.

Single CD271 marker isolates mesenchymal stem cells from human dental pulp

Mesenchymal stem cells （MSCs） are a promising tool in regenerative medicine due to their capacity to differentiate into multiple lineages. In addition to MSCs isolated from bone marrow （BMSCs）, adult MSCs are isolated from craniofacial tissues including dental pulp tissues （DPs） using various stem cell surface markers. However, there has been a lack of consensus on a set of surface makers that are reproducibly effective at isolating putative multipotent dental mesenchymal stem cel^s （~M^Cs）. II1 ~his stucly, we used clif^et（~nt combinations of surface markers （CD51/CD140a, CD271, and STRO-1/CD146） to isolate homogeneous populations of DMSCs from heterogeneous dental pulp cells （DPCs） obtained from DP and compared their capacity to undergo multilineage differentiation. Fluorescence-activated cell sorting revealed that 27.3% of DPCs were CD51＋/CD140a＋, 10.6% were CD271＋, and 0.3% were STRO-1＋/CD146＋. Under odontogenic conditions, all three subsets of isolated DMSCs exhibited differentiation capacity into odontogenic lineages. Among these isolated subsets of DMSCs, CD271＋ DMSCs demonstrated the greatest odontogenic potential. While all three combinations of surface markers in this study successfully isolated DMSCs from DPCs, the single CD271 marker presents the most effective stem cell surface marker for identification of DMSCs with high odontogenic potential. Isolated CD271＋ DMSCs could potentially be utilized for future clinical applications in dentistry and regenerative medicine.

Dental pulp stem cells, a paracrine-mediated therapy for the retina

Author contributions： Mead B was responsible for study conception and design, collection and^or assembly of data, data analysis and interpretation and manuscript writing. Logan A participated in study conception and design, data analysis and interpretation and manuscript writing. Berry M was responsible for manuscript writing. Scheven BA was in charge of study conception and design, data analysis and interpretation and manuscript writing. Leadbeater W participated in study conception and design, data analysis and interpretation and manuscript writing. All authors approved the final version of this paper.

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.

Therapeutic potential of dental pulp stem cells and their derivatives:Insights from basic research toward clinical applications

For more than 20 years,researchers have isolated and identified postnatal dental pulp stem cells(DPSCs)from different teeth,including natal teeth,exfoliated deciduous teeth,healthy teeth,and diseased teeth.Their mesenchymal stem cell(MSC)-like immunophenotypic characteristics,high proliferation rate,potential for multidirectional differentiation and biological features were demonstrated to be superior to those of bone marrow MSCs.In addition,several main application forms of DPSCs and their derivatives have been investigated,including stem cell injections,modified stem cells,stem cell sheets and stem cell spheroids.In vitro and in vivo administration of DPSCs and their derivatives exhibited beneficial effects in various disease models of different tissues and organs.Therefore,DPSCs and their derivatives are regarded as excellent candidates for stem cell-based tissue regeneration.In this review,we aim to provide an overview of the potential application of DPSCs and their derivatives in the field of regenerative medicine.We describe the similarities and differences of DPSCs isolated from donors of different ages and health conditions.The methodologies for therapeutic administration of DPSCs and their derivatives are introduced,including single injections and the transplantation of the cells with a support,as cell sheets,or as cell spheroids.We also summarize the underlying mechanisms of the regenerative potential of DPSCs.