Effects of FGF2 and TGFbeta1 on the differentiation of human dental pulp stem cells in vitro

Two crucial growth factors, FGF2 and TGFbeta1, were investigated in this study to determine their inductive effects on the odontoblastic differentiation of human dental pulp stem cells (DPSCs) in vitro. DPSCs were isolated by immunomagnetic bead selection using the STRO-1 antibody, and then co-cultured respectively with FGF2, TGFbeta1 and FGF2+TGFbeta1. The results showed that FGF2 can exert a significant effect on the cell proliferation, while TGFbeta1 or FGF2+TGFbeta1 can initiate an odontoblast-like differentiation of DPSCs. Moreover, FGF2 can synergistically upregulate the effects of TGFbeta1 on the odontoblastic differentiation of DPSCs, as indicated by the increased alkaline phosphatase activity, the polarized cell appearance and secretary ultrastructural features, the formation of mineralized nodules and the gene/protein expression of dentin sialoprotein and dentin matrix protein-1. Together, FGF2 acted primarily on the cell proliferation, while TGFbeta1 and FGF2+TGFbeta1 mainly stimulated the odontoblastic differentiation of DPSCs. This study provides interesting progress in the odontoblastic differentiation of DPSCs induced by FGF2 and TGFbeta1.     

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.     

Follicle‐stimulating hormone impairs dental pulp stem cells odontogenic differentiation

In addition to bone, the dentin‐pulp complex is also influenced by menopause, showing a decreased regenerative capacity. High levels of follicle‐stimulating hormone (FSH) during menopause could directly regulate bone metabolism. Here, the role of FSH in the odontogenic differentiation of the dentin‐pulp complex was investigated. Dental pulp stem cells (DPSCs) were isolated. CCK‐8 assays, cell apoptosis assays, Western blotting (WB), real‐time RT‐PCR, alkaline phosphatase activity assays, and Alizarin Red S staining were used to clarify the effects of FSH on the proliferation, apoptosis and odontogenic differentiation of the DPSCs. MAPK pathway‐related factors were explored by WB assays. FSH and its inhibitor were used in OVX rats combined with a direct pulp‐capping model. HE and immunohistochemistry were used to detect reparative dentin formation and related features. The results indicated that FSH significantly decreased the odontogenic differentiation of the DPSCs without affecting cell proliferation and apoptosis. Moreover, FSH significantly activated the JNK signalling pathway, and JNK inhibitor partly rescued the inhibitory effect of FSH on DPSC differentiation. In vivo, FSH treatment attenuated the dentin bridge formation and mineralization‐related protein expression in the OVX rats. Our findings indicated that FSH reduced the odontogenic capacity of the DPSCs and was involved in reparative dentinogenesis during menopause.     

The effects of platelet-rich plasma derived from human umbilical cord blood on the osteogenic differentiation of human dental stem cells

Platelet-rich plasma (PRP) is an emerging therapeutic application because PRP contains various growth factors that have beneficial effects on tissue regeneration and engineering. Mesenchymal stem cells and PRP derived from peripheral blood have been well studied. In this study, we investigated the effects of PRP derived from human umbilical cord blood (UCB-PRP) on proliferation, alkaline phosphatase (ALP) activity, and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHEDs), dental pulp stem cells (DPSCs), and periodontal ligament stem cells (PDLSCs). Three types of dental stem cells were primarily isolated and characterized by flow cytometric analysis. Dental stem cells were exposed to various concentrations of UCB-PRP, which resulted in the proliferation of dental stem cells. Treatment with 2% UCB-PRP resulted in the highest level of proliferation. The ALP activity of DPSCs and PDLSCs increased following treatment with UCB-PRP in a dose-dependent manner up to a concentration of 2%. ALP activity decreased with higher concentration of UCB-PRP. The effects of UCB-PRP on calcium deposition were similar to those on proliferation and ALP activity. Treatment with 2% UCB-PRP resulted in the highest calcium depositions in DPSCs and PDLSCs; however, treatment with 1% UCB-PRP resulted in the highest calcium deposition in SHEDs. The concentrations of platelet-derived growth factor-AB and transforming growth factor-β1 in UCB-PRP were investigated and found to be comparable to the amounts in peripheral blood. Overall, UCB-PRP had beneficial effects on the proliferation and osteogenic differentiation of dental stem cells. Determination of the optimal concentration of UCB-PRP requires further investigation for clinical applications.     

Allogenic banking of dental pulp stem cells for innovative therapeutics

Medical research in regenerative medicine and cell-based therapy has brought encouraging perspectives for the use of stem cells in clinical trials. Multiple types of stem cells, from progenitors to pluripotent stem cells, have been investigated. Among these, dental pulp stem cells (DPSCs) are mesenchymal multipotent cells coming from the dental pulp, which is the soft tissue within teeth. They represent an interesting adult stem cell source because they are recovered in large amount in dental pulps with non-invasive techniques compared to other adult stem cell sources. DPSCs can be obtained from discarded teeth, especially wisdom teeth extracted for orthodontic reasons. To shift from promising preclinical results to therapeutic applications to human, DPSCs must be prepared in clinical grade lots and transformed into advanced therapy medicinal products (ATMP). As the production of patient-specific stem cells is costly and time-consuming, allogenic biobanking of clinical grade human leukocyte antigen (HLA)-typed DPSC lines provides efficient innovative therapeutic products. DPSC biobanks represent industrial and therapeutic innovations by using discarded biological tissues (dental pulps) as a source of mesenchymal stem cells to produce and store, in good manufacturing practice (GMP) conditions, DPSC therapeutic batches. In this review, we discuss about the challenges to transfer biological samples from a donor to HLA-typed DPSC therapeutic lots, following regulations, GMP guidelines and ethical principles. We also present some clinical applications, for which there is no efficient therapeutics so far, but that DPSCs-based ATMP could potentially treat.     

牙髓干细胞的研究进展

牙髓干细胞是来源于牙髓组织中的一种成体干细胞,该种细胞具有高度增殖、自我更新的能力和多向分化潜能。牙髓干细胞的研究对牙髓再生、牙体修复等牙组织工程将产生重要的意义。该文就牙髓干细胞的研究现状作一综述,并对其应用前景进行探讨。     

Critical limb ischemia: Current and novel therapeutic strategies

Critical limb ischemia (CLI) is the advanced stage of peripheral artery disease spectrum and is defined by limb pain or impending limb loss because of compromised blood flow to the affected extremity. Current conventional therapies for CLI include amputation, bypass surgery, endovascular therapy, and pharmacological approaches. Although these conventional therapeutic strategies still remain as the mainstay of treatments for CLI, novel and promising therapeutic approaches such as proangiogenic gene/protein therapies and stem cell-based therapies have emerged to overcome, at least partially, the limitations and disadvantages of current conventional therapeutic approaches. Such novel CLI treatment options may become even more effective when other complementary approaches such as utilizing proper bioscaffolds are used to increase the survival and engraftment of delivered genes and stem cells. Therefore, herein, we address the benefits and disadvantages of current therapeutic strategies for CLI treatment and summarize the novel and promising therapeutic approaches for CLI treatment. Our analyses also suggest that these novel CLI therapeutic strategies show considerable advantages to be used when current conventional methods have failed for CLI treatment.     

Study of optimal conditions for growth and osteogenic differentiation of dental pulp stem cells based on glucose and serum content

Dental pulp stem cells (DPSCs) have shown promising characteristics in terms of their proliferation and osteogenic differentiation potential, which could be of greater benefit in regenerative dentistry. However, obstacles remain in the in vitro cultivation of DPSCs, which significantly affect their growth and differentiating ability. Therefore in this study, we demonstrated the growth and osteogenic differentiation of DPSCs in the presence of media containing different combinations of serum and glucose to get an optimized combination of both. DPSCs were cultured in media containing combinations of low glucose (LG), low serum (LS), high glucose (HG), and high serum (HS). The proliferation and osteogenic differentiation were assessed in DPSCs cultured with these different combinations of culture conditions. High glucose high serum condition significantly inhibited the proliferation of DPSCs and also affected their clonogenic potential, as evidenced by colony-forming units. Irrespective of the serum content, high glucose in the media also decreased the osteogenic potential of DPSCs confirmed by functional staining, and downregulation of osteogenesis-related genes. High glucose content in the culture media affects the growth and differentiation potential of the DPSCs. Hence, the culture conditions for the DPSCs should be reconsidered to utilize their maximum potential.     

Dental pulp stem cells (DPSCs) increase prostate cancer cell proliferation and migration under in vitro conditions

Cancer as a multistep and complicated disease is regulated by several molecular and cellular events. Cancer treatment could be managed at the early stages when the tumor is confined in the tissue. However, disseminated cancer cells metastasize to other body parts and generate new tumors resulting in mortality. Mesenchymal stem cells (MSCs) are found in different body parts and helps adult tissue regeneration. The role of MSCs in cancer progression has emerged as one of the important aspects in cancer biology and is the aim of interest in recent years. In the current study, effects of Dental Pulp Stem Cells (DPSCs) on PC-3 prostate cancer cell proliferation and migration were conducted by cell proliferation, apoptosis, gene expression and cell migration analysis in vitro. Condition medium (CM) obtained from DPSCs increased cell proliferation of PC-3 cells and decreased apoptosis. Either administration of CM or trans well co-culture of DPSCs increased cell migration in scratch assay, confirmed by gene expression analysis of migratory genes including fibronectin, laminin and collagen type I (Col I). Furthermore, DPSCs participated in a self-organized structure with PC-3 cells in co-culture conditions. Overall, results indicated that DPSCs could promote PC-3 cancer cell proliferation and metastasis in co-culture conditions in vitro.     

Downregulation of heat shock protein B8 decreases osteogenic differentiation potential of dental pulp stem cells during in vitro proliferation

Objectives

Tissue‐derived stem cells, such as dental pulp stem cells (DPSCs), reduce differentiation capability during in vitro culture. We found that cultured DPSCs reduce expression of heat shock protein B8 (HspB8) and GIPC PDZ domain containing family member 2 (Gipc2). Our objectives were to evaluate the changes in DPSC composition during in vitro proliferation and to determine whether HspB8 and Gipc2 have function in differentiation potential of DPSCs.

Materials and Methods

Different passages of rat DPSCs were evaluated for changes in CD90+ and/or CD271+ stem cells and changes in osteogenic potential. Real‐time RT‐PCR and immunostaining were conducted to determine expression of HspB8 and Gipc2. Expression of the genes in DPSCs was knocked down by siRNA, followed by osteogenic induction to evaluate the function of the genes.

Results

About 90% of cells in the DPSC cultures were CD90+ and/or CD271+ cells without dramatic change during in vitro proliferation. The DPSCs at passages 3 to 5 (P3 to P5) possess strong osteogenic potential, but such potential was greatly reduced at later passages. Expression of HspB8 and Gipc2 was significantly reduced at P11 versus P3. Knock‐down of HspB8 expression abolished osteogenic potential of the DPSCs, but knock‐down of Gipc2 had no effect.

Conclusions

CD90+ and CD271+ cells are the major components of DPSCs in in vitro culture. High‐level expression of HspB8 was critical for maintaining differentiation potential of DPSCs.

     

Mesenchymal stem cells promote lymphangiogenic properties of lymphatic endothelial cells

Lymphatic metastasis is one of the main prognostic factors concerning long‐term survival of cancer patients. In this regard, the molecular mechanisms of lymphangiogenesis are still rarely explored. Also, the interactions between stem cells and lymphatic endothelial cells (LEC) in humans have not been well examined. Therefore, the main objective of this study was to assess the interactions between mesenchymal stem cells (MSC) and LEC using in vitro angiogenesis assays. Juvenile LEC were stimulated with VEGF‐C, bFGF, MSC‐conditioned medium (MSC‐CM) or by co‐culture with MSC. LEC proliferation was assessed using a MTT assay. Migration of the cells was determined with a wound healing assay and a transmigration assay. To measure the formation of lymphatic sprouts, LEC spheroids were embedded in collagen or fibrin gels. The LEC's capacity to form capillary‐like structures was assessed by a tube formation assay on Matrigel^®. The proliferation, migration and tube formation of LEC could be significantly enhanced by MSC‐CM and by co‐culture with MSC. The effect of stimulation with MSC‐CM was stronger compared to stimulation with the growth factors VEGF‐C and bFGF in proliferation and transmigration assays. Sprouting was stimulated by VEGF‐C, bFGF and by MSC‐CM. With this study, we demonstrate the potent stimulating effect of the MSC secretome on proliferation, migration and tube formation of LEC. This indicates an important role of MSC in lymphangiogenesis in pathological as well as physiological processes.     

Notoginsenoside R1 activates the Ang2/Tie2 pathway to promote angiogenesis

BackgroundTherapeutic angiogenesis is a novel strategy for the treatment of ischemic diseases that involves promotion of angiogenesis in ischemic tissues via the use of proangiogenic agents. However, effective proangiogenic drugs that activate the Ang2/Tie2 signaling pathway remain scarce.PurposeWe aimed to investigate the proangiogenic activity of notoginsenoside R1 (NR1) isolated from total saponins of Panax notoginseng with regard to activation of the Ang2/Tie2 signaling pathway.MethodsWe examined the proangiogenic effects of NR1 by assessing the effects of NR1 on the proliferation, migration, invasion and tube formation of human umbilical vein endothelial cells (HUVECs). The aortic ring assay and vascular endothelial growth factor receptor inhibitor (VRI)-induced vascular regression in the zebrafish model were used to confirm the proangiogenic effects of NR1 ex vivo and in vivo. Furthermore, the molecular mechanism was investigated by Western blot analysis.ResultsWe found that NR1 promoted the proliferation, mobility and tube formation of HUVECs in vitro. NR1 also increased the number of sprouting vessels in rat aortic rings and rescued VRI-induced vascular regression in zebrafish. NR1-induced angiogenesis was dependent on Tie2 receptor activation mediated by increased autocrine Ang2 in HUVECs, and inhibition of the Ang2/Tie2 pathway abrogated the proangiogenic effects of NR1.ConclusionsOur results suggest that NR1 promotes angiogenesis by activating the Ang2/Tie2 signaling pathway. Thus, NR1-induced activation of the Ang2/Tie2 pathway is an effective proangiogenic approach. NR1 may be useful agent for the treatment of ischemic diseases.     

Sema4D/PlexinB1 promotes endothelial differentiation of dental pulp stem cells via activation of AKT and ERK1/2 signaling

Inducing of dental pulp stem cells (DPSCs) into endothelial cells (ECs) to prevascularize pulp tissue constructs may offer a novel and viable approach for enhancing pulp regeneration. However, there are numerous challenges in current methods for the acquisition of sufficient translational ECs. It was known that Sema4D/PlexinB1 signaling exerts profound effects on enhancing vascular endothelial growth factor (VEGF) secretion and angiogenesis. Whether Sema4D/PlexinB1 could regulate endothelial differentiation of DPSCs is not yet investigated. In this study, when DPSCs were treated with Sema4D (2 μg/mL), ECs-specific (VEGFR1, VEGFR2, CD31, and vWF), and angiogenic genes and proteins were significantly upregulated. The induced ECs exhibited similar endothelial vessel formation ability to that of human umbilical vein endothelial cells (HUVECs). Furthermore, phosphorylation of AKT increased dramatically within 5 minutes (from 0.93 to 21.8), while p-ERK1/2 was moderately elevated (from 0.94 to 2.65). In summary, our results demonstrated that Sema4D/PlexinB1 signaling induces endothelial differentiation of DPSCs. The interactions of Sema4D, VEGF, ANGPTL4, ANG1, and HIF-1α may play a crucial role in mediating the differentiation process.     

Ferutinin directs dental pulp-derived stem cells towards the osteogenic lineage by epigenetically regulating canonical Wnt signaling

Osteoporosis is a silent systemic disease that causes bone deterioration, and affects over 10 million people in the US alone. This study was undertaken to develop a potential stem cell therapy for osteoporosis. We have isolated and expanded human dental pulp-derived stem cells (DPSCs), characterized them, and confirmed their multipotential differentiation abilities. Stem cells often remain quiescent and require activation to differentiate and function. Herein, we show that ferutinin activates DPSCs by modulating the Wnt/β-catenin signaling pathway and key osteoblast-secreted proteins osteocalcin and collagen 1A1 both mRNA and protein levels. To confirm that ferutinin modulates the Wnt pathway, we inhibited glycogen synthase kinase 3 (GSK3) and found that protein expression patterns were similar to those found in ferutinin-treated DPSCs. To evaluate the role of ferutinin in epigenetic regulation of canonical Wnt signaling, the pathway molecules Wnt3a and Dvl3 were analyzed using chromatin immunoprecipitation (ChIP)-quantitative PCR approaches. We confirmed that active marks of both H3K9 acetylation and H3K4 trimethylation were significantly enhanced in the promoter sites of the WNT3A and DVL3 genes in DPSCs after addition of ferutinin. These data provide evidence that ferutinin activates and promotes osteogenic differentiation of DPSCs, and could be used as an inducer as a potentially effective stem cell therapy for osteoporosis.     

Bone tissue engineering using adipose‐derived stem cells and endothelial cells: Effects of the cell ratio

The microvascular endothelial network is essential for bone formation and regeneration. In this context, endothelial cells not only support vascularization but also influence bone physiology via cell contact‐dependent mechanisms. In order to improve vascularization and osteogenesis in tissue engineering applications, several strategies have been developed. One promising approach is the coapplication of endothelial and adipose derived stem cells (ADSCs). In this study, we aimed at investigating the best ratio of human umbilical vein endothelial cells (HUVECs) and osteogenic differentiated ADSCs with regard to proliferation, apoptosis, osteogenesis and angiogenesis. For this purpose, cocultures of ADSCs and HUVECs with ratios of 25%:75%, 50%:50% and 75%:25% were performed. We were able to prove that cocultivation supports proliferation whereas apoptosis was unidirectional decreased in cocultured HUVECs mediated by a p‐BAD‐dependent mechanism. Moreover, coculturing ADSCs and HUVECs stimulated matrix mineralization and the activity of alkaline phosphatase (ALP). Increased gene expression of the proangiogenic markers eNOS, Flt, Ang2 and MMP3 as well as sprouting phenomena in matrigel assays proved the angiogenic potential of the coculture. In summary, coculturing ADSCs and HUVECs stimulates proliferation, cell survival, osteogenesis and angiogenesis particularly in the 50%:50% coculture.     

Harnessing mesenchymal stem cell secretome: Effect of extracellular matrices on proangiogenic signaling

The low engraftment and retention rate of mesenchymal stem cells (MSCs) at the target site indicates that the potential benefits of MSC-based therapies can be attributed to their paracrine signaling. In this study, the extracellular matrices (ECMs) deposited by bone marrow-derived human MSCs in the presence and absence of ascorbic acid was characterized. MSCs were seeded on top of decellularized ECM (dECM) and the concentrations of proangiogenic and antiangiogenic molecules released in culture (conditioned) media was compared. Effects of ECM derived from MSCs with different passage numbers on MSC secretome was also investigated. Our study revealed that the expression of proangiogenesis-related factors were upregulated when MSCs were harvested on dECMs, irrespective of media supplementation, as compared with those cultured on tissue culture plates. In addition, dECM generated in the presence of ascorbic acid promoted the expression of proangiogenic molecules as compared with dECM-derived in absence of media supplementation. Further, it was observed that the effectiveness of dECM to stimulate proangiogenic signaling of MSCs was reduced as cell passage number was increased from P3 to P5. The proliferation as well as capillary morphogenesis of human umbilical vein endothelial cells (HUVECs) in the presence of conditioned media were enhanced compared with the normal HUVECs culture media. These data indicate that the secretory signatures of MSCs and consequently, the therapeutic efficacy of MSCs can be regulated by presentation of dECM composition and variation of its composition.