Annelies Bronckaers

Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate angiogenesis.

Mesenchymal stem cells or multipotent stromal cells (MSCs) have initially captured attention in the scientific world because of their differentiation potential into osteoblasts, chondroblasts and adipocytes and possible transdifferentiation into neurons, glial cells and endothelial cells. This broad plasticity was originally hypothesized as the key mechanism of their demonstrated efficacy in numerous animal models of disease as well as in clinical settings. However, there is accumulating evidence suggesting that the beneficial effects of MSCs are predominantly caused by the multitude of bioactive molecules secreted by these remarkable cells. Numerous angiogenic factors, growth factors and cytokines have been discovered in the MSC secretome, all have been demonstrated to alter endothelial cell behavior in vitro and induce angiogenesis in vivo. As a consequence, MSCs have been widely explored as a promising treatment strategy in disorders caused by insufficient angiogenesis such as chronic wounds, stroke and myocardial infarction. In this review, we will summarize into detail the angiogenic factors found in the MSC secretome and their therapeutic mode of action in pathologies caused by limited blood vessel formation. Also the application of MSC as a vehicle to deliver drugs and/or genes in (anti-)angiogenesis will be discussed. Furthermore, the literature describing MSC transdifferentiation into endothelial cells will be evaluated critically.

Angiogenic Properties of Human Dental Pulp Stem Cells

Angiogenesis, the formation of capillaries from pre-existing blood vessels, is a key process in tissue engineering. If blood supply cannot be established rapidly, there is insufficient oxygen and nutrient transport and necrosis of the implanted tissue will occur. Recent studies indicate that the human dental pulp contains precursor cells, named dental pulp stem cells (hDPSC) that show self-renewal and multilineage differentiation capacity. Since these cells can be easily isolated, cultured and cryopreserved, they represent an attractive stem cell source for tissue engineering. Until now, only little is known about the angiogenic abilities and mechanisms of the hDPSC. In this study, the angiogenic profile of both cell lysates and conditioned medium of hDPSC was determined by means of an antibody array. Numerous pro-and anti-angiogenic factors such as vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), plasminogen activator inhibitor-1 (PAI-1) and endostatin were found both at the mRNA and protein level. hDPSC had no influence on the proliferation of the human microvascular endothelial cells (HMEC-1), but were able to significantly induce HMEC-1 migration in vitro. Addition of the PI3K-inhibitor LY294002 and the MEK-inhibitor U0126 to the HMEC-1 inhibited this effect, suggesting that both Akt and ERK pathways are involved in hDPSC-mediated HMEC-1 migration. Antibodies against VEGF also abolished the chemotactic actions of hDPSC. Furthermore, in the chicken chorioallantoic membrane (CAM) assay, hDPSC were able to significantly induce blood vessel formation. In conclusion, hDPSC have the ability to induce angiogenesis, meaning that this stem cell population has a great clinical potential, not only for tissue engineering but also for the treatment of chronic wounds, stroke and myocardial infarctions.

Effect of isolation methodology on stem cell properties and multilineage differentiation potential of human dental pulp stem cells

Dental pulp stem cells (DPSCs) are an attractive alternative mesenchymal stem cell (MSC) source because of their isolation simplicity compared with the more invasive methods associated with harvesting other MSC sources. However, the isolation method to be favored for obtaining DPSC cultures remains under discussion. This study compares the stem cell properties and multilineage differentiation potential of DPSCs obtained by the two most widely adapted isolation procedures. DPSCs were isolated either by enzymatic digestion of the pulp tissue (DPSC-EZ) or by the explant method (DPSC-OG), while keeping the culture media constant throughout all experiments and in both isolation methods. Assessment of the stem cell properties of DPSC-EZ and DPSC-OG showed no significant differences between the two groups with regard to proliferation rate and colony formation. Phenotype analysis indicated that DPSC-EZ and DPSC-OG were positive for CD29, CD44, CD90, CD105, CD117 and CD146 expression without any significant differences. The multilineage differentiation potential of both stem cell types was confirmed by using standard immuno(histo/cyto)chemical staining together with an in-depth ultrastructural analysis by means of transmission electron microscopy. Our results indicate that both DPSC-EZ and DPSC-OG could be successfully differentiated into adipogenic, chrondrogenic and osteogenic cell types, although the adipogenic differentiation of both stem cell populations was incomplete. The data suggest that both the enzymatic digestion and outgrowth method can be applied to obtain a suitable autologous DPSC resource for tissue replacement therapies of both bone and cartilage.

Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite outgrowth in an aligned tissue-engineered collagen construct in vitro

In the present study, we evaluated the differentiation potential of human dental pulp stem cells (hDPSCs) toward Schwann cells, together with their functional capacity with regard to myelination and support of neurite outgrowth in vitro. Successful Schwann cell differentiation was confirmed at the morphological and ultrastructural level by transmission electron microscopy. Furthermore, compared to undifferentiated hDPSCs, immunocytochemistry and ELISA tests revealed increased glial marker expression and neurotrophic factor secretion of differentiated hDPSCs (d‐hDPSCs), which promoted survival and neurite outgrowth in 2‐dimensional dorsal root ganglia cultures. In addition, neurites were myelinated by d‐hDPSCs in a 3‐dimensional collagen type I hydrogel neural tissue construct. This engineered construct contained aligned columns of d‐hDPSCs that supported and guided neurite outgrowth. Taken together, these findings provide the first evidence that hDPSCs are able to undergo Schwann cell differentiation and support neural outgrowth in vitro, proposing them to be good candidates for cell‐based therapies as treatment for peripheral nerve injury.—Martens, W., Sanen, K., Georgiou, M., Struys, T., Bronckaers, A., Ameloot, M., Phillips, J., Lambrichts, I. Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite outgrowth in an aligned tissue‐engineered collagen construct in vitro. FASEB J. 28, 1634–1643 (2014). www.fasebj.org

Dental stem cells and their promising role in neural regeneration: an update

IntroductionStem cell-based therapies are considered to be a promising treatment method for several clinical conditions such as Alzheimers disease, Parkinsons disease, spinal cord injury, and many others. However, the ideal stem cell type for stem cell-based therapy remains to be elucidated.DiscussionStem cells are present in a variety of tissues in the embryonic and adult human body. Both embryonic and adult stem cells have their advantages and disadvantages concerning the isolation method, ethical issues, or differentiation potential. The most described adult stem cell population is the mesenchymal stem cells due to their multi-lineage (trans)differentiation potential, high proliferative capacity, and promising therapeutic values. Recently, five different cell populations with mesenchymal stem cell characteristics were identified in dental tissues: dental pulp stem cells, stem cells from human exfoliated deciduous teeth, periodontal ligament stem cells, dental follicle precursor cells, and stem cells from apical papilla.ConclusionEach dental stem cell population possesses specific characteristics and advantages which will be summarized in this review. Furthermore, the neural characteristics of dental pulp stem cells and their potential role in (peripheral) neural regeneration will be discussed.

Neurogenic Maturation of Human Dental Pulp Stem Cells Following Neurosphere Generation Induces Morphological and Electrophysiological Characteristics of Functional Neurons

Cell-based therapies are emerging as an alternative treatment option to promote functional recovery in patients suffering from neurological disorders, which are the major cause of death and permanent disability. The present study aimed to differentiate human dental pulp stem cells (hDPSCs) toward functionally active neuronal cells in vitro. hDPSCs were subjected to a two-step protocol. First, neuronal induction was acquired through the formation of neurospheres, followed by neuronal maturation, based on cAMP and neurotrophin-3 (NT-3) signaling. At the ultrastructural level, it was shown that the intra-spheral microenvironment promoted intercellular communication. hDPSCs grew out of the neurospheres in vitro and established a neurogenic differentiated hDPSC culture (d-hDPSCs) upon cAMP and NT-3 signaling. d-hDPSCs were characterized by the increased expression of neuronal markers such as neuronal nuclei, microtubule-associated protein 2, neural cell adhesion molecule, growth-associated protein 43, synapsin I, and synaptophysin compared with nondifferentiated hDPSCs. Enzyme-linked immunosorbent assay demonstrated that the secretion of brain-derived neurotrophic factor, vascular endothelial growth factor, and nerve growth factor differed between d-hDPSCs and hDPSCs. d-hDPSCs acquired neuronal features, including multiple intercommunicating cytoplasmic extensions and increased vesicular transport, as shown by the electron microscopic observation. Patch clamp analysis demonstrated the functional activity of d-hDPSCs by the presence of tetrodotoxin- and tetraethyl ammonium-sensitive voltage-gated sodium and potassium channels, respectively. A subset of d-hDPSCs was able to fire a single action potential. The results reported in this study demonstrate that hDPSCs are capable of neuronal commitment following neurosphere formation, characterized by distinct morphological and electrophysiological properties of functional neuronal cells.

Expression Pattern of Basal Markers in Human Dental Pulp Stem Cells and Tissue

Dental pulp stem cells (DPSC) have been characterized as a multipotent stem cell population, with the ability to differentiate into mesodermal and neural cell lineages. Although ‘de novo’ expression of neural markers after differentiation is mostly considered as proof of differentiation, expression of these markers in undifferentiated DPSC is not well described. Therefore, an immunocytochemical analysis was performed to evaluate the neural marker expression of undifferentiated human DPSC (hDPSC) in in vitro cultures. Undifferentiated hDPSC uniformly expressed neural markers β-III-tubulin, S100 protein and synaptophysin. A subset of the population showed a positive immune-reactivity for galactocerebroside, neurofilament and nerve growth factor receptor p75. Furthermore, the location of possible stem cell niches, present in young dental pulp tissue, was determined by means of immunohistochemistry based on mesenchymal and neural marker expression. The results demonstrated the presence of a perivascular niche and a second stem cell niche at the cervical area. In adult dental pulp, only a perivascular niche could be observed. Based on the expression of neural markers in naïve DPSC, it has to be taken into account that not only the marker expression upon neural differentiation must be analyzed, but an ultrastructural analysis of the morphological changes and functional studies must also be performed to confirm a successful differentiation.

Pro-angiogenic impact of dental stem cells in vitro and in vivo.

Within the field of dental tissue engineering, the establishment of adequate tissue vascularization is one of the most important burdens to overcome. As vascular access within the tooth is restricted by the apical foramen, it is of major importance to implement effective vascularization strategies in order to recreate viable components of teeth and periodontal tissues. However, while the current regenerative approaches focus on the use of dental stem cells (DSCs), little is known about these cells and their ability to promote angiogenesis. Therefore, the present study aimed to elucidate the paracrine angiogenic properties of postnatal DSCs, in particular dental pulp stem cells (DPSCs), stem cells from the apical papilla (SCAPs) and dental follicle precursor cells (FSCs). An antibody array, together with RT-PCR and ELISA, pointed out the differential expression of pro-angiogenic as well as anti-angiogenic factors by cultured DSCs and human gingival fibroblasts (HGF-1). Despite the secretion of proliferation-promoting factors, DSCs caused no notable increase in the proliferation of human microvascular endothelial cells (HMEC-1). With regard to other aspects of the angiogenic cascade, DPSCs, SCAPs and HGF-1 significantly promoted endothelial migration in a transwell migration assay. DPSCs also had a pronounced effect on endothelial tubulogenesis, as was shown by an in vitro Matrigel™ assay. In the last part of this study, a chorioallantoic membrane assay demonstrated a sustained pro-angiogenic impact of DPSCs and SCAPs in an in vivo setting. Collectively, these data indicate a predominant pro-angiogenic influence of DPSCs and SCAPS in vitro and in vivo in comparison to FSCs, suggesting that both stem cell populations could potentially promote the vascularization of regenerated dental tissues.

Magnetic resonance imaging of human dental pulp stem cells in vitro and in vivo.

Recent advances in stem cell research have shown the promising nature of mesenchymal stem cells as plausible candidates for cell-based regenerative medicine. Many studies reported the use of human dental pulp stem cells (hDPSCs), which possess self-renewal capacity, high proliferation potential, and the ability to undergo multilineage differentiation. Together with this therapeutic approach, development of effective, noninvasive and nontoxic imaging techniques for visualizing and tracking the cells in vivo is crucial for the evaluation and improvement of stem cell therapy. Magnetic resonance imaging (MRI) is one of the most powerful diagnostic imaging techniques currently available for in vivo diagnosis and has been proposed as the most attractive modality for monitoring stem cell migration. The aim of this study was to investigate the labeling efficiency of hDPSCs using superparamagnetic iron oxide (SPIO) particles in order to allow visualization using in vitro and in vivo MRI without influencing cellular metabolism. MRI and transmission electron microscopy (TEM) showed optimal uptake with low SPIO concentrations of 15 μg/ml in combination with 0.75 μg/ml poly-l-lysine (PLL) resulting in more than 13 pg iron/cell and an in vitro detection limit of 50 labeled cells/μl. Very low SPIO concentrations in the culture medium resulted in extremely high labeling efficiency not reported before. For these conditions, tetrazolium salt assays showed no adverse effects on cell viability. Furthermore, in vivo MRI was performed to detect labeled hDPSCs transplanted into the brain of Rag 2-γ C immune-deficient mice. Transplanted cells did not show any signs of tumorgenecity or teratoma formation during the studied time course. We have reported on a labeling and imaging strategy to visualize human dental pulp stem cells in vivo using MRI. These data provide a solid base to allow cell tracking in future regenerative studies in the brain longitudinally.

Dental Stem Cells in Pulp Regeneration: Near Future or Long Road Ahead?

Although regenerative endodontic procedures have yielded an impressive body of favorable outcomes, the treatment of necrotic immature permanent teeth in particular remains to be a challenge. Recent advances in dental stem cell (DSC) research have gained increasing insight in their regenerative potential and prospective use in the formation of viable dental tissues. Numerous studies have already reported successful dental pulp regeneration following application of dental pulp stem cells, stem cells from the apical papilla, or dental follicle precursor cells in different in vivo models. Next to responsive cells, dental tissue engineering also requires the support of an appropriate scaffold material, ranging from naturally occurring polymers to treated dentin matrix components. However, the routine use and banking of DSCs still holds some major challenges, such as culture-associated differences, patient-related variability, and the effects of culture medium additives. Only in-depth evaluation of these problems and the implementation of standardized models and protocols will effectively lead to better alternatives for patients who no longer benefit from current treatment protocols.