Cardiac atrial appendage stem cells promote angiogenesis in vitro and in vivo.

Cardiac atrial appendage stem cells (CASCs) show extraordinary myocardial differentiation properties, making them ideal candidates for myocardial regeneration. However, since the myocardium is a highly vascularized tissue, revascularization of the ischemic infarct area is essential for functional repair. Therefore, this study assessed if CASCs contribute to cardiac angiogenesis via paracrine mechanisms. First, it was demonstrated that CASCs produce and secrete high levels of numerous angiogenic growth factors, including vascular endothelial growth factor (VEGF), endothelin-1 (ET-1) and insulin-like growth factor binding protein 3 (IGFBP-3). Functional in vitro assays with a human microvascular endothelial cell line (HMEC-1) and CASC CM showed that CASCs promote endothelial cell proliferation, migration and tube formation, the most important steps of the angiogenesis process. Addition of inhibitory antibodies against identified growth factors could significantly reduce these effects, indicating their importance in CASC-induced neovascularization. The angiogenic potential of CASCs and CASC CM was also confirmed in a chorioallantoic membrane assay, demonstrating that CASCs promote blood vessel formation in vivo. In conclusion, this study shows that CASCs not only induce myocardial repair by cardiomyogenic differentiation, but also stimulate blood vessel formation by paracrine mechanisms. The angiogenic properties of CASCs further strengthen their therapeutic potential and make them an optimal stem cell source for the treatment of ischemic heart disease.

Stem Cell-Based Therapies for Ischemic Stroke: Preclinical Results and the Potential of Imaging-Assisted Evaluation of Donor Cell Fate and Mechanisms of Brain Regeneration.

Stroke is the second most common cause of death and is a major cause of permanent disability. Given the current demographic trend of an ageing population and associated increased risk, the prevalence of and socioeconomic burden caused by stroke will continue to rise. Current therapies are unable to sufficiently ameliorate the disease outcome and are not applicable to all patients. Therefore, strategies such as cell-based therapies with mesenchymal stem cell (MSC) or induced pluripotent stem cell (iPSC) pave the way for new treatment options for stroke. These cells showed great preclinical promise despite the fact that the precise mechanism of action and the optimal administration route are unknown. To gain dynamic insights into the underlying repair processes after stem cell engraftment, noninvasive imaging modalities were developed to provide detailed spatial and functional information on the donor cell fate and host microenvironment. This review will focus on MSCs and iPSCs as types of widely used stem cell sources in current (bio)medical research and compare their efficacy and potential to ameliorate the disease outcome in animal stroke models. In addition, novel noninvasive imaging strategies allowing temporospatial in vivo tracking of transplanted cells and coinciding evaluation of neuronal repair following stroke will be discussed.

Cryopreservation and Banking of Dental Stem Cells.

Over the past decade, dental tissues have become an attractive source of mesenchymal stem cells (MSCs). Dental stem cells (DSCs) are not only able to differentiate into adipogenic, chondrogenic and osteogenic lineanges, but an increasing amount of research also pointed out their potential applicability in numerous clinical disorders, such as myocardial infarction, neurodegenerative diseases and diabetes. Together with their multilineage differentiation capacity, their easy availability from extracted third molars makes these stem cells a suitable alternative for bone marrow-derived MSCs. More importantly, DSCs appear to retain their stem cell properties following cryopreservation, a key aspect in their long-term preservation and upscale production. However, the vast number of different cryopreservation protocols makes it difficult to draw definite conclusions regarding the behavior of these stem cells. The routine application and banking of DSCs is also associated with some other pitfalls, such as interdonor variability, cell culture-induced changes and the use of animal-derived culture medium additives. Only thorough assessment of these challenges and the implementation of standardized, GMP procedures will successfully lead to better treatment options for patients who no longer benefit from current stem cell therapies.

MicroRNAs targeting VEGF are related to vascular dysfunction in preeclampsia.

In preeclampsia (PE), pre-existent maternal endothelial dysfunction leads to impaired placentation and vascular maladaptation. The vascular endothelial growth factor (VEGF) pathway is essential in the placentation process and VEGF expression is regulated through post-transcriptional modification by microRNAs (miRNAs). We investigated the expression of VEGF-related circulating miR-16, miR-29b, miR-126, miR-155 and miR-200c in PE vs healthy pregnancies (HPs), and their relation with vascular function, oxidative stress (OS) and systemic inflammation. In this case-control study, 24 women with early PE (<34 weeks) were compared with 30 women with HP. Circulating microRNA levels (RT-qPCR), OS and systemic inflammation were assessed in plasma samples (PE 29.5 vs HP 25.8 weeks) and related to extensive in vivo vascular function (flow-mediated dilatation (FMD), modified FMD (mFMD), carotid-femoral pulse wave velocity (CF-PWV), heart rate corrected augmentation index (AIx75) and reactive hyperemia index (RHI)). FMD, CF-PWV, AIx75 and RHI were all significantly impaired in PE (P<0.05). PE patients had reduced levels of miR-16 (5.53 ± 0.36 vs 5.84 ± 0.61) and increased levels of miR-200c (1.34 ± 0.57 vs 0.97 ± 0.68) (P<0.05). Independent of age and parity, miR-16 was related to impaired FMD (ß 2.771, 95% C.I.: 0.023-5.519, P=0.048) and mFMD (ß 3.401, 95% C.I.: 0.201-6.602, P=0.038). Likewise, miR-200c was independently associated with CF-PWV (ß 0.513, 95% C.I.: 0.034-0.992, P=0.036). In conclusion, circulating levels of miR-16 were lower in PE, which correlated with impaired endothelial function. Circulating miR-200c was increased in PE and correlated with higher arterial stiffness. These findings suggest a post-transcriptional dysregulation of the VEGF pathway in PE and identify miR-16 and miR-200c as possible diagnostic biomarkers for PE.

Preconditioning of Human Dental Pulp Stem Cells with Leukocyte- and Platelet-Rich Fibrin-Derived Factors Does Not Enhance Their Neuroregenerative Effect.

Pathologies of the central nervous system are characterized by loss of brain tissue and neuronal function which cannot be adequately restored by endogenous repair processes. This stresses the need for novel treatment options such as cell-based therapies that are able to restore damaged tissue or stimulate repair. This study investigated the neuroregenerative potential of the conditioned medium of human dental pulp stem cells (CM-hDPSCs) on neural stem cell (NSC) proliferation and migration as well as on neurite outgrowth of primary cortical neurons (pCNs). Additionally, the effect of leukocyte- and platelet-rich fibrin (L-PRF) priming on the neuroregenerative potential of the hDPSC secretome on NSCs and pCNs was evaluated. L-PRF contains factors that enhance stem cell-induced regeneration, but its effect on hDPSC-mediated neuroregeneration is unknown. This study demonstrated that CM-hDPSCs enhanced neuritogenesis. Moreover, CM-hDPSCs had a chemoattractant effect on NSCs. Although priming hDPSCs with L-PRF increased brain-derived neurotrophic factor secretion, no additional effects on the paracrine-mediated repair mechanisms were observed. These data support the neuroregenerative potential of hDPSCs, and although priming had no additional effect, the potential of L-PRF-primed hDPSCs on distinct regenerative mechanisms remains to be clarified.

Dental Tissue and Stem Cells Revisited: New Insights From the Expression of Fibroblast Activation Protein-Alpha.

Fibroblast activation protein-α (FAPα) is a membrane protein with dipeptidyl-peptidase and type I collagenase activity and is expressed during fetal growth. At the age of adolescence, FAPα expression is greatly reduced, only emerging in pathologies associated with extracellular matrix remodeling. We determined whether FAPα is expressed in human dental tissue involved in root maturation i.e., dental follicle and apical papilla and in dental pulp tissue. The dental follicle revealed a high concentration of FAPα and vimentin-positive cells within the stromal tissue. A similar observation was made in cell culture and FACS analysis confirmed these as dental follicle stem cells. Within the remnants of the Hertwigs' epithelial root sheath, we observed FAPα staining in the E-cadherin positive and vimentin-negative epithelial islands. FAPα- and vimentin-positive cells were encountered at the periphery of the islands suggesting an epithelial mesenchymal transition process. Analysis of the apical papilla revealed two novel histological regions; the periphery with dense and parallel aligned collagen type I defined as cortex fibrosa and the inner stromal tissue composed of less compacted collagen defined as medulla. FAPα expression was highly present within the medulla suggesting a role in extracellular matrix remodeling. Dental pulp tissue uncovered a heterogeneous FAPα staining but strong staining was noted within odontoblasts. In vitro studies confirmed the presence of FAPα expression in stem cells of the apical papilla and dental pulp. This study identified the expression of FAPα expression in dental stem cells which could open new perspectives in understanding dental root maturation and odontoblast function.

Angiogenic Properties of 'Leukocyte- and Platelet-Rich Fibrin'.

Leukocyte- and Platelet-Rich Fibrin (L-PRF) is an autologous platelet concentrate, consisting of a fibrin matrix enriched with platelets, leukocytes and a plethora of cytokines and growth factors. Since L-PRF is produced bedside from whole blood without the use of an anti-coagulant, it is becoming a popular adjuvant in regenerative medicine. While other types of platelet concentrates have been described to stimulate blood vessel formation, little is known about the angiogenic capacities of L-PRF. Therefore, this study aimed to fully characterize the angiogenic potential of L-PRF. With an antibody array, the growth factors released by L-PRF were determined and high levels of CXC chemokine receptor 2 (CXCR-2) ligands and epidermal growth factor (EGF) were found. L-PRF induced in vitro key steps of the angiogenic process: endothelial proliferation, migration and tube formation. In addition, we could clearly demonstrate that L-PRF is able to induce blood vessel formation in vivo, the chorioallantoic membrane assay. In conclusion, we could demonstrate the angiogenic capacity of L-PRF both in vitro and in vivo, underlying the clinical potential of this easy-to-use platelet concentrate.

Stem Cells for Cartilage Repair: Preclinical Studies and Insights in Translational Animal Models and Outcome Measures.

Due to the restricted intrinsic capacity of resident chondrocytes to regenerate the lost cartilage postinjury, stem cell-based therapies have been proposed as a novel therapeutic approach for cartilage repair. Moreover, stem cell-based therapies using mesenchymal stem cells (MSCs) or induced pluripotent stem cells (iPSCs) have been used successfully in preclinical and clinical settings. Despite these promising reports, the exact mechanisms underlying stem cell-mediated cartilage repair remain uncertain. Stem cells can contribute to cartilage repair via chondrogenic differentiation, via immunomodulation, or by the production of paracrine factors and extracellular vesicles. But before novel cell-based therapies for cartilage repair can be introduced into the clinic, rigorous testing in preclinical animal models is required. Preclinical models used in regenerative cartilage studies include murine, lapine, caprine, ovine, porcine, canine, and equine models, each associated with its specific advantages and limitations. This review presents a summary of recent in vitro data and from in vivo preclinical studies justifying the use of MSCs and iPSCs in cartilage tissue engineering. Moreover, the advantages and disadvantages of utilizing small and large animals will be discussed, while also describing suitable outcome measures for evaluating cartilage repair.

The Angiogenic Potential of DPSCs and SCAPs in an In Vivo Model of Dental Pulp Regeneration.

Adequate vascularization, a restricting factor for the survival of engineered tissues, is often promoted by the addition of stem cells or the appropriate angiogenic growth factors. In this study, human dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAPs) were applied in an in vivo model of dental pulp regeneration in order to compare their regenerative potential and confirm their previously demonstrated paracrine angiogenic properties. 3D-printed hydroxyapatite scaffolds containing DPSCs and/or SCAPs were subcutaneously transplanted into immunocompromised mice. After twelve weeks, histological and ultrastructural analysis demonstrated the regeneration of vascularized pulp-like tissue as well as mineralized tissue formation in all stem cell constructs. Despite the secretion of vascular endothelial growth factor in vitro, the stem cell constructs did not display a higher vascularization rate in comparison to control conditions. Similar results were found after eight weeks, which suggests both osteogenic/odontogenic differentiation of the transplanted stem cells and the promotion of angiogenesis in this particular setting. In conclusion, this is the first study to demonstrate the successful formation of vascularized pulp-like tissue in 3D-printed scaffolds containing dental stem cells, emphasizing the promising role of this approach in dental tissue engineering.

Dental Pulp Stem Cells: Their Potential in Reinnervation and Angiogenesis by Using Scaffolds.

Dental pulp is a highly vascularized and innervated tissue containing a heterogeneous stem cell population with multilineage differentiation potential. Current endodontic treatments focus on the preservation of the pulp tissue and the regeneration of dental pulp after pathological insults. Human dental pulp stem cells (hDPSCs) are currently investigated as stem cell-based therapy for pulp regeneration and for peripheral nerve injury in which neurons and Schwann cells display limited regenerative capacity. We have developed a neuronal differentiation protocol for hDPSCs that requires neurosphere formation before neuronal maturation. Moreover, Schwann cell differentiation of hDPSCs in our group revealed that differentiated hDPSCs have acquired the ability to myelinate and guide neurites from dorsal root ganglia. Besides their dynamic differentiation capacity, hDPSCs were shown to exert a paracrine effect on neural and endothelial cells. Analysis of hDPSC conditioned medium revealed the secretion of a broad spectrum of growth factors including brain-derived neurotrophic factor, nerve growth factor, vascular endothelial growth factor, and glial-derived neurotrophic factor. Application of the conditioned medium to endothelial cells promoted cell migration and tubulogenesis, indicating a paracrine proangiogenic effect. This hypothesis was enforced by the enhanced formation of blood vessels in the chorioallantoic membrane assay in the presence of hDPSCs. In addition, transplantation of 3-dimensional-printed hydroxyapatite scaffolds containing peptide hydrogels and hDPSCs into immunocompromised mice revealed blood vessel ingrowth, pulplike tissue formation, and osteodentin deposition suggesting osteogenic/odontogenic differentiation of hDPSCs. Future studies in our research group will focus on the pulp regeneration capacity of hDPSCs and the role of fibroblasts within the pulp extracellular matrix.

Angiogenic Capacity of Periodontal Ligament Stem Cells Pretreated with Deferoxamine and/or Fibroblast Growth Factor-2.

Periodontal ligament stem cells (PDLSCs) represent a good source of multipotent cells for cell-based therapies in regenerative medicine. The success rate of these treatments is severely dependent on the establishment of adequate vasculature in order to provide oxygen and nutrients to the transplanted cells. Pharmacological preconditioning of stem cells has been proposed as a promising method to augment their therapeutic efficacy. In this study, the aim was to improve the intrinsic angiogenic properties of PDLSCs by in vitro pretreatment with deferoxamine (DFX; 100µM), fibroblast growth factor-2 (FGF-2; 10ng/mL) or both substances combined. An antibody array revealed the differential expression of several proteins, including vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). ELISA data confirmed a 1.5 to 1.8-fold increase in VEGF for all tested conditions. Moreover, 48 hours after the removal of DFX, VEGF levels remained elevated (1.8-fold) compared to control conditions. FGF-2 and combination treatment resulted in a 5.4 to 13.1-fold increase in PlGF secretion, whereas DFX treatment had no effect. Furthermore, both PDLSCs as pretreated PDLSCs induced endothelial migration. Despite the significant elevated VEGF levels of pretreated PDLSCs, the induced endothelial migration was not higher by pretreated PDLSCs. We find that the observed induced endothelial cell motility was not dependent on VEGF, since blocking the VEGFR1-3 with Axitinib (0.5nM) did not inhibit endothelial motility towards PDLSCs. Taken together, this study provides evidence that preconditioning with DFX and/or FGF-2 significantly improves the angiogenic secretome of PDLSCs, in particular VEGF and PlGF secretion. However, our data suggest that VEGF is not the only player when it comes to influencing endothelial behavior by the PDLSCs.

The Neurovascular Properties of Dental Stem Cells and Their Importance in Dental Tissue Engineering.

Within the field of tissue engineering, natural tissues are reconstructed by combining growth factors, stem cells, and different biomaterials to serve as a scaffold for novel tissue growth. As adequate vascularization and innervation are essential components for the viability of regenerated tissues, there is a high need for easily accessible stem cells that are capable of supporting these functions. Within the human tooth and its surrounding tissues, different stem cell populations can be distinguished, such as dental pulp stem cells, stem cells from human deciduous teeth, stem cells from the apical papilla, dental follicle stem cells, and periodontal ligament stem cells. Given their straightforward and relatively easy isolation from extracted third molars, dental stem cells (DSCs) have become an attractive source of mesenchymal-like stem cells. Over the past decade, there have been numerous studies supporting the angiogenic, neuroprotective, and neurotrophic effects of the DSC secretome. Together with their ability to differentiate into endothelial cells and neural cell types, this makes DSCs suitable candidates for dental tissue engineering and nerve injury repair.

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.

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.