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Article The ApoA-I mimetic peptide 5A enhances remyelination by promoting clearance and degradation of myelin debris Graphical abstract Highlights d ApoA-I mimetic peptide 5A enhances remyelination in a phagocyte-dependent manner d In addition to promoting lipid efflux, peptide 5A enhances clearance of myelin debris d Peptide 5A drives clearance of myelin debris via the fatty acid translocase CD36 We thank M.P. Tulleners for excellent technical assistance. The work was financially supported by the Research Foundation of Flanders (FWO Vlaanderen; 1S15519N, G099618FWO, and 12J9119N) and the Interreg V-A EMR program (EURLIPIDS, EMR23). The funding agencies had no role in the design, analysis, or writing of the article.

The apical papilla is a stem cell rich tissue located at the base of the developing dental root and is responsible for the progressive elongation and maturation of the root. The multipotent stem cells of the apical papilla (SCAP) are extensively studied in cell culture since they demonstrate a high capacity for osteogenic, adipogenic, and chondrogenic differentiation and are thus an attractive stem cell source for stem cell-based therapies. Currently, only few studies are dedicated to determining the role of the apical papilla in dental root development. In this review, we will focus on the architecture of the apical papilla and describe the specific SCAP signaling pathways involved in root maturation. Furthermore, we will explore the heterogeneity of the SCAP phenotype within the tissue and determine their micro-environmental interaction. Understanding the mechanism of postnatal dental root growth could further aid in developing novel strategies in dental root regeneration.

Stem cell-based regenerative therapies hold great promises to treat a wide spectrum of diseases. However, stem cell engraftment and survival are still challenging due to an unfavorable transplantation environment. Advanced glycation end-products (AGEs) can contribute to the generation of these harmful conditions. AGEs are a heterogeneous group of glycated products, nonenzymatically formed when proteins and/or lipids become glycated and oxidized. Our typical Western diet as well as cigarettes contain high AGEs content. AGEs are also endogenously formed in our body and accumulate with senescence and in pathological situations. Whether AGEs have an impact on stem cell viability in regenerative medicine remains unclear, and research on the effect of AGEs on stem cell proliferation and apoptosis is still ongoing. Therefore, this systematic review provides a clear overview of the effects of glycated proteins on cell viability in various types of primary isolated stem cells used in regenerative medicine. Figures were created using images from Servier Medical Art Commons Attribution 3.0 Unported License (http://smart.servier.com). Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License. This work was supported by a Bijzonder onderzoeksfonds (BOF) grant from Hasselt University (grant number: 16NI05BOF). HB benefits from an aspirant PhD mandate (grant number: 1154120N) of the `Research Foundation-Flanders' (fonds voor wetenschappelijk onderzoek (FWO)). PG is also supported by the FWO (grant numbers: 12U7718N and 1502120N). Bito, V (corresponding author), Hasselt Univ, Biomed BIOMED Res Inst, Agoralaan Bldg C, B-3590 Diepenbeek, Belgium. virginie.bito@uhasselt.be

Additional file 1. The additional data includes information regarding the TEM protocol, TEM images of the PS NPs and HPCs, data on NP characterization and cell viability assessment. Furthermore, flow cytometry gating strategies are included.

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 study was performed within the framework of the cooperation between the University of Namur and Hasselt University. Melissa Lo Monaco is funded by “Bijzonder Onderzoeksfonds” and “Fonds Spécial de Recherche” (BOF16DOCNA02-FSR-confin UHasselt-UNamur). Jessica Ratajczak, Pascal Gervois, Petra Hilkens, and Annelies Bronckaers are funded by “Fonds Wetenschappelijk Onderzoek.” Greet Merckx is funded by “Bijzonder Onderzoeksfonds.”

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.

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.

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. Esther Wolfs, Jessica Ratajczak, Tim Vangansewinkel, Petra Hilkens, and Annelies Bronckaers are funded by Fonds Wetenschappelijk Onderzoek by grants G0A7514N, G089213N, G029112N, 12D8516N, and 1508015N, respectively. Yorg Dillen is funded by Bijzonder On- ¨ derzoeksfonds by grant BOF15DOC04.

In conclusion, we state that curiosity-driven research after normal synovial folds within the PIP-joint of the finger confirms their actual location. Neurovascular bundles in the normal PIP-joint’s capsule, and neurons in lamellated corpuscles within the Proper Collateral Ligament, may shed new light on the pathogenesis of rheumatoid arthritis in human finger joints.

Objective: Current available therapies are unable to adequately promote functional recovery in patients suffering from neurological disorders such as stroke, which are a major cause of death and permanent disability. As an alternative treatment, cell-based therapies are now emerging. Human neural stem cells are the most promising candidates to induce neuronal repair, but due to ethical and practical considerations, alternative sources of progenitor cells with neuronal differentiation potential are needed. The aim of the present study was to differentiate stem cells that were isolated from the human dental pulp (hDPSCs) towards functionally active neuronal cells in vitro. Methods: hDPSCs were subjected to a two-step protocol. First, neurosphere-formation was used to acquire neuronal induction. Secondly, neuronal maturation was induced, based on cAMP and NT-3 signaling. The ultrastructural characteristics of intra-neurospheral hDPSCs and their microenvironment were determined by means of transmission electron microscopy (TEM). Neurogenic maturated hDPSCs (d-hDPSCs) were subjected to immunocytochemical, PCR, ultrastructural and electrophysiological analysis. An Enzyme-Linked Immunosorbent Assay (ELISA) was performed for VEGF, NGF, BDNF and GDNF in order to evaluate the secretome of hDPSCs before and after neurogenic differentiation. Results: Based on the ultrastructural analysis it was shown that within the neurospheres, intercellular communication was promoted. Moreover, hDPSCs grew out of the neurospheres in vitro and established a neurogenic differentiated hDPSC culture (d-hDPSCs) which was characterized by the increased expression of the neuronal markers NeuN, MAP-2 and NCAM compared to the nondifferentiated counterpart. Moreover, the secretion of BDNF, VEGF and NGF differed between dhDPSCs and hDPSCSs. Ultrastructurally, d-hDPSCs acquired neuronal features including multiple intercommunicating cytoplasmic extensions and increased vesicular transport. Finally, patch clamp analysis demonstrated the functional activity of d-hDPSCs by the presence of TTX-sensitive voltagegated sodium and TEA-sensitive potassium channels. A subset of d-hDPSCs was able to fire a single action potential. Conclusions: Although promising results were achieved, establishing a completely functional dhDPSC culture remains a challenge. The results in this study demonstrate that hDPSCs are capable of neuronal commitment with distinct features of neuronal cells.

Dental pulp stem cells (DPSC), mesenchymal stem cells (MSC) from the tooth, share phenotypical and multilineage characteristics with bone marrow derived MSCs (BMMSCs). Since DPSCs are much easier to harvest in comparison to BMMSCs they are considered as a useful alternative MSC source in stem cell research. As MSCs retain some plasticity, the neural differentiation potential of DPSCs will be evaluated. DPSCs will be labelled with the commercially available ferumoxide Endorem® to allow visualization with MRI following engraftment. This study shows expression of NeuN in neurogenic differentiated DPSCs. Future studies should include quantitative and electrophysiological experiments. This study also shows that the optimal concentration for labelling DPSCs is 0,75µg/ml PLL with 15µg/ml Endorem®. The influence of Endorem® labelling on cellular processes of DPSCs needs to be determined before these cells can be considered as a clinical applicable intracellular contrast agent for MRI analysi

Comparative observations in the hind limb of the opossum, a primate predecessor, reveal essential differences regarding lower leg coupling mechanisms. Especially the primate ankle shows cardan joint properties, causing inversion of the foot before toe-off. Inversion is turning the footsole inwards, eversion is turning it outwards. By bony landmarks, features of longitudinal rotation of bones from 3-D frames were matched to available radiocinematographs of walking stages in (a) early stance, (b) mid-stance, (c) toe-off, and (d) swing. Flemish Interuniversity Council - University Development Cooperation (VLIR-UOS), Institutional University Cooperation, ADEKUS Suriname, Project 6: Education and research programme on physical therapy, Coordinators: Yves Vanlandewijck & Tony Chang

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by focal inflammatory lesions and prominent demyelination. Even though the currently available therapies are effective in treating the initial stages of disease, they are unable to halt or reverse disease progression into the chronic progressive stage. Thus far, no repair-inducing treatments are available for progressive MS patients. Hence, there is an urgent need for the development of new therapeutic strategies either targeting the destructive immunological demyelination or boosting endogenous repair mechanisms. Using in vitro, ex vivo, and in vivo models, we demonstrate that selective inhibition of phosphodiesterase 4 (PDE4), a family of enzymes that hydrolyzes and inactivates cyclic adenosine monophosphate (cAMP), reduces inflammation and promotes myelin repair. More specifically, we segregated the myelination-promoting and anti-inflammatory effects into a PDE4Dand PDE4B-dependent process respectively. We show that inhibition of PDE4D boosts oligodendrocyte progenitor cells (OPC) differentiation and enhances (re)myelination of both murine OPCs and human iPSC-derived OPCs. In addition, PDE4D inhibition promotes in vivo remyelination in the cuprizone model, which is accompanied by improved spatial memory and reduced visual evoked potential latency times. We further identified that PDE4B-specific inhibition exerts anti-inflammatory effects since it lowers in vitro monocytic nitric oxide (NO) production and improves in vivo neurological scores during the early phase of experimental autoimmune encephalomyelitis (EAE). In contrast to the pan PDE4 inhibitor roflumilast, the therapeutic dose of both the PDE4B-specific inhibitor A33 and the PDE4D-specific inhibitor Gebr32a did not trigger emesis-like side effects in rodents. Finally, we report distinct PDE4D isoform expression patterns in human area postrema neurons and human oligodendroglia lineage cells. Using the CRISPR-Cas9 system, we confirmed that pde4d1/2 and pde4d6 are the key targets to induce OPC differentiation. Collectively, these data demonstrate that gene specific PDE4 inhibitors have potential as novel therapeutic agents for targeting the distinct disease processes of MS. This work has been supported by FWO (12G0817N, 1S57521N, G041421N, and 12G0817N), Fondation Charctot Stichting (ID2020- 0019), Nationale Belgische Multiple Sclerose Liga (Charco18VT), MS Liga Vlaanderen and Stichting MS Research (18-1016 MS). MS, EP, JP and TV have a proprietary interest in selective PDE4D inhibitors for the treatment of demyelinating disorders and neurodegenerative disorders. JP has a proprietary interest in the PDE4 inhibitor roflumilast for the treatment of cognitive impairment as well as PDE4D inhibitors for the treatment of Alzheimer’s disease. We thank Prof. Dr. O.N. Viacheslav (University Medical Center Hamburg-Eppendorf, German Center for Cardiovascular Research) and Prof. Dr. M. Conti (University of California), for providing the PDE4B KO animals. Furthermore, we thank Rewind Therapeutics for providing the visual evoked potential equipment.

Failure of remyelination underlies the progressive nature of demyelinating diseases such as multiple sclerosis. Why endogenous repair mechanisms frequently fail in these disorders is poorly understood. However, there is now evidence indicating that this is related to an overly inflammatory microenvironment combined with the intrinsic inability of oligodendrocyte precursor cells (OPCs) to differentiate into mature myelinating cells. Previously, we found that phloretin, a flavonoid abundantly present in apples and strawberries, reduces neuroinflammation by driving macrophages toward an antiinflammatory phenotype. Here, we show that phloretin also markedly stimulates remyelination in ex vivo and in vivo animal models. Improved remyelination was attributed to a direct impact of phloretin on OPC maturation and occurred independently from alterations in microglia function and inflammation. We found, mechanistically, that phloretin acts as a direct ligand for the fatty acid sensing nuclear receptor peroxisome proliferator-activated receptor gamma, thereby promoting the maturation of OPCs. Together, our findings indicate that phloretin has proregenerative properties in central nervous system disorders, with potentially broad implications for the development of therapeutic strategies and dietary interventions aimed at promoting remyelination. This work was supported by the Flemish Fund for Scientific Research (FWO Vlaanderen; G099618, 12J9119N, and 1501720N). M.K. was supported by the European Research Council under the European Union’s Horizon 2020 research and innovation program (640116), by a Salk grant from the government of Flanders, Belgium, and by the FWO (G0G1216N and G080121N)

The ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune modulation and a delivery system capable of crossing the blood-brain barrier. The recent identification and characterization of a small population of regulatory T (Treg) cells resident in the brain presents one such potential therapeutic target. Here, we identified brain interleukin 2 (IL-2) levels as a limiting factor for brain-resident Treg cells. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially direct delivery to inflammatory sites. Mice with brain-specific IL-2 delivery were protected in traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL-2 gene delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients. The work was supported by the VIB, an ERC Consolidator Grant TissueTreg (to A.L.), an ERC Proof of Concept Grant TreatBrainDamage (to A.L.), an ERC Starting Grant AstroFunc (to M.G.H.), an ERC Proof of Concept Grant AD-VIP (to M.G.H.), FWO Research Grant 1513616N (to M.G.H.), Thierry Latran Foundation Grant SOD-VIP (to M.G.H.), an ERNAET Chair (H2020-WIDESPREAD-2018-2020-6; NCBio: 951923; to M.G.H.), FWO Research Grants 1503420N (to E.P.) and 1513020N (to J.W.), an SAO-FRA pilot grant (20190032, to E.P.), and the Biotechnology and Biological Sciences Research Council through Institute Strategic Program Grant funding BBS/E/B/000C0427 and BBS/E/B/000C0428, and the Biotechnology and Biological Sciences Research Council Core Capability Grant to the Babraham Institute. E.P., V.L., M.M., P.G., J.W. and A.d.B. were supported by fellowships from the FWO. R.L. is a senior clinical investigator of FWO Flanders. P.B., O.A. and C.P.F. were supported by an ERA-NET-NEURON grant EJTC 2016 to C.P.F. and by the Netherlands Organization for Scientific research (NWO). The authors acknowledge the important contributions of J. Haughton (VIB) for mouse husbandry, M. Rincon (VIB) for advice on AAV design and production, K. Vennekens for technical support, P. -A. Penttila and the KUL FACS Core, J. Wouters and the KUL Molecular Small Animal Imaging Center (MoSAIC), S. Walker and the Babraham Institute Imaging Core, the VIB Bio-Imaging Core, the VIB Single Cell Sequencing Core, and R. Breedijk, M. Hink and the Leeuwenhoek Center for Advanced Microscopy at the University of Amsterdam.

The progressive nature of demyelinating diseases lies in the inability of the central nervous system (CNS) to induce proper remyelination. Recently, we and others demonstrated that a dysregulated innate immune response partially underlies failure of CNS remyelination. Extensive accumulation of myelin-derived lipids and an inability to process these lipids was found to induce a disease-promoting phagocyte phenotype. Hence, restoring the ability of these phagocytes to metabolize and efflux myelin-derived lipids represents a promising strategy to promote remyelination. Here, we show that ApoA-I mimetic peptide 5A, a molecule well known to promote activity of the lipid efflux transporter ABCA1, markedly enhances remyelination. Mechanistically, we find that the repair-inducing properties of 5A are attributable to increased clearance and metabolism of remyelination-inhibiting myelin debris via the fatty acid translocase protein CD36, which is transcriptionally controlled by the ABCA1-JAK2-STAT3 signaling pathway. Altogether, our findings indi-cate that 5A promotes remyelination by stimulating clearance and degradation of myelin debris. We thank M.P. Tulleners for excellent technical assistance. The work was financially supported by the Research Foundation of Flanders (FWO Vlaanderen; 1S15519N, G099618FWO, and 12J9119N) and the Interreg V-A EMR program (EURLIPIDS, EMR23). The funding agencies had no role in the design, analysis, or writing of the article.

Osteoarthritis (OA) is a degenerative and inflammatory joint disorder with cartilage loss. Dental pulp stem cells (DPSCs) can undergo chondrogenic differentiation and secrete growth factors associated with tissue repair and immunomodulation. Leukocyte- and platelet-rich fibrin (L-PRF) emerges in regenerative medicine because of its growth factor content and fibrin matrix. This study evaluates the therapeutic application of DPSCs and L-PRF in OA via immunomodulation and cartilage regeneration. Chondrogenic differentiation of DPSCs, with or without L-PRF exudate (ex) and conditioned medium (CM), and of bone marrow-mesenchymal stem cells was compared. These cells showed differential chondrogenesis. L-PRF was unable to increase cartilage-associated components. Immature murine articular chondrocytes (iMACs) were cultured with L-PRF ex, L-PRF CM, or DPSC CM. L-PRF CM had pro-survival and proliferative effects on unstimulated and cytokine-stimulated iMACs. L-PRF CM stimulated the release of IL-6 and PGE2, and increased MMP-13, TIMP-1 and IL-6 mRNA levels in cytokine-stimulated iMACs. DPSC CM increased the survival and proliferation of unstimulated iMACs. In cytokine-stimulated iMACs, DPSC CM increased TIMP-1 gene expression, whereas it inhibited nitrite release in 3D culture. We showed promising effects of DPSCs in an in vitro OA model, as they undergo chondrogenesis in vitro, stimulate the survival of chondrocytes and have immunomodulatory effects. The study was performed within the framework of the cooperation between the University of Namur and Hasselt University. Melissa Lo Monaco is funded by "Bijzonder Onderzoeksfonds" and "Fonds Special de Recherche" (BOF16DOCNA02-FSR-confin UHasselt-UNamur). Pascal Gervois (12U7718N, 1502120N), Annelies Bronckaers and Ivo Lambrichts are funded by grants of the Fonds Wetenschappelijk Onderzoek Vlaanderen. Peter Clegg is funded by the MRC-Versus Arthritis Centre for Musculoskeletal Ageing, HBLB and the Horse Trust. Jean-Michel Vandeweerd is funded by FSR (Fonds de Soutien a la Recherche) UNamur. Lo Monaco, M (corresponding author), Hasselt Univ, Biomed Res Inst BIOMED, Cardio & Organ Syst COST, B-3590 Diepenbeek, Belgium; Univ Namur, Dept Vet Med, Integrated Vet Res Unit IVRU, Namur Res Inst Life Sci NARILIS, B-5000 Namur, Belgium. melissa.lomonaco@uhasselt.be; pascal.gervois@uhasselt.be; jej.beaumont@maastrichtuniversity.nl; P.D.Clegg@liverpool.ac.uk; annelies.bronckaers@uhasselt.be; jean-michel.vandeweerd@unamur.be; ivo.lambrichts@uhasselt.be

Articular cartilage defects and tendon lesions affect millions of patients each year and are associated with a high economic burden. Furthermore, articular cartilage defects often progress into osteoarthritis (OA), a degenerative and inflammatory condition of synovial joints with associated loss of cartilage matrix. Current treatments are unable to provide long-term regeneration of the damaged tissue, stressing the need for alternative therapeutic options including stem cell-based approaches. Human dental pulp stem cells (DPSCs) can differentiate into cartilage-producing cells and secrete numerous growth factors associated with tissue repair. Moreover, leukocyte- and platelet-rich fibrin (L-PRF), an endogenous blood-derived biomaterial, has recently emerged as a promising treatment strategy due to its growth factor content and supportive fibrin matrix. We demonstrated that L-PRF does not enhance the chondrogenic differentiation of DPSCs and BM-MSCs as demonstrated by collagen type 2, aggrecan and glycosaminoglycan (GAG) production. Furthermore, differentiated DPSCs do not produce aggrecan, in contrast to BM-MSCs. Human DPSC- and L-PRF CM displayed a proliferative and a pro-survival effect on chondrocytes in vitro. Additionally, DPSCs were able to migrate towards chondrocytes. Lastly, human DPSCs and PDL-SCs formed tendon-like tissues characterized by the production of collagen and the parallel alignment of cells.