Ceramide Glycosylation by Glucosylceramide Synthase Selectively Maintains the Properties of Breast Cancer Stem Cells

Cancer stem cells are distinguished from normal adult stem cells by their stemness without tissue homeostasis control. Glycosphingolipids (GSLs), particularly globo-series GSLs, are important markers of undifferentiated embryonic stem cells, but little is known about whether or not ceramide glycosylation, which controls glycosphingolipid synthesis, plays a role in modulating stem cells. Here, we report that ceramide glycosylation catalyzed by glucosylceramide synthase, which is enhanced in breast cancer stem cells (BCSCs) but not in normal mammary epithelial stem cells, maintains tumorous pluripotency of BCSCs. Enhanced ceramide glycosylation and globotriosylceramide (Gb3) correlate well with the numbers of BCSCs in breast cancer cell lines. In BCSCs sorted with CD44⁺/ESA⁺/CD24⁻ markers, Gb3 activates c-Src/β-catenin signaling and up-regulates the expression of FGF-2, CD44, and Oct-4 enriching tumorigenesis. Conversely, silencing glucosylceramide synthase expression disrupts Gb3 synthesis and selectively kills BCSCs through deactivation of c-Src/β-catenin signaling. These findings highlight the unexploited role of ceramide glycosylation in selectively maintaining the tumorous pluripotency of cancer stem cells. It speculates that disruption of ceramide glycosylation or globo-series GSL is a useful approach to specifically target BCSCs specifically.     

Variable subcellular localization of glycosphingolipids

Although most glycosphingolipids (GSLs) are thought to be locatedin the outer leaflet of the plasma membrane, recent evidenceindicates that GSLs are also associated with intracellular organelles.We now report that the subcellular localization of GSLs variesdepending on the GSL structure and cell type. GSL localizationwas determined by indirect immunofluorescence microscopy offixed permeabilized cells. A single GSL exhibited variable subcellularlocalization in different cells. For example, antibody to GalCeris localized primarily to the plasma membrane of HaCaT II-3keratinocytes, but to intracellular organelies in other epithelialcells. GalCer is localized to small vesicles and tubulovesicularstructures in MDCK cells, and to the surface of phase-denselipid droplets in HepG2 hepatoma cells. Furthermore, withina single cell type, individual GSLs were found to exhibit differentpatterns of subcellular localization. In HepG2 cells, LacCerwas associated with small vesicles, which differed from thephase-dense vesicles stained by anti-GalCer, and Gb4Cer wasassociated with the intermediate filaments of the cytoskeleton.Both anti-GalCer and monoclonal antibody A2B5, which binds polysialogangliosides,localized to mitochondria. The distinct subcellular localizationpatterns of GSLs raise interesting questions about their functionsin different organelles. Together with published data on theenrichment of GSLs in specific organelles and in apical plasmamembrane, these findings indicate the existence of specificsorting mechanisms that regulate the intracellular transportand localization of GSLs. cytoskeleton glycosphingolipid intracellular organelles mitochondria subcellular localization     

Influence of glycosphingolipids on cancer cell energy metabolism

A growing number of studies describe a connection between glycosphingolipids (GSLs) and glutamine metabolism, glucose metabolism and mitochondrial dysfunction in cancer cells. Since deregulated cell energy metabolism is one of cancer cells hallmarks, investigating this connection is an important step in the development of anti-cancer therapies. GSL species are often aberrantly regulated in human cancers. They cluster in signaling platforms in the plasma membrane and organelle membranes in so called glycosphingolipid enriched microdomains (GEMs), thereby regulating cell signaling pathways. The most important glutamine transporter for epithelial cells, alanine-serine-cysteine transporter 2 (ASCT2) locates in GEMs and is regulated by GEM composition. The accumulation of glucosylceramide and lactosylceramide in mitochondria associated ER membranes (MAMs) leads to increased oxidative phosphorylation. This increases mitochondrial reactive oxygen species (ROS) levels and influences mitochondrial dynamics. Here, we review current knowledge about deregulated GSL species in cancer, GSL influence on glutamine and glucose metabolism. In addition, the role of GSLs in MAMs, oxidative phosphorylation (OXPHOS) and mitochondrial dynamics with a special focus on mechanistic target of rapamycin (mTOR) signaling is discussed. mTOR seems to play a pivotal role in the connection between GSLs and glutamine metabolism as well as in mitochondrial signaling.     

A Major Fraction of Glycosphingolipids in Model and Cellular Cholesterol-containing Membranes Is Undetectable by Their Binding Proteins

Glycosphingolipids (GSLs) accumulate in cholesterol-enriched cell membrane domains and provide receptors for protein ligands. Lipid-based “aglycone” interactions can influence GSL carbohydrate epitope presentation. To evaluate this relationship, Verotoxin binding its receptor GSL, globotriaosyl ceramide (Gb₃), was analyzed in simple GSL/cholesterol, detergent-resistant membrane vesicles by equilibrium density gradient centrifugation. Vesicles separated into two Gb_3/cholesterol-containing populations. The lighter, minor fraction (<5% total GSL), bound VT1, VT2, IgG/IgM mAb anti-Gb₃, HIVgp120 or Bandeiraea simplicifolia lectin. Only IgM anti-Gb₃, more tolerant of carbohydrate modification, bound both vesicle fractions. Post-embedding cryo-immuno-EM confirmed these results. This appears to be a general GSL-cholesterol property, because similar receptor-inactive vesicles were separated for other GSL-protein ligand systems; cholera toxin (CTx)-GM1, HIVgp120-galactosyl ceramide/sulfatide. Inclusion of galactosyl or glucosyl ceramide (GalCer and GlcCer) rendered VT1-unreactive Gb₃/cholesterol vesicles, VT1-reactive. We found GalCer and GlcCer bind Gb₃, suggesting GSL-GSL interaction can counter cholesterol masking of Gb₃. The similar separation of Vero cell membrane-derived vesicles into minor “binding,” and major “non-binding” fractions when probed with VT1, CTx, or anti-SSEA4 (a human GSL stem cell marker), demonstrates potential physiological relevance. Cell membrane GSL masking was cholesterol- and actin-dependent. Cholesterol depletion of Vero and HeLa cells enabled differential VT1B subunit labeling of “available” and “cholesterol-masked” plasma membrane Gb₃ pools by fluorescence microscopy. Thus, the model GSL/cholesterol vesicle studies predicted two distinct membrane GSL formats, which were demonstrated within the plasma membrane of cultured cells. Cholesterol masking of most cell membrane GSLs may impinge many GSL receptor functions.     

Adamantyl glycosphingolipids provide a new approach to the selective regulation of cellular glycosphingolipid metabolism

Mammalian glycosphingolipid (GSL) precursor monohexosylceramides are either glucosyl- or galactosylceramide (GlcCer or GalCer). Most GSLs derive from GlcCer. Substitution of the GSL fatty acid with adamantane generates amphipathic mimics of increased water solubility, retaining receptor function. We have synthesized adamantyl GlcCer (adaGlcCer) and adamantyl GalCer (adaGalCer). AdaGlcCer and adaGalCer partition into cells to alter GSL metabolism. At low dose, adaGlcCer increased cellular GSLs by inhibition of glucocerebrosidase (GCC). Recombinant GCC was inhibited at pH 7 but not pH 5. In contrast, adaGalCer stimulated GCC at pH 5 but not pH 7 and, like adaGlcCer, corrected N370S mutant GCC traffic from the endoplasmic reticulum to lysosomes. AdaGalCer reduced GlcCer levels in normal and lysosomal storage disease (LSD) cells. At 40 μM adaGlcCer, lactosylceramide (LacCer) synthase inhibition depleted LacCer (and more complex GSLs), such that only GlcCer remained. In Vero cell microsomes, 40 μM adaGlcCer was converted to adaLacCer, and LacCer synthesis was inhibited. AdaGlcCer is the first cell LacCer synthase inhibitor. At 40 μM adaGalCer, cell synthesis of only Gb(3) and Gb(4) was significantly reduced, and a novel product, adamantyl digalactosylceramide (adaGb(2)), was generated, indicating substrate competition for Gb(3) synthase. AdaGalCer also inhibited cell sulfatide synthesis. Microsomal Gb(3) synthesis was inhibited by adaGalCer. Metabolic labeling of Gb(3) in Fabry LSD cells was selectively reduced by adaGalCer, and adaGb(2) was produced. AdaGb(2) in cells was 10-fold more effectively shed into the medium than the more polar Gb(3), providing an easily eliminated "safety valve" alternative to Gb(3) accumulation. Adamantyl monohexosyl ceramides thus provide new tools to selectively manipulate normal cellular GSL metabolism and reduce GSL accumulation in cells from LSD patients.     

Structure and function of glycosphingolipids and sphingolipids: recollections and future trends

Based on development of various methodologies for isolation and characterization of glycosphingolipids (GSLs), we have identified a number of GSLs with globo-series or lacto-series structure. Many of them are tumor-associated or developmentally regulated antigens. The major question arose, what are their functions in cells and tissues? Various approaches to answer this question were undertaken. While the method is different for each approach, we have continuously studied GSL or glycosyl epitope interaction with functional membrane components, which include tetraspanins, growth factor receptors, integrins, and signal transducer molecules. Often, GSLs were found to interact with other carbohydrates within a specific membrane microdomain termed "glycosynapse", which mediates cell adhesion with concurrent signal transduction. Future trends in GSL and glycosyl epitope research are considered, including stem cell biology and epithelial-mesenchymal transition (EMT) process.     

VIP21/caveolin, glycosphingolipid clusters and the sorting of glycosylphosphatidylinositol-anchored proteins in epithelial cells.

We studied the role of the association between glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipid (GSL) clusters in apical targeting using gD1-DAF, a GPI-anchored protein that is differentially sorted by three epithelial cell lines. Differently from MDCK cells, where both gD1-DAF and glucosylceramide (GlcCer) are sorted to the apical membrane, in MDCK Concanavalin A-resistant cells (MDCK-ConAr) gD1-DAF was mis-sorted to both surfaces, but GlcCer was still targeted to the apical surface. In both MDCK and MDCK-ConAr cells, gD1-DAF became associated with TX-100-insoluble GSL clusters during transport to the cell surface. In dramatic contrast with MDCK cells, the Fischer rat thyroid (FRT) cell line targeted both gD1-DAF and GlcCer basolaterally. The targeting differences for GSLs in FRT and MDCK cells cannot be accounted for by a differential ability to form clusters because, in spite of major differences in the GSL composition, both cell lines assembled GSLs into TX-100-insoluble complexes with identical isopycnic densities. Surprisingly, in FRT cells, gD1-DAF did not form clusters with GSLs and, therefore, remained completely soluble. This clustering defect in FRT cells correlated with the lack of expression of VIP21/caveolin, a protein localized to both the plasma membrane caveolae and the trans Golgi network. This suggests that VIP21/caveolin may have an important role in recruiting GPI-anchored proteins into GSL complexes necessary for their apical sorting. However, since MDCK-ConAr cells expressed caveolin and clustered GPI-anchored proteins normally, yet mis-sorted them, our results also indicate that clustering and caveolin are not sufficient for apical targeting, and that additional factors are required for the accurate apical sorting of GPI-anchored proteins.     

Glycosphingolipids during skeletal muscle cell differentiation: Comparison of normal and fusion-defective myoblasts

The regulation of glycosphingolipid (GSL) synthesis in culture by fusion-competent (E63) myoblasts and fusion-defective (fu-1) cells was examined. Upon reaching confluency E63 cells fused to form multinucleated myotubes and demonstrated many characteristics of developing skeletal muscle including induction of creatine kinase activity and a shift in creatine kinase isozymes to the MM isoform. The fu-1 cells displayed none of these characteristics, despite the fact that both cells were cloned from the same parental myoblast line (rat L8). There was a transient increase in the synthesis of total neutral GSLs by E63 cells at the time of membrane fusion. In contrast, neutral GSL synthesis by fu-1 cells gradually decreased with time in culture. The major GSLs synthesized by both cell types were lactosylceramide and ganghoside GM3, with more complex structures being observed with prolonged time in culture. Several glycosyltransferase activities were assayed at varying times in culture. Generally, the changes in activities fell into three groups. One group was maximally activated at the end of the culture period (GalT-3, GalNAcT-1 and GalT-6). Another group was maximally activated during the time of active membrane fusion (GlcT and SAT-1). A third group was maximally activated at the time of cell contact and the beginning of membrane fusion (GlcNAcT-1 and GalT-2). In terms of the times of maximal activation there were few differences between E63 and fu-1 cells, with one notable exception. The activity of GalT-2 (lactosylceramide synthase) in E63 cells increased dramatically upon contact and the beginning of membrane fusion, whereas there were no changes in GalT-2 activity in fu-1 cells during time in culture. These results support our hypothesis that membrane glycosphingolipids play an important role in the differentiation of skeletal muscle cells.Abbreviations GSL glycosphingolipid - CK creatine kinase - HPTLC high performance thin layer chromatography - PMSF phenylmethylsulfonyl fluoride - CTH ceramide trihexoside (GbOse₃Cer) - GlcCer glycosylceramide - LacC N-acetylglucosamine - NeuNAc N-acetylneuraminic acid (sialic acid)     

Pathophysiological roles and applications of glycosphingolipids in the diagnosis and treatment of cancer diseases

Glycosphingolipids (GSLs) are major amphiphilic glycolipids present on the surface of living cell membranes. They have important biological functions, including maintaining plasma membrane stability, regulating signal transduction, and mediating cell recognition and adhesion. Specific GSLs and related enzymes are abnormally expressed in many cancer diseases and affect the malignant characteristics of tumors. The regulatory roles of GSLs in signaling pathways suggest that they are involved in tumor pathogenesis. GSLs have therefore been widely studied as diagnostic markers of cancer diseases and important targets of immunotherapy. This review describes the tumor-related biological functions of GSLs and systematically introduces recent progress in using diverse GSLs and related enzymes to diagnose and treat tumor diseases. Development of drugs and biomarkers for personalized cancer therapy based on GSL structure is also discussed. These advances, combined with recent progress in the preparation of GSLs derivatives through synthetic biology technologies, suggest a strong future for the use of customized GSL libraries in treating human diseases.     

Fibroblast growth factor 10 protects against UVB‐induced skin injury by activating the ERK/YAP signalling pathway

ObjectivesUltraviolet light B (UVB) irradiation can induce skin injury and result in keratinocytes proliferation inhibition. However, the molecular understanding of the repair during UVB‐induced cell proliferation inhibition remains poorly understood. The purpose of this study was to explore the role and potential mechanism of FGF10 in promoting keratinocytes cell cycle and proliferation after UVB injury.Materials and MethodsExpression of FGF10 protein was analysed in skin treated with UVB radiation by immunohistochemistry. The proliferation potential was examined by Immunofluorescence, Western Blot and RT‐PCR under UVB radiation, treated with FGF10 protein or overexpression of FGF10 using adeno‐associated virus. CCK8 kit was used to further detect cell proliferation ability.ResultsWe found that FGF10 is highly expressed in skin treated with UVB. Overexpression of FGF10 has a protective effect against UVB‐induced skin damage by balancing epidermal thickness and enhancing epidermal keratinocytes proliferation. Importantly, FGF10 is found to alleviate UVB‐induced downregulation of YAP activity, then promoting keratinocytes proliferation. Disruption of YAP function, either with the small molecule YAP inhibitor Verteporfin (VP) or YAP small‐interfering RNA (siRNA), largely abolishes the protective activity of FGF10 on epidermal keratinocytes proliferation. Meanwhile, disruption of ERK kinase (MEK) activity with U0126 or ERK siRNA hinder the positive influence of FGF10 on UVB‐induced skin injury.ConclusionFGF10 promotes epidermal keratinocytes proliferation during UVB‐induced skin injury in an ERK/YAP‐dependent manner.

Schematic illustration of the protective effects of FGF10 on keratinocytes under UVB radiation. UVB radiation would cause skin injury and proliferation inhibition. In this study, we revealed that FGF10 promotes keratinocytes cell cycle and proliferation via ERK/YAP signalling pathway.     

How Cholesterol Constrains Glycolipid Conformation for Optimal Recognition of Alzheimer's β Amyloid Peptide (Aβ1-40)

Membrane lipids play a pivotal role in the pathogenesis of Alzheimer''s disease, which is associated with conformational changes, oligomerization and/or aggregation of Alzheimer''s β-amyloid (Aβ) peptides. Yet conflicting data have been reported on the respective effect of cholesterol and glycosphingolipids (GSLs) on the supramolecular assembly of Aβ peptides. The aim of the present study was to unravel the molecular mechanisms by which cholesterol modulates the interaction between Aβ_1–40 and chemically defined GSLs (GalCer, LacCer, GM1, GM3). Using the Langmuir monolayer technique, we show that Aβ_1–40 selectively binds to GSLs containing a 2-OH group in the acyl chain of the ceramide backbone (HFA-GSLs). In contrast, Aβ_1–40 did not interact with GSLs containing a nonhydroxylated fatty acid (NFA-GSLs). Cholesterol inhibited the interaction of Aβ_1–40 with HFA-GSLs, through dilution of the GSL in the monolayer, but rendered the initially inactive NFA-GSLs competent for Aβ_1–40 binding. Both crystallographic data and molecular dynamics simulations suggested that the active conformation of HFA-GSL involves a H-bond network that restricts the orientation of the sugar group of GSLs in a parallel orientation with respect to the membrane. This particular conformation is stabilized by the 2-OH group of the GSL. Correspondingly, the interaction of Aβ_1–40 with HFA-GSLs is strongly inhibited by NaF, an efficient competitor of H-bond formation. For NFA-GSLs, this is the OH group of cholesterol that constrains the glycolipid to adopt the active L-shape conformation compatible with sugar-aromatic CH-π stacking interactions involving residue Y10 of Aβ_1–40. We conclude that cholesterol can either inhibit or facilitate membrane-Aβ interactions through fine tuning of glycosphingolipid conformation. These data shed some light on the complex molecular interplay between cell surface GSLs, cholesterol and Aβ peptides, and on the influence of this molecular ballet on Aβ-membrane interactions.     

GRASP55 regulates intra‐Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis

The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis‐trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI‐based retrograde transport vesicles, thus concentrating them in the trans‐Golgi. In genome‐edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis‐Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.     

Preparation of homogenous oligosaccharide chains from glycosphingolipids

After the discovery of glycosphingolipid (GSL) glycan detaching enzymes, Rhodococcal endoglycoceramidase (EGCase) and leech ceramide glycanase (CGase), the method for enzymatically releasing glycans from GSLs has become the method of choice for preparing intact ceramide-free oligosaccharide chains from GSLs. This paper describes (1) the preparation of the intact oligosaccharides from GM1 (II³NeuAcGgOse₄Cer) and GbOse₄Cer as examples to show the use of CGase to prepare intact glycan chains from GSLs, and (2) the specificity and detergent requirements of Rhodococcal EGCases for the release of glycan chains from different GSLs.     

Glycosphingolipids in endocytic membrane transport

Endocytosis leads to the internalisation of both lipids and proteins and their delivery to specific subcellular locations. This involves sorting processes that are not completely understood, but may involve interactions between lipids and proteins as well as pH and calcium gradients. This article discusses the importance of endocytosis in glycosphingolipid (GSL) synthesis as well as the potential roles of GSLs in endocytic membrane transport. Although the accumulation of GSLs in storage diseases clearly disrupts endocytic transport, increasing evidence also supports a role for GSLs in endocytosis in normal cells.     

Structural studies on the neutral glycosphingolipids of Manduca sexta

Glycosphingolipids (GSLs) have been implicated as playing major roles in cellular interactions and control of cell proliferation in muticellular organisms. Moreover GSLs and other sphingolipids such as sphingomyelins, ceramides and sphingosines serve a variety of roles in signal transduction. Hence, identification of structures of GSLs in different biota will shed light in understanding their physiological role. During this study, the major glycosphingolipid component present in the extracts of stage-12 and stage-17/18 metamorphosing adults of Manduca sexta was identified as mactosyl ceramide. We report the isolation of several ceramide disaccharides, a ceramide trisaccharide and a ceramide tetrasaccharide. The GSL structures were confirmed by high-resolution mass spectrometry and tandem mass spectrometry. The identity of the monosaccharides was proved using exoglycosidases. The predominant sphingosine chain-length varied from C-14 (tetradecasphing-4-enine) to C-16 (hexadecasphing-4-enine) in these GSLs. Sphingosines of both chain lengths were accompanied by their doubly unsaturated counterparts tetradecasphinga-4,6-diene and hexadecasphinga-4,6-diene. It is also interesting to note the presence of tetradecasphinganine and hexadecasphinganine in minute amounts in the form of a GSL in the extracts of M. sexta. The varying degrees of unsaturation in the sphingosine moiety of GSLs in M. sexta may be biologically significant in insect metamorphosis. The ceramide trisaccharides and ceramide tetrasaccharide belong to the arthro-series, The observation of fucose in the M. sexta GSLs is the first report of the presence of fucose in an arthroseries GSL.     

Activation of Wnt signalling reduces the population of cancer stem cells in ameloblastoma

ObjectivesThe treatment of ameloblastoma, an odontogenic epithelial tumour destroying jawbone, mainly depends on radical destructive resections. Other therapeutic options are limited by the characteristics of ameloblastoma, such as high recurrence rates and resistance to radiation and chemotherapy, which implies possible existence of cancer stem cells (CSCs) in ameloblastoma. Here, we identified a putative CSC population in immortalized and primary human ameloblastoma cells and examined possible therapeutic reagents to reduce the CSC population.MethodsWe investigated subpopulations of AM‐1 cell line and human ameloblastoma cells using immunocytochemistry and flow cytometry and the effects of Wnt signalling activators on the 2‐ and 3‐dimensional cultured ameloblastoma cells using molecular biological analyses.ResultAmong heterogenous ameloblastoma cells, small‐sized and round‐shaped cells were found to be proliferative and expressed a marker of dental epithelial stem cells, SRY‐box 2 (Sox2). Exogenous activation of Wnt signalling using glycogen synthase kinase 3β inhibitors, lithium chloride (LiCl) and valproic acid (VPA), increased the cell size and decreased proliferation of cells and expression of Sox2 in 2 dimensionally cultured AM‐1 and human primary ameloblastoma cells. Furthermore, the growth of 3 dimensionally cultured AM‐1 cells as suspended or embedded in gel was suppressed by treatment with Wnt signalling activators, VPA and CHIR99021, or antibodies to sclerostin, an antagonist of Wnt signalling.ConclusionWe suggest that Wnt signalling activators are potential drug candidates to suppress CSCs in ameloblastoma.     

Conditioned medium derived from 3D tooth germs: A novel cocktail for stem cell priming and early in vivo pulp regeneration

ObjectivesConditioned medium (CM) from 2D cell culture can mitigate the weakened regenerative capacity of the implanted stem cells. However, the capacity of 3D CM to prime dental pulp stem cells (DPSCs) for pulp regeneration and its protein profile are still elusive. We aim to investigate the protein profile of CM derived from 3D tooth germs, and to unveil its potential for DPSCs‐based pulp regeneration.Materials and MethodsWe prepared CM of 3D ex vivo cultured tooth germ organs (3D TGO‐CM) and CM of 2D cultured tooth germ cells (2D TGC‐CM) and applied them to prime DPSCs. Influences on cell behaviours and protein profiles of CMs were compared. In vivo pulp regeneration of CMs‐primed DPSCs was explored using a tooth root fragment model on nude mice.ResultsTGO‐CM enhanced DPSCs proliferation, migration, in vitro mineralization, odontogenic differentiation, and angiogenesis performances. The TGO‐CM group generated superior pulp structures, more odontogenic cells attachment, and enhanced vasculature at 4 weeks post‐surgery, compared with the TGC‐CM group. Secretome analysis revealed that TGO‐CM contained more odontogenic and angiogenic growth factors and fewer pro‐inflammatory cytokines. Mechanisms leading to the differential CM profiles may be attributed to the cytokine–cytokine receptor interaction and PI3K‐Akt signalling pathway.ConclusionsThe unique secretome profile of 3D TGO‐CM made it a successful priming cocktail to enhance DPSCs‐based early pulp regeneration.     

SHED aggregate exosomes shuttled miR‐26a promote angiogenesis in pulp regeneration via TGF‐β/SMAD2/3 signalling

ObjectivesPulp regeneration brings big challenges for clinicians, and vascularization is considered as its determining factor. We previously accomplished pulp regeneration with autologous stem cells from deciduous teeth (SHED) aggregates implantation in teenager patients, however, the underlying mechanism needs to be clarified for regenerating pulp in adults. Serving as an important effector of mesenchymal stem cells (MSCs), exosomes have been reported to promote angiogenesis and tissue regeneration effectively. Here, we aimed to investigate the role of SHED aggregate‐derived exosomes (SA‐Exo) in the angiogenesis of pulp regeneration.Materials and MethodsWe extracted exosomes from SHED aggregates and utilized them in the pulp regeneration animal model. The pro‐angiogenetic effects of SA‐Exo on SHED and human umbilical vein endothelial cells (HUVECs) were evaluated. The related mechanisms were further investigated.ResultsWe firstly found that SA‐Exo significantly improved pulp tissue regeneration and angiogenesis in vivo. Next, we found that SA‐Exo promoted SHED endothelial differentiation and enhanced the angiogenic ability of HUVECs, as indicated by the in vitro tube formation assay. Mechanistically, miR‐26a, which is enriched in SA‐Exo, improved angiogenesis both in SHED and HUVECs via regulating TGF‐β/SMAD2/3 signalling.ConclusionsIn summary, these data reveal that SA‐Exo shuttled miR‐26a promotes angiogenesis via TGF‐β/SMAD2/3 signalling contributing to SHED aggregate‐based pulp tissue regeneration. These novel insights into SA‐Exo may facilitate the development of new strategies for pulp regeneration.