Notch and Wnt Signaling Modulation to Enhance DPSC Stemness and Therapeutic Potential.

The Dental Pulp of permanent human teeth is home to stem cells with remarkable multilineage differentiation ability: human Dental Pulp Stem Cells (DPSCs). These cells display a very notorious expression of pluripotency core factors, and the ability to give rise to mature cell lineages belonging to the three embryonic layers. For these reasons, several researchers in the field have long considered human DPSCs as pluripotent-like cells. Notably, some signaling pathways such as Notch and Wnt contribute to maintaining the stemness of these cells through a complex network involving metabolic and epigenetic regulatory mechanisms. The use of recombinant proteins and selective pharmacological modulators of Notch and Wnt pathways, together with serum-free media and appropriate scaffolds that allow the maintenance of the non-differentiated state of hDPSC cultures could be an interesting approach to optimize the potency of these stem cells, without a need for genetic modification. In this review, we describe and integrate findings that shed light on the mechanisms responsible for stemness maintenance of hDPSCs, and how these are regulated by Notch/Wnt activation, drawing some interesting parallelisms with pluripotent stem cells. We summarize previous work on the stem cell field that includes interactions between epigenetics, metabolic regulations, and pluripotency core factor expression in hDPSCs and other stem cell types.

Stromal DLK1 promotes proliferation and inhibits differentiation of the intestinal epithelium during development.

The stem/progenitor cells of the developing intestine are biologically distinct from their adult counterparts. Here, we examine the microenvironmental cues that regulate the embryonic stem/progenitor population, focusing on the role of Notch pathway factor delta-like protein-1 (DLK1). mRNA-seq analyses of intestinal mesenchymal cells (IMCs) collected from embryonic day 14.5 (E14.5) or adult IMCs and a novel coculture system with E14.5 intestinal epithelial organoids were used. Following addition of recombinant DLK1 (rDLK) or Dlk1 siRNA (siDlk1), epithelial characteristics were compared using imaging, replating efficiency assays, qPCR, and immunocytochemistry. The intestinal phenotypes of littermate Dlk1+/+ and Dlk1-/- mice were compared using immunohistochemistry. Using transcriptomic analyses, we identified morphogens derived from the embryonic mesenchyme that potentially regulate the developing epithelial cells, to focus on Notch family candidate DLK1. Immunohistochemistry indicated that DLK1 was expressed exclusively in the intestinal stroma at E14.5 at the top of emerging villi, decreased after birth, and shifted to the intestinal epithelium in adulthood. In coculture experiments, addition of rDLK1 to adult IMCs inhibited organoid differentiation, whereas Dlk1 knockdown in embryonic IMCs increased epithelial differentiation to secretory lineage cells. Dlk1-/- mice had restricted Ki67+ cells in the villi base and increased secretory lineage cells compared with Dlk1+/+ embryos. Mesenchyme-derived DLK1 plays an important role in the promotion of epithelial stem/precursor expansion and prevention of differentiation to secretory lineages in the developing intestine.NEW & NOTEWORTHY Using a novel coculture system, transcriptomics, and transgenic mice, we investigated differential molecular signaling between the intestinal epithelium and mesenchyme during development and in the adult. We show that the Notch pathway factor delta-like protein-1 (DLK1) is stromally produced during development and uncover a new role for DLK1 in the regulation of intestinal epithelial stem/precursor expansion and differentiation to secretory lineages.

Nanostructured scaffolds based on bioresorbable polymers and graphene oxide induce the aligned migration and accelerate the neuronal differentiation of neural stem cells.

Within the field of neural tissue engineering, there is a huge need for the development of materials that promote the adhesion, aligned migration and differentiation of stem cells into neuronal and supportive glial cells. In this study, we have fabricated bioresorbable elastomeric scaffolds combining an ordered nanopatterned topography together with a surface functionalization with graphene oxide (GO) in mild conditions. These scaffolds allowed the attachment of murine neural stem cells (NSCs) without the need of any further coating of its surface with extracellular matrix adhesion proteins. The NSCs were able to give rise to both immature neurons and supporting glial cells over the nanostructured scaffolds in vitro, promoting their aligned migration in cell clusters following the nanostructured grooves. This system has the potential to reestablish spatially oriented neural precursor cell connectivity, constituting a promising tool for future cellular therapy including nerve tissue regeneration.

Advances and Perspectives in Dental Pulp Stem Cell Based Neuroregeneration Therapies.

Human dental pulp stem cells (hDPSCs) are some of the most promising stem cell types for regenerative therapies given their ability to grow in the absence of serum and their realistic possibility to be used in autologous grafts. In this review, we describe the particular advantages of hDPSCs for neuroregenerative cell therapies. We thoroughly discuss the knowledge about their embryonic origin and characteristics of their postnatal niche, as well as the current status of cell culture protocols to maximize their multilineage differentiation potential, highlighting some common issues when assessing neuronal differentiation fates of hDPSCs. We also review the recent progress on neuroprotective and immunomodulatory capacity of hDPSCs and their secreted extracellular vesicles, as well as their combination with scaffold materials to improve their functional integration on the injured central nervous system (CNS) and peripheral nervous system (PNS). Finally, we offer some perspectives on the current and possible future applications of hDPSCs in neuroregenerative cell therapies.

Wnt signaling reprograms metabolism in dental pulp stem cells.

Human dental pulp stem cells (DPSCs) can differentiate to a wide range of different cell lineages, and share some gene expression and functional similarities with pluripotent stem cells. The stemness of DPSCs can also be pharmacologically enhanced by the activation of canonical Wnt signaling. Here, we examined the metabolic profile of DPSCs during reprogramming linked to Wnt activation, by a short (48 hr) exposure to either the GSK3-ß inhibitor BIO (6-bromoindirubin-3´-oxine) or human recombinant protein WNT-3A. Both treatments largely increased glucose consumption, and induced a gene overexpression of pyruvate and mitochondrial acetyl-coA producing enzymes, thus activating mitochondrial tricarboxylic acid cycle (TCA) metabolism in DPSCs. This ultimately led to an accumulation of reducing power and a mitochondrial hyperpolarization in DPSCs. Interestingly, Nile Red staining showed that lipid fuel reserves were being stored in Wnt-activated DPSCs. We associate this metabolic reprogramming with an energy-priming state allowing DPSCs to better respond to subsequent high demands of energy and biosynthesis metabolites for cellular growth. These results show that enhancement of the stemness of DPSCs by Wnt activation comes along with a profound metabolic remodeling, which is distinctly characterized by a crucial participation of mitochondrial metabolism.

BDNF and NT3 Reprogram Human Ectomesenchymal Dental Pulp Stem Cells to Neurogenic and Gliogenic Neural Crest Progenitors Cultured in Serum-Free Medium.

BACKGROUND/AIMS: Human Dental Pulp Stem Cells (hDPSCs) are one of the most promising types of cells to regenerate nerve tissues. Standard DMEM+10% fetal bovine serum (FBS) culture medium allows a fast expansion of hDPSC as a surface-adherent cell monolayer. However, the use of FBS also compromises the clinical use of these protocols, and its longterm presence favors hDPSCs differentiation toward mesenchymal cell-derived lineages, at the expense of a reduced capability to generate neural cells. The objective of this work was to characterize the role of neurotrophin signaling on hDPSCs using a serum-free culture protocol, and to assess the neurogenic and gliogenic capacity of hDPSCs for future nerve tissue bioengineering and regeneration. METHODS: We compared the different expression of neurotrophin receptors by RT-PCR, Q-PCR, and IF of hDPSCs cultured with different growth media in the presence or absence of serum. Moreover, we assessed the response of hDPSCs to stimulation of neurotransmitter receptors by live cell calcium imaging under these different media. Finally, we compared the osteogenic potential of hDPSCs by Alizarin red staining, and the differentiation to gliogenic/neurogenic fates by immunostaining for Schwann lineage and neuronal lineage markers. We tested a commercial serum-free medium designed for the growth of mesenchymal stem cells: StemPro MSCTM (STP). RESULTS: hDPSCs cultured in STP generated small non-adherent floating dentospheres that showed very low proliferation rates, in contrast to standard FBS-containing medium. We found that hDPSCs grown in STP conditions overexpressed neurotrophin receptor genes NTRK2 (TrkB) and NTRK3 (TrkC). Interestingly, the stimulation of these receptors by adding their respective ligands BDNF and NT-3 to STP medium enhanced the neural crest (NC) progenitor features of cultured hDPSCs. We observed a 10 to 100-fold increase of migratory NC cell markers HNK1 and P75NTR, and a significant overexpression of pluripotency core factors SOX2, OCT4 and NANOG. Moreover, hDPSCs cultured in BDNF/NT-3 supplemented STP showed a largely increased potential to differentiate towards neuronal and Schwann glial lineage cells, assessed by positive immunostaining for DCX, NeuN and S100ß, p75NTR markers, respectively. CONCLUSION: Our results demonstrate that the use of BDNF and NT-3 combined with STP induced the partial reprogramming of ectomesenchymal hDPSCs to generate early NC progenitor cells, which are far more competent for neuronal and glial differentiation than hDPSCs grown in the presence of FBS.

Targeting liver sinusoidal endothelial cells with miR-20a-loaded nanoparticles reduces murine colon cancer metastasis to the liver.

Phenotypic transformation of liver sinusoidal endothelial cells is one of the most important stages of liver metastasis progression. The miRNA effects on liver sinusoidal endothelial cells during liver metastasis have not yet been studied. Herein, whole genome analysis of miRNA expression in these cells during colorectal liver metastasis revealed repressed expression of microRNA-20a. Importantly, downregulation of miR-20a occurs in parallel with upregulation of its known protein targets. To restore normal miR-20a levels in liver sinusoidal endothelial cells, we developed chondroitin sulfate-sorbitan ester nanoparticles conjugated with miR-20a in a delivery system that specifically targets liver sinusoidal endothelial cells. The restoration of normal mir-20a levels in these cells induced downregulation of the expression of its protein targets, and this also resulted in a reduction of in vitro LSEC migration and a reduction of in vivo activation and tumor-infiltrating capacity and ability of the tumor decreased by ∼80% in a murine liver metastasis model.

DLK1 regulates branching morphogenesis and parasympathetic innervation of salivary glands through inhibition of NOTCH signalling.

BACKGROUND INFORMATION: Delta-like proteins 1 and 2 (DLK1, 2) are NOTCH receptor ligands containing epidermal growth factor-like repeats, which regulate NOTCH signalling. We investigated the role of DLK and the NOTCH pathway in the morphogenesis of the submandibular salivary glands (SMGs), using in vitro organotypic cultures. RESULTS: DLK1 and 2 were present in all stages of SMG morphogenesis, where DLK1 inhibited both NOTCH activity and SMG branching. The addition of NOTCH inhibitory agents, either soluble DLK1 (sDLK1) or N-[N-(3, 5-difluorophenacetyl-L-alanyl]-S-phenylglycine t-buthyl ester (DAPT), to the SMG culture medium did not affect the rate of cell proliferation, but induced a strong reduction in SMG branching, increased epithelial apoptosis, and impaired innervation of the epithelial end buds by local parasympathetic ganglion neurons. SMG innervation could be restored by the acetylcholine analog carbachol (CCh), which also rescued cytokeratin 5 (CK5(+))-expressing epithelial progenitor cells. Despite this, CCh failed to restore normal branching morphogenesis in the presence of either sDLK1 or DAPT. However, it improved recovery of branching morphogenesis in SMGs, once DLK1 or DAPT were removed from the medium. CONCLUSIONS: Our data suggest that DLK1 regulates SMGs morphogenesis and parasympathetic nerve fibre outgrowth through inhibition of NOTCH signalling.

Disrupted odontoblast differentiation and dentin dysplasia in Epiprofin-deficient mice.

Tooth formation is a process tightly regulated by reciprocal interactions between epithelial and mesenchymal tissues. These epithelial-mesenchyme interactions regulate the expression of target genes via transcription factors. Among the regulatory elements governing this process, Epiprofin/Sp6 is a zinc finger transcription factor which is expressed in the embryonic dental epithelium and in differentiating pre-odontoblasts. Epiprofin knockout (Epfn-/-) mice present severe dental abnormalities, such as supernumerary teeth and enamel hypoplasia. Here, we describe dentin defects in molars and incisors of Epfn-/- mice. We observed that in the absence of Epfn, markers of early odontoblast differentiation, such as alkaline phosphatase activity, Dsp/Dpp expression, and Collagen Type I deposition, are downregulated. In addition, the expression of tight and gap junction proteins was severely impaired in the predontoblastic cell layer of developing Epfn-/- molars. Altogether, our data shows that Epfn is crucial for the proper differentiation of dental mesenchymal cells towards functional odontoblasts and subsequent dentin-matrix deposition.

Intracranial graft of bioresorbable polymer scaffolds loaded with human Dental Pulp Stem Cells in stab wound murine injury model.

The prevalence of central nervous system (CNS) dysfunction as a result of disease or trauma remains a clinically unsolved problem which is raising increased awareness in our aging society. Human Dental Pulp Stem Cells (hDPSCs) are excellent candidates to be used in tissue engineering and regenerative therapies of the CNS due to their neural differentiation ability and lack of tumorigenicity. Accordingly, they have been successfully used in animal models of spinal cord injury, stroke and peripheral neuropathies. The ideal therapy in brain injury should combine strategies aiming to protect the damaged lesion and, at the same time, accelerate brain tissue regeneration, thus promoting fast recovery while minimizing side or long-term effects. The use of bioresorbable nanopatterned poly(lactide-co-ɛ-caprolactone) (PLCL) polymeric scaffolds as hDPCSs carriers can represent an advantage for tissue regeneration. In this chapter, we describe the surgical procedures to implant functionalized bioresorbable scaffolds loaded with hDPSCs to improve the brain lesion microenvironment in an intracranial stab wound injury model severing the rostral migratory stream (RMS) that connects the brain subventricular zone (SVZ) and the olfactory bulb in nude mice. Additionally, we also describe the technical steps after animal sacrifice for histological tissue observation and characterization.

Enhanced Wnt/ß-catenin signalling during tooth morphogenesis impedes cell differentiation and leads to alterations in the structure and mineralisation of the adult tooth.

BACKGROUND INFORMATION: Previous studies have indicated that over-activation of the wingless interaction site (Wnt)/ß-catenin signalling pathway has important implications for tooth development, at the level of cell differentiation and morphology, as well as for the production of supernumerary teeth. Here, we provide evidence for a crucial role of this signalling pathway during the stage of tooth morphogenesis. We have developed an in vitro model consisting of 14.5-day-old mouse embryo first molars, in which the Wnt pathway is overactivated by the glycogen synthase kinase-3 inhibitor 6-bromoindirubin-3'-oxime (BIO; 20 µM). RESULTS: We found that over-activation of the Wnt/ß-catenin pathway delayed the differentiation and growth of the inner dental epithelium. In addition, in contrast to controls in which Nestin protein expression was restricted to differentiated odontoblasts, in BIO-treated molars, Nestin expression spread through sub-odontoblastic cellular layers. This alteration appears to be related to: (i) the over-expression of Bmp4 in the same region, (ii) the delay in odontoblast precursor cell differentiation and (iii) increased proliferation of mesenchymal cells. Furthermore, treatments longer than 6 days induced the malformation of typical dental structures and led to a total lack of cell differentiation. Finally, over-activation of the Wnt route during odontogenesis resulted in adult teeth which presented altered size, morphology and mineralisation. CONCLUSIONS: Our results indicate that Wnt/ß-catenin over-activation during tooth morphogenesis is sufficient to cause dramatic alterations in the adult tooth, by delaying cellular differentiation and stimulating proliferation of the dental mesenchyme of developing teeth.

Stem Cell Therapy and Rejuvenation, and Their Impact on Society.

In his worldwide best-seller Homo Deus [...].

Self-assembled three-dimensional hydrogels based on graphene derivatives and cerium oxide nanoparticles: scaffolds for co-culture of oligodendrocytes and neurons derived from neural stem cells.

Stem cell-based therapies have shown promising results for the regeneration of the nervous system. However, the survival and integration of the stem cells in the neural circuitry is suboptimal and might compromise the therapeutic outcomes of this approach. The development of functional scaffolds capable of actively interacting with stem cells may overcome the current limitations of stem cell-based therapies. In this study, three-dimensional hydrogels based on graphene derivatives and cerium oxide (CeO2) nanoparticles are presented as prospective supports allowing neural stem cell adhesion, migration and differentiation. The morphological, mechanical and electrical properties of the resulting hydrogels can be finely tuned by controlling several parameters of the self-assembly of graphene oxide sheets, namely the amount of incorporated reducing agent (ascorbic acid) and CeO2 nanoparticles. The intrinsic properties of the hydrogels, as well as the presence of CeO2 nanoparticles, clearly influence the cell fate. Thus, stiffer adhesion substrates promote differentiation to glial cell lineages, while softer substrates enhance mature neuronal differentiation. Remarkably, CeO2 nanoparticle-containing hydrogels support the differentiation of neural stem cells to neuronal, astroglial and oligodendroglial lineage cells, promoting the in vitro generation of nerve tissue grafts that might be employed in neuroregenerative cell therapies.

Epiprofin/Sp6 regulates Wnt-BMP signaling and the establishment of cellular junctions during the bell stage of tooth development.

Epiprofin/Specificity Protein 6 (Epfn) is a Krüppel-like family (KLF) transcription factor that is critically involved in tooth morphogenesis and dental cell differentiation. However, its mechanism of action is still not fully understood. We have employed both loss-of-function and gain-of-function approaches to address the role of Epfn in the formation of cell junctions in dental cells and in the regulation of junction-associated signal transduction pathways. We have evaluated the expression of junction proteins in bell-stage incisor and molar tooth sections from Epfn(-/-) mice and in dental pulp MDPC-23 cells overexpressing Epfn. In Epfn(-/-) mice, a dramatic reduction occurs in the expression of tight junction and adherens junction proteins and of the adherens-junction-associated ß-catenin protein, a major effector of canonical Wnt signaling. Loss of cell junctions and ß-catenin in Epfn(-/-) mice is correlated with a clear decrease in bone morphogenetic protein 4 (BMP-4) expression, a decrease in nestin in the tooth mesenchyme, altered cell proliferation, and failure of ameloblast cell differentiation. Overexpression of Epfn in MDPC-23 cells results in an increased cellular accumulation of ß-catenin protein, indicative of upregulation of canonical Wnt signaling. Together, these results suggest that Epfn enhances canonical Wnt/ß-catenin signaling in the developing dental pulp mesenchyme, a condition that promotes the activity of other downstream signaling pathways, such as BMP, which are fundamental for cellular induction and ameloblast differentiation. These altered signaling events might underlie some of the most prominent dental defects observed in Epfn(-/-) mice, such as the absence of ameloblasts and enamel, and might throw light on developmental malformations of the tooth, including hyperdontia.

Bioactive Materials for Next-Generation Dentistry.

Teeth were some of the first organs whose function was effectively restored by inert refilling materials that have become widely known to the general public; amalgams, polymeric resin composites, and gutta-percha are some such examples [...].

In vitro preparation of human Dental Pulp Stem Cell grafts with biodegradable polymer scaffolds for nerve tissue engineering.

Human Dental Pulp Stem Cells (hDPSCs) are one of the most promising stem cell sources for tissue engineering and regeneration, due to their extraordinary multi-lineage differentiation ability, ease of extraction from biological waste in dental clinics, safe non-tumorigenic phenotype, immune-tolerance upon in vivo transplantation, and great possibilities of application in autologous tissue reconstruction. The in vitro manipulation of hDPSCs paves the way for drug screening and tailor-made regeneration of damaged tissues, in the context of personalized medicine. The neural crest phenotype of these stem cells gives them the capacity to differentiate to a large variety of cell types, including neural-lineage cells. In this chapter, we describe various culture methods to generate different cellular phenotypes from hDPSCs, which can not only grow as mesenchymal-like plastic adherent cells, but also as non-adherent neurogenic dentospheres in serum-free medium. Floating dentospheres can be used to generate large populations of mature neuronal and glial marker expressing cells, which may be cultured over a substrate of nanopatterned scaffold based on biodegradable poly(lactide-co-caprolactone) (PLCL) to induce a controlled alignment of neurites and cell migration, to generate in vivo biocompatible constructs for nerve tissue bioengineering.

Enhanced Adipogenic Differentiation of Human Dental Pulp Stem Cells in Enzymatically Decellularized Adipose Tissue Solid Foams.

Engineered 3D human adipose tissue models and the development of physiological human 3D in vitro models to test new therapeutic compounds and advance in the study of pathophysiological mechanisms of disease is still technically challenging and expensive. To reduce costs and develop new technologies to study human adipogenesis and stem cell differentiation in a controlled in vitro system, here we report the design, characterization, and validation of extracellular matrix (ECM)-based materials of decellularized human adipose tissue (hDAT) or bovine collagen-I (bCOL-I) for 3D adipogenic stem cell culture. We aimed at recapitulating the dynamics, composition, and structure of the native ECM to optimize the adipogenic differentiation of human mesenchymal stem cells. hDAT was obtained by a two-enzymatic step decellularization protocol and post-processed by freeze-drying to produce 3D solid foams. These solid foams were employed either as pure hDAT, or combined with bCOL-I in a 3:1 proportion, to recreate a microenvironment compatible with stem cell survival and differentiation. We sought to investigate the effect of the adipogenic inductive extracellular 3D-microenvironment on human multipotent dental pulp stem cells (hDPSCs). We found that solid foams supported hDPSC viability and proliferation. Incubation of hDPSCs with adipogenic medium in hDAT-based solid foams increased the expression of mature adipocyte LPL and c/EBP gene markers as determined by RT-qPCR, with respect to bCOL-I solid foams. Moreover, hDPSC capability to differentiate towards adipocytes was assessed by PPAR-γ immunostaining and Oil-red lipid droplet staining. We found out that both hDAT and mixed 3:1 hDAT-COL-I solid foams could support adipogenesis in 3D-hDPSC stem cell cultures significantly more efficiently than solid foams of bCOL-I, opening the possibility to obtain hDAT-based solid foams with customized properties. The combination of human-derived ECM biomaterials with synthetic proteins can, thus, be envisaged to reduce fabrication costs, thus facilitating the widespread use of autologous stem cells and biomaterials for personalized medicine.

Stem and Cancer Stem Cell Identities, Cellular Markers, Niche Environment and Response to Treatments to Unravel New Therapeutic Targets.

Adult stem cells are a partially quiescent cell population responsible for natural cell renewal and are found in many different regions of the body, including the brain, teeth, bones, muscles, skin, and diverse epithelia, such as the epidermal or intestinal epithelium, among others [...].

Identification of sugar moieties in chief cells of the rat fundic gastric glands.

Many studies have been conducted to determine the composition of the glycoconjugates of the mucus-secreting cells of the fundic glands of the stomach. However, the chief cells of these glands have been largely ignored because they secrete mainly zymogens with a lower glycosylation. The aim of this work was to analyze the glycoconjugates of the gastric chief cells by a battery of 17 different lectins, recognizing Fucose, N-acetylgalactosamine, Galactose, N-acetylneuraminic acid, N-acetylglucosamine and Mannose containing oligosaccharides. Histochemical techniques were performed with several lectins and also combined with two pre-treatments; ß-elimination, which removes O-linked oligosaccharides, and incubation with Peptide-N-Gycosidase F, which removes N-linked oligosaccharides. In addition, acid hydrolysis was performed before WGA histochemistry, and incubation with glucose oxidase before Con A labeling. Many lectins did not stain the chief cells. In addition, the presence of O-glycans in the apical cell membrane was demonstrated with the lectins AAL, HPA, MPA/MPL, PNA, RCA-I, and WGA. Some of these O-glycans were resistant to short-term ß-elimination pre-treatments. Mannose-binding lectins stained the basal cytoplasm of the chief cells. The level of glycosylation of the chief cells was lower than that of the mucous cells. The presence of O-glycans in the apical cell membrane is consistent with the presence of mucins such as MUC1 in the apical membrane of chief cells. Moreover, Mannose-binding lectins revealed N-glycosylation in the basal cytoplasm. The knowledge of gastric chief cell glycoconjugates is relevant because of their potential involvement not only in in physiological but also in pathological processes, such as cancer.

Vasculogenesis from Human Dental Pulp Stem Cells Grown in Matrigel with Fully Defined Serum-Free Culture Media.

The generation of vasculature is one of the most important challenges in tissue engineering and regeneration. Human dental pulp stem cells (hDPSCs) are some of the most promising stem cell types to induce vasculogenesis and angiogenesis as they not only secrete vascular endothelial growth factor (VEGF) but can also differentiate in vitro into both endotheliocytes and pericytes in serum-free culture media. Moreover, hDPSCs can generate complete blood vessels containing both endothelial and mural layers in vivo, upon transplantation into the adult brain. However, many of the serum free media employed for the growth of hDPSCs contain supplements of an undisclosed composition. This generates uncertainty as to which of its precise components are necessary and which are dispensable for the vascular differentiation of hDPSCs, and also hinders the transfer of basic research findings to clinical cell therapy. In this work, we designed and tested new endothelial differentiation media with a fully defined composition using standard basal culture media supplemented with a mixture of B27, heparin and growth factors, including VEGF-A165 at different concentrations. We also optimized an in vitro Matrigel assay to characterize both the ability of hDPSCs to differentiate to vascular cells and their capacity to generate vascular tubules in 3D cultures. The description of a fully defined serum-free culture medium for the induction of vasculogenesis using human adult stem cells highlights its potential as a relevant innovation for tissue engineering applications. In conclusion, we achieved efficient vasculogenesis starting from hDPSCs using serum-free culture media with a fully defined composition, which is applicable for human cell therapy purposes.