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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.

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.

3D cell culture systems based on biological scaffold materials obtainable from both animal and human tissues constitute very interesting tools for cell therapy and personalised medicine applications. The white adipose tissue (AT) extracellular matrix (ECM) is a very promising biomaterial for tissue engineering due to its easy accessibility, malleability and proven biological activity. In the present study, human dental pulp stem cells (hDPSCs) were combined in vitro with ECM scaffolds from porcine and human decellularised adipose tissues (pDAT, hDAT) processed as 3D solid foams, to investigate their effects on the osteogenic differentiation capacity and bone matrix production of hDPSCs, compared to single-protein-based 3D solid foams of collagen type I and conventional 2D tissue-culture-treated polystyrene plates. pDAT solid foams supported the osteogenic differentiation of hDPSCs to similar levels to collagen type I, as assessed by alkaline phosphatase and alizarin red stainings, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and osteocalcin/bone gamma-carboxyglutamate protein (BGLAP) immunostaining. Interestingly, hDAT solid foams showed a markedly lower capacity to sustain hDPSC osteogenic differentiation and matrix calcification and a higher capacity to support adipogenesis, as assessed by RT-qPCR and oil red O staining. White ATs from both human and porcine origins are relatively abundant and available sources of raw material to obtain high quality ECM-derived biomedical products. These biomaterials could have promising applications in tissue engineering and personalised clinical therapy for the healing and regeneration of lesions involving not only a loss of calcified bone but also its associated soft non-calcified tissues.

340 p., We studied the human Dental Pulp Stem Cells (hDPSCs) adherence, proliferation, viability and osteo-differentiation potential when cultured on widely used Ti6AL4V titanium surface and a new biomimetic porous surface (BASTM), in the presence or absence of osteoblastic differentiation media supplemented with plasma rich in growth factors (PRGF) and platelet rich fibrin (PRF). Once we studied the effects of these two titanium surfaces on hDPSCs, we performed a comparative study of viability, proliferation and osteoblastic differentiation potential between the most promising mesenchymal stem cells for bone regeneration therapies, hDPSCs and hBMSCs. Lastly, another problem in dental implantology is the loss of periodontal ligament. The function of this tissue is to act like a cushion to absorb part of the masticatory mechanical forces to protect the alveolar bone. Nowadays, the periodontal regenerative therapy is based on the use of barrier membranes in alveolar ridge defects to enhance the bone growth on the surrounding affected area before the placement of dental implants. For this purpose, we studied the porcine decellularized adipose tissue (pDAT).

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.

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.

The conversion of healthy stem cells into cancer stem cells (CSCs) is believed to underlie tumor relapse after surgical removal and fuel tumor growth and invasiveness. CSCs often arise from the malignant transformation of resident multipotent stem cells, which are present in most human tissues. Some organs, such as the gut and the brain, can give rise to very aggressive types of cancers, contrary to the dental pulp, which is a tissue with a very remarkable resistance to oncogenesis. In this review, we focus on the similarities and differences between gut, brain and dental pulp stem cells and their related CSCs, placing a particular emphasis on both their shared and distinctive cell markers, including the expression of pluripotency core factors. We discuss some of their similarities and differences with regard to oncogenic signaling, telomerase activity and their intrinsic propensity to degenerate to CSCs. We also explore the characteristics of the events and mutations leading to malignant transformation in each case. Importantly, healthy dental pulp stem cells (DPSCs) share a great deal of features with many of the so far reported CSC phenotypes found in malignant neoplasms. However, there exist literally no reports about the contribution of DPSCs to malignant tumors. This raises the question about the particularities of the dental pulp and what specific barriers to malignancy might be present in the case of this tissue. These notable differences warrant further research to decipher the singular properties of DPSCs that make them resistant to transformation, and to unravel new therapeutic targets to treat deadly tumors.

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.

Dental pulp stem cells (DPSCs) from adult teeth show the expression of a very complete repertoire of stem pluripotency core factors and a high plasticity for cell reprogramming. Canonical Wnt and Notch signaling pathways regulate stemness and the expression of pluripotency core factors in DPSCs, and even very short-term (48 h) activations of the Wnt pathway induce a profound remodeling of DPSCs at the physiologic and metabolic levels. In this work, DPSC cultures were exposed to treatments modulating Notch and Wnt signaling, and also induced to differentiate to osteo/adipocytes. DNA methylation, histone acetylation, histone methylation, and core factor expression levels where assessed by mass spectroscopy, Western blot, and qPCR. A short-term activation of Wnt signaling by WNT-3A induced a genomic DNA demethylation, and increased histone acetylation and histone methylation in DPSCs. The efficiency of cell reprogramming methods relies on the ability to surpass the epigenetic barrier, which determines cell lineage specificity. This study brings important information about the regulation of the epigenetic barrier by Wnt signaling in DPSCs, which could contribute to the development of safer and less aggressive reprogramming methodologies with a view to cell therapy.

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 (48hr) exposure to either the GSK3- 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.

222 p., Las Células Stem de Pulpa Dental (DPSCs; en inglés) son células madre extraídas de la pulpa dental de terceros molares de pacientes. Presentan unas propiedades que las convierte en un recurso especialmente interesante para la terapia celular, además de un gran potencial de diferenciación a distintos tipos de linajes y tejidos incluido hueso, cartílago, músculo y tejido adiposo. Este tipo de células madre son fáciles de obtener de pacientes, cultivar en forma de cultivo primario y amplificar gracias a su alta tasa de renovación. También presentan propiedades inmunosupresoras y buena tolerancia a los biomateriales, lo que las hace especialmente atractivas para su uso en clínica (ANEXO 1).Las vías de señalización Notch y Wnt/ß-Catenina son cruciales en el mantenimiento y la diferenciación de diferentes tipos de células madre y su conocimiento sería indispensable paraconocer qué procesos gobiernan estos acontecimientos. El uso de proteínas recombinantes para la modulación de estas vías a diferentes tiempos y concentraciones podría ayudar en el control sobre el mantenimiento de estas poblaciones, sin que ello suponga una alteración definitiva o errores en el DNA, como ocurre con otras metodologías. Además, la modulación en la troncalidad viene acompañada de una remodelación del metabolismo y de la epigenética necesaria para garantizar la eficiencia de la reprogramación. El estudio de todos estos cambios fisiológicos en las DPSCs podría ser necesario para asegurar una terapia celular efectiva, precisa y segura (ANEXO 2).Las vías de señalización Notch y Wntß-Catenina tienen un papel importante en el mantenimiento y diferenciación de las poblaciones de células madre. En el caso de las DPSCs las vías Notch y Wntß-Catenina, conjuntamente, son indispensables en la expresión de marcadores de pluripotencia (Oct4, Nanog, Sox2, cMyc, Lin28, Rex1, Stella y Ssea1) y en el mantenimiento de la proliferación de células madre. Además, los tratamientos previos con BIO y WNT-3A mejoraron

Dental pulp stem cells (DPSCs) have the capacity to give rise to cells with neuronal-like phenotypes, suggesting their use in brain cell therapies. In the present work, we wanted to address the phenotypic fate of adult genetically unmodified human DPSCs cultured in Neurocult (TM) (Stem Cell Technologies), a cell culture medium without serum which can be alternatively supplemented for the expansion and/or differentiation of adult neural stem cells (NSCs). Our results show that non-genetically modified human adult DPSCs cultured with Neurocult NS-A proliferation supplement generated neurosphere-like dentospheres expressing the NSC markers Nestin and glial fibrillary acidic protein (GFAP), but also the vascular endothelial cell marker CD31. Remarkably, 1 month after intracranial graft into athymic nude mice, human CD31+/CD146+ and Nestin+ DPSC-derived cells were found tightly associated with both the endothelial and pericyte layers of brain vasculature, forming full blood vessels of human origin which showed an increased laminin staining. These results are the first demonstration that DPSC-derived cells contributed to the generation of neovasculature within brain tissue, and that Neurocult and other related serum-free cell culture media may constitute a fast and efficient way to obtain endothelial cells from human DPSCs.

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

El capítulo II (Results and Discussion) está sujeto a confidencialidad por el autor. 112 p., Ezaguna da hortz muin helduak zelula ama populazio garrantzitsu bat duela barnean, hauei, hortz muineko zelula amak deritze (ingelesez, dental pulp stem cells, DPSCs). Oinarrizko egoeran aurkezten dituzten zelula neuralen markatzaileak, zelula ama neuralek baino irisgarritasun handiagoa, jatorrizko organoaz kanpo hedatzeko ahalmena eta neurodesberdintzapen ahalmenak, bakarrak bilakatzen dituzte giza DPSCs-ak (gDPSCs-ak) gorputz helduan aurkitu daitezkeen beste zelulekiko. Izan ere, birsorkuntza neuraleko terapia zelular autologoetan erabilgarriak izan daitezke. Normalean erabiltzen diren hazkuntza eta mantentze medioek behi serum fetala (BFS) dute konposatu garrantzitsuena bezala, zeinek gDPSCs-en hazkuntza azkarra eta azalerako geruza bakar itxurako hazkuntza zelularra eragiten duen. Era berean, BFS-aren presentziak alergiak, zein erreakzio immunearen aktibazioa eragin ditzakela deskribatua dagoin vivo, konposatu honen erabilera tratamendu klinikoetatik baztertzea ezinbestekoa delarik. BFS-ak sor ditzakeen arazo medikotik at erabilgarritasun arazoak ere sor ditzake, izan ere, serumaren presentzian gDPSCs-ak leinu mesenkimaletara bideratzen direla, eta beraz, potentzial neurogenikoa galera dagoela aztertu dute zenbait autorek literaturan. BFS-aren erabilerak dakartzan eragozpen guztiek ezinbestekoa egiten dute serum gabeko eragile neurogeniko berritzaileen diseinua eta hobekuntza.Hurrengo lanean, serum gabeko hazkuntza mediotan ereindako zelulen ezaugarriak ezagutzea izan da gure xedea. Lortutako emaitzek inolako aldaketa genetiko edo epigenetikorik jasan izan ez duten gDPSCs-ak, zelula ama neuralek (ingelesez, neural stem cells, NSCs) sortzen dituzten neuro-esfera egituren antzeko hortz-esferak sortzeko duten ahalmena berretsi zuten. Hortaz aparte, bai serum gabeko zein serum-dun hazkuntza mediotan hazitako gDPSCs-ek zelula ama mesenkimal izaera eta ezaugarri pluripotentzialak mantentzeko gai zirela frogatu genuen. Hala ere, erabili genituen serum gabeko hazkuntza

El contenido de los capítulos 2,3 y 4 están sujetos a confidencialidad por la autora. 76 p., En esta tesis se aborda el diseño y caracterización de una vacuna basada en nanoparticulas de óxido de hierro para su aplicación en inmunoterapia del cáncer. Concretamente, las nanoparticulas están biofuncionalizadas con una combinación de agonistas sintéticos de TLR (Poly(I:C) e imiquimod), que activan de manera sinérgica una respuesta inmune innata. Las células dendríticas, que son las principales células presentadoras de antígeno, reconocen dichos ligandos como señales de peligro y en consecuencia se produce su activación y maduración. Estas células a su vez son capaces de generar y coordinar una respuesta inmune adaptativa específica de antígeno. Esta capacidad inmunoestimuladora de las nanoparticulas, junto con su particular biodistribución, permite crear una potente respuesta de forma dirigida contra el tumor. La inmunización de ratones que portan el modelo tumoral de melanoma B16F10 demuestra que esta `nanovacuna¿ es capaz de retrasar o incluso impedir el crecimiento tumoral, además de prolongar la supervivencia de los animales.

Dental pulp stem cells (DPSCs) from adult teeth express neural crest (NC) markers together with core transcriptional factors associated with stem cell pluripotency, such as Oct4a, Sox2, c-Myc, Rex1, Stella/Dppa3, Ssea1/Fut4, Lin28 and Nanog. The possibility to boost the natural stemness features of DPSCs by mild methods, that do not involve gene and/or chromatin modification or gene transfection, is highly desirable for cell therapy. Canonical Wnt and Notch are two highly conserved developmental signalling pathways that are involved in NC emergence and stem cell self-renewal. We determined that both pathways coordinate to regulate the expression of core pluripotency and NC factors in DPSCs. Pharmacological inhibition of the Notch pathway for 48 h, by the gamma-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), abolished the expression of NC and core factors. In addition, it induced a silencing of the canonical Wnt signalling and a clear reduction in the stemness potential of DPSCs, as shown by a reduced ability to generate mature, fully differentiated osteoblasts and adipocytes. Conversely, pharmacological activation of the Wnt pathway for 48 h, by either the glycogen synthase kinase 3 beta (GSK3-beta) inhibitor 6-bromoindirubin-3'-oxime (BIO) or the human recombinant protein Wnt-3a, not only largely increased the expression of NC and core factors, but also increased the efficiency of DPSCs to differentiate into mature osteoblasts and adipocytes. These results showed that a short preconditioning activation of Wnt/Notch signalling by small molecules and/or recombinant proteins enhanced the stemness and potency of DPSCs in culture, which could be useful for optimising the therapeutic use of these and other tissue-specific stem cells.

103 p.; 102 p., Organogenesia seinalizazio bide ezberdinez gidatutako prozesu konplexua da. Askotan organu ezberdinen garapena seinalizazio faktore berdinez dago erregulatuta. Gainera, seinalizazio bideen ikerketa garapena ezagutarazteko erabiltzeaz aparte, minbizia eta ehunen birsorkuntza zelan gertatzen diren jakiteko ere balio digu.Lan honetan, listu-guruinaren garapena aztertu dugu. Listu-guruinaren organogenesia adrakatze morfogenesia deituriko prozesuaren bidez ematen da. Garapen prozesu hau beste organo batzuetan ere ematen da, esaterako, birikietan, giltzurrunetan eta ugatz guruinetan.NOTCH seinale bidea, garapenean dauden ehunetan, seinalerik aztertuenetako bat da, non orain dela gutxi deskribatu diren DLK1 eta DLK2 lotugai ez kanonikoak dauden. Gure lanaren helbururik nagusiena, DLK1en eta DLK2ren funtzioa ezagutzea izan zen. Batetik NOTCH seinalizazioan duten efektuak argituz eta bestetik baraila azpiko listu-guruinaren (SMG: submandibular salivary gland) garapenean jokatzen duen rola aztertuz. Hau egiteko, sagu enbrioien SMGen hazkunde organotipikoak egin genituen, sDLK1 edo DAPTrekin ( ¿-sekretasaren inibitzaile erreaktiboa). Bestalde, Dlk1 (-/-) saguarekin ere esperimentuak egin genituen. Gure emaitzek DLK1 eta DLK2 lotugaiak garapeneko listu-guruinaren maila altuetan adierazten direla erakutsi zuten. Aldiz, sagu helduko listu-guruinetan DLK1-2ren adierazpen mailak murriztu egiten dira, emaitza hauek DLK1-2 SMGaren morfogenesian parte hartu dezaketelaren ideia eman zigun. Bestalde, enbrioien SMG hazkuntzetan, sDLK1 edo DAPTrekin inhibituriko NOTCH seinale bideak, listu-guruinaren adarkatze morfogenesia kaltetzeaz aparte, inerbazioa murriztu eta azinoen barneko zelula progenitore epitelialen apoptosia eragiten zuen. NOTCH seinalearen inhibitzaileek SMGaren inerbazioa oztopatzen dutenez, karbakola (CCh) erabili genuen, inerbazio kolinergikoaren ordezko gisa bezala, modu honetan SMGaren garapena berreskuratzeko. CChak SMGaren adarkadura morfogenesia partzialki laguntzen

8 p., The procurement of induced pluripotent stem cells, or IPS cells, from adult differentiated animal cells has the potential to revolutionize future medicine, where reprogrammed IPS cells may be used to repair disease-affected tissues on demand. The potential of IPS cell technology is tremendous, but it will be essential to improve the methodologies for IPS cell generation and to precisely evaluate each clone and subclone of IPS cells for their safety and efficacy. Additionally, the current state of knowledge on IPS cells advises that research on their regenerative properties is carried out in appropriate tissue and organ systems that permit a safe assessment of the long-term behavior of these reprogrammed cells. In the present paper, we discuss the mechanisms of cell reprogramming, current technical limitations of IPS cells for their use in human tissue engineering, and possibilities to overcome them in the particular case of dental regeneration.