HB-EGF activates EGFR to induce reactive neural stem cells in the mouse hippocampus after seizures.

Hippocampal seizures mimicking mesial temporal lobe epilepsy cause a profound disruption of the adult neurogenic niche in mice. Seizures provoke neural stem cells to switch to a reactive phenotype (reactive neural stem cells, React-NSCs) characterized by multibranched hypertrophic morphology, massive activation to enter mitosis, symmetric division, and final differentiation into reactive astrocytes. As a result, neurogenesis is chronically impaired. Here, using a mouse model of mesial temporal lobe epilepsy, we show that the epidermal growth factor receptor (EGFR) signaling pathway is key for the induction of React-NSCs and that its inhibition exerts a beneficial effect on the neurogenic niche. We show that during the initial days after the induction of seizures by a single intrahippocampal injection of kainic acid, a strong release of zinc and heparin-binding epidermal growth factor, both activators of the EGFR signaling pathway in neural stem cells, is produced. Administration of the EGFR inhibitor gefitinib, a chemotherapeutic in clinical phase IV, prevents the induction of React-NSCs and preserves neurogenesis.

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.

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.

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.

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.

A Comparative Study of Cell Culture Conditions during Conversion from Primed to Naive Human Pluripotent Stem Cells.

The successful reprogramming of human somatic cells into induced pluripotent stem cells (hiPSCs) represented a turning point in the stem cell research field, owing to their ability to differentiate into any cell type with fewer ethical issues than human embryonic stem cells (hESCs). In mice, PSCs are thought to exist in a naive state, the cell culture equivalent of the immature pre-implantation embryo, whereas in humans, PSCs are in a primed state, which is a more committed pluripotent state than a naive state. Recent studies have focused on capturing a similar cell stage in human cells. Given their earlier developmental stage and therefore lack of cell-of-origin epigenetic memory, these cells would be better candidates for further re-differentiation, use in disease modeling, regenerative medicine and drug discovery. In this study, we used primed hiPSCs and hESCs to evaluate the successful establishment and maintenance of a naive cell stage using three different naive-conversion media, both in the feeder and feeder-free cells conditions. In addition, we compared the directed differentiation capacity of primed and naive cells into the three germ layers and characterized these different cell stages with commonly used pluripotent and lineage-specific markers. Our results show that, in general, naive culture NHSM medium (in both feeder and feeder-free systems) confers greater hiPSCs and hESCs viability and the highest naive pluripotency markers expression. This medium also allows better cell differentiation cells toward endoderm and mesoderm.

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.

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.

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.

Human Dental Pulp Stem Cells Grown in Neurogenic Media Differentiate Into Endothelial Cells and Promote Neovasculogenesis in the Mouse Brain.

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 NeurocultTM (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.

A preclinical mouse model of glioma with an alternative mechanism of telomere maintenance (ALT).

Glioblastoma multiforme is the most aggressive primary tumor of the central nervous system. Glioma stem cells (GSCs), a small population of tumor cells with stem-like properties, are supposedly responsible for glioblastoma multiforme relapse after current therapies. In approximately thirty percent of glioblastoma multiforme tumors, telomeres are not maintained by telomerase but through an alternative mechanism, termed alternative lengthening of telomere (ALT), suggesting potential interest in developing specific therapeutic strategies. However, no preclinical model of ALT glioma was available until the isolation of TG20 cells from a human ALT glioma. Herein, we show that TG20 cells exhibit a high level of telomeric recombination but a stable karyotype, indicating that their telomeres retain their protective function against chromosomal instability. TG20 cells possess all of the characteristic features of GSCs: the expression of neural stem cell markers, the generation of intracerebral tumors in NOD-SCID-IL2Rγ (NSG) mice as well as in nude mice, and the ability to sustain serial intracerebral transplantations without expressing telomerase, demonstrating the stability of the ALT phenotype in vivo. Furthermore, we also demonstrate that 360B, a G-quadruplex ligand of the pyridine derivative series that impairs telomere replication and mitotic progression in cancer cells, prevents the development of TG20 tumors. Together, our results show that intracerebral grafts of TG20 cells in immunodeficient mice constitute an efficient preclinical model of ALT glioblastoma multiforme and that G-quadruplex ligands are a potential therapy for this specific type of tumor.

Ikaros-1 couples cell cycle arrest of late striatal precursors with neurogenesis of enkephalinergic neurons.

During central nervous system development, several transcription factors regulate the differentiation of progenitor cells to postmitotic neurons. Here we describe a novel role for Ikaros-1 in the generation of late-born striatal neurons. Our results show that Ikaros-1 is expressed in the boundary of the striatal germinal zone (GZ)/mantle zone (MZ), where it induces cell cycle arrest of neural progenitors by up-regulation of the cyclin-dependent kinase inhibitor (CDKi) p21(Cip1/Waf1). This effect is coupled with the neuronal differentiation of late precursors, which in turn is critical for the second wave of striatal neurogenesis that gives rise to matrix neurons. Consistently, Ikaros(-/-) mice had fewer striatal projecting neurons and, in particular, enkephalin (ENK)-positive neurons. In addition, overexpression of Ikaros-1 in primary striatal cultures increases the number of calbindin- and ENK-positive neurons. Our results also show that Ikaros-1 acts downstream of the Dlx family of transcription factors, insofar as its expression is lost in Dlx1/2 double knockout mice. However, we demonstrate that Ikaros-1 and Ebf-1 independently regulate the final determination of the two populations of striatal projection neurons of the matrix compartment, ENK- and substance P-positive neurons. In conclusion, our findings identify Ikaros-1 as a modulator of cell cycle exit of neural progenitors that gives rise to the neurogenesis of ENK-positive striatal neurons.

Interplay of leukemia inhibitory factor and retinoic acid on neural differentiation of mouse embryonic stem cells.

Embryonic stem (ES) cells have great potential for cell replacement in neurodegenerative disorders. Implantation of these cells into the brain, however, requires their prior differentiation. We examined the interplay between leukemia inhibitory factor (LIF) and retinoic acid (RA) on neural differentiation of mouse ES (mES) cells. Mouse embryonic stem cells were allowed to form cell aggregates, the so-called embryoid bodies (EBs), in the absence or presence of LIF. In the absence of LIF, mES cells downregulated the expression of the undifferentiated mES cell marker Oct-3/4, and increased mRNA levels of two neural precursor markers, Sox-1 and Nestin, as well as the neuronal marker beta-tubulin III. This neuronal differentiation was enhanced by treating EBs with RA. Moreover, RA irreversibly increased the number of postmitotic neurons in culture, as shown by the reduction of proliferating mES cells and the increase in beta-tubulin III-positive cells 6 days after RA removal, which in turn affected mES cell viability. The addition of LIF during EBs formation, however, blocked completely this neuronal differentiation. Our findings also showed that pre-differentiation of mES cells in vitro avoided the teratocarcinoma formation observed when proliferating mES cells were grafted into the brain. In addition, mES cells pre-differentiated with RA in culture showed a reduction in proliferation and the presence of neural phenotypes after grafting. In conclusion, the present results indicate that RA enhances neuronal differentiation of mES cells in the absence of LIF, although it compromises cell viability and transplantation.