The classical model of tissue renewal posits that small numbers of quiescent stem cells (SCs) give rise to proliferating transit-amplifying cells before terminal differentiation. However, many organs house pools of SCs with proliferative and differentiation potentials that diverge from this template. Resolving SC identity and organization is therefore central to understanding tissue renewal. Here, using a combination of single-cell RNA sequencing (scRNA-seq), mouse genetics and tissue injury approaches, we uncover cellular hierarchies and mechanisms that underlie the maintenance and repair of the continuously growing mouse incisor. Our results reveal that, during homeostasis, a group of actively cycling epithelial progenitors generates enamel-producing ameloblasts and adjacent layers of non-ameloblast cells. After injury, tissue repair was achieved through transient increases in progenitor-cell proliferation and through direct conversion of Notch1-expressing cells to ameloblasts. We elucidate epithelial SC identity, position and function, providing a mechanistic basis for the homeostasis and repair of a fast-turnover ectodermal appendage.

The stem cell-determining transcription factor Sox2 is required for the maintenance of normal neural stem cells. In this study, we investigated the requirement for Sox2 in neural cancer stem-like cells using a conditional genetic deletion mutant in a mouse model of platelet-derived growth factor-induced malignant oligodendroglioma. Transplanting wild-type oligodendroglioma cells into the brain generated lethal tumors, but mice transplanted with Sox2-deleted cells remained free of tumors. Loss of the tumor-initiating ability of Sox2-deleted cells was reversed by lentiviral-mediated expression of Sox2. In cell culture, Sox2-deleted tumor cells were highly sensitive to differentiation stimuli, displaying impaired proliferation, increased cell death, and aberrant differentiation. Gene expression analysis revealed an early transcriptional response to Sox2 loss. The observed requirement of oligodendroglioma stem cells for Sox2 suggested its relevance as a target for therapy. In support of this possibility, an immunotherapeutic approach based on immunization of mice with SOX2 peptides delayed tumor development and prolonged survival. Taken together, our results showed that Sox2 is essential for tumor initiation by mouse oligodendroglioma cells, and they illustrated a Sox2-directed strategy of immunotherapy to eradicate tumor-initiating cells. Cancer Res; 74(6); 1833-44. Ó2014 AACR.

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The transcription factor Sox2 has been shown to play essential roles during embryonic development as well as in cancer. To more precisely understand tumor biology and to identify potential therapeutical targets, we thoroughly investigated the expression and function of Sox2 in medulloblastoma, a malignant embryonic brain tumor that initiates in the posterior fossa and eventually spreads throughout the entire cerebrospinal axis. We examined a large series of tumor samples (n ¼ 188) to show that SOX2 is specifically expressed in Sonic hedgehog (SHH)-associated medulloblastoma with an interesting preponderance in adolescent and adult cases. We further show that cerebellar granule neuron precursors (CGNP), which are believed to serve as the cell of origin for this medulloblastoma subgroup, express Sox2 in early stages. Also, Shh-associated medulloblastoma can be initiated from such Sox2-positive CGNPs in mice. Independent of their endogenous Sox2 expression, constitutive activation of Shh signaling in CGNPs resulted in significantly enhanced proliferation and ectopic expression of Sox2 in vitro and Sox2-positive medulloblastoma in vivo. Genetic ablation of Sox2 from murine medulloblastoma did not affect survival, most likely due to a compensatory overexpression of Sox3. However, acute deletion of Sox2 from primary cultures of CGNPs with constitutive Shh signaling significantly decreased proliferation, whereas overexpression of Sox2 enhanced proliferation of murine medulloblastoma cells. We conclude that Sox2 is a marker for Shhdependent medulloblastomas where it is required and sufficient to drive tumor cell proliferation. Cancer Res; 73(12); 3796-807. Ó2013 AACR.

Stem cell-based mediated therapies represent very promising approaches for tissue regeneration and are already applied with success in clinics. These therapeutic approaches consist of the in vitro manipulation of stem cells and their consequent administration to patients as living and dynamic biological agents. Nevertheless, the deregulation of stem cells function might result in the generation of pathologies such as tumours or accelerated senescence. Moreover, different stem cells sources are needed for regeneration of specific tissues. It is thus fundamental to understand the mechanisms regulating the physiology of stem cells. Microfluidic technology can be used to mimic in vivo scenarios and allow the study of stem cell physiology at both single cell and whole stem cell niche levels. This review focuses on the potential sources of stem and progenitor cells for orofacial regeneration and the use of microfluidic technologies for the study of stem cells behaviour and stem cell niches, in the light of regenerative medicine. Keywords:Microfluidics, stem cells, stem cell niches, orofacial regeneration, tooth, innervation, trigeminal ganglia, regenerative medicine. IntroductionThe development of organs and tissues that belong to the orofacial complex proceeds through a series of inductive interactions between cells originated from the epithelium, mesoderm and cranial neural crest-derived mesenchyme (Mao et al., 2012;Mitsiadis and Graf, 2009;Mitsiadis and Papagerakis, 2011). Orofacial organs are highly diverse and exert fundamental and specific functions such as breathing, chewing, speech, smell, and sight (Mao et al., 2012). The physiological functions of these organs are compromised by traumatic injuries, congenital and infectious diseases, and cancer (Mao et al., 2012;Scheller et al., 2009). Furthermore, these pathologies are often accompanied by intensive pain and aesthetic deformities. Therefore, the treatment of compromised pathological orofacial tissues and organs should guarantee restoration of both functionality and aesthetics, which constitutes an enormous clinical challenge. Moreover, organ structure, function, aesthetics, and pain should be managed simultaneously during the regenerative care, a situation that is more complex than in other compartments of the body (Scheller et al., 2009).Biological regeneration is proving an increasingly attractive alternative and complement to traditional surgical techniques for prosthetic replacement of tissues and organs. Cell-based therapeutic approaches are already applied with success in clinics and consist of in vitro manipulation of stem cells and their consequent administration to patients as living and dynamic biological agents. Stem cells are characterised by their potential to self-replicate and their capacity to differentiate into a vast variety of cell types that form the diverse tissues. Therefore, stem cells guarantee tissue repair and regeneration throughout life. During the last decades, a plethora of adult stem cell populations have been isolate...

Transcriptional cofactors that participate in Wnt/β-catenin signaling act in the cytoplasm to promote proper development of tooth enamel.

Human teeth contain a variety of mesenchymal stem cell populations that could be used for cell-based regenerative therapies. However, the isolation and potential use of these cells in the clinics require the extraction of functional teeth, a process that may represent a significant barrier to such treatments. Fibroblasts are highly accessible and might represent a viable alternative to dental stem cells. We thus investigated and compared the in vitro differentiation potential of human dental pulp stem cells (hDPSCs), gingival fibroblasts (hGFs) and foreskin fibroblasts (hFFs). These cell populations were cultured in osteogenic and adipogenic differentiation media, followed by Alizarin Red S and Oil Red O staining to visualize cytodifferentiation. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) was performed to assess the expression of markers specific for stem cells (NANOG, OCT-4), osteogenic (RUNX2, ALP, SP7/OSX) and adipogenic (PPAR-γ2, LPL) differentiation. While fibroblasts are more prone towards adipogenic differentiation, hDPSCs exhibit a higher osteogenic potential. These results indicate that although fibroblasts possess a certain mineralization capability, hDPSCs represent the most appropriate cell population for regenerative purposes involving bone and dental tissues.

Teeth exert fundamental functions related to mastication and speech. Despite their great biomedical importance, an overall picture of their cellular and molecular composition is still missing. In this study, we have mapped the transcriptional landscape of the various cell populations that compose human teeth at single-cell resolution, and we analyzed in deeper detail their stem cell populations and their microenvironment. Our study identified great cellular heterogeneity in the dental pulp and the periodontium. Unexpectedly, we found that the molecular signatures of the stem cell populations were very similar, and that their distinctive behavior could be due to substantial differences between their microenvironments. Our findings suggest that the microenvironmental specificity is the potential source for the major functional differences of the stem cells located in the various tooth compartments and open new perspectives towards cell-based dental therapeutic approaches.