Adult Mammalian Tissues Research Articles

Deep Mouse Brain Two-Photon Near-Infrared Fluorescence Imaging Using a Superconducting Nanowire Single-Photon Detector Array.

Two-photon microscopy (2PM) has become an important tool in biology to study the structure and function of intact tissues in vivo. However, adult mammalian tissues such as the mouse brain are highly scattering, thereby putting fundamental limits on the achievable imaging depth, which typically reside at around 600-800 μm. In principle, shifting both the excitation as well as (fluorescence) emission light to the shortwave near-infrared (SWIR, 1000-1700 nm) region promises substantially deeper imaging in 2PM, yet this shift has proven challenging in the past due to the limited availability of detectors and probes in this wavelength region. To overcome these limitations and fully capitalize on the SWIR region, in this work, we introduce a novel array of superconducting nanowire single-photon detectors (SNSPDs) and associated custom detection electronics for use in near-infrared 2PM. The SNSPD array exhibits high efficiency and dynamic range as well as low dark-count rates over a wide wavelength range. Additionally, the electronics and software permit a seamless integration into typical 2PM systems. Together with an organic fluorescent dye emitting at 1105 nm, we report imaging depth of >1.1 mm in the in vivo mouse brain, limited mostly by available labeling density and laser properties. Our work establishes a promising, and ultimately scalable, new detector technology for SWIR 2PM that facilitates deep tissue biological imaging.

PARKIN is not required to sustain OXPHOS function in adult mammalian tissues

Loss-of-function variants in the PRKN gene encoding the ubiquitin E3 ligase PARKIN cause autosomal recessive early-onset Parkinson’s disease (PD). Extensive in vitro and in vivo studies have reported that PARKIN is involved in multiple pathways of mitochondrial quality control, including mitochondrial degradation and biogenesis. However, these findings are surrounded by substantial controversy due to conflicting experimental data. In addition, the existing PARKIN-deficient mouse models have failed to faithfully recapitulate PD phenotypes. Therefore, we have investigated the mitochondrial role of PARKIN during ageing and in response to stress by employing a series of conditional Parkin knockout mice. We report that PARKIN loss does not affect oxidative phosphorylation (OXPHOS) capacity and mitochondrial DNA (mtDNA) levels in the brain, heart, and skeletal muscle of aged mice. We also demonstrate that PARKIN deficiency does not exacerbate the brain defects and the pro-inflammatory phenotype observed in mice carrying high levels of mtDNA mutations. To rule out compensatory mechanisms activated during embryonic development of Parkin-deficient mice, we generated a mouse model where loss of PARKIN was induced in adult dopaminergic (DA) neurons. Surprisingly, also these mice did not show motor impairment or neurodegeneration, and no major transcriptional changes were found in isolated midbrain DA neurons. Finally, we report a patient with compound heterozygous PRKN pathogenic variants that lacks PARKIN and has developed PD. The PARKIN deficiency did not impair OXPHOS activities or induce mitochondrial pathology in skeletal muscle from the patient. Altogether, our results argue that PARKIN is dispensable for OXPHOS function in adult mammalian tissues.

Metabolic "Sense Relay" in Stem Cells: A Short But Impactful Life of PAS Kinase Balancing Stem Cell Fates.

Tissue regeneration is a complex molecular and biochemical symphony. Signaling pathways establish the rhythmic proliferation and differentiation cadence of participating cells to repair the damaged tissues and repopulate the tissue-resident stem cells. Sensory proteins form a critical bridge between the environment and cellular response machinery, enabling precise spatiotemporal control of stem cell fate. Of many sensory modules found in proteins from prokaryotes to mammals, Per-Arnt-Sim (PAS) domains are one of the most ancient and found in the most diverse physiological context. In metazoa, PAS domains are found in many transcription factors and ion channels; however, PAS domain-containing Kinase (PASK) is the only metazoan kinase where the PAS sensory domain is connected to a signaling kinase domain. PASK is predominantly expressed in undifferentiated, self-renewing embryonic and adult stem cells, and its expression is rapidly lost upon differentiation, resulting in its nearly complete absence from the adult mammalian tissues. Thus, PASK is expressed within a narrow but critical temporal window when stem cell fate is established. In this review, we discuss the emerging insight into the sensory and signaling functions of PASK as an integrator of metabolic and nutrient signaling information that serves to balance self-renewal and differentiation programs during mammalian tissue regeneration.

Cartilage regeneration in zebrafish depends on Nrg1/ErbB signaling pathway.

Objective: Cartilage, as the majority of adult mammalian tissues, has limited regeneration capacity. Cartilage degradation consecutive to joint injury or aging then leads to irreversible joint damage and diseases. In contrast, several vertebrate species such as the zebrafish have the remarkable capacity to spontaneously regenerate skeletal structures after severe injuries. The objective of our study was to test the regenerative capacity of Meckel's cartilage (MC) upon mechanical injury in zebrafish and to identify the mechanisms underlying this process. Methods and Results: Cartilage regenerative capacity in zebrafish larvae was investigated after mechanical injuries of the lower jaw MC in TgBAC(col2a1a:mCherry), to visualize the loss and recovery of cartilage. Confocal analysis revealed the formation of new chondrocytes and complete regeneration of MC at 14days post-injury (dpi) via chondrocyte cell cycle re-entry and proliferation of pre-existing MC chondrocytes near the wound. Through expression analyses, we showed an increase of nrg1 expression in the regenerating lower jaw, which also expresses Nrg1 receptors, ErbB3 and ErbB2. Pharmacological inhibition of the ErbB pathway and specific knockdown of Nrg1 affected MC regeneration indicating the pivotal role of this pathway for cartilage regeneration. Finally, addition of exogenous NRG1 in an in vitro model of osteoarthritic (OA)-like chondrocytes induced by IL1β suggests that Nrg1/ErbB pathway is functional in mammalian chondrocytes and alleviates the increased expression of catabolic markers characteristic of OA-like chondrocytes. Conclusion: Our results show that the Nrg1/ErbB pathway is required for spontaneous cartilage regeneration in zebrafish and is of interest to design new therapeutic approaches to promote cartilage regeneration in mammals.

The atypical CDK activator RingoA/Spy1 regulates exit from quiescence in neural stem cells

In the adult mammalian brain, most neural stem cells (NSCs) are held in a reversible state of quiescence, which is essential to avoid NSC exhaustion and determine the appropriate neurogenesis rate. NSCs of the mouse adult subependymal niche provide neurons for olfactory circuits and can be found at different depths of quiescence, but very little is known on how their quiescence-to-activation transition is controlled. Here, we identify the atypical cyclin-dependent kinase (CDK) activator RingoA as a regulator of this process. We show that the expression of RingoA increases the levels of CDK activity and facilitates cell cycle entry of a subset of NSCs that divide slowly. Accordingly, RingoA-deficient mice exhibit reduced olfactory neurogenesis with an accumulation of quiescent NSCs. Our results indicate that RingoA plays an important role in setting the threshold of CDK activity required for adult NSCs to exit quiescence and may represent a dormancy regulator in adult mammalian tissues.

Reindeer light the way to scarless wound healing

Wound healing in adult mammalian tissues generally involves scarring instead of tissue regeneration. A study in this issue of Cell reveals that after injury, reindeer antler skin regenerates by priming regenerative genes in wound fibroblasts instead of forming a scar through an inflammatory gene program.

Abstract P1034: Neurohormonal Pathways Drive Postnatal Thermogenesis And Loss Of Cardiac Regenerative Potential

Why do mammalian organs including the heart lose regenerative potential during the perinatal window remains enigmatic. Through phylogenetic analysis of vertebrate cardiomyocyte ploidy as a proxy for cardiac regenerative potential, we uncover that certain monotreme, edentate, cetacean, chiropteran species have unusually high percentages of diploid cardiomyocytes in the adult heart. Cardiomyocyte abundance across 41 vertebrate species conforms to Kleiber's law, the 3/4-power law scaling of metabolism with bodyweight, and decreases when the standard metabolic rate and body temperature increase during the ectotherm-to-endotherm transition. Recently, following the fractal feature of the cardiovascular system, we further strengthen the link between endothermy and heart physiology. Thermogenesis increases by more than 10-fold during the ectotherm-to-endotherm transition requiring similar increases in blood flow and cardiac function that may impede heart regenerative potential. Moreover, we report sympathetic nerve-adrenergic receptor and thyroid hormone signaling as two major pathways promoting both thermogenesis and cardiomyocyte cell cycle arrest. Combined inhibition of postnatal adrenergic and thyroid hormone signaling results in juvenile mice with strikingly low body temperatures (~25 ° C), significantly elevated abundances of diploid cardiomyocytes (~60%), dramatically increased cardiomyocyte proliferation, and enhanced cardiac regenerative abilities when analyzed at postnatal day 14. Intriguingly, both neurohormonal pathways seem to be inactive in adult zebrafish hearts. Altogether, our findings support critical roles of two major thermogenic pathways in suppressing heart regenerative capacity, and implicate that the limited regenerative capacity in various adult mammalian tissues may be a tradeoff for the acquisition of endothermy in ontogeny and phylogeny.

The transcription factor LEF1 interacts with NFIX and switches isoforms during adult hippocampal neural stem cell quiescence.

Stem cells in adult mammalian tissues are held in a reversible resting state, known as quiescence, for prolonged periods of time. Recent studies have greatly increased our understanding of the epigenetic and transcriptional landscapes that underlie stem cell quiescence. However, the transcription factor code that actively maintains the quiescence program remains poorly defined. Similarly, alternative splicing events affecting transcription factors in stem cell quiescence have been overlooked. Here we show that the transcription factor T-cell factor/lymphoid enhancer factor LEF1, a central player in canonical β-catenin-dependent Wnt signalling, undergoes alternative splicing and switches isoforms in quiescent neural stem cells. We found that active β-catenin and its partner LEF1 accumulated in quiescent hippocampal neural stem and progenitor cell (Q-NSPC) cultures. Accordingly, Q-NSPCs showed enhanced TCF/LEF1-driven transcription and a basal Wnt activity that conferred a functional advantage to the cultured cells in a Wnt-dependent assay. At a mechanistic level, we found a fine regulation of Lef1 gene expression. The coordinate upregulation of Lef1 transcription and retention of alternative spliced exon 6 (E6) led to the accumulation of a full-length protein isoform (LEF1-FL) that displayed increased stability in the quiescent state. Prospectively isolated GLAST + cells from the postnatal hippocampus also underwent E6 retention at the time quiescence is established in vivo. Interestingly, LEF1 motif was enriched in quiescence-associated enhancers of genes upregulated in Q-NSPCs and quiescence-related NFIX transcription factor motifs flanked the LEF1 binding sites. We further show that LEF1 interacts with NFIX and identify putative LEF1/NFIX targets. Together, our results uncover an unexpected role for LEF1 in gene regulation in quiescent NSPCs, and highlight alternative splicing as a post-transcriptional regulatory mechanism in the transition from stem cell activation to quiescence.

Enhancer decommissioning imposes an epigenetic barrier to sensory hair cell regeneration.

Adult mammalian tissues such as heart, brain, retina, and the sensory structures of the inner ear do not effectively regenerate, although a latent capacity for regeneration exists at embryonic and perinatal times. We explored the epigenetic basis for this latent regenerative potential in the mouse inner ear and its rapid loss during maturation. In perinatal supporting cells, whose fate is maintained by Notch-mediated lateral inhibition, the hair cell enhancer network is epigenetically primed (H3K4me1) but silenced (active H3K27 de-acetylation and trimethylation). Blocking Notch signaling during the perinatal period of plasticity rapidly eliminates epigenetic silencing and allows supporting cells to transdifferentiate into hair cells. Importantly, H3K4me1 priming of the hair cell enhancers in supporting cells is removed during the first post-natal week, coinciding with the loss of transdifferentiation potential. We hypothesize that enhancer decommissioning during cochlear maturation contributes to the failure of hair cell regeneration in the mature organ of Corti.

608 Neuroimmune control of adult mammalian scarless skin regeneration

Adult mammalian wounds, with rare exception, heal with fibrotic scars that severely disrupt tissue architecture and function. Regenerative medicine seeks methods to avoid scar formation and restore the original tissue structures. We show in three adult mouse models that pharmacologic activation of the nociceptor TRPA1 on cutaneous sensory neurons reduces scar formation and can also promote tissue regeneration. Local activation of TRPA1 induces tissue regeneration on distant untreated areas of injury, demonstrating a systemic effect. Activated TRPA1 stimulates local production of interleukin-23 (IL-23) by dermal dendritic cells, leading to activation of circulating dermal IL-17–producing gd T cells. Genetic ablation of TRPA1, IL-23, dermal dendritic cells, or gd T cells prevents TRPA1-mediated tissue regeneration. These results reveal a cutaneous neuroimmune-regeneration cascade triggered by topical TRPA1 activators that promotes adult mammalian tissue regeneration, presenting a new avenue for research and development of therapies for wounds and scars.

A Photodynamic Tool to Promote a Sustained, ROS-Dependent Growth of Human Hair Follicles in Ex Vivo Culture.

Reactive oxygen species (ROS) may severely affect the biochemical viability of most cells. However, ROS may act also as key second messengers regulating important physiological functions in eukaryotic organisms. Of special interest is the potential role of ROS in the regulation of stem cell function and tissue homeostasis and regeneration in adult mammalian tissues. In this context, the hair follicle constitutes an excellent experimental model to study this aspect of ROS biology.Here we present a robust protocol to promote a sustained growth of ex vivo cultured human hair follicles based on the induction of a transient/modulable production of nonlethal endogenous ROS levels in the tissue through a protoporphyrin IX-dependent photodynamic procedure. The light-switchable ROS production activates hair follicle stem cell niches, induces cell proliferation, and maintains the growth/anagen phase for long time. This approach constitutes a complementary experimental tool to study the physiological roles of ROS in human tissues.

Brain-specific angiogenesis inhibitor 1 is expressed in the Myo/Nog cell lineage

The Myo/Nog cell lineage was discovered in the chick embryo and is also present in adult mammalian tissues. The cells are named for their expression of mRNA for the skeletal muscle specific transcription factor MyoD and bone morphogenetic protein inhibitor Noggin. A third marker for Myo/Nog cells is the cell surface molecule recognized by the G8 monoclonal antibody (mAb). G8 has been used to detect, track, isolate and kill Myo/Nog cells. In this study, we screened a membrane proteome array for the target of the G8 mAb. The array consisted of >5,000 molecules, each synthesized in their native confirmation with appropriate post-translational modifications in a single clone of HEK-293T cells. G8 mAb binding to the clone expressing brain-specific angiogenesis inhibitor 1 (BAI1) was detected by flow cytometry, re-verified by sequencing and validated by transfection with the plasmid construct for BAI1. Further validation of the G8 target was provided by enzyme-linked immunosorbent assay. The G8 epitope was identified by screening a high-throughput, site directed mutagenesis library designed to cover 95–100% of the 954 amino acids of the extracellular domain of the BAI1 protein. The G8 mAb binds within the third thrombospondin repeat of the extracellular domain of human BAI1. Immunofluorescence localization experiments revealed that G8 and a commercially available BAI1 mAb co-localize to the subpopulation of Myo/Nog cells in the skin, eyes and brain. Expression of the multi-functional BAI1 protein in Myo/Nog cells introduces new possibilities for the roles of Myo/Nog cells in normal and diseased tissues.

Sonic Hedgehog Signaling in Organogenesis, Tumors, and Tumor Microenvironments.

During mammalian embryonic development, primary cilia transduce and regulate several signaling pathways. Among the various pathways, Sonic hedgehog (SHH) is one of the most significant. SHH signaling remains quiescent in adult mammalian tissues. However, in multiple adult tissues, it becomes active during differentiation, proliferation, and maintenance. Moreover, aberrant activation of SHH signaling occurs in cancers of the skin, brain, liver, gallbladder, pancreas, stomach, colon, breast, lung, prostate, and hematological malignancies. Recent studies have shown that the tumor microenvironment or stroma could affect tumor development and metastasis. One hypothesis has been proposed, claiming that the pancreatic epithelia secretes SHH that is essential in establishing and regulating the pancreatic tumor microenvironment in promoting cancer progression. The SHH signaling pathway is also activated in the cancer stem cells (CSC) of several neoplasms. The self-renewal of CSC is regulated by the SHH/Smoothened receptor (SMO)/Glioma-associated oncogene homolog I (GLI) signaling pathway. Combined use of SHH signaling inhibitors and chemotherapy/radiation therapy/immunotherapy is therefore key in targeting CSCs.

Induction of Fibrogenic Phenotype in Human Mesenchymal Stem Cells by Connective Tissue Growth Factor in a Hydrogel Model of Soft Connective Tissue.

Scar formation is the typical endpoint of wound healing in adult mammalian tissues. An overactive or prolonged fibrogenic response following injury leads to excessive deposition of fibrotic proteins that promote tissue contraction and scar formation. Although well-defined in the dermal tissue, the progression of fibrosis is less explored in other connective tissues, such as the vocal fold. To establish a physiologically relevant 3D model of loose connective tissue fibrosis, we have developed a synthetic extracellular matrix using hyaluronic acid (HA) and peptidic building blocks carrying complementary functional groups. The resultant network was cell adhesive and protease degradable, exhibiting viscoelastic properties similar to the human vocal fold. Human mesenchymal stem cells (hMSCs) were encapsulated in the HA matrix as single cells or multicellular aggregates and cultured in pro-fibrotic media containing connective tissue growth factor (CTGF) for up to 21 days. hMSCs treated with CTGF-supplemented media exhibited an increased expression of fibrogenic markers and ECM proteins associated with scarring. Incorporation of α-smooth muscle actin into F-actin stress fibers was also observed. Furthermore, CTGF treatment increased the migratory capacity of hMSCs as compared to the CTGF-free control groups, indicative of the development of a myofibroblast phenotype. Addition of an inhibitor of the mitogen-activated protein kinase (MAPK) pathway attenuated cellular expression of fibrotic markers and related ECM proteins. Overall, this study demonstrates that CTGF promotes the development of a fibrogenic phenotype in hMSCs encapsulated within an HA matrix and that the MAPK pathway is a potential target for future therapeutic endeavors towards limiting scar formation in loose connective tissues.

Bisphenol A exposure under metabolic stress induces accelerated cellular senescence in vivo in a p53 independent manner

Senescence is an irreversible process that is a characteristic of age-associated disease like Type 2 diabetes (T2D). Bisphenol-A (BPA), one of the most common endocrine disruptor chemicals, received special attention in the development of insulin resistance and T2D. To understand the role played by BPA in cellular senescence under metabolic stress, zebrafish embryos were exposed to BPA in the absence and presence of hyperglycaemia. Transcriptional levels of the senescence markers p15, p53, Rb1 and β-galactosidase were increased when BPA was combined with metabolic stress. In addition, zebrafish embryos that were exposed to combination of hyperglycaemia and BPA exhibited increased levels of apoptosis. However, cellular senescence remained induced by a combination of hyperglycaemia and BPA exposure even in the absence of a translated p53 protein suggesting that senescence is primarily independent of it but dependent on the p15-Rb1 pathway under our experimental conditions. To confirm that our results hold true in adult mammalian tissues, we validated our embryonic experiments in an adult mammalian metabolic model of skeletal muscle cells. Our work reveals a novel and unique converging role of senescence and apoptosis axis contributing to glucose dyshomeostasis. Thus, we conclude that BPA exposure can exacerbate existing metabolic stress to increase cellular senescence that leads to aggravation of disease phenotype in age-associated diseases like type 2 diabetes.

Replication-Independent Histone Turnover Underlines the Epigenetic Homeostasis in Adult Heart.

Replication-independent histone turnover has been linked to cis-regulatory chromatin domains in cultured cell lines, but its molecular underpinnings and functional relevance in adult mammalian tissues remain yet to be defined. We investigated regulatory functions of replication-independent histone turnover in chromatin states of postmitotic cardiomyocytes from adult mouse heart. We used H2B-GFP (histone 2B-green fluorescent protein) fusion protein pulse-and-chase approaches to measure histone turnover rate in mouse cardiomyocytes. Surprisingly, we found that the short histone half-life (≈2 weeks) contrasted dramatically with the long lifetime of cardiomyocytes, and rapid histone turnover regions corresponded to cis-regulatory domains of heart genes. Interestingly, recruitment of chromatin modifiers, including Polycomb EED (embryonic ectoderm development), was positively correlated with histone turnover rate at enhancers. Mechanistically, through directly interacting with and engaging the BAF (BRG1 [Brahma-related gene-1]-associated factor) complex for nucleosome exchange for stereotyped histone modifications from the free histone pool, EED augmented histone turnover to restrain enhancer overactivation. We propose a model in which replication-independent histone turnover reinforces robustness of local chromatin states for adult tissue homeostasis.

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues.

The analysis of single cell gene expression across thousands of individual cells within a tissue or microenvironment is a valuable tool for identifying cell composition, discrimination of functional states, and molecular pathways underlying observed tissue functions and animal behaviors. However, the isolation of intact, healthy single cells from adult mammalian tissues for subsequent downstream single cell molecular analysis can be challenging. This protocol describes the general processes and quality control checks necessary to obtain high-quality adult single cell preparations from the nervous system or skin that enabled subsequent unbiased single cell RNA sequencing and analysis. Guidelines for downstream bioinformatic analysis are also provided.

Structural evidence for a new elaborate 3D-organization of the cardiomyocyte lateral membrane in adult mammalian cardiac tissues.

This study explored the lateral crest structures of adult cardiomyocytes (CMs) within healthy and diseased cardiac tissue. Using high-resolution electron and atomic force microscopy, we performed an exhaustive quantitative analysis of the three-dimensional (3D) structure of the CM lateral surface in different cardiac compartments from various mammalian species (mouse, rat, cow, and human) and determined the technical pitfalls that limit its observation. Although crests were observed in nearly all CMs from all heart compartments in all species, we showed that their heights, dictated by the subsarcolemmal mitochondria number, substantially differ between compartments from one species to another and tightly correlate with the sarcomere length. Differences in crest heights also exist between species; for example, the similar cardiac compartments in cows and humans exhibit higher crests than rodents. Unexpectedly, we found that lateral surface crests establish tight junctional contacts with crests from neighbouring CMs. Consistently, super-resolution SIM or STED-based immunofluorescence imaging of the cardiac tissue revealed intermittent claudin-5-claudin-5 interactions in trans via their extracellular part and crossing the basement membrane. Finally, we found a loss of crest structures and crest-crest contacts in diseased human CMs and in an experimental mouse model of left ventricle barometric overload. Overall, these results provide the first evidence for the existence of differential CM surface crests in the cardiac tissue as well as the existence of CM-CM direct physical contacts at their lateral face through crest-crest interactions. We propose a model in which this specific 3D organization of the CM lateral membrane ensures the myofibril/myofiber alignment and the overall cardiac tissue cohesion. A potential role in the control of sarcomere relaxation and of diastolic ventricular dysfunction is also discussed. Whether the loss of CM surface crests constitutes an initial and common event leading to the CM degeneration and the setting of heart failure will need further investigation.

PO-333 Somatic engineering of mammary gland epithelial cells using CRISPR/Cas9 for rapid testing of breast cancer susceptibility genes in mouse models

IntroductionBreast cancer is one of the most commonly diagnosed malignancies in women. In Western countries it is the second leading cause of cancer death among women. Five to ten percent of all breast cancers have a hereditary component, and approximately twenty five percent of all hereditary breast and ovarian cases are attributable to mutations in the breast cancer 1 or breast cancer 2 (BRCA1 or BRCA2) genes. In vivo studies represent a highly comprehensive and relevant setting for studying the complexity of this human disease. Genetically engineered germline mouse models of breast cancer paved the way for improved basic and translational research, but their generation is slow and expensive.Material and methodsTo overcome these limitations, our lab previously developed CRISPR/Cas9-mediated somatic genome editing approaches to test the role of candidate factors in the adult mammary tissue. In particular, a conditional Cas9-expressing mouse model was used for intraductal injection of sgRNAs encoding lentiviruses (LV-sgRNA), which allowed for somatic gene disruption of tumour suppressor genes in mammary epithelial cells and for evaluating the effect on tumour formation and disease progression (Annunziato et al. 2016).Results and discussionsWe will extend this approach to analyse candidate susceptibility genes in triple negative breast cancer. Using a K14Cre;p53F/F;Cas9 mouse model, this LV-sgRNA intraductal platform provides the opportunity to test in vivo several new (candidate) susceptibility genes and study the effects of tumour suppressor domain deletions.ConclusionThrough these studies, we hope to shed light on several genetic alterations and how they affect tumour suppression and drug response.

Isolation and Characterization of Neural Crest-Derived Stem Cells From Adult Ovine Palatal Tissue.

Adult mammalian craniofacial tissues contain limited numbers of post-migratory neural crest-derived stem cells. Similar to their embryonic counterparts, these adult multipotent stem cells can undergo multi-lineage differentiation and are capable of contributing to regeneration of mesodermal and ectodermal cells and tissues in vivo. In the present study, we describe for the first time the presence of Nestin-positive neural crest-derived stem cells (NCSCs) within the ovine hard palate. We show that these cells can be isolated from the palatal tissue and are able to form neurospheres. Ovine NCSCs express the typical neural crest markers Slug and Twist, exhibit high proliferative and migratory activity and are able to differentiate into α smooth muscle cells and β-III-tubulin expressing ectodermal cells. Finally, we demonstrate that oNCSCs are capable of differentiating into osteogenic, adipogenic and chondrogenic cells. Taken together, our results suggest that oNCSCs could be used as model cells to assess the efficacy and safety of autologous NCSC transplantation in a large animal model.