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In JoVE (1)
Other Publications (18)
- Developmental Biology
- Journal of Photochemistry and Photobiology. B, Biology
- Developmental Neuroscience
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- The Anatomical Record. Part A, Discoveries in Molecular, Cellular, and Evolutionary Biology
- Progress in Brain Research
- Birth Defects Research. Part C, Embryo Today : Reviews
- Molecular and Cellular Neurosciences
- Stem Cells (Dayton, Ohio)
- Stem Cell Reviews
- Developmental Neuroscience
- BMC Genomics
- Biomedical Sciences Instrumentation
- Stem Cell Reviews
- Brain Research Bulletin
- Stem Cell Reviews
- The Journal of Pathology
- Stem Cell Research & Therapy
Articles by Maya Sieber-Blum in JoVE
JoVE General
Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures
1Institute of Human Genetics and Northeast England Stem Cell Institute, Newcastle University, 2Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin
Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.
Other articles by Maya Sieber-Blum on PubMed
Neural Crest Origin of Mammalian Merkel Cells
Here, we provide evidence for the neural crest origin of mammalian Merkel cells. Together with nerve terminals, Merkel cells form slowly adapting cutaneous mechanoreceptors that transduce steady indentation in hairy and glabrous skin. We have determined the ontogenetic origin of Merkel cells in Wnt1-cre/R26R compound transgenic mice, in which neural crest cells are marked indelibly. Merkel cells in whiskers and interfollicular locations express the transgene, beta-galactosidase, identifying them as neural crest descendants. We thus conclude that murine Merkel cells originate from the neural crest.
Crystal Violet Combined with Merocyanine 540 for the Ex Vivo Purging of Hematopoietic Stem Cell Grafts
The purpose of this study was to determine in a preclinical purging model, how effective crystal violet-mediated photodynamic therapy (CV-PDT) is against solid tumor and drug-resistant mutant tumor cells, and if certain limitations of CV-PDT can be overcome by using crystal violet (CV) in combination with the membrane-active photosensitizer, Merocyanine 540 (MC540). When used under conditions that preserved an adequate fraction of normal human granulocyte/macrophage progenitors (CFU-GM), CV-PDT failed to achieve meaningful reductions of DU145 prostate, H69 small cell lung cancer, and MDA-MB-435S breast cancer cells. Melphalan-resistant L1210/L-PAM1, adriamycin-resistant P388/ADR, and adriamycin-resistant HL-60/ADR leukemia cells were markedly less sensitive to CV-PDT than their wild-type counterparts, whereas cisplatin-resistant H69/CDDP cells were more sensitive than wild-type H69 cells. Sequential exposure to MC540- and CV-PDT under conditions that preserved an adequate fraction (73% and 29%, respectively) of normal CD34-positive hematopoietic stem cells and granulocyte/macrophage progenitors was highly effective against H69 (99.997% reduction) and H69/CDDP (99.999% reduction) cells, but ineffective against HL-60/ADR, MDA-MB-435S, and DU145 cells. CV thus shows only limited promise as a single-modality purging agent. However, in certain situations, clinically meaningful tumor cell depletions can be obtained by using CV in combination with a second photosensitizer such as MC540.
Ontogeny and Plasticity of Adult Hippocampal Neural Stem Cells
We have investigated the ontogenetic origin and the degree of plasticity of adult hippocampal neural stem cells. Wnt1-expressing cells are located at the dorsal aspect of the embryonic neural tube and some of them are predestined to give rise to neural crest stem cells. Whereas the majority of adult hippocampal neural stem cells do not originate from cells that express Wnt1, a subset does express Wnt1 transiently during embryogenesis, as determined in the double transgenic mouse, Wnt1-cre/R26R. Hippocampal stem cells from adult ROSA 26 mice differentiate into chondrocytes, melanocytes (pigment cells) and smooth muscle cells when cocultured with neural crest cells from quail embryos. Neural crest cell-generated stimuli have a short-range of action and are recognized across species. These observations provide evidence for the heterogeneity in the hippocampal neural stem cell pool with regard to Wnt1 expression. Furthermore, they show plasticity and a remarkably wide range of developmental options of adult hippocampal stem cells.
Neurotrophin-3 Signaling in Mammalian Merkel Cell Development
Merkel cells are sensory cells of neural crest origin. Because little is known about the mechanisms that direct their differentiation, we have investigated the potential role of a candidate regulatory factor, neurotrophin-3 (NT-3). At embryonic day 16.5 (E 16.5), neither NT-3 nor its primary receptors, TrkC and p75NTR are expressed by Merkel cells in the murine whisker. At the time of birth, however, Merkel cells are immunoreactive for NT-3, TrkC and p75NTR. In TrkC null and NT-3 null mice, Merkel cells differentiate initially, but undergo apoptosis perinatally. These results show that NT-3 signaling is not required for the differentiation of Merkel cells, but that it is essential for their postnatal survival.
Cardiac Neural Crest Stem Cells
Whereas the heart itself is of mesodermal origin, components of the cardiac outflow tract are formed by the neural crest, an ectodermal derivative that gives rise to the peripheral nervous system, endocrine cells, melanocytes of the skin and internal organs, and connective tissue, bone, and cartilage of the face and ventral neck, among other tissues. Cardiac neural crest cells participate in the septation of the cardiac outflow tract into aorta and pulmonary artery. The migratory cardiac neural crest consists of stem cells, fate-restricted cells, and cells that are committed to the smooth muscle cell lineage. During their migration within the posterior branchial arches, the developmental potentials of pluripotent neural crest cells become restricted. Conversely, neural crest stem cells persist at many locations, including in the cardiac outflow tract. Many aspects of neural crest cell differentiation are driven by growth factor action. Neurotrophin-3 (NT-3) and its preferred receptor, TrkC, play important roles not only in nervous system development and function, but also in cardiac development as deletion of these genes causes outflow tract malformations. In vitro clonal analysis has shown a premature commitment of cardiac neural crest stem cells in TrkC null mice and a perturbed morphology of the endothelial tube. Norepinephrine transporter (NET) function promotes the differentiation of neural crest stem cells into noradrenergic neurons. Surprisingly, many diverse nonneuronal embryonic tissues, in particular in the cardiovascular system, express NET also. It will be of interest to determine whether norepinephrine transport plays a role also in cardiovascular development.
The Role of NT-3 Signaling in Merkel Cell Development
Merkel cells originate from the neural crest. They are located in hairy and glabrous skin and have neuroendocrine characteristics. Together with A beta afferents, Merkel cells form a slowly adapting mechanoreceptor, the Merkel nerve ending, which transduces steady skin indentation. Neurotphin-3 (NT-3) plays important roles in neural crest cell development. We thus sought to determine whether neurotrophin signaling is essential for Merkel cell development in the whisker pad of the mouse. Our data indicate that at embryonic day 16.5 (E 16.5), NT-3 and its receptors, p75 neurotrophin receptor (p75NTR) and tyrosine kinase receptor, TrkC are not expressed at detectable levels in Merkel cells. After a perinatal switch, however, Merkel cells in whiskers of newborn mice are immunoreactive for p75NTR, TrkC and NT-3. Immunoreactivity of all three markers persists into adulthood. By contrast, innervating fibers are intensely p75NTR-immunoreactive in E16.5 whiskers, but no TrkC immunoreactivity is detected. At birth, and at 6 weeks of age, afferent fibers are intensely immunoreactive for both p75NTR and TrkC. In TrkC null whiskers, numerous Merkel cells are present at E16.5, and they are innervated. We draw three major conclusions from these observations: (i) NT-3 signaling through p75NTR or TrkC is not required for the development and prenatal survival of either a major subset or of all Merkel cells, (ii) the postnatal survival of Merkel cells is supported by autocrine or paracrine NT-3, rather than by neuron-derived NT-3, and (iii) Merkel cell-derived NT-3 is not a chemoattractant for innervating A beta fibers, but is likely to be involved in maintaining Merkel cell innervation postnatally.
The Adult Hair Follicle: Cradle for Pluripotent Neural Crest Stem Cells
This review focuses on the recent identification of two novel neural crest-derived cells in the adult mammalian hair follicle, pluripotent stem cells, and Merkel cells. Wnt1-cre/R26R compound transgenic mice, which in the periphery express beta-galactosidase in a neural crest-specific manner, were used to trace neural crest cells. Neural crest cells invade the facial epidermis as early as embryonic day 9.5. Neural crest-derived cells are present along the entire extent of the whisker follicle. This includes the bulge area, an epidermal niche for keratinocyte stem cells, as well as the matrix at the base of the hair follicle. We have determined by in vitro clonal analysis that the bulge area of the adult whisker follicle contains pluripotent neural crest stem cells. In culture, beta-galactosidase-positive cells emigrate from bulge explants, identifying them as neural crest-derived cells. When these cells are resuspended and grown in clonal culture, they give rise to colonies that contain multiple differentiated cell types, including neurons, Schwann cells, smooth muscle cells, pigment cells, chondrocytes, and possibly other types of cells. This result provides evidence for the pluripotentiality of the clone-forming cell. Serial cloning showed that bulge-derived neural crest cells undergo self-renewal, which identifies them as stem cells. Pluripotent neural crest cells are also localized in the back skin hair of adult mice. The bulge area of the whisker follicle is surrounded by numerous Merkel cells, which together with innervating nerve endings form slowly adapting mechanoreceptors that transduce steady skin indentation. Merkel cells express beta-galactosidase in double transgenic mice, which confirms their neural crest origin. Taken together, our data indicate that the epidermis of the adult hair follicle contains pluripotent neural crest stem cells, termed epidermal neural crest stem cells (eNCSCs), and one newly identified neural crest derivative, the Merkel cell. The intrinsic high degree of plasticity of eNCSCs and the fact that they are easily accessible in the skin make them attractive candidates for diverse autologous cell therapy strategies.
Characterization of Epidermal Neural Crest Stem Cell (EPI-NCSC) Grafts in the Lesioned Spinal Cord
We have characterized in the contusion-lesioned murine spinal cord the behavior of acutely implanted epidermal neural crest stem cells (EPI-NCSC, formerly eNCSC). EPI-NCSC, a novel type of multipotent adult stem cell, are remnants of the embryonic neural crest. They reside in the bulge of hair follicles and have the ability to differentiate into all major neural crest derivatives (Sieber-Blum, M., Grim, M., Hu, Y.F., Szeder, V., 2004. Pluripotent neural crest stem cells in the adult hair follicle. Dev. Dyn. 231, 258-269). Grafted EPI-NCSC survived, integrated, and intermingled with host neurites in the lesioned spinal cord. EPI-NCSC were non-migratory. They did not proliferate and did not form tumors. Significant subsets expressed neuron-specific beta-III tubulin, the GABAergic marker glutamate decarboxylase 67 (GAD67), the oligodendrocyte marker, RIP, or myelin basic protein (MBP). Close physical association of non-neuronal EPI-NCSC with host neurites was observed. Glial fibrillary acidic protein (GFAP) immunofluorescence was not detected. Collectively, our data indicate that intraspinal EPI-NCSC demonstrate several desirable characteristics that may include local neural replacement and re-myelination.
An Epidermal Neural Crest Stem Cell (EPI-NCSC) Molecular Signature
Here, we report the first transcriptome for mouse epidermal neural crest stem cells (EPI-NCSC, formerly eNCSCs). In addition, our study resolves conflicting opinions in the literature by showing that EPI-NCSC are distinct from other types of skin-resident stem cells/progenitors. Finally, with the three gene profiles, we have established a foundation and provide a valuable resource for future mouse NCSC research. EPI-NCSC represent a novel type of multipotent adult stem cell that originates from the embryonic neural crest and resides in the bulge of hair follicles. We performed gene profiling by LongSAGE (long serial analysis of gene expression) with mRNA from EPI-NCSC, embryonic NCSC, and in vitro differentiated embryonic neural crest progeny. We have identified important differentially expressed genes, including novel genes and disease genes. Furthermore, using stringent criteria, we have defined an NCSC molecular signature that consists of a panel of 19 genes and is representative of both EPI-NCSC and NCSC. EPI-NCSC have characteristics that combine advantages of embryonic and adult stem cells. Similar to embryonic stem cells, EPI-NCSC have a high degree of innate plasticity, they can be isolated at high levels of purity, and they can be expanded in vitro. Similar to other types of adult stem cell, EPI-NCSC are readily accessible by minimal invasive procedure. Multipotent adult mammalian stem cells are of great interest because of their potential value in future cell replacement therapy by autologous transplantation, which avoids graft rejection.
Epidermal Neural Crest Stem Cells (EPI-NCSC) and Pluripotency
This article serves three purposes. We summarize current knowledge of the origin and characteristics of EPI-NCSC, review their application in a mouse model of spinal cord injury, and we present new data that highlight aspects of pluripotency of EPI-NCSC. EPI-NCSC are multipotent stem cells, which are derived from the embryonic neural crest and are located in the bulge of hair follicles. EPI-NCSC can undergo self-renewal and they are able to generate all major neural crest derivatives, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. Despite their ectodermal origin, neural crest cells can also generate cell types that typically are derived from mesoderm. We were therefore interested in exploring aspects of EPI-NCSC pluripotency. We here show that EPI-NCSC can fuse with adult skeletal muscle fibers and that incorporated EPI-NCSC nuclei are functional. Furthermore, we show that adult skeletal muscle represents an environment conducive to long-term survival of neurogenic EPI-NCSC. Genes used to create induced pluripotent stem (iPS) cells are present in our EPI-NCSC longSAGE gene expression library. Here we have corroborated this notion by real-time PCR. Our results show similarities in the expression of Myc, Klf4, Sox2 and Lin28 genes between EPI-NCSC and embryonic stem cells (ESC). In contrast there were major differences in Nanog and Pou5f1 (Oct-4) expression levels between EPI-NCSC and ESC, possibly explaining why EPI-NCSC are not tumorigenic. Overall, as embryonic remnants in an adult location EPI-NCSC show several attractive characteristics for future cell replacement therapy and/or biomedical engineering: Due to their ability to migrate, EPI-NCSC can be isolated as a highly pure population of multipotent stem cells by minimally-invasive procedures. The cells can be expanded in vitro into millions of stem cells/progenitors and they share some characteristics with pluripotent stem cells without being tumorigenic. Since the patients' own EPI-NCSC could be used for autologous transplantation, this would avoid graft rejection.
Essential Role of Stem Cell Factor Signaling in Primary Sensory Neuron Development
Here we show that stem cell factor (SCF) signaling through its receptor, c-kit, is essential for the development of c-kit-expressing small- and medium-diameter primary sensory neurons. We used the W mouse, which is c-kit deficient and has a perinatal lethal phenotype due to a naturally occurring point mutation in the c-kit gene. In c-kit-null newborn mice, 52.5% of substance P immunoreactive and 31.4% of calcitonin gene-related peptide (CGRP) immunoreactive small- and medium-diameter sensory neurons were absent, whereas large-diameter sensory neurons were unaffected. Equivalent deficits occurred during embryogenesis. There was neither a developmental delay nor degeneration of differentiated neurons. We thus conclude that, in the absence of SCF signaling, neural crest-derived progenitors do not differentiate into c-kit-expressing visceral and somatic afferent neurons.
Norepinephrine Transport-mediated Gene Expression in Noradrenergic Neurogenesis
We have identified a differential gene expression profile in neural crest stem cells that is due to deletion of the norepinephrine transporter (NET) gene. NET is the target of psychotropic substances, such as tricyclic antidepressants and the drug of abuse, cocaine. NET mutations have been implicated in depression, anxiety, orthostatic intolerance and attention deficit hyperactivity disorder (ADHD). NET function in adult noradrenergic neurons of the peripheral and central nervous systems is to internalize norepinephrine from the synaptic cleft. By contrast, during embryogenesis norepinephrine (NE) transport promotes differentiation of neural crest stem cells and locus ceruleus progenitors into noradrenergic neurons, whereas NET inhibitors block noradrenergic differentiation. While the structure of NET und the regulation of NET function are well described, little is known about downstream target genes of norepinephrine (NE) transport.
Diffusion Heterogeneity Tensor MRI (?-Dti): Mathematics and Initial Applications in Spinal Cord Regeneration After Trauma - Biomed 2009
Diffusion weighted magnetic resonance imaging (DWI) is a powerful tool for evaluation of microstructural anomalies in numerous central nervous system pathologies. Diffusion tensor imaging (DTI) allows for the magnitude and direction of water self diffusion to be estimated by sampling the apparent diffusion coefficient (ADC) in various directions. Clinical DWI and DTI performed at a single level of diffusion weighting, however, does not allow for multiple diffusion compartments to be elicited. Furthermore, assumptions made regarding the precise number of diffusion compartments intrinsic to the tissue of interest have resulted in a lack of consensus between investigations. To overcome these challenges, a stretched-exponential model of diffusion was applied to examine the diffusion coefficient and "heterogeneity index" within highly compartmentalized brain tumors. The purpose of the current study is to expand on the stretched-exponential model of diffusion to include directionality of both diffusion heterogeneity and apparent diffusion coefficient. This study develops the mathematics of this new technique along with an initial application in quantifying spinal cord regeneration following acute injection of epidermal neural crest stem cell (EPI-NCSC) grafts.
Epidermal Neural Crest Stem Cell (EPI-NCSC)--mediated Recovery of Sensory Function in a Mouse Model of Spinal Cord Injury
Here we show that epidermal neural crest stem cell (EPI-NCSC) transplants in the contused spinal cord caused a 24% improvement in sensory connectivity and a substantial recovery of touch perception. Furthermore we present a novel method for the ex vivo expansion of EPI-NCSC into millions of stem cells that takes advantage of the migratory ability of neural crest stem cells and is based on a new culture medium and the use of microcarriers. Functional improvement was shown by two independent methods, spinal somatosensory evoked potentials (SpSEP) and the Semmes-Weinstein touch test. Subsets of transplanted cells differentiated into myelinating oligodendrocytes. Unilateral injections of EPI-NCSC into the lesion of midline contused mouse spinal cords elicited bilateral improvements. Intraspinal EPI-NCSC did not migrate laterally in the spinal cord or invade the spinal roots and dorsal root ganglia, thus implicating diffusible factors. EPI-NCSC expressed neurotrophic factors, angiogenic factors, and metalloproteases. The strength of EPI-NCSC thus is that they can exert a combination of pertinent functions in the contused spinal cord, including cell replacement, neuroprotection, angiogenesis and modulation of scar formation. EPI-NCSC are uniquely qualified for cell-based therapy in spinal cord injury, as neural crest cells and neural tube stem cells share a higher order stem cell and are thus ontologically closely related.
Epidermal Neural Crest Stem Cells and Their Use in Mouse Models of Spinal Cord Injury
Epidermal neural crest stem cell (EPI-NCSC) grafts cause a significant improvement in sensory connectivity and touch perception in the contused mouse spinal cord. EPI-NCSC are derived from the embryonic neural crest but reside in a postnatal location, the bulge of hair follicles. Both mouse and human EPI-NCSC are multipotent adult stem cells capable of generating all major neural crest derivatives. EPI-NCSC of mouse and human origin express the neural crest stem cell molecular signature, genes that were initially used to create induced pluripotent stem (iPS) cells, and other neural crest and global stem cell genes. Due to their origin in the neural folds and because they share a higher order stem cell, neural crest cells, and thus EPI-NCSC, are closely related to neural tube stem cells. This close ontological relationship with the spinal cord makes EPI-NCSC attractive candidates for cell-based therapy in spinal cord injury. In two different contusion models of spinal cord injury, we have shown that EPI-NCSC integrate into the murine spinal cord tissue and that subsets differentiate into GABAergic neurons and myelinating oligodendrocytes. Intraspinal EPI-NCSC do not form tumours. In the presence of EPI-NCSC grafts, but not in control animals, there is a 24% improvement of sensory connectivity and a substantial improvement in touch perception. Unilateral transplants leading to bilateral functional improvements suggest that underlying mechanisms include diffusible molecules. EPI-NCSC indeed express genes that encode neurotrophins, other trophic factors, angiogenic factors and metalloproteases. Intraspinal EPI-NCSC thus have multiple effects in the contused spinal cord, the sum of which can explain the observed functional improvements.
Human Epidermal Neural Crest Stem Cells (hEPI-NCSC)--characterization and Directed Differentiation into Osteocytes and Melanocytes
Here we describe the isolation, characterisation and ex-vivo expansion of human epidermal neural crest stem cells (hEPI-NCSC) and we provide protocols for their directed differentiation into osteocytes and melanocytes. hEPI-NCSC are neural crest-derived multipotent stem cells that persist into adulthood in the bulge of hair follicles. Multipotency and self-renewal were determined by in vitro clonal analyses. hEPI-NCSC generate all major neural crest derivatives, including bone/cartilage cells, neurons, Schwann cells, myofibroblasts and melanocytes. Furthermore, hEPI-NCSC express additional neural crest stem cell markers and global stem cell genes. To variable degrees and in a donor-dependent manner, hEPI-NCSC express the six essential pluripotency genes C-MYC, KLF4, SOX2, LIN28, OCT-4/POU5F1 and NANOG. hEPI-NCSC can be expanded ex vivo into millions of stem cells that remain mulitpotent and continue to express stem cell genes. The novelty of hEPI-NCSC lies in the combination of their highly desirable traits. hEPI-NCSC are embryonic remnants in a postnatal location, the bulge of hair follicles. Therefore they are readily accessible in the hairy skin by minimal invasive procedure. hEPI-NCSC are multipotent somatic stem cells that can be isolated reproducibly and with high yield. By taking advantage of their migratory ability, hEPI-NCSC can be isolated as a highly pure population of stem cells. hEPI-NCSC can undergo robust ex vivo expansion and directed differentiation. As somatic stem cells, hEPI-NCSC are conducive to autologous transplantation, which avoids graft rejection. Together, these traits make hEPI-NCSC novel and attractive candidates for future cell-based therapies and regenerative medicine.
Transition from Cylindroma to Spiradenoma in CYLD-defective Tumours is Associated with Reduced DKK2 Expression
Patients carrying heterozygous germline truncating mutations in the CYLD gene develop multiple primary hair follicle-related tumours. A highly patterned tumour, termed cylindroma, and a highly disorganized tumour, termed spiradenoma, may both develop in the same patient. Furthermore, histological features of both tumour types have been described within the same tumour specimen. We used three-dimensional computer-aided reconstruction of these tumours to demonstrate contiguous growth of cylindromas into spiradenomas, thus suggesting a transition between the two tumour types. To explore factors that may influence cutaneous tumour patterning, genome-wide transcriptomic analysis of 32 CYLD-defective tumours was performed. Overexpression of the Wnt/β-catenin signalling pathway was observed relative to normal perilesional tissue. Morphometric analysis was used to investigate the relationship between Wnt pathway-related gene expression and tumour organization. This revealed an association between reduced Dickkopf 2 (DKK2-a negative regulator of the Wnt/β-catenin signalling pathway) expression and loss of tumour patterning. Reduced DKK2 expression was associated with methylation of the DKK2 gene promoter in the majority of tumour samples assayed. RNA interference-mediated silencing of DKK2 expression in cylindroma primary cell cultures caused an increase in colony formation, cell viability, and anchorage-independent growth. Using these data, we propose a model where epigenetic programming may influence tumour patterning in patients with CYLD mutations.
Epidermal Stem Cell Dynamics
Wong and Reiter have explored the possibility that hair follicle stem cells can give rise to basal cell carcinoma (BCC). They expressed in mice an inducible human BCC-derived oncogenic allele of Smoothened, SmoM2, under the control of either the cytokeratin 14 (K14) or cytokeratin 15 (K15) promoter. Smoothened encodes a G-protein-coupled receptor protein in the hedgehog pathway, the misregulation of which is implicated in BCC and other human cancers. Chronic injury is thought to be a contributing factor. The authors used K14 as a marker for stem cells in the basal layer of the epidermis and K15 as a marker for epidermal stem cells in the bulge of hair follicles. Upon activation, K14 construct-bearing mice readily formed BCC-like tumours, whereas this was not the case in K15:SmoM2-carrying mice. Upon wounding the epidermis, however, there was widespread BCC-like tumour formation in the skin of K15:SmoM2 mice. The authors conclude that wounding recruited bulge epidermal stem cells to the surface, allowing the cells to escape quiescence in the stem cell niche and to arrive in an environment where the hedgehog pathway becomes activated and therefore tumorigenesis is elicited. While this is a provocative result and the authors' conclusion may well be correct, there are alternative explanations.
