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An epidermal neural crest stem cell (EPI-NCSC) molecular signature
Abstract
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.
... A number of laboratories have performed gene-expression analysis on the hair-follicle stem cells, identifying stem-cellrelated gene expression, such as nestin, Nanog, OCT4, Sox-2 as well as differentiation markers 19,34 including human hair follicles stem cells. 35 The gene-expression data are complimentary to the immunohistochemistry data describe above. ...
... There is controversy regarding "pluripotent" vs "multipotent" designations. We have used the term "pluripotent" since numerous cell types and lineages have shown to differentiate from nestin-expressing hair follicle stem cells including adipocytes, smooth muscle cells, melanocytes, neurons, glial cells, epithelial cells, vascular cells, 2,10,17,[34][35][36] osteocytes and chondrocytes, 19,20 and heart muscle cells. [25][26][27] In addition, as briefly mentioned above, the hair follicle cells express transcription factors found in embryonic stem (ES) cells that are considered "pluripotent" transcription factors by some authors. ...
... These transcription factors included OCT4, Nanog, Sox-2, AKP, and TERT. 19,34,35 It should be noted that pioneering authors in the field have termed hair-follicle stem cells "pluripotent." 17 Other authors may prefer the term "multipotent." ...
Various types of stem cells reside in the skin, including keratinocyte progenitor cells, melanocyte progenitor cells, and skin-derived precursors (SKPs), and nestin-expressing hair follicle-associated-pluripotent (HAP) stem cells. HAP stem cells, located in the bulge area of the hair follicle, have been shown to differentiate to nerve cells, glial cells, keratinocytes, smooth muscle cells, cardiac muscle cells, and melanocytes. HAP stem cells are positive forthe stem cell marker CD34, as well as K15-negative, suggestingtheir relatively undifferentiated state and therefore, HAP stem cells may be the most primitive stem cells in the skin. Moreover, HAP stem cells can regenerate the epidermis and at least parts of the hair follicle. These results suggest that HAP stem cells may be the origin of other stem cells in the skin. Transplanted HAP stem cells promote the recovery of peripheral nerve and spinal cord injuries and have the potential for heart regeneration as well. HAP stem cells are readily accessible from everyone, do not form tumors, and can be cryopreserved without loss of differentiation potential. These results suggest that HAP stem cells may have greater potential than iPS or ES cells for regenerative medicine.
... Previous reports have suggested the hippocampal synaptic plasticity improvement following stem cell transplantation in 2VO and myocardial infarction models [11,43]. It has been indicated that EPI-NCSCs could express the vascular endothelial growth factor (VEGF), neurotrophic growth factor (NGF), and brain-derived neurotrophic factor (BDNF), [24,44,45]. BDNF can enhance synaptic transmission [46] and synaptogenesis [47] and improve BST, LTP [32], and synaptic interactions [48]. ...
... Several trophic factors can influence hippocampal neurogenesis including BDNF [69], insulinlike growth factor-I (IGF-I) [70], NGF [71], VEGF, and fibroblast growth factor-2 (FGF-2). EPI-NCSCs-derived growth factors include BDNF, NGF, and VEGF [24,44,45]. ...
This study aimed to evaluate the efficacy and treatment mechanism of platelet-rich plasma (PRP) and neural crest-derived epidermal stem cells (ESCs) in their administration alone and combination in vascular dementia (VaD) model by two-vessel occlusion (2VO). Methods. Sixty-six rats were divided into six groups: the control, sham, 2VO + vehicle, 2VO + PRP, 2VO + ESC, and 2VO + ESC + PRP. The treated groups received 1 million cells on days 4, 14, and 21 with or without 500 µl PRP (twice a week) after 2VO. The memory performance and anxiety were evaluated by behavioral tests including open field, passive avoidance, and Morris water maze. The basal-synaptic transmission (BST) and long-term potentiation (LTP) were assessed through field-potential recordings of the CA1. The mRNA expression levels of IGF-1, TGF-β1, PSD-95, and GSk-3β were measured in the rat hippocampus by quantitative reverse transcription polymerase chain reaction. Results. The results demonstrated impaired learning, memory, and synaptic plasticity in the 2VO rats, along with a significant decrease in the expression of IGF-1, TGF-β1, PSD-95, and upregulation of GSK-3β. Treatment with ESC alone and ESC + PRP showed similar improvements in spatial memory and LTP induction, with associated upregulation of PSD-95 and downregulation of GSK-3β. However, only the ESC + PRP group showed recovery in BST. Furthermore, combination therapy was more effective than PRP monotherapy for LTP and memory. Conclusions. The transplantation of ESC showed better effects than PRP alone, and combination therapy increased the treatment efficacy with the recovery of BST. This finding may be a clue for the combination therapy of ESC and PRP for VaD.
... The proliferation of neuronal progenitor in the hippocampus is retained throughout life (Eriksson et al., 1998;Kuhn et al., 1996). Previous studies demonstrated that the EPI-NCSCs could express the Neurotrophic growth factor (NGF), BDNF, and Vascular endothelial growth factor (VEGF) (Hu et al., 2006;Pandamooz et al., 2019;Salehi et al., 2020). Hippocampal neurogenesis can be influenced by several trophic factors including Insulin-like growth factor-I (IGF-I) (Åberg et al., 2000), NGF (Cuello and Bruno, 2007), VEGF, Fibroblast growth factor-2 (FGF-2), and BDNF (Zigova et al., 1998). ...
... Therefore, the high level of neurotrophic factors in the brain following transplantation could be the main cause of the LTP recovery. Furthermore, a variety of neurotrophic factors are expressed by EPI-NCSCs (Hu et al., 2006;Pandamooz et al., 2019;Salehi et al., 2020). Salehi et al. in 2020 also found that 7 days after EPI-NCSCs implantation in the ischemic stroke model the expression levels of BDNF, Sox10, nestin, doublecortin, β-III tubulin, GFAP, and interleukin-6 were increased, whereas neurotrophin-3 and interleukin-10 decreased (Salehi et al., 2020) BDNF with an important neuroprotective effect plays a key role in the regulation of synaptogenesis (Gonzalez et al., 2016), long-term increases in synaptic transmission, and long-term potentiation in the hippocampus (Gomez-Palacio Schjetnan and Escobar-Rodriguez, 2007). ...
Introduction
The incidence rate of senile dementia is rising, and there is no definite cure for it yet. Cell therapy, as a new investigational approach, has shown promising results. Hair bulges with abundant easily accessible neural stem cells permit autologous implantation in irreversible neurodegenerative disorders.
Methods
Fifty rats were randomly divided into 5 groups of control, sham-operation, two-common carotid vessel-occlusion rats that received vehicle (2VO + V), 2VO rats that received 1×10⁶ epidermal stem cells (2VO + ESC1), and 2VO rats that received 2.5×10⁶ epidermal stem cells (2VO + ESC2) in 300µl PBS intravenously on days 4, 9, and 14 after surgery. The epidermal neural crest stem cells (EPI‐NCSCs) were isolated from hair follicles of rat whiskers. The open-field, passive avoidance, and Morris water maze were used as behavioral tests. The basal-synaptic transmission, long-term potentiation (LTP), and short-term synaptic plasticity were evaluated by field-potential recording of the CA1 hippocampal area.
Results
30 days after the first transplantation in the 2VO + ESC1 group, functional recovery was prominent in anxiety and fear memory compared to the 2VO + ESC2 group, while LTP induction was recovered in both groups of grafted animals without improvement in basal synaptic transmission. These positive recoveries may be related to the release of different neurotrophic factors from grafted cells that can stimulate endogenous neurogenesis and synaptic plasticity.
Conclusions
Our results showed that EPI-NCSCs implantation could rescue LTP and cognitive disability in 2VO rats, while transplantation of 1 million cells showed better performance relative to 2.5 million cells.
... While SKPs were reported to express Nestin, SNAIL, SLUG, TWIST and SOX9 (Toma et al., 2001;Fernandes et al., 2004), EPI-NCSCs were shown to lack expression of Slug, Snail and Twist. On the contrary, EPI-NCSCs were shown to display expression of Msx2 and SOX10 Hu et al., 2006), emphasizing the transcriptional heterogeneity of NCSCs even between bulk populations located close to each other. Interestingly, an assessment of the adult murine epidermis on single level by Joost et al. (2016) suggested that self-renewing cells in the adult hair follicle lack a distinct stemness gene expression signature. ...
... These findings suggest a great cellular plasticity of single LGR5-positive hair follicle bulge stem cells allowing rapid transcriptional adaptations during wound healing (Joost et al., 2018). Accordingly, EPI-NCSCs were also described to show great cellular plasticity Hu et al., 2006;Hu et al., 2010) but no reports addressing their intrapopulational heterogeneity are available so far. Single SKPs from human foreskin were shown to give rise to mesenchymal and ectodermal cell types like neurons or smooth muscle cells (Toma et al., 2001), although Dai et al. (2018) suggested SKP-spheres to contain a heterogeneous mixture of stem and progenitor cells. ...
Stem cells of the neural crest (NC) vitally participate to embryonic development, but also remain in distinct niches as quiescent neural crest-derived stem cell (NCSC) pools into adulthood. Although NCSC-populations share a high capacity for self-renewal and differentiation resulting in promising preclinical applications within the last two decades, inter- and intrapopulational differences exist in terms of their expression signatures and regenerative capability. Differentiation and self-renewal of stem cells in developmental and regenerative contexts are partially regulated by the niche or culture condition and further influenced by single cell decision processes, making cell-to-cell variation and heterogeneity critical for understanding adult stem cell populations. The present review summarizes current knowledge of the cellular heterogeneity within NCSC-populations located in distinct craniofacial and trunk niches including the nasal cavity, olfactory bulb, oral tissues or skin. We shed light on the impact of intrapopulational heterogeneity on fate specifications and plasticity of NCSCs in their niches in vivo as well as during in vitro culture. We further discuss underlying molecular regulators determining fate specifications of NCSCs, suggesting a regulatory network including NF-κB and NC-related transcription factors like SLUG and SOX9 accompanied by Wnt- and MAPK-signaling to orchestrate NCSC stemness and differentiation. In summary, adult NCSCs show a broad heterogeneity on the level of the donor and the donors’ sex, the cell population and the single stem cell directly impacting their differentiation capability and fate choices in vivo and in vitro. The findings discussed here emphasize heterogeneity of NCSCs as a crucial parameter for understanding their role in tissue homeostasis and regeneration and for improving their applicability in regenerative medicine.
... Recent reports have demonstrated that NCSC are present not only in the neural crest in the embryo but also in their target tissues in fetal and adult tissues; human epidermal NCSC are multipotent remnants in a postnatal location. For example, NCSC can be found in the bulge of hair follicles where they persist postnatally and into adulthood [8][9][10]. In addition, many reports have identified the presence of stem cells in NCSC-derived dental tissues such as dental follicle, apical papilla, pulp, exfoliated deciduous teeth, and PDL, and numerous in vitro and in vivo studies [16-22, 24-45, 49-69]. ...
... The presence of NCSC was demonstrated not only in the embryo but also in the adult tissues [8][9][10], and our group investigated the presence of NCSC in the adult PDL tissue using two immortalized clonal PDL cell lines with multipotency [63]. One of these, cell line 1-11, had the capacity to differentiate into osteoblasts and adipocyte, whereas the other clone, cell line 1-17, could differentiate into osteoblasts, chondrocytes, adipocytes, and neural cell. ...
The ultimate goal of periodontal treatment is not only the stabilization of periodontal disease but also regeneration of the periodontal tissues destroyed by severe periodontitis. The tissue engineering triad of stem cells, biological signals, and cell-seeded scaffold provides a biological template for tissue regeneration. Neural crest stem cells (NCSC) comprise a transient multipotent stem cell population that initially emerges from the junction of the epidermal and neural ectoderm through reciprocal interactions between these tissues and signals from the mesoderm during embryonic development of vertebrates. The cells then migrate to their destination in the embryo and differentiate into various dental mesenchymal stem cells which give rise to most components of the dental tissues, including periodontal ligament (PDL), alveolar bone, cementum, dental pulp, and dentin. The crucial role that NCSC play in embryonic development makes them a highly promising stem cell population for use in the regeneration of periodontal tissues. Here, we review the current understanding of the features and functions of NCSC-derived cells in dental tissues. We additionally discuss the possibility that NCSC contribute to the regeneration of periodontal tissues.
... Neural crest cells become migratory and translocate away from the forming neural tube to various locations within the embryo, where they give rise to a wide array of cell types and tissues, including the Schwann cells of the peripheral nervous system (Le Douarin and Kalcheim, 1999). A subset of multipotent embryonic neural crest cells invade the ectoderm early in development (Richardson and Sieber-Blum, 1993;Narytnyk et al., 2014a), some of which become located in a stem cell niche of the hair follicle, the 'bulge', where they persist postnatally and into adulthood Hu et al., 2006;Clewes et al., 2011;Sieber-Blum, 2014). Another type of neural crest-derived skin progenitor cell is a Schwann cell precursor that arrives in the skin via projecting nerves and gives rise to melanocytes (Adameyko et al., 2009). ...
... Furthermore, hEPI-NCSC-derived Schwann cells express neurotrophins and other factors essential for nerve regeneration. Similar to mouse EPI-NCSC (mEPI-NCSC; GEO accession number GSE4680; Hu et al., 2006;Sieber-Blum et al., 2006) andcEPI-NCSC (McMahill et al., 2014;McMahill et al., 2015), hEPI-NCSC and Schwann cells derived therefrom express the VEGFA and VEGFB genes (GEO accession number GSE61273). This is an important aspect, as angiogenesis is crucial for nerve repair (Kolar and Kingham, 2014). ...
We show that highly pure populations of human Schwann cells can be derived rapidly and in a straightforward way, without the need for genetic manipulation, from human epidermal neural crest stem cells [hEPI-NCSC(s)] present in the bulge of hair follicles. These human Schwann cells promise to be a useful tool for cell-based therapies, disease modelling and drug discovery. Schwann cells are glia that support axons of peripheral nerves and are direct descendants of the embryonic neural crest. Peripheral nerves are damaged in various conditions, including through trauma or tumour-related surgery, and Schwann cells are required for their repair and regeneration. Schwann cells also promise to be useful for treating spinal cord injuries. Ex vivo expansion of hEPI-NCSC isolated from hair bulge explants, manipulating the WNT, sonic hedgehog and TGFβ signalling pathways, and exposure of the cells to pertinent growth factors led to the expression of the Schwann cell markers SOX10, KROX20 (EGR2), p75NTR (NGFR), MBP and S100B by day 4 in virtually all cells, and maturation was completed by 2 weeks of differentiation. Gene expression profiling demonstrated expression of transcripts for neurotrophic and angiogenic factors, as well as JUN, all of which are essential for nerve regeneration. Co-culture of hEPI-NCSC-derived human Schwann cells with rodent dorsal root ganglia showed interaction of the Schwann cells with axons, providing evidence of Schwann cell functionality. We conclude that hEPI-NCSCs are a biologically relevant source for generating large and highly pure populations of human Schwann cells.
© 2015. Published by The Company of Biologists Ltd.
... Stammzellen besitzen nach Filip (Filip et al., 2004) Talgdrüsen und aller Zelltypen des Haarfollikels regenerieren zu können (Cotsarelis et al., 1990;Lyle et al., 1998;Oshima et al., 2001;Liu et al., 2003;Trempus et al., 2003;Blanpain et al., 2004;Ito et al., 2004;Tumbar et al., 2004;Morris et al., 2004;Amoh et al., 2005;Roh et al., 2005;Hu et al., 2006;Yu et al., 2006;Zhang et al., 2006). Dagegen wurde berichtet, dass sich auf ein Signal hin aus der dermalen Papille die Zellen der interfollikulären Epidermis (IFE) in Haarfollikel und Talgdrüsen differenzieren können (Niemann und Watt, 2002 (Hunt et al., 2008). ...
... Zellen war eine schwache Expression des α6-Integrins (CD49f) detektierbar; (β1und α2-Integrine, sowie der zusätzlich getestete Transferrin-Rezeptor CD71, waren nicht nachweisbar. CD71 wird als Marker proliferativ aktiver Zellen beschrieben (Li et al., 1998;Fathke et al., 2004 Grinnell, 2004;Hu et al., 2006;Lorenz et al., 2008;Aberdam et al., 2008). Merkelzellen der Epidermis exprimieren CK18, CK19 und CK20 (Eispert et al., 2009 Neuralleistenstammzellen (Widera et al., 2009). ...
... After hair bulge explantation, EPI-NCSCs with stellate morphology, characteristic of neural crest stem cells, started emigration from the bulge explants 3-4 days later, as mentioned in earlier studies PMID: 19,066,583. This emigration happens because the bulge area is a niche for EPI-NCSCs [27]. It is important to note that no specific migration test was conducted in this study; rather, the mention of migration pertains to the natural migratory behavior of EPI-NCSCs observed during the culture process. ...
Aims
Epidermal Neural Crest Stem Cells (EPI-NCSCs) have emerged as prospective ideal candidates to meet the fundamental requirements of cell-based therapies in neurodegenerative disorders. The present study aimed to identify the potential of metformin in driving EPI-NCSCs to neuronal/glial differentiation and express neurotrophic factors as well as assess their therapeutic potential for mitigating the main behavioral manifestations of chemotherapy-induced neurotoxicity (CIN).
Main Methods
EPI-NCSCs were extracted from the bulge region of hair follicle. Following expansion, transcript and protein expression profiles of key markers for stemness (Nestin, EGR-1, SOX-2 and 10), neurotrophic activity (BDNF, GDNF, NGF, FGF-2, and IL-6), and neuronal (TUB3, DCX, NRF and NeuN) and glial (PDGFRα, NG2, GFAP, and MBP) differentiation were determined on days 1 and 7 post-treatment with 10 and 100 μM metformin using real time-PCR and immunocytochemistry methods. Then, the in vivo function of metformin-treated stem cells was evaluated in the context of paclitaxel CIN. To do so, thermal hyperalgesia, mechanical allodynia, and spatial learning and memory tests were evaluated by Hotplate, Von Frey, and Morris water maze tests.
Key Findings
Our result indicated that exposure of EPI-NCSCs to metformin was associated with progressive decline in stemness markers and enhanced expression levels of several neurotrophic, neuron and oligodendrocyte-specific markers. Further, it was observed that intranasal metformin-treated EPI-NCSCs improved the cognitive impairment, and mechanical and thermal hypersensitivity induced by paclitaxel in rats.
Significance
Collectively, we reasoned that metformin pretreatment of EPI-NCSCs might further enhance their therapeutic benefits against CIN.
Graphical Abstract
... Since 2013, our research group has been studying the characteristics and regenerative potential of hair follicle-derived stem cells (HFSCs) of rats and human [13][14][15][16][17][18]. These multipotent stem cells are located in the bulge region of hair follicles throughout adulthood, are ontologically related to the nervous system and show a high level of physiological plasticity [19]. In the current study, we investigated the expression of oxytocin receptor (OXTR) in HFSCs migrated from the bulge region of rat hair follicles. ...
The intricate nature of the human brain and the limitations of existing model systems to study molecular and cellular causes of neuropsychiatric disorders represent a major challenge for basic research. The promising progress in patient-derived stem cell technology and in our knowledge on the role of the brain oxytocin (OXT) system in health and disease offer new possibilities in that direction. In this study, the rat hair follicle stem cells (HFSCs) were isolated and expanded in vitro. The expression of oxytocin receptors (OXTR) was evaluated in these cells. The cellular viability was assessed 12 h post stimulation with OXT. The activation of OXTR-coupled intracellular signaling cascades, following OXT treatment was determined. Also, the influence of OXT on neurite outgrowth and cytoskeletal rearrangement were defined. The assessment of OXTR protein expression revealed this receptor is expressed abundantly in HFSCs. As evidenced by the cell viability assay, no adverse or cytotoxic effects were detected following 12 h treatment with different concentrations of OXT. Moreover, OXTR stimulation by OXT resulted in ERK1/2, CREB, and eEF2 activation, neurite length alterations, and cytoskeletal rearrangements that reveal the functionality of this receptor in HFSCs. Here, we introduced the rat HFSCs as an easy-to-obtain stem cell model that express functional OXTR. This cell-based model can contribute to our understanding of the progression and treatment of neuropsychiatric disorders with oxytocinergic system deficiency.
Graphical Abstract
... Previous reports have suggested the hippocampal synaptic plasticity improvement following stem cell transplantation in 2VO and myocardial infarction models 11,31 . It has been indicated that EPI-NCSCs could express the vascular endothelial growth factor (VEGF), Neurotrophic growth factor (NGF), and Brain-derived neurotrophic factor (BDNF), [32][33][34] . BDNF can enhance synaptic transmission 35 , and synaptogenesis 36 , and improve BST, LTP, 20 , and synaptic interactions 37 . ...
This study aimed to evaluate effects of platelet-rich plasma (PRP) and neural crest-derived epidermal stem cells (ESC) in alone administration and their combination in vascular dementia (VaD) model by tow-vessels occlusion (2VO).
Methods
the rats were divided into control, sham, 2VO + vehicle, 2VO + PRP, 2VO + ESC, and 2VO + ESC + PRP groups. The treated-groups received one million cells on days 4, 14, and 21 with or without 500µl PRP (twice a week). The behavioral performance was evaluated by open-field, passive-avoidance, and Morris-water maze. The basal-synaptic transmission (BST) and long-term potentiation (LTP) were assessed by field-potential recording. The mRNA expression levels of IGF-1, TGF-β1, PSD-95, and GSk-3β were measured in rat hippocampus by qRT-PCR.
Results
The results showed a learning, memory, and synaptic plasticity impairment with a significant decrease in IGF1, TGF-β1, PSD-95, and increase in expression of GSk-3β 2VO rats. Although treatment with ESC alone and ESC + PRP show comparable improvement effects on spatial memory and LTP induction associated with PSD-95 up-regulation and GSK-3β down-regulation, the BST only recovered in the ESC + PRP group. Moreover, combination therapy was superior to monotherapy with PRP.
Conclusions
The transplantation of ESC showed better effects than PRP alone, and combination therapy increased treatment efficacy with the recovery of BST.
... CEE is a complex cocktail of growth factors that has been widely used to supplement the growth media of various cell types, such as neural crest stem cells (NCSCs). [34][35][36] In the present study, CEE was collected and used to determine whether its presence can stimulate migration of stem cells from the hair bulge, proliferation rate of migrated stem cells, and alter their morphology. Also, the influence of CEE treatment on expression of key cell surface markers, differentiation into osteoblast and adipocyte as well as colony-forming efficiency were assessed. ...
The beneficial effects of hair follicle stem cells in different animal models of nervous system conditions have been extensively studied. While chick embryo extract (CEE) has been used as a growth medium supplement for these stem cells, this is the first study to show the effect of CEE on them. The rat hair follicle stem cells were isolated and supplemented with 10% fetal bovine serum plus 10% CEE. The migration rate, proliferative capacity and multipotency were evaluated along with morphometric alteration and differentiation direction. The proteome analysis of CEE content identified effective factors of CEE that probably regulate fate and function of stem cells. The CEE enhances the migration rate of stem cells from explanted bulges as well as their proliferation, likely due to activation of AP-1 and translationally controlled tumour protein (TCTP) by thioredoxin found in CEE. The increased length of outgrowth may be the result of cyclic AMP response element binding protein (CREB) phosphorylation triggered by active CamKII contained in CEE. Further, CEE supplementation upregulates the expression of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor. The elevated expression of target genes and proteins may be due to CREB, AP-1 and c-Myc activation in these stem cells. Given the increased transcript levels of neurotrophins, VEGF, and the expression of PDGFR-α, S100B, MBP and SOX-10 protein, it is possible that CEE promotes the fate of these stem cells towards Schwann cells.
... Although all of these factors are initially important for the innervation of dental tissues, they can play fundamental roles in the restoration of neural tissues. Similarly, HFSCs express a variety of trophic factors such as insulin-like growth factor, fibroblast growth factor, transforming growth factor, VEGF, NT-3, NGF, GDNF, and BDNF [20,21,57,58]. To the best of our knowledge, no study has compared the secretome of DPSCs and HFSCs. ...
During the last 20 years, stem cell therapy has been considered as an effective approach for regenerative medicine. Due to poor ability of stem cells to survive following transplantation, it has been proposed that beneficial effects of stem cells mainly depend on paracrine function. Therefore, the present study was designed to reinforce mesenchymal stem cells (MSCs) to express higher levels of trophic factors especially the ones with the neurotrophic properties. Here, bone marrow (BM)-MSCs and adipose-MSCs were treated with conditioned medium (CM) of dental pulp stem cells (DPSCs) or hair follicle stem cells (HFSCs) for up to three days. The relative expression of five key trophic factors that have critical effects on the central nervous system regeneration were evaluated using qRT-PCR technique. Furthermore, to assess the impacts of conditioned mediums on the fate of MSCs, expression of seven neuronal/glial markers were evaluated 3 days after the treatments. The obtained data revealed priming of BM-MSCs with HFSC-CM or DPSC-CM increases the BDNF expression over time. Such effect was also observed in adipose-MSCs following DPSC-CM treatment. Secretome preconditioning remarkably increased NGF expression in the adipose-MSCs. In addition, although priming of adipose-MSCs with HFSC-CM increased GDNF expression one day after the treatment, DPSC-CM enhanced GDNF mRNA in BM-MSCs at a later time point. It seemed priming of BM-MSCs with HFSC-CM, promoted differentiation into the glial lineage. Our findings showed that MSCs preconditioning with secretome of neural crest-derived stem cells could be a promising approach to enhance the neurotrophic potential of these stem cells.
... EPI-NCSCs have an ectodermic origin, separated from the neural crest during embryonic development.They can generate all major neural crest derivatives, such as neurons, nerve-supporting cells, melanocytes, and smooth muscle cells. EPI-NCSCs provide a combination of embryonic and adult stem cells benefit (10,11). These cells exhibit a high level of plasticity and are easily expanded into millions of cells in vitro.EPI-NCSCs have also no immunological incompatibility and their isolation method is non-invasive (8). ...
Neurodegenerative diseases are incurable and debilitating conditions that result in progressive degeneration of nerve cells. Due to the complexity of conditions in neurodegenerative diseases, combination therapy, including cell and drug therapy is important as a new therapeutic strategy. Epidermal neural crest stem cells (EPI-NCSCs) are among the best choices in cell therapy for various neurological diseases. In this study, the effect of Lithium carbonate and Crocin, considering their effects on cellular signaling pathways and neuroprotective properties were investigated on the expression of neurotrophic factors BDNF and GDNF in EPI-NCSCs. EPI-NCSCs were isolated from the hair follicle and treated with different concentrations of drugs [Lithium, Crocin, and lithium + Crocin] for 72h. Then, trial concentrations were selected by MTT assay. The cells were treated with selected concentrations (Lithium 1 mM, Crocin 1.5 mM, and for co-treatment Lithium 1 mM and Crocin 1 mM) for 7 days. The Real-Time PCR results indicated an increasing in expression of BDNF and GDNF in treated cells as compared with control (* p < 0.05, ** p < 0.01 and *** p < 0.001). The results in this study confirmed and supported the neuroprotective/neurogenesis effects of Lithium and Crocin. It also showed that the proposed protocol could be used to increase EPI-NCSCs differentiation potential into neural cells in cell therapy and combination therapy of neurodegenerative diseases.
... Epidermal neural crest stem cells (EPI-NCSCs) are adult stem cells preserving their neural crest origin neurologic differentiation potentials. Due to their unique properties, including multipotency, physiological plasticity, easily accessible autologous source (the bulge of hair follicles), no graft rejection, and lack of tumorigenicity, they are considered precious cellular source for cell-based therapy purposes [13,14]. ...
Schwann cells (SCs) are considered potentially attractive candidates for transplantation therapies in neurodegenerative diseases. However, problems arising from the isolation and expansion of the SCs restrict their clinical applications. Establishing an alternative Schwann-like cell type is a prerequisite. Epidermal neural crest stem cells (EPI-NCSCs) are well studied for their autologous accessibility, along with the ability to produce major neural crest derivatives and neurotrophic factors. In the current study, we explored insulin influence, a well-known growth factor, on directing EPI-NCSCs into the Schwann cell (SC) lineage. EPI-NCSCs were isolated from rat hair bulge explants. The viability of cells treated with a range of insulin concentrations (0.05–100 μg/ml) was defined by MTT assay at 24, 48, and 72 h. The gene expression profiles of neurotrophic factors (BDNF, FGF-2, and IL-6), key regulators involved in the development of SC (EGR-1, SOX-10, c-JUN, GFAP, OCT-6, EGR-2, and MBP), and oligodendrocyte (PDGFR-α and NG-2) were quantified 1 and 9 days post-treatment with 0.05 and 5 μg/ml insulin. Furthermore, the protein expression of nestin (stemness marker), SOX-10, PDGFR-α, and MBP was analyzed following the long-term insulin treatment. Insulin downregulated the early-stage SC differentiation marker (EGR-1) and increased neurotrophins (BDNF and IL-6) and pro-myelinating genes, including OCT-6, SOX-10, EGR-2, and MBP, as well as oligodendrocyte differentiation markers, upon exposure for 9 days. Insulin can promote EPI-NCSC differentiation toward SC lineage and possibly oligodendrocytes. Thus, employing insulin might enhance the EPI-NCSCs efficiency in cell transplantation strategies.
Graphical abstract
... Our research group has been studying the regenerative potential of epidermal neural crest stem cells (EPI-NCSCs) derived from hair follicles of rat and human since 2013 [3,4]. These multipotent stem cells are located in the bulge region of hair follicles throughout adulthood ( Figure 1A), are ontologically related to the nervous system and present a high level of physiological plasticity [5]. Recently, we showed the therapeutic effect of transplanted EPI-NCSCs in a rat model of ischemic stroke, most likely through the simultaneous induction of neuronal and glial formation, as well as Bdnf overexpression. ...
In the recent review published in Brain Sciences, Othman and Tan suggested several
preconditioning strategies to improve stem cell therapy after ischemic brain injury [1].
They explored the regeneration efficacy of neural stem cells and introduced methods to
optimize cell-based therapies. Due to some limitations related to the usage of neural
stem cells, application of other pretreated cell types can also be beneficial for the ischemic
brain [2]. Our research group has been studying the regenerative potential of epidermal
neural crest stem cells (EPI-NCSCs) derived from hair follicles of rat and human since
2013 [3,4]. These multipotent stem cells are located in the bulge region of hair follicles
throughout adulthood (Figure 1A), are ontologically related to the nervous system and
present a high level of physiological plasticity [5]. Recently, we showed the therapeutic
effect of transplanted EPI-NCSCs in a rat model of ischemic stroke, most likely through the
simultaneous induction of neuronal and glial formation, as well as Bdnf overexpression.
In this study, administration of EPI-NCSCs via intra-arterial or intravenous routes immediately
after reperfusion, resulted in reduced infarct size seven days post transplantation
(Figure 1B) [6]. Our previous findings also demonstrated that cerebrospinal fluid can
be a practical route for the delivery of EPI-NCSCs [3]. Currently, our research group is
focusing on the intranasal administration of EPI-NCSCs and their conditioned medium in
the context of stroke. The preliminary data revealed that intranasal application of rat and
human derived EPI-NCSCs and their secretome could alleviate the devastating condition
following cerebral ischemia.
... Our research group has been studying the regenerative potential of epidermal neural crest stem cells (EPI-NCSCs) derived from hair follicles of rat and human since 2013 [3,4]. These multipotent stem cells are located in the bulge region of hair follicles throughout adulthood ( Figure 1A), are ontologically related to the nervous system and present a high level of physiological plasticity [5]. Recently, we showed the therapeutic effect of transplanted EPI-NCSCs in a rat model of ischemic stroke, most likely through the simultaneous induction of neuronal and glial formation, as well as Bdnf overexpression. ...
In the recent review published in Brain Sciences, Othman and Tan suggested several preconditioning strategies to improve stem cell therapy after ischemic brain injury [...]
... Many other subpopulations of NCSCs also persist in late gestation, with some even detected at postnatal stages, including adulthood [30,31]. Such postnatal NCSCs have been detected in both mice and humans (Figure 1), within a wide variety of niches such as in the skin [32,33], bowel [34], dorsal root ganglia [35], heart [36], carotid body [37], bone marrow [38], eyes [39,40], teeth [41][42][43], palate [44], and oral mucosa [45]. ...
Neurocristopathies form a heterogeneous group of rare diseases caused by abnormal development of neural crest cells. Heterogeneity of neurocristopathies directly relates to the nature of these migratory and multipotent cells, which generate dozens of specialized cell types throughout the body. Neurocristopathies are thus characterized by congenital malformations of tissues/organs that otherwise appear to have very little in common, such as the craniofacial skeleton and enteric nervous system. Treatment options are currently very limited, mainly consisting of corrective surgeries. Yet, as reviewed here, analyses of normal and pathological neural crest development in model organisms have opened up the possibility for better treatment options involving cellular and molecular approaches. These approaches provide hope that some neurocristopathies might soon be curable or preventable.
(https://authors.elsevier.com/a/1cdGn5Eb1BS5BR)
... More recently, we showed that these cells are localized to the bulge region of hair follicles. 118 These findings are consistent with data from animal model studies, 119 such as multipotent nestin-positive cells or neural crest epidermal stem cells isolated from the mouse bulge area. Therefore, it is clear that there are several potential stem cell populations in hair follicles, with the capacity to give rise to melanocytes that are potential targets for melanomagenesis. ...
Recent advances in molecular genetics and cancer stem cell biology have shed some light on the molecular basis of melanomagenesis. In this review, we will focus on major genetic alterations in the melanoma, particularly pathways involved in cell proliferation, apoptosis, and tumor suppression. The potential role of melanoma-initiating cells during melanomagenesis and progression will also be discussed. Understanding pathogenesis of melanoma may uncover new diagnostic clues and therapeutic targets for this increasingly prevalent disease.
... Although NCSC and NCLSC appear to share common characteristics, such as multipotency, NC marker expression (eg, Sox10, p75 NTR , AP-2, and Nestin) and pluripotent gene expression, and a molecular signature representative of EPI-NCSC and embryonic NCSC has been defined, 202 there also are some apparent differences. For instance, long SAGEtranscriptome profiling revealed that human NCSCs exhibited a unique NC molecular signature, expressed pluripotent genes (Nanog, POU5F1. ...
Neural crest stem cells (NCSCs) are a transient population of cells that arise during early vertebrate development and harbor stem cell properties, such as self-renewal and multipotency. These cells form at the interface of non-neuronal ectoderm and neural tube and undergo extensive migration whereupon they contribute to a diverse array of cell and tissue derivatives, ranging from craniofacial tissues to cells of the peripheral nervous system. Neural crest-like stem cells (NCLSCs) can be derived from pluripotent stem cells, placental tissues, adult tissues, and somatic cell reprogramming. NCLSCs have a differentiation capability similar to NCSCs, and possess great potential for regenerative medicine applications. In this review, we present recent developments on the various approaches to derive NCLSCs and the therapeutic application of these cells for tissue regeneration.
... Previous investigations on these stem cells showed a high level of physiological plasticity when cultured in presence of different growth factors (Clewes et al., 2011;Narytnyk et al., 2014;Sieber-Blum & Grim, 2004). It has been reported that EPI-NCSCs can differentiate into various cell types, including different neural lineages, chondrocytes, and osteocytes (Hu, Zhang, & Sieber-Blum, 2006). Remarkably, these adult multipotent stem cells are relatively abundant and accessible in the bulge of hairy skin and they can be isolated through the minimally invasive procedure. ...
According to the intrinsic plasticity of stem cells, controlling their fate is a critical issue in cell‐based therapies. Recently, a growing body of evidence has suggested that substrate stiffness can affect the fate decisions of various stem cells. Epidermal neural crest stem cells as one of the main neural crest cell derivatives hold great promise for cell therapies due to presenting a high level of plasticity. This study was conducted to define the influence of substrate stiffness on the lineage commitment of these cells. Here, four different polyacrylamide hydrogels with elastic modulus in the range of 0.7–30 kPa were synthesized and coated with collagen and stem cells were seeded on them for 24 hr. The obtained data showed that cells can attach faster to hydrogels compared with culture plate and cells on <1 kPa stiffness show more neuronal‐like morphology as they presented several branches and extended longer neurites over time. Moreover, the transcription of actin downregulated on all hydrogels, while the expression of Nestin, Tubulin, and PDGFR‐α increased on all of them and SOX‐10 and doublecortin gene expression were higher only on <1 kPa. Also, it was revealed that soft hydrogels can enhance the expression of glial cell line‐derived neurotrophic factor, neurotrophin‐3, and vascular endothelial growth factor in these stem cells. On the basis of the results, these cells can respond to the substrate stiffness in the short term culture and soft hydrogels can alter their morphology and gene expression. These findings suggested that employing proper substrate stiffness might result in cells with more natural profiles similar to the nervous system and superior usefulness in therapeutic applications.
... Heart muscle repair with adult stem cells rise to nervous tissue(47). Stem cells are under active consideration as a source of donor tissues for neuronal cell therapy for Parkinson's disease(48), Huntington's disease(49), spinal cord injury ...
... Although, hair follicle is taken as an epidermal stem cell source, Àespecially the bulge regionemerge as a key source for the regeneration of both epidermal and dermal cell populations (Cheng et al. 2018). It was demonstrated that the hair bulge contains stem cells of neural origin such as the human epidermal neural crest stem cells (EPI-NCSCs), most likely by the epidermal ORS being in strong association with DS of the hair bulge (Vapniarsky et al. 2015;Hu et al. 2006). These cells display multipotency covering major neural derivatives such as the bone, cartilage, neurons, Schwann cells, myofibroblasts, and melanocytes (Vapniarsky et al. 2015). ...
Maintaining integrity of the skin and its appendages still preserves its top-ranking in priorities of survival for the modern human as it probably once did for the ancient individual, −not only- because it is the primary barrier to external assaults, but also because of social and psychological impact of healthy skin during their life-span. Healing wounds in order to shield off the internal organs from infections and damage, restoring its ability to adapt to various environmental stimuli, and slowing-down and reversing aging of the skin in the quest for an everlasting youth can be named as a few of the main drivers behind the multi-million investments dedicated to the advancement of our understanding of skin’s physiology. Over the years, these tremendous efforts culminated in the breakthrough discovery of skin stem cells the regenerative capacity of which accounted for the resilience of the skin through their unique capacity as a special cell type that can both self-renew and differentiate into various lineages. In this review, first we summarize the current knowledge on this amazing organ both at a structural and functional level. Next, we provide a comprehensive -in depth- discussion on epidermal as well as dermal stem cells in terms of the key regulatory pathways as well as the main genetic factors that have been implicated in the orchestration of the skin stem cell biology in regards to the shifts between quiescence and entry into distinct differentiation programs.
... Previous investigations on these stem cells showed a high level of physiological plasticity when cultured in presence of different growth factors (Clewes et al., 2011;Narytnyk et al., 2014;Sieber-Blum & Grim, 2004). It has been reported that EPI-NCSCs can differentiate into various cell types, including different neural lineages, chondrocytes, and osteocytes (Hu, Zhang, & Sieber-Blum, 2006). Remarkably, these adult multipotent stem cells are relatively abundant and accessible in the bulge of hairy skin and they can be isolated through the minimally invasive procedure. ...
According to the intrinsic plasticity of the stem cells, controlling their fate is a critical issue in cell‐based therapies. Recently, a growing body of evidence has suggested that substrate stiffness can affect fate decisions of various stem cells. Epidermal neural crest stem cells as one of the main neural crest cell derivatives hold great promise for cell therapies due to presenting high level of plasticity. This study was conducted to define the influence of substrate stiffness on lineage commitment of these cells. Here, four different polyacrylamide hydrogels with elastic modules in the range of 0.2‐10 kPa were synthetized and coated with collagen and stem cells were seeded on them for 24 h. The obtained data showed that cells can attach faster to hydrogels compared to culture plate and cells on <1 kPa stiffness show more neuronal‐like morphology as they presented several branches and extended longer neurites over time. Moreover, the transcription of actin downregulated on all hydrogels, while the expression of Nestin, Tubulin and PDGFR‐α increased on all of them and SOX‐10 and DCX gene expression was higher only on <1 kPa. Also, it was revealed that soft hydrogels can enhance the expression of GDNF, NT‐3 and VEGF in these stem cells. Based on the results, these cells can respond to the substrate stiffness in the short term culture and soft hydrogels can alter their morphology and gene expression. These findings suggested that employing proper substrate stiffness might result in cells with more natural profile similar to nervous system and superior usefulness in therapeutic applications. This article is protected by copyright. All rights reserved.
... Seed cells, as an important component of NTE, provide an optional strategy for NTE with an enhanced ability to repair PNI. Attractively, EPI-NCSCs, combined with the advantage of adult stem cell and embryonic stem cell, represent a unusual type of multipotent adult stem cell and is a suitable candidate for cell-based therapy ( Hu et al., 2006;Sieber-Blum et al., 2006). Accumulating evidences ( Amoh et al., 2005) suggest that EPI-NCSCs can be induced to differentiate into Schwann cells and might secrete growth factors to modulate the behavior of Schwann cells. ...
Cell-based therapy is an alternative strategy to improve outcomes of peripheral nerve injury (PNI). Epidermal neural crest stem cell (EPI-NCSC) is obtained from autologous tissue without immunological rejection, which could expand quickly in vitro and is suitable candidate for cell-based therapy. Olfactory ensheathing cell (OEC) could secrete multiple neurotrophic factors (NTFs), which is often used to repair PNI individually. However, whether the combination of EPI-NCSC and OEC have better effects on PNI repair remains unclear. Here we use EPI-NCSC and OEC co-transplantation in a rat sciatic nerve defect model to ascertain the effects and potential mechanisms of cells co-transplantation on PNI. The effect of EPI-NCSC and OEC co-transplantation on PNI is assessed by using a combination of immunohistochemistry (IHC), electrophysiological recording and neural function test. Co-transplantation of EPI-NCSC and OEC exerts a beneficial effect upon PNI such as better organized structure, nerve function recovery, and lower motoneuron apoptosis. IHC and enzyme-linked immuno sorbent assay (ELISA) further demonstrate that cells co-transplantation may improve PNI via the expression of brain derived growth factor (BDNF) and nerve growth factor (NGF) up-regulated by EPI-NCSC and OEC synergistically. Eventually, the results from this study reveal that EPI-NCSC and OEC co-transplantation effectively repairs PNI through enhancing the level of BDNF and NGF, indicating that cells co-transplantation may serve as a fruitful avenue for PNI in clinic treatment.
... Scientific REPORTS | (2019) 9:597 | DOI:10.1038/s41598-018-37160-y neural crest cells 57 and has been reported to be a signature gene of epidermal neural crest stem cells 58 . However, aside from white belly spots, Myo10 tm2/tm2 mice did not exhibit major neural crest-related disorders, such as craniofacial defects or megacolon (intestinal aganglionosis) 59 . ...
We investigated the physiological functions of Myo10 (myosin X) using Myo10 reporter knockout (Myo10tm2) mice. Full-length (motorized) Myo10 protein was deleted, but the brain-specific headless (Hdl) isoform (Hdl-Myo10) was still expressed in homozygous mutants. In vitro, we confirmed that Hdl-Myo10 does not induce filopodia, but it strongly localized to the plasma membrane independent of the MyTH4-FERM domain. Filopodia-inducing Myo10 is implicated in axon guidance and mice lacking the Myo10 cargo protein DCC (deleted in colorectal cancer) have severe commissural defects, whereas MRI (magnetic resonance imaging) of isolated brains revealed intact commissures in Myo10tm2/tm2 mice. However, reminiscent of Waardenburg syndrome, a neural crest disorder, Myo10tm2/tm2 mice exhibited pigmentation defects (white belly spots) and simple syndactyly with high penetrance (>95%), and 24% of mutant embryos developed exencephalus, a neural tube closure defect. Furthermore, Myo10tm2/tm2 mice consistently displayed bilateral persistence of the hyaloid vasculature, revealed by MRI and retinal whole-mount preparations. In principle, impaired tissue clearance could contribute to persistence of hyaloid vasculature and syndactyly. However, Myo10-deficient macrophages exhibited no defects in the phagocytosis of apoptotic or IgG-opsonized cells. RNA sequence analysis showed that Myo10 was the most strongly expressed unconventional myosin in retinal vascular endothelial cells and expression levels increased 4-fold between P6 and P15, when vertical sprouting angiogenesis gives rise to deeper layers. Nevertheless, imaging of isolated adult mutant retinas did not reveal vascularization defects. In summary, Myo10 is important for both prenatal (neural tube closure and digit formation) and postnatal development (hyaloid regression, but not retinal vascularization).
... Intermixed with epidermal stem cells are neural crest derived multipotent stem cells, as we have described in the mouse [32], in the dog [33] and with human tissue [27]. The neural crest origin was first determined in the WNT1-cre/R26R mouse in which all neural crest derived cells are indelibly marked by their expression of beta-galactosidase [32] and by LongSAGE gene expression profiling [34]. The neural crest origin of canine and human EPI-NCSC was determined by gene expression profiling and quantitative polymerase gene reaction (qPCR) [27,33]. ...
Here we provide new technology for generating human peptidergic nociceptive sensory neurons in a straightforward and efficient way. The cellular source, human epidermal neural crest stem cells (hEPI-NCSC), consists of multipotent somatic stem cells that reside in the bulge of hair follicles. hEPI-NCSC and primary sensory neurons have a common origin, the embryonic neural crest. For directed differentiation, hEPI-NCSC were exposed to pertinent growth factors and small molecules in order to modulate master signalling networks involved in differentiation of neural crest cells into postmitotic peptidergic sensory neurons during embryonic development. The neuronal populations were homogenous in regard to antibody marker expression. Cells were immunoreactive for essential master regulatory genes, including NGN1/2, SOX10, and BRN3a among others, and for the pain-mediating genes substance P (SP), calcitonin gene related protein (CGRP) and the TRPV1 channel. Approximately 30% of total cells responded to capsaicin, indicating that they expressed an active TRPV1 channel. In summary, hEPI-NCSC are a biologically relevant and easily available source of somatic stem cells for generating human peptidergic nociceptive neurons without the need for genetic manipulation and cell purification. As no analgesics exist that specifically target TRPV1, a ready supply of high-quality human peptidergic nociceptive sensory neurons could open the way for new approaches, in a biologically relevant cellular context, to drug discovery and patient-specific disease modelling that is aimed at pain control, and as such is highly desirable.
... In such a model, the fate of NCSC was mapped in vivo by mating ROSA26 Cre reporter (R26R) mice, expressing beta-galactosidase upon Cre-mediated recombination, with mice expressing Cre recombinase under the control of the Wnt1 promoter. Using this transgenic model (or a similar one), the presence of multipotent NC-derived cells was confirmed in adult hair follicles [10], adult skin [11][12][13][14][15][16], adult bone marrow [17,18], the dental pulp [19][20][21], the cornea [22], the olfactory epithelium [23] and more recently in the adipose tissue [24][25][26]. ...
Adult neural crest stem-derived cells (NCSC) are of extraordinary high plasticity and promising candidates for use in regenerative medicine. Several locations such as skin, adipose tissue, dental pulp or bone marrow have been described in rodent, as sources of NCSC. However, very little information is available concerning their correspondence in human tissues, and more precisely for human bone marrow. The main objective of this study was therefore to characterize NCSC from adult human bone marrow. In this purpose, we compared human bone marrow stromal cells to human adipose tissue and dermis, already described for containing NCSC. We performed comparative analyses in terms of gene and protein expression as well as functional characterizations. It appeared that human bone marrow, similarly to adipose tissue and dermis, contains NESTIN⁺ / SOX9⁺ / TWIST⁺ / SLUG⁺ / P75NTR+/ BRN3A⁺/ MSI1⁺/ SNAIL1⁺ cells and were able to differentiate into melanocytes, Schwann cells and neurons. Moreover, when injected into chicken embryos, all those cells were able to migrate and follow endogenous neural crest migration pathways. Altogether, the phenotypic characterization and migration abilities strongly suggest the presence of neural crest-derived cells in human adult bone marrow.
... It has been reported previously that EPI-NCSCs can directly or indirectly influence expression and secretion of numerous neurotrophic factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), fibroblast growth factors (FGF), insulin-like growth factor-1 (IGF-1; Hu et al., 2010), transforming growth factor-β (TGF-β) and their cognate receptors (Ren et al., 2001;Hu et al., 2006). Recently, studies have focused on the potential of EPI-NCSCs to provide neuroprotection for damaged neurons, stimulate local axonal sprouting, and induce or facilitate neovascularization (Kocsis, 2009). ...
Cell-based therapy is a promising strategy for the repair of peripheral nerve injuries (PNIs). epidermal neural crest stems cells (EPI-NCSCs) are thought to be important donor cells for repairing PNI in different animal models. Following PNI, inflammatory response is important to regulate the repair process. However, the effects of EPI-NCSCs on regulation of local inflammation microenviroment have not been investigated extensively. In the present study, these effects were studied by using 10 mm defected sciatic nerve, which was bridged with 15 mm artificial nerve composed of EPI-NCSCs, extracellular matrix (ECM) and poly (lactide-co-glycolide) (PLGA). Then the expression of pro- and anti-inflammatory cytokines, polarization of macrophages, regulation of fibroblasts and shwann cells (SCs) were assessed by western blot, immunohistochemistry, immunofluorescence staining at 1, 3, 7 and 21 days after bridging. The structure and the function of the bridged nerve were determined by observation under light microscope and by examination of right lateral foot retraction time (LFRT), sciatic function index (SFI), gastrocnemius wet weight and electrophysiology at 9 weeks. After bridging with EPI-NCSCs, the expression of anti-inflammatory cytokines (IL-4 and IL-13) was increased, but decreased for pro-inflammatory cytokines (IL-6 and TNF-α) compared to the control bridging, which was consistent with increase of M2 macrophages and decrease of M1 macrophages at 7 days after transplantation. Likewise, myelin-formed SCs were significantly increased, but decreased for the activated fibroblasts in their number at 21 days. The recovery of structure and function of nerve bridged with EPI-NCSCs was significantly superior to that of DMEM. These results indicated that EPI-NCSCs could be able to regulate and provide more suitable inflammation microenvironment for the repair of defected sciatic nerve.
... Other genes are involved in neural/neural-crest related diseases (Thop1, Adam12, Ube4b, and Vars2) (Baertling et al., 2016;Caren et al., 2006;Pollio et al., 2008;Shao et al., 2014), regulation of Wnt signaling pathways (Pygo2) (Belenkaya et al., 2002), and invasiveness (Crmp1, Adam12) (Cai et al., 2016;Shao et al., 2014). Interestingly, a detailed comparison of gene expression revealed epidermal NCSCs did not express any NC marker genes (Snail1/2, Twist, Pax3, Sox9, nexin, Nestin, fibronectin, Wnt5a, Sca-1, Shox2, and Dermo-1) in SKPs (Fernandes et al., 2004;Hu et al., 2006), suggesting at least two molecularly distinct populations of NCSCs are located in the hair follicle. ...
... Other genes are involved in neural/neural-crest related diseases (Thop1, Adam12, Ube4b, and Vars2) (Baertling et al., 2016;Caren et al., 2006;Pollio et al., 2008;Shao et al., 2014), regulation of Wnt signaling pathways (Pygo2) (Belenkaya et al., 2002), and invasiveness (Crmp1, Adam12) (Cai et al., 2016;Shao et al., 2014). Interestingly, a detailed comparison of gene expression revealed epidermal NCSCs did not express any NC marker genes (Snail1/2, Twist, Pax3, Sox9, nexin, Nestin, fibronectin, Wnt5a, Sca-1, Shox2, and Dermo-1) in SKPs (Fernandes et al., 2004;Hu et al., 2006), suggesting at least two molecularly distinct populations of NCSCs are located in the hair follicle. ...
... Differentiation potential of NFs and AFs. Both NF as well as AF RNA profiles were consistent with the molecular signature of epidermal neural crest stem cells (26) (Fig. 1). However, these two cell types significantly differed in the differentiation potential. ...
Clinical evidence suggests that healing is faster and almost scarless at an early neonatal age in comparison with that in adults. In this study, the phenotypes of neonatal and adult dermal fibroblasts and keratinocytes (nestin, smooth muscle actin, keratin types 8, 14 and 19, and fibronectin) were compared. Furthermore, functional assays (proliferation, migration, scratch wound closure) including mutual epithelial‑mesenchymal interactions were also performed to complete the series of experiments. Positivity for nestin and α smooth muscle actin was higher in neonatal fibroblasts (NFs) when compared with their adult counterparts (adult fibroblasts; AFs). Although the proliferation of NFs and AFs was similar, they significantly differed in their migration potential. The keratinocyte experiments revealed small, poorly differentiated cells (positive for keratins 8, 14 and 19) in primary cultures isolated from neonatal tissues. Moreover, the neonatal keratinocytes exhibited significantly faster rates of healing the experimentally induced in vitro defects in comparison with adult cells. Notably, the epithelial/mesenchymal interaction studies showed that NFs in co-culture with adult keratinocytes significantly stimulated the adult epithelial cells to acquire the phenotype of small, non-confluent cells expressing markers of poor differentiation. These results indicate the important differences between neonatal and adult cells that may be associated with improved wound healing during the early neonatal period.
... Unlike NPCs which are localized within the CNS and hard to isolate, neural crest stem cells can also be isolated from various adult tissues, making them an accessible stem cell population to isolate and use in therapy. These cells have been successfully isolated from the sciatic nerve, intestines, skin, cornea, heart, dental pulp, the carotid body, the dorsal root ganglion, and bone marrow (Arthur, Rychkov, Shi, Koblar, & Gronthos, 2008;Hu, Zhang, & Sieber-Blum, 2006;Kruger et al., 2002;Morikawa et al., 2009;Morrison, White, Zock, & Anderson, 1999;Pardal, Ortega-Saenz, Duran, & Lopez-Barneo, 2007;Toma, McKenzie, Bagli, & Miller, 2005;Tomita et al., 2005;Widera et al., 2009;Yoshida et al., 2006). ...
Multiple sclerosis (MS) is predominantly considered an immune-mediated chronic disease, with complicated pathogenesis and clinical signs of paralysis. MS is an inflammatory disease of the central nervous system in which T lymphocytes show a second phase of activation. This activation is followed by axonal demyelination and neurological disorder manifestations. In most patients, the disease is associated with axonal loss and progressive disabilities.
... Our findings show that the migratory cells from bulge area did not express CD34. Although in one study the expression of this marker was negative in collagen-coated plates (Hu et al. 2006), there were a number of published data regarding expression of CD34 in HAP stem cells (Amoh et al. 2005b;Liu et al. 2011), and demands further studies. ...
An embryo has the capability to accept allo- or xeno-geneic cells, which probably makes it an ideal candidate for stem cell transplantation of various cerebral cortex abnormalities, such as cortical dysplasia. The aim of this study was to determine hair follicle-associated pluripotent (HAP) stem cells homing into various organs of mother and fetus. Cells were obtained, analyzed for immunophenotypic features, and then labelled with CM-Dil; nestin(+)HAP stem cells or media phosphate-buffered saline (PBS) were intravenously delivered on day 16 of gestation in BALB/c mice, which intraperitoneally received methylazoxymethanol (MAM) one day in advance, and homing was assessed at 24 h after cell injection. Flow cytometry and immunocytochemistry manifested positive expression of nestin in HAP stem cells. For both mother and fetus, brain, lungs, liver, and spleen were the host organs for cell implants. For the brain, the figure was considerably higher in fetus, 4.05 ± 0.5% (p ≤ 0.05 vs. mother). MAM-injected mice had a downward trend for SDF-1α and CXCR4 (p ≤ 0.05 vs. control), but HAP stem cells group showed an upward trend for CXCR4 (p ≤ 0.05 vs. MAM). We conclude the HAP stem cells show homing potential in experimental cortical dysplasia, which may permit these cells to be a target in future work on prenatal therapy of neural disorders.
... Various groups have performed extensive work focusing on isolation, characterization, and in vitro propagation of DSCs [2][3][4][5][6]. Excitingly, it has been demonstrated that DSCs have potential to differentiate not only along mesenchymal lineages [2][3][4][5][6] but also along the ectodermal [4,7] and endodermal lineages [8]. Differentiation toward these lineages, however, has been shown mostly at the individual cell level or, at most, on small clusters of cells. ...
Significance:
Autologous dermis-derived stem cells are generating great excitement and efforts in the field of regenerative medicine and tissue engineering. The substantial impact of this review lies in its critical coverage of the available literature and in providing insight regarding niches, characteristics, and isolation methods of stem cells derived from the human dermis. Furthermore, it provides analysis of the current state-of-the-art regenerative approaches using human-derived dermal stem cells, with consideration of current guidelines, to assist translation toward therapeutic use.
... Walking scale, limb coordination, and plantar stepping improved following HFSC transplantation [8] . Consistent with this study, Hu et al [50] , also demonstrated that unilateral bulge-derived Epi-NCSC transplantation into a contused spinal cord promoted a 24% recovery in sensory connectivity and touch perception. Moreover, some grafted Epi-NCSCs differentiated into functional motor neurons. ...
Conflict-of-interest: The authors declare that there are no conflicts of interest. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. Abstract Hair follicle stem cells (HFSCs) normally give rise to keratinocytes, sebocytes, and transient amplifying progenitor cells. Along with the capacity to proliferate rapidly, HFSCs provide the basis for establishing a putative source of stem cells for cell therapy. HFSCs are multipotent stem cells originating from the bulge area. The importance of these cells arises from two important characteristics, distinguishing them from all other adult stem cells. First, they are accessible and proliferate for long periods. Second, they are multipotent, possessing the ability to differentiate into mesodermal and ectodermal cell types. In addition to a developmental capacity in vitro , HFSCs display an ability to form differentiated cells in vivo. During the last two decades, numerous studies have led to the development of an appropriate culture condition for producing various cell lineages from HFSCs. Therefore, these stem cells are considered as a novel source for cell therapy of a broad spectrum of neurodegenerative disorders. This review presents the current status of human, rat, and mouse HFSCs from both the cellular and molecular biology and cell therapy perspectives. The first section of this review highlights the importance of HFSCs and in vitro differentiation, while the final section emphasizes the significance of cell differentiation in vivo .
... [3] Under specific differentiation conditions, NCSCs generates S100+ melanocytes, neurofilament+ neuron-like cells, smooth muscle actin+ smooth muscle cells, adipocytes, osteocytes, and chondrocytes. [8] Stem Cells in Vitiligo Re-pigmentation Evolutionarily, follicular and epidermal melanocytes are closely related with epidermal melanocytes evolving from the follicular ones. [9] Hair follicle melanocytes play an important role in re-pigmentation of vitiliginous lesion. ...
Skin is an easily accessible source of various sub population of stem cells including epidermal stem cells, hair follicle stem cells (HFSCs) and dermal mesenchymal stem cells. The outer root sheath (ORS) of the hair follicle is a rich source of a type of HFSCs called the melanocytes stem cells (MelSCs). These HFSCs have a vast, unexplored potential in the treatment of vitiligo as initial re-pigmentation often occurs around the hair follicles. Common therapeutic modalities such as tacrolimus, phototherapy and dermabrasion acts through MelSCs. Newer cellular techniques have explored the use of ORS hair follicle suspension in surgical treatment of vitiligo. Advancement in melanocyte and stem cell research has identified various cytokines, growth factors and regulators involved in proliferation and differentiation of melanoblasts, which can be used for autologous in situ melanocyte regeneration. In this review, we briefly discuss the current position and future prospects of stem cells in vitiligo.
... To date, the embryonic CNS is considered to be the main source of NSCs. NSCs derived from the embryonic neural tube present a unique advantage, as they have the same origin as the cells of the SCI lesion (13)(14)(15). Increasing the number of NSCs can be achieved via the expansion of NSCs in vitro, of which there are two methods that are currently used: Neurosphere culture (2) and adherent culture (16). ...
The aim of the present study was to investigate the therapeutic effect of a combined treatment of Buyang Huanwu decoction (BYHWD), a well-known formula of traditional Chinese medicine, and neural stem cells (NSCs) on spinal cord injury (SCI) and the associated underlying mechanisms. A SCI model was established by surgery via a complete transection of the T10 vertebra of female Sprague-Dawley rats. Gelatin sponges were used to absorb NSCs labeled with the thymidine analog, 5-bromo-2-deoxyuridine (BrdU), and were transferred into the transected spinal cords. BYHWD was administered once a day by introgastric infusion. Motor functions of the hind limbs were evaluated using the 21-point locomotor rating scale developed by Basso, Beattie and Bresnahan (BBB). The fate of the transplanted NSCs under the various conditions was examined by double immunofluorescence staining, using markers for neurons, astrocytes and oligodendrocytes, with BrdU. Ultrastructural changes of the SCI site following the various treatments were examined under a transmission electron microscope. The number of double positive cells for glial fibrillary acidic protein and BrdU in the BYHWD + NSC group was significantly decreased when compared with that in the NSC group (P<0.05). However, the number of cells that were labeled double positive for myelin basic protein and BrdU, as well as neuron specific enolase and BrdU, was greater in the BYHWD + NSC group when compared with the NSC group. Electron microscopy demonstrated that treatment with BYHWD combined with NSCs significantly alleviated demyelination. Results from the BBB motor function test exhibited a significant improvement in the BYHWD + NSC group when compared with the SCI, BYHWD and NSC only groups. In conclusion, the results demonstrated that the traditional Chinese medicine formula, BYHWD, exerted an effect on the differentiation and migration of NSCs. Combining the administration of BYHWD with NSCs was shown to have a synergistic effect on the recovery of neurological function, mitigating the progress of demyelination or ameliorating the recovery of myelination.
... Heart muscle repair with adult stem cells rise to nervous tissue(47). Stem cells are under active consideration as a source of donor tissues for neuronal cell therapy for Parkinson's disease(48), Huntington's disease(49), spinal cord injury(45), stroke (50) and multiple sclerosis(51). ...
ABSTRACT: The new millennium promises to guide in the age of human genome. So far, a different area of biology, the stem cell biology – has captured both scientific and international news headlines. Stem cells are generally very early stage cells that have the ability to differentiate into other specialized types of cells. Stem cells hoist the prospect of regenerating failed body parts and curing diseases that have so far defined drug – based therapy. This review attempts to give an overview of stem cell biology and scientific factors surrounding it.
Keywords: Embryonic stem cells; Adult stem cells; Stem
cell markers; Hematopoietic stem cells
Hair analysis is a reliable and widely used tool to evaluate drug exposure in many fields, including workplace testing, drug abuse history and withdrawal control, post-mortem toxicology, doping control, therapeutic drug monitoring of pharmaceuticals and even environmental exposure to toxic agents. Compounds incorporated into the hair structure resist hair growth and regular washing for several months, leading to a potential chronological trace of exposure, with farther periods corresponding to the hair segments more distant from the hair root. The relentless improvement of analytical procedures and instrumental technologies, together with the continuous introduction of new psychoactive substances, have led to an increasing number of studies and practical applications of hair analysis.
This book is a comprehensive guide to hair analysis from general concepts, ideal for students and those new to the field, to interpretation and advanced methods for experts working in the area. With contributions from world-leading scientists in each field, this book describes state-of-the-art, emerging issues and recent analytical approaches to hair analysis that will serve as an essential tool to clinical and forensic toxicology laboratories across the globe.
Following nerve tissue damage, various events, such as inflammatory responses, microglial activation, endoplasmic reticulum stress, and apoptosis, can occur, which all lead to cell death, prevent axonal growth, and cause axonal circumvolution. So far, several researchers have tended to adopt strategies to reduce the harmful conditions associated with neurological disorders, and stem‑cell‑based therapy is one of those promising strategies. Epidermal neural crest stem cells (EPI‑NCSCs) are a type of stem cell that has widely been employed for the treatment of various neurological disorders. It has been suggested that these stem cells perform their supportive actions primarily through the release of different neurotrophic factors. Hence, in this study, the neuroprotective impacts of valproic acid (VPA) and crocin were evaluated on the mRNA expression levels of brain‑derived neurotrophic factor (BDNF) and glial‑cell‑derived neurotrophic factor (GDNF) in EPI‑NCSCs. In this research, we isolated EPI‑NCSCs from the hair follicles of the rat whisker pad. Then, the cells were treated with different concentrations of VPA and crocin for 72 h. Subsequently, an MTT assay was performed to define the suitable concentrations of drugs. Finally, real‑time PCR was performed to evaluate the mRNA expression levels of BDNF and GDNF in these stem cells. The results of the MTT assay showed that the treatment of EPI‑NCSCs with 1 mM VPA and 1.5 mM crocin, and the co‑treatment with 1 mM VPA and 500 μM crocin, led to the survival and proliferation of these stem cells. Moreover, the real‑time PCR results revealed that both VPA and crocin, both individually and in combination, can significantly increase the expression levels of BDNF and GDNF in EPI‑NCSCs. According to the findings of this study, both VPA and crocin, alone and in combination, are potential candidates for enhancing the capacity of EPI‑NCSCs to differentiate into neural lineages. Therefore, the co‑treatment of EPI‑NCSCs with these drugs can be employed for the treatment of various neurological disorders, such as spinal cord injury.
Hair follicle stem cells (HFSCs) are able to differentiate into neurons and glial cells. Distinct microRNAs (miRNAs) regulate the proliferation and differentiation of HFSCs. However, the exact role of miR‐124 in the neural differentiation of HFSCs has not been elucidated. HFSCs were isolated from mouse whisker follicles. miR‐9, let‐7b, and miR‐124, Ptbp1 , and Sox9 expression levels were detected by real‐time polymerase chain reaction (RT‐PCR). The influence of miR‐124 transfection was evaluated using immunostaining. We demonstrated that miR‐124 and let‐7b expression levels were significantly increased after the neural differentiation. Sox9 and Ptbp1 were identified as the target of miR‐124 in the HFSCs. During neural differentiation and miR‐124 mimicking, Ptbp1 and Sox9 levels were decreased. Moreover, the miR‐124 overexpression increased MAP2 (58.43 ± 11.26) and NeuN (48.34 ± 11.15) proteins expression. The results demonstrated that miR‐124 may promote the differentiation of HFSCs into neuronal cells by targeting Sox9 and Ptbp1.
Outcomes following peripheral nerve injury remain poor despite the regenerative capacity displayed by the peripheral nervous system. Current therapies are limited and do not provide satisfactory functional recovery in a multitude of cases. Biomaterials have decreased the need for nerve autograft across small nerve gaps in small-caliber nerves, but the lack of a cellular substrate presents a limiting factor to the effectiveness of this therapy. Schwann cells are the supportive cells in the peripheral nervous system and play an integral role in the physiological response and regeneration following nerve injury. Limitations to autologous Schwann cells include donor site morbidity during harvesting, limited expansion capability, and finite source. Stem cells are multipotent or pluripotent cells with self-renewing capabilities that show promise to improve functional recovery following nerve injury. Differentiation of stem cells into supportive Schwann cells could provide additional trophic support without the disadvantages of autologous Schwann cells, providing an avenue to improve existing therapies. A variety of stem cells have been evaluated in animal models for this clinical application; the current options, along with their clinical feasibility, are summarized in this article.
Significance:
It has been established that mouse and human epidermal neural crest stem cells are somatic multipotent stem cells with proved innovative potential in a mouse model of spinal cord injury (SCI) offering promise of a valid treatment for SCI. Traumatic SCI is a common neurological problem in dogs with marked similarities, clinically and pathologically, to the syndrome in people. For this reason, dogs provide a readily accessible, clinically realistic, spontaneous model for evaluation of epidermal neural crest stem cells therapeutic intervention. The results of this study are expected to give the baseline data for a future clinical trial in dogs with traumatic SCI.
Neural crest cells (NCC) are migratory multipotent cells that give rise to diverse derivatives. They generate various cell types during embryonic development, including neurons and glial cells of the peripheral sensory and autonomic ganglia, Schwann cells, melanocytes, endocrine cells, smooth muscle, and skeletal and connective tissue cells of the craniofacial complex. The multipotency of NCC is thought to be transient at the early stage of NCC generation; once NCC emerge from the neural tube, they change into lineage-restricted precursors. Although many studies have described the clear segregation of NCC lineages right after their delamination from the neural tube, recent reports suggest that multipotent neural crest stem cells (NCSC) are present not only in migrating NCC in the embryo, but also in their target tissues in the fetus and adult. Furthermore, fully differentiated NCC-derived cells such as glial cells and melanocytes have been shown to dedifferentiate or transdifferentiate into other NCC derivatives. The multipotency of migratory and postmigratory NCC-derived cells was found to be similar to that of NCSC. Collectively, these findings support the multipotency or plasticity of NCC and NCC-derived cells.
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Background & objectives: During lifetime, hair follicles undergo three cyclic changes:
anagen, catagen, telogen. In hair follicles, stem cells located in Bulge area, which is part of
the outer root sheath. Bulge cells proliferate new cells in anagen phase. Bulge region of hair
follicle indicated as a source of stem cell for many years, but little studies done in vitro to
characterize rat bulge region hair follicle stem cells.
Methods: In this study Bulge cells of rat hair follicle were isolated and cultured, then
morphological features of these cells surveyed. Nestin and CD34 as hair follicle stem cell
markers, and K15 as a keratinocyte marker assessed by immunocytochemistry after one to
three weeks.
Results: In this study, we found that, 2 days after attachment of cells to floor of plates, the
cells were initiated to proliferation and migration. These cells had good nestin and CD34
immunostaining, but after three week differentiation,were nestin and CD34 negative. Also
these cells couldn’t express K15.
Conclusion: Results showed that cultivated rat bulge cells, have high proliferative potential,
and also could express nestin and CD34 as stem cell factors.
Key words: Nestin, CD34; K15; Bulge Region; Hair Follicle; Rat
A culture method for neural crest cells of mouse embryo is described. Trunk neural tubes were dissected from 9-day mouse embryos and explanted in culture dishes. The developmental potential of mouse neural crest in vitro was shown to be essentially similar to that of avian neural crest. In the mouse, however, melanocytes always appeared in association with the epithelial sheet close to the explant. Neural crest cells surrounding the epithelial sheet, which probably migrated from the neural tubes in the early culture phase, never differentiated into melanocytes. The bimodal behaviour of mouse crest cells seems to be due to the heterogenous potency of the crest cells and the interaction of these cells with the surrounding microenvironment. This culture system is well suited for various experiments including the analysis of gene control on the differentiation of neural crest cells.
The characteristics of an organism are determined by the genes expressed within it. A method was developed, called serial
analysis of gene expression (SAGE), that allows the quantitative and simultaneous analysis of a large number of transcripts.
To demonstrate this strategy, short diagnostic sequence tags were isolated from pancreas, concatenated, and cloned. Manual
sequencing of 1000 tags revealed a gene expression pattern characteristic of pancreatic function. New pancreatic transcripts
corresponding to novel tags were identified. SAGE should provide a broadly applicable means for the quantitative cataloging
and comparison of expressed genes in a variety of normal, developmental, and disease states.
The protooncogene Wnt-1 encodes a short-range signal which is first expressed in, and appears to demarcate, the presumptive midbrain. Absence of Wnt-1 expression leads to the loss of this region of the brain. By the end of neural tube closure, expression of Wnt-1 extends down much of the dorsal midline of the central nervous system (CNS). Expression is exclusively limited to the CNS at this and later stages. We have investigated the regulation of Wnt-1 during mouse development. Analysis of the embryonic expression of Wnt-1-lacZ reporter constructs spanning nearly 30 kb of the Wnt-1 locus identified a 5.5 kb cis-acting 3' enhancer element which confers correct temporal and spatial expression on the lacZ gene. Interestingly embryos express Wnt-1-lacZ transgenes in migrating neural crest cells which are derived from the dorsal CNS. Ectopic expression of the Wnt-1-lacZ transgenes may result from perdurance of beta-galactosidase activity in migrating neural crest cells originating from a Wnt-1-expressing region of the dorsal CNS. Alternatively, ectopic expression may arise from transient de novo activation of the transgenes in this cell population. These results are a first step towards addressing how regional cell signaling is established in the mammalian CNS. In addition, transgene expression provides a new tool for the analysis of neural crest development in normal and mutant mouse embryos.
This article reviews recent molecular genetic findings in autosomal dominant craniosynostotic syndromes. A mutation in the homeotic gene MSX2 was the first genetic defect identified in an autosomal dominant primary craniosynostosis, i.e. in craniosynostosis type 2 (Boston type). In the more common syndromes of Crouzon, Pfeiffer, Jackson-Weiss, and Apert, mutations were found in the gene coding for fibroblast growth factor receptor (FGFR) 2. Less frequently, mutations are observed in FGFR1 and FGFR3 in some cases of Crouzon and Pfeiffer syndrome. The mutations identified in FGFR2 are located in exons 5 and 7 of the gene that code for immunoglobulin (Ig)-like chain III and the region linking Ig II and Ig III of the receptor. These domains of the receptor are important for ligand binding. Apart from Apert syndrome, identical mutations are found in the clinically distinct syndromes of Crouzon, Pfeiffer, and Jackson-Weiss. Furthermore, the same gene defect can result in a highly variable phenotype even within one family. Therefore, the clinically distinct craniosynostotic syndromes are extremes of a spectrum of craniofacial abnormalities and not nosologic entities. In Saethre-Chotzen syndrome, the gene coding for transcription factor TWIST is mutated. The disease genes identified in craniosynostotic syndromes to date either regulate transcription or are required for signal transduction and play a central role in the development of the calvarial sutures.
Stem cells are vital for the homeostasis of self-renewing tissues such as the hair follicle. Epithelial stem cells have been implicated in tumorigenesis and wound healing, and their manipulation may have wide ranging applications including gene therapy and tissue transplantation. Rodent hair follicle stem cells have been localized to an area of the follicle called the bulge, however, the identification and characterization of human hair follicle stem cells has been hampered by a lack of cellular markers for this area. We have determined that the C8/144B monoclonal antibody, originally generated against a short intracytoplasmic peptide of CD8, preferentially immunostains hair follicle bulge keratinocytes without staining the remaining hair follicle. Using expression cloning, we identified cytokeratin 15 as the keratinocyte protein recognized by the C8/144B monoclonal antibody. By delineating the bulge using this antibody, we demonstrated that bulge cells possess a stem cell phenotype characterized by their slowly-cycling nature, preferential proliferation at the onset of new hair follicle growth, high level of beta1 integrin expression, and expression of cytokeratin 19.
Calreticulin is a ubiquitous Ca2+ binding protein, located in the endoplasmic reticulum lumen, which has been implicated in many diverse functions including: regulation of intracellular Ca2+ homeostasis, chaperone activity, steroid-mediated gene regulation, and cell adhesion. To understand the physiological function of calreticulin we used gene targeting to create a knockout mouse for calreticulin. Mice homozygous for the calreticulin gene disruption developed omphalocele (failure of absorption of the umbilical hernia) and showed a marked decrease in ventricular wall thickness and deep intertrabecular recesses in the ventricular walls. Transgenic mice expressing a green fluorescent protein reporter gene under the control of the calreticulin promoter were used to show that the calreticulin gene is highly activated in the cardiovascular system during the early stages of cardiac development. Calreticulin protein is also highly expressed in the developing heart, but it is only a minor component of the mature heart. Bradykinin-induced Ca2+ release by the InsP3-dependent pathway was inhibited in crt-/- cells, suggesting that calreticulin plays a role in Ca2+ homeostasis. Calreticulin-deficient cells also exhibited impaired nuclear import of nuclear factor of activated T cell (NF-AT3) transcription factor indicating that calreticulin plays a role in cardiac development as a component of the Ca2+/calcineurin/NF-AT/GATA-4 transcription pathway.
The composite structure of the mammalian skull, which forms predominantly via intramembranous ossification, requires precise pre- and post-natal growth regulation of individual calvarial elements. Disturbances of this process frequently cause severe clinical manifestations in humans. Enhanced DNA binding by a mutant MSX2 homeodomain results in a gain of function and produces craniosynostosis in humans. Here we show that Msx2-deficient mice have defects of skull ossification and persistent calvarial foramen. This phenotype results from defective proliferation of osteoprogenitors at the osteogenic front during calvarial morphogenesis, and closely resembles that associated with human MSX2 haploinsufficiency in parietal foramina (PFM). Msx2-/- mice also have defects in endochondral bone formation. In the axial and appendicular skeleton, post-natal deficits in Pth/Pthrp receptor (Pthr) signalling and in expression of marker genes for bone differentiation indicate that Msx2 is required for both chondrogenesis and osteogenesis. Consistent with phenotypes associated with PFM, Msx2-mutant mice also display defective tooth, hair follicle and mammary gland development, and seizures, the latter accompanied by abnormal development of the cerebellum. Most Msx2-mutant phenotypes, including calvarial defects, are enhanced by genetic combination with Msx1 loss of function, indicating that Msx gene dosage can modify expression of the PFM phenotype. Our results provide a developmental basis for PFM and demonstrate that Msx2 is essential at multiple sites during organogenesis.
The neural crest plays a crucial part in cardiac development. Cells of the cardiac subpopulation of cranial neural crest migrate from the hindbrain into the outflow tract of the heart where they contribute to the septum that divides the pulmonary and aortic channels. In Splotch mutant mice, which lack a functional Pax3 gene, migration of cardiac neural crest is deficient and aorticopulmonary septation does not occur. Downstream genes through which Pax3 regulates cardiac neural crest development are unknown. Here, using a combination of genetic and molecular approaches, we show that the deficiency of cardiac neural crest development in the Splotch mutant is caused by upregulation of Msx2, a homeobox gene with a well-documented role as a regulator of BMP signaling. We provide evidence, moreover, that Pax3 represses Msx2 expression via a direct effect on a conserved Pax3 binding site in the Msx2 promoter. These results establish Msx2 as an effector of Pax3 in cardiac neural crest development.
Human CD34 (hCD34)-positive cells are used currently as a source for hematopoietic transplantation in humans. However, in steady-state murine hematopoiesis, hematopoietic stem cells (HSCs) with long-term reconstitution activity are found almost exclusively in the murine CD34 (mCD34)-negative to low fraction. To evaluate the possible differences in hCD34 and mCD34 gene expression in hematopoiesis, we made transgenic mouse strains with human genomic P1 artificial chromosome clones spanning the entire hCD34 genomic locus. In all transgenic mouse strains, a vast majority of phenotypic and functional HSC populations including mCD34(-/lo) express the hCD34 transgene. These data strongly support the notion that hCD34(+) human bone marrow cells contain long-term HSCs that can maintain hematopoiesis throughout life.
We found neural crest stem cells (NCSCs) in the adult gut. Postnatal gut NCSCs were isolated by flow-cytometry and compared to fetal gut NCSCs. They self-renewed extensively in culture but less than fetal gut NCSCs. Postnatal gut NCSCs made neurons that expressed a variety of neurotransmitters but lost the ability to make certain subtypes of neurons that are generated during fetal development. Postnatal gut NCSCs also differed in their responsiveness to lineage determination factors, affecting cell fate determination in vivo and possibly explaining their reduced neuronal subtype potential. These perinatal changes in gut NCSCs parallel perinatal changes in hematopoietic stem cells, suggesting that stem cells in different tissues undergo similar developmental transitions. The persistence of NCSCs in the adult PNS opens up new possibilities for regeneration after injury or disease.
CD34 is a 90- to 120-kDa cell surface sialomucin that is widely used for the enrichment of human hematopoietic stem cells (HSCs) because of its selective expression on progenitor cells and absence on mature hematopoietic cells. Recently we found that CD34 is the prototypic member of a family of three proteins with similar structure and gene organization. In light of this observation, we further examined the distribution of CD34 family members in the mouse.
Hematopoietic cell lines and primary tissues were evaluated for CD34 mRNA expression by Northern blot and protein expression by cell surface immunofluorescence. To confirm specific reactivity of the CD34 antibody, cells from CD34-deficient mice were used as controls.
Although CD34 mRNA was undetectable in all murine progenitor cell lines tested, high level expression was detected for bone marrow-derived mast cells (BMMCs). Likewise, cell surface immunofluorescence confirmed that CD34 is expressed by BMMCs and by in vivo peritoneal mast cells. No protein expression was observed for CD34-deficient mast cells. In addition, our data show that mast cells highly express the stem cell antigen Sca-1 and the well-known stem cell and mast cell antigen c-kit.
Our results demonstrate that, contrary to current dogma, CD34 is expressed by one mature hematopoietic lineage: mast cells. Our data also demonstrate that antigenically, murine mast cells and their precursors closely resemble HSCs and suggest caution should be used in the phenotypic characterization of HSCs to prevent mast cell contamination of stem cell preparations.
Meltrin α (ADAM12) is a metalloprotease-disintegrin whose specific expression patterns during development suggest that it
is involved in myogenesis and the development of other organs. To determine the roles Meltrin α plays in vivo, we generated
Meltrin α-deficient mice by gene targeting. Although the number of homozygous embryos are close to the expected Mendelian
ratio at embryonic days 17 to 18, ca. 30% of the null pups born die before weaning, mostly within 1 week of birth. The viable
homozygous mutants appear normal and are fertile. Most of the muscles in the homozygous mutants appear normal, and regeneration
in experimentally damaged skeletal muscle is unimpeded. In some Meltrin α-deficient pups, the interscapular brown adipose
tissue is reduced, although the penetrance of this phenotype is low. Impaired formation of the neck and interscapular muscles
is also seen in some homozygotes. These observations suggest Meltrin α may be involved in regulating adipogenesis and myogenesis
through a linked developmental pathway. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a candidate
substrate of Meltrin α, and we found that TPA (12-O-tetradecanoylphorbol-13-acetate)-induced ectodomain shedding of HB-EGF is markedly reduced in embryonic fibroblasts prepared
from Meltrin α-deficient mice. We also report here the chromosomal locations of Meltrin α in the mouse and rat.
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.
Many adult regenerative cells divide infrequently but have high proliferative capacity. We developed a strategy to fluorescently label slow-cycling cells in a cell type-specific fashion. We used this method to purify the label-retaining cells (LRCs) that mark the skin stem cell (SC) niche. We found that these cells rarely divide within their niche but change properties abruptly when stimulated to exit. We determined their transcriptional profile, which, when compared to progeny and other SCs, defines the niche. Many of the >100 messenger RNAs preferentially expressed in the niche encode surface receptors and secreted proteins, enabling LRCs to signal and respond to their environment.
Unconventional myosins are actin-based motors with a growing number of attributed functions. Interestingly, it has been proposed that integrins are transported by unidentified myosins to facilitate cellular remodelling. Here we present an interaction between the unconventional myosin-X (Myo10) FERM (band 4.1/ezrin/radixin/moesin) domain and an NPXY motif within beta-integrin cytoplasmic domains. Importantly, knock-down of Myo10 by short interfering RNA impaired integrin function in cell adhesion, whereas overexpression of Myo10 stimulated the formation and elongation of filopodia in an integrin-dependent manner and relocalized integrins together with Myo10 to the tips of filopodia. This integrin relocalization and filopodia elongation did not occur with Myo10 mutants deficient in integrin binding or with a beta(1)-integrin point mutant deficient in Myo10 binding. Taken together, these results indicate that Myo10-mediated relocalization of integrins might serve to form adhesive structures and thereby promote filopodial extension.
We used microarray hybridization to identify genes induced in the dermal papilla (DP) during anagen as a result of the interaction with epithelial matrix cells. We identified inhibitors of the bone morphogenetic protein (BMP) and transforming growth factor beta (TGFbeta)-signalling pathway, as well as the rat homologue of the Xenopus-secreted WNT modulator Wise. A large number of genes previously determined to be expressed in the DP were shown to be expressed in both the DP and dermal sheath (DS). Genes induced in the DP during anagen included modulators of genes expressed additionally in the DS as well as specialized extracellular matrix components. Expression of some of these genes were lost when the DP cells were cultured, suggesting that their expression was interaction dependent. One such gene, the WNT-signalling modulator Wise, was expressed in the DP and not in the non-inductive DS and was additionally expressed at high levels in the precortex and in the putative bulge region. In addition to the reported WNT-signalling modulation role, we show that Wise reduced both BMP and TGFbeta signalling in transformed fibroblasts. We speculate that loss of gene expression in cultured cells is a model for the loss of gene expression observed at catagen.
Neural crest stem cells (NCSCs) persist in peripheral nerves throughout late gestation but their function is unknown. Current models of nerve development only consider the generation of Schwann cells from neural crest, but the presence of NCSCs raises the possibility of multilineage differentiation. We performed Cre-recombinase fate mapping to determine which nerve cells are neural crest derived. Endoneurial fibroblasts, in addition to myelinating and non-myelinating Schwann cells, were neural crest derived, whereas perineurial cells, pericytes and endothelial cells were not. This identified endoneurial fibroblasts as a novel neural crest derivative, and demonstrated that trunk neural crest does give rise to fibroblasts in vivo, consistent with previous studies of trunk NCSCs in culture. The multilineage differentiation of NCSCs into glial and non-glial derivatives in the developing nerve appears to be regulated by neuregulin, notch ligands, and bone morphogenic proteins, as these factors are expressed in the developing nerve, and cause nerve NCSCs to generate Schwann cells and fibroblasts, but not neurons, in culture. Nerve development is thus more complex than was previously thought, involving NCSC self-renewal, lineage commitment and multilineage differentiation.
Stem cells are vital for the homeostasis of self-renewing tissues such as the hair follicle. Epithelial stem cells have been implicated in tumorigenesis and wound healing, and their manipulation may have wide ranging applications including gene therapy and tissue transplantation. Rodent hair follicle stem cells have been localized to an area of the follicle called the bulge, however, the identification and characterization of human hair follicle stem cells has been hampered by a lack of cellular markers for this area. We have determined that the C8/144B monoclonal antibody, originally generated against a short intracytoplasmic peptide of CD8, preferentially immunostains hair follicle bulge keratinocytes without staining the remaining hair follicle. Using expression cloning, we identified cytokeratin 15 as the keratinocyte protein recognized by the C8/144B monoclonal antibody. By delineating the bulge using this antibody, we demonstrated that bulge cells possess a stem cell phenotype characterized by their slowly-cycling nature, preferential proliferation at the onset of new hair follicle growth, high level of beta1 integrin expression, and expression of cytokeratin 19.
This 1999 edition of The Neural Crest contains comprehensive information about the neural crest, a structure unique to the vertebrate embryo, which has only a transient existence in early embryonic life. The ontogeny of the neural crest embodies the most important issues in developmental biology, as the neural crest is considered to have played a crucial role in evolution of the vertebrate phylum. Data that analyse neural crest ontogeny in murine and zebrafish embryos have been included in this revision. This revised edition also takes advantage of recent advances in our understanding of markers of neural crest cell subpopulations, and a full chapter is now devoted to cell lineage analysis. The major research breakthrough since the first edition has been the introduction of molecular biology to neural crest research, enabling an elucidation of many molecular mechanisms of neural crest development. This book is essential reading for students and researchers in developmental biology, cell biology, and neuroscience.
We report the presence of pluripotent neural crest stem cells in the adult mammalian hair follicle. Numerous neural crest cells reside in the outer root sheath from the bulge to the matrix at the base of the follicle. Bulge explants from adult mouse whisker follicles yield migratory neural crest cells, which in clonal culture form colonies consisting of over a thousand cells. Clones contain neurons, smooth muscle cells, rare Schwann cells and melanocytes, demonstrating pluripotency of the clone-forming cell. Targeted differentiation into Schwann cells and chondrocytes was achieved with neuregulin-1 and bone morphogenetic protein-2, respectively. Serial cloning in vitro demonstrated self-renewal capability. Together, the data show that the adult mouse whisker follicle contains pluripotent neural crest stem cells, termed epidermal neural crest cells (eNCSC). eNCSC are promising candidates for diverse cell therapy paradigms because of their high degree of inherent plasticity and due to their easy accessibility in the skin. Developmental Dynamics 231:258–269, 2004. © 2004 Wiley-Liss, Inc.
The possible presence of pluripotent cells in dorsal root (DRG) and sympathetic ganglia (SG) of the quail embryo has been investigated by in vitro clonal analysis. Both types of ganglia originate from the neural crest. At, or soon after, initiation of emigration from the neural tube, the neural crest appears as a mixed population of pluripotent cells and cells with more restricted developmental capacities. In the present study it was determined that pluripotent cells and precursor cells with restricted developmental potentials are also present in early DRG and SG, and that their proportions change with progressing age of the embryo. As in the neural crest, cells in one class are at least tripotent, able to give rise to pigment cells, sensory neurons, and cells in the sympathoadrenal lineage. Cells in the other class appear to have lost the melanogenic potential, but generate cells in the sensory neuron and sympathoadrenal lineages. In addition, there are two types of cells that seem to be committed to the melanogenic and sensory neuron lineages, respectively. Apparently committed melanogenic cells within the DRG are not detected after the first third of embryonic development, whereas precursor cells that are at least bipotent and generate both types of neurons persist in both DRG and SG at least through the first half of embryonic development. Neurogenic cells that are apparently committed to the sensory neuron or sympathoadrenal lineages were observed in the appropriate type of ganglion only, suggesting that location-specific cues influence the choice of phenotype generated by pluripotent neural crest cells.
The developmental potentials of cardiac neural crest cells were investigated by in vitro clonal analysis. Five morphologically distinct types of clones were observed: (1) "pigmented" clones contained melanocytes only; (2) "mixed" clones consisted of pigmented and unpigmented cells; (3) "unpigmented dense" clones consisted of flattened, closely aligned unpigmented cells; (4) "unpigmented loose" clones consisted of a few loosely arranged, flattened cells; and (5) "unpigmented large" clones included a large number of small, stellate cells that were highly proliferative. The binding patterns of antibodies against lineage-specific markers showed that cells in the different clones expressed characteristic phenotypes. The following phenotypes were expressed in addition to pigment cells: smooth muscle cells, connective tissue cells, chondrocytes, and cells in the sensory neuron lineage. Mixed clones expressed all five phenotypes. Unpigmented dense clones contained smooth muscle cells, connective tissue cells, chondrocytes, and sensory neurons. Unpigmented loose clones exclusively consisted of smooth muscle cells, whereas unpigmented large clones contained chondrocytes and sensory neuron precursors. Based on these results, the following conclusions can be drawn: (1) Pigmented and unpigmented loose clones are most likely formed by precursors that are committed to the melanogenic and myogenic cell lineages, respectively. (2) Mixed and unpigmented dense clones are derived from pluripotent cells with the capacity to give rise to four or five phenotypes. (3) Unpigmented large clones originate from progenitor cells that appear to have a partially restricted developmental potential, that is, these cells are capable of generating two phenotypes in clonal cultures. Thus, the data indicate that the early migratory cardiac neural crest is a heterogeneous population of cells, consisting of pluripotent cells, cells with a partially restricted developmental potential, and cells committed to a particular cell lineage.
The purpose of the present study is to analyze the effect of serum or chick embryo extract (CEE) on the neuronal differentiation of the mouse neural crest cells. When the crest cells were cultured in the medium containing serum at low concentration (5% calf serum), neurite outgrowth was observed. The active outgrowth was detected at 3-4 days in culture. However, in the medium supplemented with 20% calf serum, no neurite appeared, and the crest cells remained fibroblast-like. The differentiation of adrenergic neurons was observed when the crest cells were cultured in the medium containing CEE along with serum.
The early migratory cells of the posterior rhombencephalic neural crest consist of a heterogeneous population of pluripotent and developmentally restricted neural crest cells (Ito and Sieber-Blum, Dev. Biol. 148, 95-106, 1991). To determine if changes in developmental capacities occur during migration, the developmental potentials of posterior visceral arch mesenchymal cells were investigated by in vitro clonal analysis. Most of these cells consisted of the post-migratory cells of the posterior rhombencephalic neural crest. Four morphologically distinct types of clones were observed, and the cells within these clones expressed characteristic phenotypes as shown by their binding of antibodies against cell type-specific markers: (1) "DP" clones consisted of densely packed polygonal cells, with flattened large cells located predominantly at the periphery of these clones. Immunocytochemical analyses showed that DP clones contained smooth muscle cells, connective tissue cells, chondrocytes, and serotonin (5-HT)-positive neurons, and over 90% of the cells per clone were HNK-1 positive. This suggests that DP clone-forming cells are pluripotent neural-crest-derived cells with the capacity to develop into ectomesenchymal derivatives and serotonergic neurons. (2) "DS" clones consisted of densely packed spindle-shaped cells. These clones included smooth muscle cells, connective tissue cells, and chondrocytes. By contrast, neuronal phenotypes were not present. An average of 0.4% of the cells per clone were HNK-1 positive. DS clones appear to be formed by neural-crest-derived cells that are partially restricted, expressing ectomesenchymal phenotypes only. (3) "DF" clones consisted of densely packed small cells and flattened large cells. Although no HNK-1-positive cells were found in DF clones, these clones contained connective tissue cells and/or smooth muscle cells. DF clones appear to be derived from bipotent cells with the ability to differentiate into connective tissue cells and smooth muscle cells, or cells committed to the connective tissue cell lineage. (4) "LF" clones consisted of loosely arranged, flattened large cells. These clones did not contain HNK-1-positive cells. The clones consisted entirely of smooth muscle cells. Therefore, LF clones are most likely formed by precursors that are committed to the smooth muscle cell lineage. These results indicate the presence of pluripotent neural-crest-derived cells, cells with a restricted developmental potential, and apparently committed cells in the posterior visceral arch. Pluripotent cells can generate up to four neuronal and non-neuronal phenotypes. Other cells are restricted to ectomesenchymal cell types, and the proportion of these cells in the posterior visceral arch changes with progressing embryonic development.(ABSTRACT TRUNCATED AT 400 WORDS)
Analysis of lineage segregation during mammalian neural crest development has not been sufficiently performed due to technical difficulties. In the present study, therefore, we established a clonal culture system of mouse neural crest cells in order to analyze developmental potentials of individual neural crest cells and their patterns of lineage segregation. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and cholera toxin (CT) were applied to culture medium to trigger melanogenic differentiation of mouse neural crest cells. Three morphologically distinct types of clones were observed. (1) "Pigmented clones" consisted of melanocytes only, suggesting that the clone-forming cells were committed to the melanogenic lineage. These clones were observed only in the presence of TPA and CT. The proportion of this type of clone (8%) was much lower than that of the equivalent type of clone in quail trunk neural crest (40-60%; Sieber-Blum and Cohen, 1980, Dev. Biol. 80, 96-106). It therefore appears that the segregation pattern to the melanogenic lineage during mouse neural crest development in vitro differs quantitatively from that in the quail. (2) "Mixed clones" consisted of pigmented and unpigmented cells. Like pigmented clones, they were observed only in the presence of TPA and CT. The clones contained up to four types of cells: melanocytes, S100-positive cells (Schwann cells or melanogenic precursor cells), serotonin (5-HT)-positive autonomic neuron-like cells, and substance P (SP)-immunoreactive sensory neuron-like cells. Thus, at least some mixed clone-forming cells are pluripotent. (3) Two classes of "unpigmented clones" were observed that consisted of unpigmented cells only. These clones developed in the presence and absence of TPA and CT. Unpigmented clones in one class contained up to three types of cells as well as other, as yet unidentified cells: S100-, 5-HT-, and SP-positive cells. This observation suggests that at least some of these clones originate from cells with a partially restricted developmental potential. Clones in another class consisted of S100- or SP-positive cells only. These clones might be derived from cells restricted to the SP-positive neuronal cell or melanocyte/Schwann cell lineage. The present data indicate that at initiation of migration, the mouse neural crest of the trunk region is a heterogeneous population of cells containing pluripotent cells, cells with a restricted developmental potential, and cells apparently committed to the melanogenic cell lineage.
The neural crest, a migratory population of embryonic cells, gives rise to a wide range of differentiated cell types in the mature vertebrate organism, including the melanocytes of the skin. Little is known about the developmental potentials of neural crest cells toward the end of their migratory phase. We have therefore used in vitro analysis to examine the developmental potential of mesenchymal cells derived from explants of trunk epidermal ectoderm of the quail embryo. Melanocytes which differentiated in the cultures could be identified by their content of melanin granules. To detect different neuronal cells, the cultures were stained with antibodies including anti-dopamine-beta-hydroxylase (anti-DBH), which characterizes sympathoadrenal cells, and AC4, an antibody which recognizes the stage-specific embryonic antigen-1 (SSEA-1) that is expressed by cells in the sensory neuron lineage of the quail embryo, but not by sympathoadrenal cells. Seventy-eight percent of the population of neural crest-derived cells seeding the ectoderm around stage 21 gave rise to colonies containing melanocytes only. Twenty percent, however, generated mixed colonies that contained melanocytes, DBH+ cells, SSEA-1+ cells, and unidentified, unpigmented cells. Small numbers of colonies containing fewer phenotypes were also seen. With increasing embryonic age, the number of colonies containing multiple phenotypes declined, until by stage 30 all neural crest colonies contained melanocytes only. Some colonies had been marked at the single-cell stage, and this provided additional confirmation that each colony-type could be generated from a single cell. Thus the significant finding in this study is that a substantial fraction of the neural crest cells arriving early in the ectoderm are pluripotent cells that are able to give rise to pigment cells, to sympathoadrenal cells, to primary sensory neuron precursors, and possibly to other cells which were not identified here. This observation may have implications for our understanding of the mechanisms that control neural crest cell migration and differentiation.
Craniosynostosis, the premature fusion of calvarial sutures, is a common developmental anomaly that causes abnormal skull shape. The locus for one autosomal dominant form of craniosynostosis has been mapped to chromosome 5qter. The human MSX2 gene localizes to chromosome 5, and a polymorphic marker in the MSX2 intron segregates in a kindred with the disorder with no recombination. Moreover, a histidine substitutes for a highly conserved proline at position 7 of the MSX2 homeodomain exclusively in affected members. In the mouse, transcripts of the Msx2 gene are localized to calvarial sutures. These results provide compelling evidence that the mutation causes this craniosynostosis syndrome.
We have examined the growth capacity of keratinocytes isolated from human scalp hair follicles. Like the keratinocytes of glabrous epidermis, most of the colony-forming cells are classified as holoclones or meroclones when analyzed in a clonal assay. Some of them have extensive growth potential, as they are able to undergo at least 130 doublings. Therefore, the hair follicle, like the epidermis, contains keratinocytes with the expected property of stem cells: an extensive proliferative capacity permitting the generation of a large amount of epithelium. We have also examined the distribution of clonogenic keratinocytes within the hair follicle. Several hundred colony-forming cells are concentrated at a region below the midpoint of the follicle and outside the hair bulb. This region lies deeper than the site of insertion of the arrector pili muscle, which corresponds with the position of the bulge when the latter can be identified. In contrast, few colony-forming cells are present in the hair bulb, where most of the mitotic activity is observed during the active growth phase of the follicle. Paraclones, which are present both in the midregion and in the bulb of hair follicles, are unlikely to be the transient amplifying cells expected from kinetic studies.
The epidermis and its related appendages such as the hair follicle constitute the epithelial compartment of the skin. The exact location and distribution of the keratinocyte colony-forming cells within the epidermis or its appendages are unknown. We report that in the rat vibrissa, keratinocyte colony-forming cells are highly clustered in the bulge-containing region. Approximately 95% of the total colonies formed in culture from fractionated vibrissae were in this location and fewer than 4% were located in the matrix area of the follicle. Finer dissection of the bulge-containing region located the colony-forming cells in the small part containing the bulge itself. The segregation of keratinocyte colony-forming cells in the bulge confirms the hypothesis that the bulge is the reservoir of the stem cells responsible for the long-term growth of the hair follicle and perhaps of the epidermis as well.
The neural crest is a transitory and pluripotent structure of the vertebrate embryo composed of cells endowed with developmentally regulated migratory properties. We review here a series of studies carried out both in vivo and in vitro on the ontogeny of the neural crest in the avian embryo. Through in vivo studies we established the fate map of the neural crest along the neuraxis prior to the onset of the migration and we demonstrated the crucial role played by the tissue environment in which the crest cells migrate in determining their fate. Moreover, the pathways of neural crest cell migration could also be traced by the quail-chick marker system and the use of the HNK1/NC1 monoclonal antibody (Mab).
A large series of clonal cultures of isolated neural crest cells showed that, at migration time, most crest cells are pluripotent. Some, however, are already committed to a particular pathway of differentiation. The differentiation capacities of the pluripotent progenitors are highly variable from one to the other cell. Rare totipotent progenitors able to give rise to representatives of all the phenotypes (neuronal, glial, melanocytic, and mesectodermal) encountered in neural crest derivatives were also found. As a whole we propose a model according to which totipotent neural crest cells become progressively restricted (according to a stochastic rather than a sequentially ordered mechanism) in their potentialities, while they actively divide during the migration process. At the sites of gangliogenesis, selective forces allow only certain crest cells potentialities to be expressed in each type of peripheral nervous system (PNS) ganglia.
Enlargement of the skull vault occurs by appositional growth at the fibrous joints between the bones, termed cranial sutures. Relatively little is known about the developmental biology of this process, but genetically determined disorders of premature cranial suture fusion (craniosynostosis) provide one route to the identification of some of the key molecules involved. Mutations of the MSX2, FGFR1, FGFR2, FGFR3 and TWIST genes yield new insights, both into normal and abnormal cranial suture biogenesis and into problems of broad interest, such as the conservation of molecular pathways in development, and mechanisms of mutation and dominance.
Genes differentially expressed in different tissues, during development, or during specific pathologies are of foremost interest to both basic and pharmaceutical research. "Transcript profiles" or "digital Northerns" are generated routinely by partially sequencing thousands of randomly selected clones from relevant cDNA libraries. Differentially expressed genes can then be detected from variations in the counts of their cognate sequence tags. Here we present the first systematic study on the influence of random fluctuations and sampling size on the reliability of this kind of data. We establish a rigorous significance test and demonstrate its use on publicly available transcript profiles. The theory links the threshold of selection of putatively regulated genes (e.g., the number of pharmaceutical leads) to the fraction of false positive clones one is willing to risk. Our results delineate more precisely and extend the limits within which digital Northern data can be used.
Calreticulin is a ubiquitously expressed protein, which has been implicated in a large number of cellular functions, including calcium storage and signaling, protein folding, and cell attachment. To examine the role of calreticulin during in vivo development, mice deficient in calreticulin were generated by targeted inactivation of the calreticulin gene. Calreticulin-deficient mutants die in utero, mostly in late gestation. Half of these embryos had decreased cardiac cell mass, associated with increased apoptosis of cardiac myocytes. In vitro differentiation cultures of calreticulin-deficient embryonic stem cells resulted in fewer embryoid bodies with contractile activity than cultures derived from calreticulin +/- stem cells (P < 0.001). Sixteen percent of the mutants exhibited exencephaly secondary to a defect in neural tube closure. Embryos surviving until Embryonic Day 16.5 had omphalocele. Lack of calreticulin did not influence survival of embryonic fibroblasts under various endoplasmic reticulum stress conditions. However, calreticulin did influence cell migration in a calcium- and substrate-dependent manner. We conclude that calreticulin is not essential during the early stages of embryonic development, but is important for the development of heart and brain and for ventral body wall closure. The observed abnormalities are compatible with a role of calreticulin in the modulation of cellular calcium signaling.
The roles in the nervous system of the receptor tyrosine kinase Kit and its ligand, Steel factor, are unclear. We have now found first, that sensory nerve populations are reduced in mutant Kit and Steel mice, implicating Steel-Kit interactions in neuronal development. Second, sensory axonal regeneration (which occurs independently of nerve growth factor, or NGF) is impaired, while collateral sprouting (NGF dependent) is normal. Therefore, there is a selective involvement of Kit signal transduction pathways in nerve growth; supporting this, in wild-type animals Kit was up-regulated in regenerating, but unchanged in sprouting, sensory neurons. The receptor tyrosine kinase Kit thus contrasts with the receptor tyrosine kinase trkA, which is activated by the sprouting stimulus (NGF) but not by the axonal regeneration signal.
The norepinephrine transporter (NET) is a neurotransmitter scavenger and site of drug action in noradrenergic neurons. The aim of this study was to identify mechanisms that regulate NET expression during the development of quail (q) sympathetic neuroblasts, which develop from neural crest stem cells. Neurotrophin-3 (NT-3) and transforming growth factor beta1 (TGF-beta1) cause an increase of qNET mRNA levels in neural crest cells. When combined, the growth factors are additive in increasing qNET mRNA levels. Both NT-3 and TGF-beta1 are synthesized by neural crest cells. Onset of NET expression precedes the onset of neural crest stem cell emigration from the neural tube. In older embryos, qNET is expressed by several crest-derived and noncrest tissues. The data show that qNET expression in presumptive sympathetic neurons is initiated early in embryonic development by growth factors that are produced by neural crest cells themselves. Moreover, the results support our previous observations that norepinephrine transport contributes to the regulation of the differentiation of neural crest stem cells into sympathetic neurons.
Serial Analysis of Gene Expression (SAGE) is becoming a widely used gene expression profiling method for the study of development,
cancer and other human diseases. Investigators using SAGE rely heavily on the quantitative aspect of this method for cataloging
gene expression and comparing multiple SAGE libraries. We have developed additional computational and statistical tools to
assess the quality and reproducibility of a SAGE library. Using these methods, a critical variable in the SAGE protocol was
identified that has the potential to bias the Tag distribution relative to the GC content of the 10 bp SAGE Tag DNA sequence.
We also detected this bias in a number of publicly available SAGE libraries. It is important to note that the GC content bias
went undetected by quality control procedures in the current SAGE protocol and was only identified with the use of these statistical
analyses on as few as 750 SAGE Tags. In addition to keeping any solution of free DiTags on ice, an analysis of the GC content
should be performed before sequencing large numbers of SAGE Tags to be confident that SAGE libraries are free from experimental
bias.
A series of experimental bioassays has shown that the dermal papilla of the adult rodent vibrissa hair follicle retains unique inductive properties. In view of the many phenotypic and functional differences between specific hair follicle types, and the growing interest in hair follicle biology and disease, it remains important to establish that the human hair follicle dermal papilla has equivalent capabilities. In this study we tested the ability of human hair follicle papillae to induce hair growth when implanted into transected, athymic mouse vibrissa follicles. The implanted papillae that interacted with mouse follicle epithelium created new fibre-producing follicle end bulbs. The origin of the papillae in the recombinant structures was confirmed using laser capture microdissection and human specific gender determination by PCR. The demonstration that intact adult human dermal papillae can induce hair growth has implications for molecular analysis of basic hair growth mechanisms, particularly since the study involved common epithelial-mesenchymal signalling and recognition properties across species. It also improves the prospects for a cell-based clinical approach to hair follicle disorders.
We describe here the isolation of stem cells from juvenile and adult rodent skin. These cells derive from the dermis, and clones of individual cells can proliferate and differentiate in culture to produce neurons, glia, smooth muscle cells and adipocytes. Similar precursors that produce neuron-specific proteins upon differentiation can be isolated from adult human scalp. Because these cells (termed SKPs for skin-derived precursors) generate both neural and mesodermal progeny, we propose that they represent a novel multipotent adult stem cell and suggest that skin may provide an accessible, autologous source of stem cells for transplantation.
Stem cells and neuroblasts derived from mouse embryos undergo repeated asymmetric cell divisions, generating neural lineage trees similar to those of invertebrates. In Drosophila, unequal distribution of Numb protein during mitosis produces asymmetric cell divisions and consequently diverse neural cell fates. We investigated whether a mouse homologue m-numb had a similar role during mouse cortical development.
Progenitor cells isolated from the embryonic mouse cortex were followed as they underwent their next cell division in vitro. Numb distribution was predominantly asymmetric during asymmetric cell divisions yielding a β-tubulin III− progenitor and a β-tubulin III+ neuronal cell (P/N divisions) and predominantly symmetric during divisions producing two neurons (N/N divisions). Cells from the numb knockout mouse underwent significantly fewer asymmetric P/N divisions compared to wild type, indicating a causal role for Numb.
When progenitor cells derived from early (E10) cortex undergo P/N divisions, both daughters express the progenitor marker Nestin, indicating their immature state, and Numb segregates into the P or N daughter with similar frequency. In contrast, when progenitor cells derived from later E13 cortex (during active neurogenesis in vivo) undergo P/N divisions they produce a Nestin+ progenitor and a Nestin– neuronal daughter, and Numb segregates preferentially into the neuronal daughter. Thus during mouse cortical neurogenesis, as in Drosophila neurogenesis, asymmetric segregation of Numb could inhibit Notch activity in one daughter to induce neuronal differentiation.
At terminal divisions generating two neurons, Numb was symmetrically distributed in approximately 80% of pairs and asymmetrically in 20%. We found a significant association between Numb distribution and morphology: most sisters of neuron pairs with symmetric Numb were similar and most with asymmetric Numb were different. Developing cortical neurons with Numb had longer processes than those without.
Numb is expressed by neuroblasts and stem cells and can be asymmetrically segregated by both. These data indicate Numb has an important role in generating asymmetric cell divisions and diverse cell fates during mouse cortical development.
Kit-ligand (Kitl) also known as steel factor, stem cell factor and mast cell growth factor plays a crucial role in the development and maintenance of the melanocyte lineage in adult skin. Kitl exerts permanent survival, proliferation and migration functions in Kit receptor-expressing melanocytes. A comprehensive overview over the differential roles of Kitl in melanocyte development and homeostasis is provided. I discuss species-specific differences of the Kitl/Kit signalling system, regulation at the transcriptional level and also covering the regulation of cell surface Kitl presentation by cytoplasmic targeting sequences. In addition, recent studies evoked the importance of Kitl misexpression in some hyperpigmented lesions that may open the avenue for Kitl-dependent treatment of pathological skin conditions.
Subcellular fractionation experiments with mouse hepatocytes, combined with sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)-immunoblot analysis using antibodies against two different tail regions of mouse myosin-X demonstrated a 240 kDa molecular mass to be associated with the plasma membrane-rich P2 fraction. The basolateral plasma membrane fraction, but not the brush border fraction, isolated from renal cortices also contained the 240 kDa form of myosin-X. In an attempt to assess relative contributions of possible functional domains in the tail of myosin-X to localization and function, cDNA corresponding to all three pleckstrin homology (PH) domains and different regions (PH1, 2 and 3, and the two subdomains of PH1: PHS1 and PHS2), as well as the myosin tail homology 4 domain (MyTH4) and the band4.1/ezrin/radixin/moesin-like domain (FERM) were separately inserted into the pEGFP vector and expressed in cultured COS-1 cells. As a result, two distinct regions responsible for localization were identified with regard to PH: one covers all three forms that tends to localize to regions of dynamic actin, such as membrane ruffles, lamellipodia and thick cortical actin bundles at the sites of cell-cell adhesion in a Rac- and Cdc42-dependent manner. The other covers PHS1 and PH2 that localizes to filopodia, filopodial puncta and the sites of intercellular adhesion in a Cdc42-dependent manner. Expression of green fluorescent protein (GFP)-MyTH4 fusion protein resulted in formation of phalloidin-positive granules, while GFP-FERM affected the actin cytoskeletal system in a distinctly different way. Taken altogether, the results lend support to the view that myosin-X is involved in cell-cell adhesion-associated signaling-linked membrane and/or cytoskeleton reorganization.
In mammals, hair follicles produce hairs that fulfill a number of functions including thermoregulation, collecting sensory information, protection against environmental trauma, social communication, and mimicry. Hair follicles develop as a result of epithelial-mesenchymal interactions between epidermal keratinocytes committed to hair-specific differentiation and cluster of dermal fibroblasts that form follicular papilla. During postnatal life, hair follicles show patterns of cyclic activity with periods of active growth and hair production (anagen), apoptosis-driven involution (catagen), and relative resting (telogen). During last decade, substantial progress has been achieved in delineating molecular mechanisms that control hair follicle development and cyclic activity. In this review, we summarize the data demonstrating that regulation of hair follicle development in the embryo and control of hair follicle growth during postnatal life are highly conserved and both require involvement of similar molecular mechanisms. Since many of the molecules that control hair follicle development and cycling are also involved in regulating morphogenesis and postnatal biology of other ectodermal derivatives, such as teeth, feathers, and mammary glands, basic principles and molecular mechanisms that govern hair follicle development and growth may also be applicable for other developmental systems.
The flat bones of the vertebrate skull vault develop from two migratory mesenchymal cell populations, the cranial neural crest and paraxial mesoderm. At the onset of skull vault development, these mesenchymal cells emigrate from their sites of origin to positions between the ectoderm and the developing cerebral hemispheres. There they combine, proliferate and differentiate along an osteogenic pathway. Anomalies in skull vault development are relatively common in humans. One such anomaly is familial calvarial foramina, persistent unossified areas within the skull vault. Mutations in MSX2 and TWIST are known to cause calvarial foramina in humans. Little is known of the cellular and developmental processes underlying this defect. Neither is it known whether MSX2 and TWIST function in the same or distinct pathways. We trace the origin of the calvarial foramen defect in Msx2 mutant mice to a group of skeletogenic mesenchyme cells that compose the frontal bone rudiment. We show that this cell population is reduced not because of apoptosis or deficient migration of neural crest-derived precursor cells, but because of defects in its differentiation and proliferation. We demonstrate, in addition, that heterozygous loss of Twist function causes a foramen in the skull vault similar to that caused by loss of Msx2 function. Both the quantity and proliferation of the frontal bone skeletogenic mesenchyme are reduced in Msx2-Twist double mutants compared with individual mutants. Thus Msx2 and Twist cooperate in the control of the differentiation and proliferation of skeletogenic mesenchyme. Molecular epistasis analysis suggests that Msx2 and Twist do not act in tandem to control osteoblast differentiation, but function at the same epistatic level.
Little is known about the molecular mechanism underlying specification and differentiation of smooth muscle (SM), and this is, at least in part, because of the few cellular systems available to study the acquisition of a SM phenotype in vitro. Mesoangioblasts are vessel-derived stem cells that can be induced to differentiate into different cell types of the mesoderm, including SM. We performed a DNA microarray analysis of a mesoangioblast clone that spontaneously expresses an immature SM phenotype and compared it with a sister clone mainly composed of undifferentiated progenitor cells. This study allowed us to define a gene expression profile for "stem" cells versus smooth muscle cells (SMCs) in the absence of differentiation inducers such as transforming growth factor beta. Two transcription factors, msx2 and necdin, are expressed at least 100 times more in SMCs than in stem cells, are coexpressed in all SMCs and tissues, are induced by transforming growth factor beta, and, when coexpressed, induce a number of SM markers in mesoangioblast, fibroblast, and endothelial cell lines. Conversely, their downregulation through RNA interference results in a decreased expression of SM markers. These data support the hypothesis that Msx2 and necdin act as master genes regulating SM differentiation in at least a subset of SMCs.
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.
A fundamental question in stem cell research is whether cultured multipotent adult stem cells represent endogenous multipotent precursor cells. Here we address this question, focusing on SKPs, a cultured adult stem cell from the dermis that generates both neural and mesodermal progeny. We show that SKPs derive from endogenous adult dermal precursors that exhibit properties similar to embryonic neural-crest stem cells. We demonstrate that these endogenous SKPs can first be isolated from skin during embryogenesis and that they persist into adulthood, with a niche in the papillae of hair and whisker follicles. Furthermore, lineage analysis indicates that both hair and whisker follicle dermal papillae contain neural-crest-derived cells, and that SKPs from the whisker pad are of neural-crest origin. We propose that SKPs represent an endogenous embryonic precursor cell that arises in peripheral tissues such as skin during development and maintains multipotency into adulthood.
The lowermost portion of the resting (telogen) follicle consists of the bulge and secondary hair germ. We previously showed that the progeny of stem cells in the bulge form the lower follicle and hair, but the relationship of the bulge cells with the secondary hair germ cells, which are also involved in the generation of the new hair at the onset of the hair growth cycle (anagen), remains unclear. Here we address whether secondary hair germ cells are derived directly from epithelial stem cells in the adjacent bulge or whether they arise from cells within the lower follicle that survive the degenerative phase of the hair cycle (catagen). We use 5-bromo-2'-deoxyuridine to label bulge cells at anagen onset, and demonstrate that the lowermost portion of the bulge collapses around the hair and forms the secondary hair germ during late catagen. During the first six days of anagen onset bulge cells proliferate and self-renew. Bulge cell proliferation at this time also generates cells that form the future secondary germ. As bulge cells form the secondary germ cells at the end of catagen, they lose expression of a biochemical marker, S100A6. Remarkably, however, following injury of bulge cells by hair depilation, progenitor cells in the secondary hair germ repopulate the bulge and re-express bulge cell markers. These findings support the notion that keratinocytes can "dedifferentiate" to a stem cell state in response to wounding, perhaps related to signals from the stem cell niche. Finally, we also present evidence that quiescent bulge cells undergo apoptosis during follicle remodeling in catagen, indicating that a subpopulation of bulge cells is not permanent.