Enrichment and identification of human 'fetal' epidermal stem cells

BACKGROUND: Human epidermis, a continuously renewing tissue, is maintained throughout life by stem cells that proliferate and replenish worn-out or damaged cells in the tissue. Cultured human epidermal stem cells have great potential in clinical application. However, isolating and culturing a pure population of epidermal stem cells has proven to be challenging. METHODS AND RESULTS: We show that p63, a new marker for epidermal stem cells, is expressed in the basal layer of human fetal epidermis using immunohistochemistry, and that keratinocytes with the characteristics of stem cells can be isolated from the epidermis of aborted human fetuses aged >/=20 weeks based on high expression of beta(1) integrins by fluorescence-activated cell sorting. Furthermore, the enriched population showed the expression of molecular markers of putative human epidermal stem cells under a confocal microscope and a high colony formation efficiency when it was cultured at a clonal density. Under an electron microscope the sorted stem cells exhibited a high nuclear:cytoplasmic ratio and fewer organelles than the transit amplifying cells. The cultured epidermal stem cells can also be amplified and induced to terminal differentiation by suspension in vitro. CONCLUSIONS: Human 'fetal' epidermal stem cells have been successfully isolated and cultured in vitro. The cultured human epidermal stem cells could be used as a tool for studying stem cell biology and testing stem cell therapy.     

Acceleration of the formation of cultured epithelium using the sonic hedgehog expressing feeder cells.

Sonic hedgehog (Shh) regulates the principal possesses in many developmental stages, including the epithelial-mesenchymal interaction. The extraordinary acceleration of signaling by Shh is responsible for the development of human basal cell carcinomas and trichoepitheliomas; they might originate from the very immature keratinocytes, including the stem cells. We tried to utilize the mitogenic effect of Shh to accelerate the formation of cultured epithelium, which is already used in the medical field practically. To this end, we transfected shh cDNA into a Swiss-3T3 cell line, widely used as a feeder for keratinocytes, and established a Shh expressing cell line. The lethally irradiated Shh expressing feeder cells remarkably accelerated the growth of keratinocyte colonies obtained from the human neonatal foreskin, and the formation of well-stratified cultured epithelium, which is rich in immature small keratinocytes, expressing cytokeratin 14. This acceleration was suppressed by the addition of cyclopamine, a specific inhibitor of Shh signaling. These data indicate that the Shh is a promising mitogen to improve the technology for cultured epithelium formation.     

Sonic hedgehog induces the proliferation of primitive human hematopoietic cells via BMP regulation

A pool of stem cells that arise from the mesoderm during embryogenesis initiates hematopoiesis. However, factors that regulate the expansion of blood stem cells are poorly understood. We show here that cytokine-induced proliferation of primitive human hematopoietic cells could be inhibited with antibodies to hedgehog (Hh). Conversely, Sonic hedgehog (Shh) treatment induced the expansion of pluripotent human hematopoietic repopulating cells detected in immunodeficient mice. Noggin, a specific inhibitor of bone morphogenetic protein 4 (BMP-4), was capable of inhibiting Shh-induced proliferation in a similar manner to anti-Hh; however, anti-Hh had no effect on BMP-4-induced proliferation. Our study shows that Shh functions as a regulator of primitive hematopoietic cells via mechanisms that are dependent on downstream BMP signals.     

Commercially available recombinant sonic hedgehog up-regulates Ptc and modulates the cytokine and chemokine expression of human macrophages: an effect mediated by endotoxin contamination?

The Sonic hedgehog (Shh) signalling pathway plays an important role in developmental patterning and proliferation. Recent evidence suggests that Shh also plays a role in the development of the immune system. Here, we demonstrate that components of the Shh signalling pathway are expressed in human macrophages and that the receptor for Shh, Ptc, is up-regulated by a commercially available recombinant preparation of Shh (CArShh). Further, we report that the addition of CArShh up-regulates the production of IL-6, IL-8, MCP-1, IP-10, MIG and RANTES by macrophages, an effect enhanced by the presence of fetal calf serum in the culture medium. In contrast, TGF-beta, TNF-alpha, IL-1b, IL-12 and IL-10 production were not modulated by CArShh and VEGF was minimally up-regulated even in the presence of serum. The up-regulation of these cytokines and chemokines was abrogated by CD14 inhibition and polymixin B, but not reliably inhibited by the specific Shh pathway inhibitor cyclopamine. These results suggest that, although components of the Shh signalling pathway are expressed in macrophages, the modulation of macrophage cytokine and chemokine effector function seen in response to commercially available rShh results from low levels of endotoxin contained within the CArShh preparations employed to explore the effects of Shh in vitro.     

Sonic hedgehog regulates adult neural progenitor proliferation in vitro and in vivo

Neural stem cells exist in the developing and adult nervous systems of all mammals, but the basic mechanisms that control their behavior are not yet well understood. Here, we investigated the role of Sonic hedgehog (Shh), a factor vital for neural development, in regulating adult hippocampal neural stem cells. We found high expression of the Shh receptor Patched in both the adult rat hippocampus and neural progenitor cells isolated from this region. In addition, Shh elicited a strong, dose-dependent proliferative response in progenitors in vitro. Furthermore, adeno-associated viral vector delivery of shh cDNA to the hippocampus elicited a 3.3-fold increase in cell proliferation. Finally, the pharmacological inhibitor of Shh signaling cyclopamine reduced hippocampal neural progenitor proliferation in vivo. This work identifies Shh as a regulator of adult hippocampal neural stem cells.     

Monoclonal antibodies (BL7 and BL9) having crossed reactivity with cells of the human epidermis

The relationships between epithelial cells and immunocompetent cells could be approached by studying of the common antigens expressed by these two cell types. This paper reports the study of two monoclonal antibodies (BL7 and BL9) which react with epidermal cells. BL7 was obtained after immunization of mice with human thymic cell suspensions and BL9, after immunization with Raji cells. BL7 stained the epithelial network of the thymus and the basal cell layer of the epidermis. BL7 also reacted with the endothelial cells of the vessels of the dermis. This reactivity against the basal cell layer of the epidermis was observed in man, mice and rabbit. BL9 showed a reactivity against thymic epithelial cells and stained the membrane of the keratinocytes of the human epidermis. The antigenic expression revealed by BL9 decreased during the epidermal cell differentiation and disappeared in the horny layer. BL9 showed no reactivity with the epidermis of mice and rabbit. These two monoclonal antibodies are new tools in cutaneous immunopathology: BL7 is the first monoclonal experimental marker which identifies the basal cells of the epidermis, BL9 identifies an antigen related to the human epidermal cell differentiation.     

Nucleotides and epidermal growth factor induce parallel cytoskeletal rearrangements and migration in cultured adult murine neural stem cells

Aim: The adult subventricular zone (SVZ) contains neural stem cells that generate neuroblasts migrating to the olfactory bulb (OB) and differentiating into interneurones. The molecular cues controlling essential functions within the neurogenesis pathway such as proliferation, short and long distance migration, functional integration and cell survival are poorly understood. We have previously shown that cultured adult neural stem cells express a considerable variety of nucleotide receptors and that nucleotides and epidermal growth factor (EGF) induce converging intracellular signalling pathways that carry potential for synergism in the control of neural stem cell proliferation and cell survival. Here we investigate the role of EGF and the nucleotides ATP, ADPβS and UTP in neural stem cell migration. Methods: Neural stem cells were prepared from adult mice and subjected to adherent culture. Labelling of F-actin was performed with tetramethylrhodamine isothiocyanate-phalloidin. Images were processed for quantitative evaluation of fluorescence labelling. Agonist-induced phosphorylation of AKT and focal adhesion kinase was analysed by quantitative Western blotting. Agonist-dependent cell migration was assayed using 48-well microchemotaxis chambers. Results: Nucleotides and EGF induce the formation of stress fibres, an increase in the cortical actin cytoskeleton and in cell spreading. This is associated with increased phosphorylation of AKT and focal adhesion kinase. Using microchemotaxis chambers we demonstrate a parallel increase in cell migration. Conclusion: Our results suggest that nucleotides and EGF acting as paracrine or autocrine signalling substances can be of relevance for structuring and maintaining the cytoarchitecture of the SVZ and the stream of neuroblasts migrating to the OB.     

Characterization of bipotential epidermal progenitors derived from human sebaceous gland: contrasting roles of c-Myc and beta-catenin

The current belief is that the epidermal sebaceous gland (SG) is maintained by unipotent stem cells that are replenished by multipotent stem cells in the hair follicle (HF) bulge. However, sebocytes can be induced by c-Myc (Myc) activation in interfollicular epidermis (IFE), suggesting the existence of bipotential stem cells. We found that every SZ95 immortalized human sebocyte that underwent clonal growth in culture generated progeny that differentiated into both sebocytes and cells expressing involucrin and cornifin, markers of IFE and HF inner root sheath differentiation. The ability to generate involucrin positive cells was also observed in a new human sebocyte line, Seb-E6E7. SZ95 xenografts differentiated into SG and IFE but not HF. SZ95 cells that expressed involucrin had reduced Myc levels; however, this did not correlate with increased expression of the Myc repressor Blimp1, and Blimp1 expression did not distinguish cells undergoing SG, IFE, or HF differentiation in vivo. Overexpression of Myc stimulated sebocyte differentiation, whereas overexpression of beta-catenin stimulated involucrin and cornifin expression. In transgenic mice simultaneous activation of Myc and beta-catenin revealed mutual antagonism: Myc blocked ectopic HF formation and beta-catenin reduced SG differentiation. Overexpression of the Myc target gene Indian hedgehog did not promote sebocyte differentiation in culture and cyclopamine treatment, while reducing proliferation, did not block Myc induced sebocyte differentiation in vivo. Our studies provide evidence for a bipotential epidermal stem cell population in an in vitro model of human epidermal lineage selection and highlight the importance of Myc as a regulator of sebocyte differentiation.     

Expression and regulation of Sprouty-2 in the granulosa-lutein cells of the corpus luteum

Growth factor signalling has important modulatory roles in the process of human follicular growth, oocyte maturation and corpus luteum (CL) formation. Recently, Sprouty-2, an inhibitor of receptor tyrosine kinase (RTK) signalling pathway was advocated as a marker of oocyte competence in the bovine ovary. We sought to study Sprouty-2 expression and regulation in the human ovary. RT-PCR was used to detect Sprouty-2 mRNA in human granulosa-lutein cells (GLC) collected from follicular aspiration of IVF patients. The effect of epidermal and fibroblast growth factors (EGF and FGF) on Sprouty-2 mRNA expression in GLC was studied using quantitative real-time PCR. Immunohistochemistry was performed on cultured GLC, human CL and stimulated rat ovary sections. Sprouty-2 mRNA was expressed in human GLC. EGF and basic FGF, but not FGF4 and FGF10, increased Sprouty-2 mRNA expression in GLC. The Sprouty protein was localized to GLC of early and late human CL but not to the theca cell layer. Immunostaining of developing rat CL confirmed the temporal and spatial expression of Sprouty in humans. The detection of Sprouty-2 mRNA and protein in human GLC may suggest a role for Sprouty-2 during the final stages of follicle maturation and CL formation.     

Cell proliferation during blastema formation in the regenerating teleost fin.

Epimorphic regeneration in teleost fins occurs through the establishment of a balanced growth state in which a blastema gives rise to all the mesenchymal cells, whereas definite areas of the epidermis proliferate leading to its extension, thus, allowing the enlargement of the whole structure. This type of regeneration involves specific mechanisms that temporally and spatially regulate cell proliferation. To understand how the blastema is formed and how this growth situation is set up, we investigated cell proliferation patterns in the regenerating fin of the goldfish Carassius auratus from the time of amputation to that of blastema formation by using proliferating cell nuclear antigen immunostaining and bromodeoxyuridine labeling. Wound closure and apical epidermal cap formation took place by epidermal migration and re-arrangement, without the contribution of cell proliferation. As soon as the apical cap had formed, the epidermis started to proliferate at its lateral surfaces, in which all layers maintained cycling for the duration of the studied process. The distal epidermal cap, on the contrary, presented very few cycling cells, and its cytoarchitecture was indicative of continuous remodeling due to ray growth. The basal layer of this epidermal cap showed a typical morphology and remained nonproliferative whilst in contact with the proliferating blastema. Proliferation in the mesenchymal compartment of the ray started far from the amputation plane. Subsequently, cycling cells approached that location, until they formed the blastema in contact with the apical epidermal cap. Differences observed between the epidermis and mesenchyma, regarding activation of the cell cycle and the establishment of proliferative patterns, suggest that differential mechanisms regulate cell proliferation in each of these compartments during the initial stages of regeneration.     

A stable niche supports long-term maintenance of human epidermal stem cells in organotypic cultures

Stem cells in human interfollicular epidermis are still difficult to identify, mainly because of a lack of definitive markers and the inability to label human beings for label-retaining cells (LRCs). Here, we report that LRCs could be identified and localized in organotypic cultures (OTCs) made with human cells. Labeling cultures for 2 weeks with iododeoxyuridine (IdU) and then chasing for 6-10 weeks left <1% of basal cells retaining IdU label. Whole mounts demonstrated that LRCs were individually dispersed in the epidermal basal layer. Some LRCs, but not all, colocalized with cells expressing melanoma chondroitin sulfate proteoglycan, a putative stem cell marker. Although we found LRCs in both collagen- and scaffold-based OTCs, only the scaffold-OTCs supported long-term survival and regeneration. LRCs ' short survival in collagen-OTCs was not due to loss of appropriate growth factors from fibroblasts. Instead, it was due to expression of metalloproteinases, especially matrix metalloproteinase (MMP)-2 and MMP-14, which caused collagen fragmentation, matrix degradation, and dislocation of specific basement membrane components bound to epidermal integrins. Blocking MMP activation not only abrogated MMP-dependent matrix degradation but also increased longevity of the epidermis and the LRCs in these cultures. Such findings indicate that the stem cell niche, the microenvironment surrounding and influencing the stem cell, is essential for stem cell survival and function, including long-term tissue regeneration. Disclosure of potential conflicts of interest is found at the end of this article.     

人胎脑神经干细胞的体外培养

目的从人胎脑纹状体中分离培养并鉴定神经干细胞。方法采用包含碱性成纤维细胞生长因子(bFGF)和表皮细胞生长因子(EGF)的无血清培养和单细胞克隆技术,从36周自愿水囊引产人胎脑纹状体中分离出神经干细胞,并观察神经干细胞体外培养、传代、分化潜能。结果从36周人胎脑纹状体中成功分离出具有自我更新和多分化潜能的神经干细胞,在无血清培养时细胞呈悬浮状态生长,形成神经球,该细胞具有连续克隆能力,可传代培养,呈Nestin免疫反应阳性细胞;在含血清培养时诱导神经干细胞分化,分化后的细胞表达神经元细胞、星形胶质细胞和少突胶质细胞的特异性抗原。结论神经干细胞的存活和分裂有赖于EGF和bFGF的共同作用;36周人胎脑纹状体仍能培养出具有自我复制和分化潜能的神经干细胞。     

Tiers of Clonal Organization in the Epidermis: The Epidermal Proliferation Unit Revisited

As one of the most proliferative tissues in adult mammals, the epidermis is a good example of the precise regulation necessary between stem cell self-renewal and differentiation. The epidermis is derived from ectodermal progenitor cells and contains three distinct classes of cells: epidermal stem cells which are capable of infinite rounds of cell division; their immediate descendants, transient amplifying cells, which are capable of numerous but finite rounds of cell division; and finally, non-dividing, differentiating cells (Aberdam in Cell and Tissue Research 331:103–107, 2008). This proliferative hierarchy must be tightly regulated both temporally and spatially during epidermal development and homeostasis in order to prevent uncontrolled growth leading to hyperproliferative states and/or tumorigenesis. Historically, the most basic unit of epidermal proliferation has been described as the epidermal proliferation unit (EPU). The EPU, as originally characterized by Christophers, Potten and Mackenzie, is a proliferation unit consisting of approximately 10 basal cells with a clonogenic cell in the center and overlaid by the suprabasal and corneocyte progeny (reviewed in Potten, C. S. (1974). The epidermal proliferative unit: the possible role of the central basal cell. Cell and Tissue Kinetics, 7(1), 77–88). Numerous researchers have identified this classical EPU structure, consisting of approximately 20 cells, in a variety of mammalian skin sources. Recently however, lineage analyses have provided evidence for much larger clonal epidermal units consisting of hundreds to thousands of cells. Furthermore, cutaneous mosaicism as well as a variety of cutaneous pathologies indicate that clonal areas extend to whole patches of mammalian skin many centimeters across. In this review we revisit four decades of experimental evidence and put forward a model of clonal units derived from multiple classes of epidermal progenitors ranging from the largest and most primitive units, clonal ectodermal units, to epidermal stem cell units, and finally, to the most basic structural unit, the EPU.     

Enhanced proliferation of neural stem cells in a collagen hydrogel incorporating engineered epidermal growth factor

Neural stem cells (NSCs) have received much attention in cell-transplantation therapy for central nervous disorders such as Parkinson's disease. However, poor engraftment of transplanted cells limits the efficacy of the treatments. To overcome this problem, collagen-based hydrogels were designed in this study to provide microenvironments for embedded cells to survive and proliferate. Our approach was to incorporate epidermal growth factor (EGF), known as a mitogen for NSCs, into a collagen hydrogel. For the stable binding of EGF with collagen under mild conditions, EGF was fused with a collagen-binding polypeptide domain by recombinant DNA technology. A cell population containing NSCs was derived from the fetal rat brain and cultured in the composite hydrogels for 7 d followed by analysis for cell proliferation. It was shown that the number of living cells was significantly higher in hydrogels incorporating collagen-binding EGF. This effect is largely owing to the collagen-binding domain that serves to sustain presentation of EGF toward cells within the hydrogel. It is further revealed by gene expression analysis that cells proliferated in the EGF-incorporating collagen hydrogel contained subpopulations expressing the marker of stem cells, neurons, astrocytes, or oligodendrocytes.     

Sonic hedgehog regulates proliferation of the retinal ciliary marginal zone in posthatch chicks.

The ciliary marginal zone (CMZ) has long been known to be a source of postembryonic neuronal production in the retinas of fish and amphibians, and more recently, birds. However, there is little known about the factors that are required for the maintenance of this neural stem cell zone. The cells of the CMZ respond to mitogens such as endothelial growth factor, insulin-like growth factor-1, and insulin, factors that are also mitogenic for embryonic retinal progenitors, suggesting that the continued expression of embryonic mitogenic factors might be required to maintain the postembryonic proliferative potential of the CMZ. To test this hypothesis, we examined the expression and functional role of a critical embryonic retinal progenitor mitogen, Sonic hedgehog (Shh) in the regulation of proliferation of the cells of the CMZ. We have found that Shh is concentrated at the retinal margin of postembryonic chicks. Moreover, we report that intraocular injection of Shh stimulates proliferation of the CMZ cells, whereas cyclopamine, an inhibitor of the Shh pathway, inhibits CMZ proliferation. We conclude that Shh signaling is an important factor in the maintenance of postembryonic retinal neurogenesis.     

Fungiform papilla pattern: EGF regulates inter-papilla lingual epithelium and decreases papilla number by means of PI3K/Akt, MEK/ERK, and p38 MAPK signaling

Fungiform papillae are epithelial taste organs that form on the tongue, requiring differentiation of papillae and inter-papilla epithelium. We tested roles of epidermal growth factor (EGF) and the receptor EGFR in papilla development. Developmentally, EGF was localized within and between papillae whereas EGFR was progressively restricted to inter-papilla epithelium. In tongue cultures, EGF decreased papillae and increased cell proliferation in inter-papilla epithelium in a concentration-dependent manner, whereas EGFR inhibitor increased and fused papillae. EGF preincubation could over-ride disruption of Shh signaling that ordinarily would effect a doubling of fungiform papillae. With EGF-induced activation of EGFR, we demonstrated phosphorylation in PI3K/Akt, MEK/ERK, and p38 MAPK pathways; with pathway inhibitors (LY294002, U0126, SB203580) the EGF-mediated decrease in papillae was reversed, and synergistic actions were shown. Thus, EGF/EGFR signaling by means of PI3K/Akt, MEK/ERK, and p38 MAPK contributes to epithelial cell proliferation between papillae; this biases against papilla differentiation and reduces numbers of papillae.     

Effects of all-trans retinoic acid and Ca++ on human skin in organ culture.

In this study, we have established an organ culture model of human skin and examined the effects of both all-trans retinoic acid (RA) and extracellular Ca++ on the epidermal and dermal components of the organ-cultured skin. Our data show that while organ cultures maintained in serum-free, growth factor-free culture medium containing 0.15 mM Ca++ degenerated rapidly, those treated with concentrations of RA that have been shown previously to stimulate fibroblast and keratinocyte proliferation in monolayer culture (J Invest Dermatol 1989, 93:449; 1990, 94:717; Am J Pathol 1990, 136:1275) demonstrated a healthy appearance for up to 12 days. Degeneration of the control cultures was characterized by separation of the epidermis from the underlying dermis, progressive cell necrosis leading to a complete absence of viable cells from both the dermal and epidermal compartments, disintegration and fibrillation of the dermal connective tissue, and a cessation of protein synthesis. RA-treated organ cultures contained large numbers of healthy-appearing cells in both the epidermal and dermal compartments. One or several layers of viable basal cells in the epidermis could be seen at least through day 12. However, the upper layers of the epidermis frequently separated from the cells in the basal layer. The dermal connective tissue was histologically well-preserved. Furthermore, the level of protein synthesis was higher in the RA-treated cultures than in the control cultures. In addition to treating organ cultures with RA, other cultures were exposed to serum-free, growth factor-free culture medium containing 1.4 mM Ca++. The presence of the elevated Ca++ concentration also preserved cellular and connective tissue structures in the dermal and epidermal compartments. In comparison to RA there was better preservation of the overall epidermal structure. The upper layers of epidermal cells did not separate from the basal cells, and the various stages of epithelial differentiation could be seen. Histologically, the dermis was well-preserved in the presence of elevated extracellular Ca++. Specimens treated with a combination of Ca++ and RA demonstrated features consistent with the features induced by each treatment separately. This included an expanded basal layer of epithelial cells and a prominent keratotic layer with a fairly orderly pattern of differentiation. The tendency of the upper epidermis to separate from the basal cells was partially mitigated. Taken together, these data indicate that both RA and extracellular Ca++ act to prevent the degeneration of human skin in organ culture but probably do so through different mechanisms.