A comparison of primary oesophageal squamous epithelial cells with HET‐1A in organotypic culture

Background Information. Carcinoma of the oesophagus is the sixth leading cause of cancer death in the western world and is associated with a 5‐year survival of less than 15%. Recent evidence suggests that stromal—epithelial interactions are fundamental in carcinogenesis. The advent of co‐culture techniques permits the investigation of cross‐talk between the stroma and epithelium in a physiological setting. We have characterized a histologically representative oesophageal organotypic model and have used it to compare the most commonly used squamous oesophageal cell line, HET‐1A, with primary oesophageal squamous cells for use in studies of the oesophageal epithelium in vitro. Results. When grown in an organotypic culture with normal fibroblasts, the oesophageal carcinoma cell lines OE21 (squamous) and OE19 (adenocarcinoma) morphologically resembled the tumour of origin with evidence of stromal invasion and mucus production, respectively. However, HET‐1A cells, which were derived from normal squamous oesophageal cells, appeared dysplastic and failed to display evidence of squamous differentiation. By comparison with primary oesophageal epithelial cells, the HET‐1A cells were highly proliferative and did not express the epithelial markers E‐cadherin or CK5/6 (casein kinase 5/6), or the stratified epithelial marker ΔNp63, but did express the mesenchymal markers vimentin and N‐cadherin. Conclusion. Studies of epithelial carcinogenesis will benefit from culture systems which allow manipulation of the stromal and epithelial layers independently. We have developed an organotypic culture using primary oesophageal squamous cells and fibroblasts in which a stratified epithelium with a proliferative basal layer that stains strongly for ΔNp63 develops. This model will be suitable for the study of the molecular events in the development of Barrett's oesophagus. The most commonly used normal oesophageal squamous cell line, HET‐1A, does not have the characteristics of normal oesophageal squamous cells and should not be used in models of the normal oesophageal epithelium. Until more representative cell lines are available, future studies in oesophageal cancer will be reliant on the availability and manipulation of primary tissue.     

Organotypic culture of human amnion cells in air-liquid interface as a potential substitute for skin regeneration

Background aimsThe aim of the present study was to evaluate the effects of air-liquid interface on the differentiation potential of human amnion epithelial cells (HAECs) to skin-like substitute in organotypic culture.MethodsHAECs at passage 1–2 were seeded onto a fibrin layer populated with human amnion mesenchymal cells to form the organotypic cultures. The organotypic HAECs were then cultured for 7, 14 and 21 d in two types of culture system: the submerged culture and the air-liquid interface culture. Cell morphogenesis was examined under the light and electron microscopes (transmission and scanning) and analyzed by immunohistochemistry.ResultsOrganotypic HAECs formed a single layer epithelium after 3 wk in submerged as well as air-liquid interface cultures. Ultrastructurally, desmosomes were observed in organotypic HAECs cultured in the air-liquid interface but not in the submerged culture. The presence of desmosomes marked the onset of early epidermal differentiation. Organotypic HAECs were positive against anti-CK18 and anti-CK14 in both the submerged and the air-liquid interface cultures. The co-expression of CK14 and CK18 suggested that differentiation of HAECs into skin may follow the process of embryonic skin development. However, weak expression of CK14 was observed after 2 and 3 wk of culture in air-liquid interface. CK10, involucrin, type IV collagen and laminin-5 expression was absent in organotypic HAECs. This observation reflects the initial process of embryonic epidermal differentiation and stratification.ConclusionsResults from the present study suggest that the air-liquid interface could stimulate early differentiation of organotypic HAECs to epidermal cells, with a potential use for skin regeneration.     

Characterization of organotypic keratinocyte cultures on de-epithelialized bovine tongue mucosa

Organotypic cultures have been used to study epithelial cell behavior for many years. The aim of this study was to develop an organotypic culture method that better mimics the three-dimensional morphology of interdigitating rete ridges and connective tissue papillae and that also conserves the basement membrane zone (BMZ). Bovine tongue mucosa connective tissue, separated from epithelium after 1 M NaCl incubation, was used as organotypic culture substratum for different human keratinocyte cell lines. Organotypic cultures were characterized by electron and immunofluorescence microscopy for expression of integrin subunits and extracellular matrix components. While spontaneously immortalized mucosal keratinocytes produced highly irregular stratified organotypic cultures, the normal human epidermal keratinocytes (NHEK) demonstrated culture morphology that resembled in vivo epidermis. However, in this model, the histomorphology, expression of differentiation markers involucrin, keratin 10 and 14, and integrins varied significantly between the cell lines. Some cultures appeared to have an extended survival since they were maintained up to 40 days without histological signs of degeneration. The ultrastructure of the BMZ including hemidesmosomes was similar to the normal dermo-epidermal junction. Extracellular matrix molecules, including tenascin, laminin-1 and -5, were expressed in the cultures demonstrating their secretion solely by keratinocytes. Distribution and expression of integrins in NHEK cultures was similar to that seen in vivo skin with the exception of additional expression of alpha5beta1 and alpha(v)beta6 integrins. Organotypic NHEK cultures show similarities to normal stratified epithelium and are potentially useful for multiple applications for studies on epithelial cell behavior in vitro.     

Combination of Low Calcium with Y-27632 Rock Inhibitor Increases the Proliferative Capacity,Expansion Potential and Lifespan of Primary Human Keratinocytes while Retaining Their Capacity to Differentiate into Stratified Epidermis in a 3D Skin Model

Human keratinocytes are difficult to isolate and have a limited lifespan. Traditionally, immortalised keratinocyte cell lines are used in vitro due to their ability to bypass senescence and survive indefinitely. However these cells do not fully retain their ability to differentiate in vitro and they are unable to form a normal stratum corneum in organotypic culture. Here we aimed to generate a pool of phenotypically similar keratinocytes from human donors that could be used in monolayer culture, without a fibroblast feeder layer, and in 3D human skin equivalent models. Primary human neonatal epidermal keratinocytes (HEKn) were cultured in low calcium, (0.07mM) media, +/-10μM Y-27632 ROCK inhibitor (HEKn-CaY). mRNA and protein was extracted and expression of differentiation markers Keratin 14 (K14), Keratin 10 (K10) and Involucrin (Inv) assessed by qRT-PCR and Western blotting. The differentiation potential of the HEKn-CaY cultures was assessed by increasing calcium levels and removing the Y-27632 for 72hrs prior to assessment of K14, K10 and Inv. The ability of the HEKn-CaY, to form a stratified epithelium was assessed using a human skin equivalent (HSE) model in the absence of Y-27632. Increased proliferative capacity, expansion potential and lifespan of HEKn was observed with the combination of low calcium and 10μM ROCK inhibitor Y-27632. The removal of Y-27632 and the addition of high calcium to induce differentiation allowed the cells to behave as primary keratinocytes even after extended serial passaging. Prolonged lifespan HEK-CaYs were capable of forming an organised stratified epidermis in 3D HSE cultures, demonstrating their ability to fully stratify and retain their original, primary characteristics. In conclusion, the use of 0.07mM Calcium and 10μM Y-27632 in HEKn monocultures provides the opportunity to culture primary human keratinocytes without a cell feeder layer for extended periods of culture whilst retaining their ability to differentiate and form a stratified epithelium.     

Outer root sheath (ORS) cells organize into epidermoid cyst-like spheroids when cultured inside Matrigel: a light-microscopic and immunohistological comparison between human ORS cells and interfollicular keratinocytes

In organotypic cultures, outer root sheath (ORS) cells of the human hair follicle develop into a stratified epithelium largely reminiscent of the epidermis; this apparently reflects their importance during wound healing. In the present study, ORS cells were grown inside a three-dimensional network of extracellular matrix proteins (Matrigel), together with different mesenchymal cells, in an attempt to mimic their follicular environment. Thus, inside Matrigel, ORS cells formed spheroids differentiating toward the center and showing all the markers of epidermal keratinization. Under identical conditions, normal epidermal keratinocytes developed similar spheroids, but of a significantly smaller size. Human dermal fibroblasts and dermal papilla cells, cocultured in the matrix, had a positive influence on both the proliferation and differentiation within both types of spheroids. Epidermal differentiation markers, such as suprabasal keratins, involucrin, filaggrin, gp80 and pemphigoid antigen, were readily expressed in ORS spheroids, whereas hard (hair) keratins were not detectable by immunostaining. Cells positive for an epithelial membrane antigen, strongly expressed in sebaceous glands, were seen in numerous spheroids. In contrast to organotypic “surface” epithelia, the expression and location of different integrin chains was normalized in ORS spheroids, indicating an enhanced mesenchymal influence in this in vitro system.     

Growth of intestinal epithelium in organ culture is dependent on EGF signalling

Differentiation of endoderm into intestinal epithelium is initiated at E13.5 of mouse development when there are significant changes in morphology resulting in the conversion of undifferentiated stratified epithelium into a mature epithelial monolayer. Here we demonstrate that monolayer formation is associated with the selective apoptosis of superficial cells lining the lumen while cell proliferation is progressively restricted to cells adjacent to the basement membrane. We describe an innovative embryonic gut culture system that maintains the three-dimensional architecture of gut and in which these processes are recapitulated in vitro. Explants taken from specific regions of the gut and placed into organ culture develop and express molecular markers (Cdx1, Cdx2 and A33 antigen) in the same spatial and temporal pattern observed in vivo indicating that regional specification is maintained. Inhibition of the epidermal growth factor receptor (EGFR) tyrosine kinase using the specific inhibitor AG1478 significantly reduced the proliferation and survival of cells within the epithelial cell layer of cultured gut explants. This demonstrates an essential role for the EGF signalling pathway during the early stages of intestinal development.     

Outer root sheath (ORS) cells organize into epidermoid cyst-like spheroids when cultured inside Matrigel: a light-microscopic and immunohistological comparison between human ORS cells and interfollicular keratinocytes

In organotypic cultures, outer root sheath (ORS) cells of the human hair follicle develop into a stratified epithelium largely reminiscent of the epidermis; this apparently reflects their importance during wound healing. In the present study, ORS cells were grown inside a three-dimensional network of extracellular matrix proteins (Matrigel), together with different mesenchymal cells, in an attempt to mimic their follicular environment. Thus, inside Matrigel, ORS cells formed spheroids differentiating toward the center and showing all the markers of epidermal keratinization. Under identical conditions, normal epidermal keratinocytes developed similar spheroids, but of a significantly smaller size. Human dermal fibroblasts and dermal papilla cells, cocultured in the matrix, had a positive influence on both the proliferation and differentiation within both types of spheroids. Epidermal differentiation markers, such as suprabasal keratins, involucrin, filaggrin, gp80 and pemphigoid antigen, were readily expressed in ORS spheroids, whereas hard (hair) keratins were not detectable by immunostaining. Cells positive for an epithelial membrane antigen, strongly expressed in sebaceous glands, were seen in numerous spheroids. In contrast to organotypic surface epithelia, the expression and location of different integrin chains was normalized in ORS spheroids, indicating an enhanced mesenchymal influence in this in vitro system.     

Interpreting epithelial cancer biology in the context of stem cells: tumor properties and therapeutic implications

Over 90% of all human neoplasia is derived from epithelia. Significant progress has been made in the identification of stem cells of many epithelia. In general, epithelial stem cells lack differentiation markers, have superior in vivo and in vitro proliferative potential, form clusters in association with a specialized mesenchymal environment (the 'niche'), are located in well-protected and nourished sites, and are slow-cycling and thus can be experimentally identified as 'label-retaining cells'. Stem cells may divide symmetrically giving rise to two identical stem cell progeny. Any stem cells in the niche, which defines the size of the stem cell pool, may be randomly expelled from the niche due to population pressure (the stochastic model). Alternatively, a stem cell may divide asymmetrically yielding one stem cell and one non-stem cell that is destined to exit from the stem cell niche (asymmetric division model). Stem cells separated from their niche lose their stemness, although such a loss may be reversible, becoming 'transit-amplifying cells' that are rapidly proliferating but have a more limited proliferative potential, and can give rise to terminally differentiated cells. The identification of the stem cell subpopulation in a normal epithelium leads to a better understanding of many previously enigmatic properties of an epithelium including the preferential sites of carcinoma formation, as exemplified by the almost exclusive association of corneal epithelial carcinoma with the limbus, the corneal epithelial stem cell zone. Being long-term residents in an epithelium, stem cells are uniquely susceptible to the accumulation of multiple, oncogenic changes giving rise to tumors. The application of the stem cell concept can explain many important carcinoma features including the clonal origin and heterogeneity of tumors, the occasional formation of tumors from the transit amplifying cells or progenitor cells, the formation of precancerous 'patches' and 'fields', the mesenchymal influence on carcinoma formation and behavior, and the plasticity of tumor cells. While the concept of cancer stem cells is extremely useful and it is generally assumed that such cells are derived from normal stem cells, more work is needed to identify and characterize epithelial cancer stem cells, to address their precise relationship with normal stem cells, to study their markers and their proliferative and differentiation properties and to design new therapies that can overcome their unusual resistance to chemotherapy and other conventional tumor modalities.     

Potential localization of putative stem/progenitor cells in human bulbar conjunctival epithelium

Although the conjunctival fornix appears to contain the greatest proportion of stem cells, it is likely that pockets of conjunctival epithelial stem cells may also exist throughout the conjunctival epithelium. This study was to investigate the potential localization of putative stem/progenitor cells in the human bulbar conjunctival epithelium by evaluating 6 keratins and 13 molecules that have been previously proposed stem cell associated or differentiation markers. We found that cornea specific cytokeratin (CK) 3 was not expressed by the bulbar conjunctival epithelial cells. In contrast, CK4 and CK7 were expressed by the superficial cells of bulbar conjunctival epithelium. CK14 and CK15 were confined to the basal cell layer. CK19 was strongly expressed by all layers of the bulbar conjunctival epithelium. The expression patterns of molecular markers in the basal cells of human bulbar conjunctival epithelium were found to be similar to the corneal epithelium. Basal conjunctival epithelial cells strongly expressed stem cell associated markers, including ABCG2, p63, nerve growth factor (NGF) with its receptors tyrosine kinase receptor A (TrkA) and neurotrophin low‐affinity receptor p75NTR, glial cell‐derived neurotrophic factor (GDNF) with its receptor GDNF family receptor alpha 1 (GFRα‐1), integrin β1, α‐enolase, and epidermal growth factor receptor (EGFR). The differentiation associated markers nestin, E‐cadherin and involucrin were not expressed by these cells. These findings indicate that the basal cells of bulbar conjunctival epithelium shares a similar expression pattern of stem cell associated markers to the corneal epithelium, but has a unique pattern of differentiation associated cytokeratin expression. J. Cell. Physiol. 225: 180–185, 2010. © 2010 Wiley‐Liss, Inc.     

Epidermal keratinocyte stem cells: their maintenance and regulation

The epidermis is a stratified epithelium consisting of inter follicular regions and appendages (hair follicles, sweat glands, sebaceous glands). The dominant cell type (the keratinocyte) is arranged in groups of cells termed epidermal proliferative units (EPUs), and one centrally-located clonogenic stem cell is ultimately responsible for replacing the remainder of the cells in the unit. Evidence is reviewed which indicates that the epidermal Langerhan's cell (ELC), and the cells comprising the dermis, may modify the keratinocyte microenvironment to create stem cell ‘niches’ and cellular diversity within the basal layer.     

Putative epidermal stem cell convert into corneal epithelium-like cell under corneal tissue <Emphasis Type="Italic">in vitro</Emphasis>

Rhesus putative epidermal stem cells are being investigated for their potential use in regenerative corneal epithelium-like cells, which may provide a practical source of autologous seed cells for the construction of bioengineered corneas. The goal of this study was to investigate the potential of epidermal stem cells for trans-differentiation into corneal epithelium-like cells. Rhesus putative epidermal stem cells were isolated by type IV collagen attachment method. Flow cytometry analysis, immunohistology and RT-PCR were conducted to identify the expression of specific markers (β1m α6 integrin, K15, K1/K10, K3/K12 and CD71) on the isolated rapid attaching cells. The isolated cells were cocultured with human corneal limbal stroma and corneal epithelial cells. After coculture, the expression of the same specific markers was evaluated in order to identify expression difference caused by the coculture conditions. K3/K12 expression was analyzed in coculture cells on day 2, 4, 6, 8 and 10. Putative epidermal stem cells in conditioned culture media were used as control. Putative epidermal stem cells were predominant in rapid attaching cells by type IV collagen attachment isolation. Before being cocultured, the rhesus putative epidermal stem cells expressed K15, α6 and β1 integrin, but no CD71, K1/K10 and K3/K12. After coculture, these cells expressed K3/K12 (a marker of corneal epithelial cells), K15 and β1 integrin, but no K1/K10. Cells being not coculture converted into terminally differentiated cells expressing K1/K10. These results indicate that rhesus putative epidermal stem cells can trans-differentiate into corneal epithelium-like cells and, therefore, may have potential therapeutic application as autologous seed cells for the construction of bioengineered corneas.     

Differentiation of Human Umbilical Cord Mesenchymal Stem Cells into Prostate-Like Epithelial Cells In Vivo

Although human umbilical cord mesenchymal stem cells (hUC-MSCs) have been identified as a new source of MSCs for potential application in regenerative medicine, their full potential of differentiation has not been determined. In particular, whether they have the capability to differentiate into epithelial cells of endodermal origin such as the prostate epithelial cells is unknown. Here we report that when hUC-MSCs were combined with rat urogenital sinus stromal cells (rUGSSs) and transplanted into the renal capsule in vivo, they could differentiate into prostate epithelial-like cells that could be verified by prostate epithelial cell-specific markers including the prostate specific antigen. The prostatic glandular structures formed in vivo displayed similar cellular architecture with lumens and branching features as seen for a normal prostate. In addition, the human origin of the hUC-MSCs was confirmed by immunocytochemistry for human nuclear antigen. These findings together indicate that hUC-MSCs have the capability to differentiate into epithelial-like cells that are normally derived from the endoderm, implicating their potential applications in tissue repair and regeneration of many endoderm-derived internal organs.     

Human vaginal epithelial multilayer tissue culture

Summary Fragments of normal human adult vagina, when explanted onto glass slides gave rise to outgrowing sheets of pure epithelium, which had microscopic morphological features in common with normal vaginal epithelium. Infrequent fibroblast contamination was observed. Proliferating epithelial cells formedmultilayers of stratified squamous epithelium and demonstrated a progressive decrease in proliferative activity after 14 days. Continuous lines of epithelial cells were not obtained. Even in the absence of estrogens, transmission electron microscopy revealed evidence of keratinization of the superficial cells of the multilayer. Scanning electron microscopy of the surface of mature epithelial cells in culture revealed ultrastructural features that closely resembled those present on the surface of exfoliated cells obtained by scraping the vagina in vivo. This in vitro tissue culture model of human vaginal epithelium may provide a simple method of studying factors that influence vaginal epithelium growth, maturation and function.     

Long-term survival and characterisation of human umbilical cord-derived mesenchymal stem cells on dermal equivalents

During early embryogenesis, mesenchymal cells arise from the primitive epithelium and can revert to an epithelial phenotype by passing through mesenchymal-to-epithelial transition (MET). Mesenchymal stem cells (MSC) of the Wharton's Jelly of the umbilical cord (UC-MSC) express pluripotency markers underlining their primitive developmental state. As mesenchymal stem cells from bone marrow (BM-MSC) possess a strong propensity to ameliorate mesenchymal tissue damage, UC-MSC might also be able to differentiate into cells apart from the mesoderm, allowing replacement of ectodermal and mesodermal tissues.     

Establishment and optimization of epithelial cell cultures from human ectocervix,transformation zone,and endocervix optimization of epithelial cell cultures

Cervical cancer is a major public health problem and research using cell culture models has improved understanding of this disease. The human cervix contains three anatomic regions; ectocervix with stratified squamous epithelium, endocervix with secretory epithelium, and transformation zone (TZ) with metaplastic cells. Most cervical cancers originate within the TZ. However, little is known about the biology of TZ cells or why they are highly susceptible to carcinogenesis. The goal of this study was to develop and optimize methods to compare growth and differentiation of cells cultured from ectocervix, TZ or endocervix. We examined the effects of different serum-free media on cell attachment, cell growth and differentiation, and cell population doublings in monolayer culture. We also optimized conditions for organotypic culture of cervical epithelial cells using collagen rafts with human cervical stromal cells. Finally, we present a step-by-step protocol for culturing cells from each region of human cervix.