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REVIEW

Models of epithelial histogenesis
A. Casasco, M. Casasco, A. Icaro Cornaglia, F. Riva, A. Calligaro
Department of Experimental Medicine, Histology and Embryology Unit, University of Pavia, Pavia Italy

©2007, European Journal of Histochemistry
Epithelial histogenesis: a chance to unveil the
face of human stem cells
Epithelial tissues emerge from coordinated sequences of cell Although epithelial cells are characterized by
renewal, specialization and assembly. Like corresponding imma- common structural features, especially their
ture tissues, adult epithelial tissues are provided by stem cells
which are responsible for tissue homeostasis. Advances in arrangement into cohesive sheets or thee-dimen-
epithelial histogenesis has permitted to clarify several aspects sional aggregates, they provide an enormous variety
related to stem cell identification and dynamics and to under- of biological functions, including protection, absorp-
stand how stem cells interact with their environment, the so-
called stem cell niche. The development and maintenance of tion, secretion, gametogenesis and special senses.
epithelial tissues involves epithelial-mesenchymal signalling A major difference between developing and
pathways and cell-matrix interactions which control target nuclear mature epithelial tissues is that differentiated
factors and genes. The tooth germ is a prototype for such induc- epithelial cells that are not prone to locomotion. On
tive tissue interactions and provides a powerful experimental sys-
tem for the study of genetic pathways during development. the other hand, the migration of immature epithelial
Clonogenic epithelial cells isolated from developing as well cells is essential for the development of many
mature epithelial tissues has been used to engineer epithelial tis- organs, including most of exocrine and endocrine
sue-equivalents, e.g. epidermal constructs, that are used in clin-
ical practise and biomedical research. Information on molecular glands, skin appendages and teeth. However, most of
mechanisms which regulate epithelial histogenesis, including the adult epithelial tissues are dynamic as to cell renew-
role of specific growth/differentiation factors and cognate recep- al and cell cycle activity. In fact, many processes
tors, is essential to improve epithelial tissue engineering.
that are observed during development are operating
Key words: epithelial histogenesis – developmental biology – also in mature state, thus ensuring tissue homeosta-
stem cell – differentiation – epithelial-mesenchymal interac- sis. Accordingly, most – if not all - of adult epithe-
tions – tissue engineering. lial tissues are provided by specific stem cells and
Correspondence: Casasco Andrea, may be regarded to as useful models to study tissue
Department of Experimental Medicine formation and repair.The occurrence of stem cells in
Histology and Embryology Unit adult epithelial tissues has permitted the generation
Via Forlanini, 10 27100 PAVIA, Italy
Tel: +39.0382.987272. of bioengineered epithelial constructs that can be
Fax: +39.0382.528330. applied in cell and tissue therapy.
E-mail: andrea.casasco@unipv.it Epithelial stem cells, like other adult stem cells,
European Journal of Histochemistry are thought to be slow- or rarely-cycling cells,
2007; vol. 51 supplement 1:93-100 which retain clonogenicity and proliferative capac-
ity for a long time. According to current models of
tissue homeostasis, the division of a stem cell gives
rise to another stem cell and one transient amplify-
ing cell. Such a cell, after exhausting its prolifera-
tive potential, undergoes terminal differentiation,
thus generating functional cells which are not fur-
ther capable of proliferation (Leblond, 1981;
Potten, 1983; Fuchs, 1990; Potten and Loeffler,
1990; Jones et al., 1995). Since stem cells are slow
cycling, they minimise DNA replication-related
errors. Stemness properties are greatly conditioned
by the microenvironment and positional creden-

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and epidermal construct (F). x 400 (C.B.F). Li et al. it is possible to observe a thin basement membrane at the dermo-epidermal junction (G).g. been proposed which. thus suggesting the existence of a specific cation of molecular markers which allow the niche for each stem cells (Fuchs et al. x 300 (A. bioengineered skin (E). Tissue architecture of human nat- ural skin (A). x 20000 (G). compared to other tissues. Figure 1. A challenge in stem cell research is the identifi- 94 . major advantage of studying epithelial histogene. bilayered human skin equivalent (B). Casasco et al. 1993. tials. 2004).E). Candidate markers for epithelial stem cells have sis is that stem cells are confined in discrete posi.. 2004. In human skin equivalent. x 26000 (H).D. e. well-developed desmosomes between cells of the suprabasal layer (H) and irregular keratohyalin granules comparable to those observed in natural skin (I). correlated with cytokinetic tions. the basal layer of stratified epithelia. 2004). Aspects of histogenesis and cell differentiation in models of epidermis engineered in vitro.. Magn. A recognition of immature cell populations in tissues. have permitted the isolation and thus making easier the identification of their niche cloning of epithelial stem cells (Jones and Watt. parameters.A.. x 18000 (I). and simple epidermal construct (C). whereas connective tissue cells in the dermis (D) and dermal equivalent (E) are negative. Blanpain et al. Cytokeratins are immunodetected thoroughout the cytoplasm of keratinocytes in natural skin (D).

it is possible to show that the secretion of Tooth morphogenesis proceeds through charac. in the matura- is possible to monitor continuously cell differenti. ectomesenchyme cells. 2003). 1993. 1987. being 1985.. the production cessing of enamel matrix which will result in the and secretion of specific molecules. during odontogenesis. 2000. bud. (Figure 2). tiated cells. tion which immediately precede the secretion of late the initiation of tooth formation. will die as soon as tooth erupts. ameloblast differentiation is shown in Figure 3. the differen. Review Enamel epithelium: the histogenesis of the secretory stage. 1988). Bronckers et al. the cells of the inner enamel hardest tissue epithelium proliferate and acquire terminal cytod- The tooth germ represents a powerful model to ifferentiation. ameloblasts are involved in the pro- ation in relative spatial positions.. 1976. phases of odontoblast and ameloblast differentia- Epithelial-mesenchymal interactions that regu. 1992. Casasco et al. 1985. specific dentine and enamel matrices (Slavkin et tiation of odontoblasts and ameloblasts and the al. it has also been suggested that cells of the tory and maturation phases (Warshawsky and Hertwig sheath may undergo epithelial-mesenchy- Smith. The interaction of a cell with the surrounding odontoblasts and cementoblasts (reviewed by extracellular matrix influences cell proliferation Sharpe. the earliest evidence of cross the basement membrane in the epithelial- tooth development is an epithelial thickening of mesenchymal interface (i. later replaced by the cementum. Smith and Warshawsky. Thesleff. (reviewed by Bosshardt and Schroeder. secre.e. Jernval and Thesleff. the inner enamel epithelium undergoes a and temporal aspects of odontoblast and precise developmental program. initiation. Jernval and Thesleff.. sion of enamel specific genes is restricted to deter- 1968. enchyme. Cobourne and Sharpe. and differentiation gene expression via specific These two cells types interact to control the entire membrane receptors which activate downstream process of tooth initiation. become functional and secrete spe- mal interactions. It has been shown that expres- studies on tissue recombination (Kollar and Baird. Much interest has been given to the cytodifferentiation.Thesleff. it cific enamel matrix components. 2003). Indeed. which can be properly called esis which are mediated by epithelial-mesenchy.. Although it is gen- The differentiation programme of the cells of the erally believed that cementoblasts differentiate from inner enamel epithelium can be summarized in dental follicle. 1990. ameloblasts. which form after which time the enamel cannot be replaced by ameloblasts. mined enamel epithelium cells that have with- Furthermore. and corre. mal transformation and give rise to cementoblasts Nanci et al. drawn from the cell cycle and have undergone ter- nalling pathways and related target nuclear fac. 2003). 1998). enamel specific proteins immediately precedes teristic stages. Thesleff and Mikkola. As in many organs. 2000. epithelium forms the so-called Hertwig root sheath ucts which direct enamel biomineralization (Ruch. including pre-secretory. cap and bell dentine mineralization and that enamel proteins stages. 1994). which are matrix. ultimately differ. 2001. 1985. 1996). 1991. morphogenesis and target genes. and cranial neural-crest derived new synthesis. during the secretory stage. which derive from cranial ectomes- three main phases. Couwenhoven acquisition of shape have been characterized by and Snead. 1975. cells: stomodeal ectoderm cells. 95 . (Inai et al. ameloblasts. proposed as candidate regulatory molecules in Slavkin. formation of the hardest tissue of human body. As sponding modifications of the extracellular in a romantic drama. i. tion stage. located at the surface of the tooth crown and have Mammalian teeth develop from two types of fulfilled their task. differen- understand molecular mechanisms of organogen. A simplified scheme describing spatial enchyme.. During pre. Under the enamel junction) and reach the odontoblasts layer instructive influence of the odontogenic mes. 1996). dental epithelial-mesenchymal interactions. cell-to-cell and cell-matrix sig. the future dentine- the stomodeal lining epithelium. cal communication between dental epithelial and Dentine and enamel specific proteins have been mesenchymal cells (reviewed by Ruch. 1974. which defines the final size of the tooth root. minal differentiation to the ameloblast phenotype tors have been identified as mediators of recipro. In fact.e. 2002. Lumsden. entiates to the ameloblast phenotype and initiate The extension downward of cells of the enamel the expression of tissue-specific enamel gene prod. which form pulp cells.

2002). Replacement of peridermal cells.. TP. The primordium of the epidermis is a single layer of surface ectodermal cells. x 25000 (D). In the adult. entiation. epidermis is a dynamic tissue in which terminally differentiated keratinocytes are replaced by the proliferation of new epithelial cells that undergo differentiation. including 96 . In the epider- x 500 (A). characteristic cytokeratin expression. Intracytoplasmic localization of enam. x 400 (B). thereafter the periderm disap- pears and the stratum corneum forms. which are part of the vernix caseosa. 2004). Golgi apparatus. integrin) has permitted the isolation of subpopula- latory molecules of odontogenesis may find a role in tions of epidermal cells showing stemness properties regenerative periodontal therapy (Gestrelius et al. F: immunogold detection of enamel matrix proteins within dermal stem cells (Blanpain et al. Recent data support the view that keratinisation may be regarded to as a specialized form of apop- tosis that produces the stratum corneum concomi- tant with keratinocyte cell death (Hathaway and Kuechle. since it is expressed in all basal cells as 1998). skin develops from the interaction of surface ectoderm and underlying mesenchyme.. keratins 5 and 14 are expressed in the basal layer. while keratins 1 and 10 are found in the suprabasal layer. continues until about the 21st week. D: immunogold detection of brome-deoxyur. Keratinocyte exhibit DP.Tumbar et al. 2004). C: Cell prolif- cells undergoes an irreversible commitment to differ- eration in tooth germ as observed by immudetection of brome. clinical studies have demonstrated well as a significant number of suprabasal cells. 2004. p63 is not restricted development (discussed in Bosshardt and Nanci. inner enamel epithelium.A. transferrin receptor CD71 and α-6 esis and new bone deposition. Li et al. Tomes process. stem cell properties are lost Figure 2. which is not cellular but composed of intracytoplasmic remnants bound to the skin surface after the death of keratinocytes. Keratinocytes express several integrins. that the application of enamel proteins in bone Interestingly. x 150 (C). Aspects of histogenesis and cell differentiation in rat gradually through successive rounds of division. odontoblast layer. These cells proliferate and differentiate to form a layer of squa- mous epithelium. OBL. Specific microenvironmental factors that deoxyuridine: the number of positive cells in the inner enamel epithelium decreases from the cervical loop (CL) toward the regulate the growth and differentiation of ker- forming cusp (FC). According to the spiral model of stemness pro- posed by Potten (1990). Nevertheless.F). inner enamel epithelium. 2004. suggesting that regu. 2000). whereas more committed progeny of epidermal stem el matrix proteins (A) and 28 Kda-calciun binding protein (B) during early stage of ameloblast differentiation... IEE. Terminal differentia- tion of epidermal keratinocytes leads to the forma- tion of the stratum corneum. important role in cementogenesis during tooth McKeon. Fuchs et the cytoplasm of a secretory amelobast as well as the forming enamel. and a basal ger- minative layer. dental pulp. Koster and Roop.g. al. (Jones and Watt. as unequivocal criteria for the identification of epi- E. markers (e.. 2001. Recently. to stem cells.. atinocyte progenitors remain poorly defined as well dine within the nucleus of an immature cell of the inner enam- el epithelium which is traversing the S phase of the cell cycle. 2004. 2004). Magn. GA. called periderm. Casasco et al. Skin histogenesis: the story of the first bioengineered organ In developing embryo. 1993. x 40000 (E. a combined identification of specific defects around human teeth stimulates cementogen. mis. The transcription factor p63 has Enamel-related proteins secreted by epithelial cells been proposed as a marker for keratinocyte stem of the Hertwig sheath are supposed to have an cells (Pellegrini et al.

It has been shown that integrins not only ing have permitted the generation of skin and epi- mediate adhesion to the underlying extracellular dermal substitutes in vitro. 1997). fibronectin. sional models. Recent advances in tissue engineer- receptors. 2004). Li et al. collagen-. Subsequently. the future dentine-enamel junc- tion) and reach the odonto- blast layer. x 1000. Interestingly. as well as enamel formation is delayed compared to dentine forma- tion. 1975).. Marchisio et al. including devel- (Adams and Watt. but also regulate keratinocyte differentiation been conceived to engineer such substitutes and to (Watt et al. laminin-.e. Magn. The microscopical picture shows the stages of the cells of the odontoblast layer (OBL) and of the inner enamel epithelium (IEE) which precede and go along with ini- tial deposition of dentine and enamel. dermal constructs have been combined with dermal 97 .. epithelial layers similar to natural epidermis logical experiments and eliminate the flat biology of (Rheinwald and Green. Different strategies have matrix.and vitronectin. oping and adult tissues. the secretion of enamel specific proteins imme- diately precedes dentine min- eralization and enamel pro- teins cross the basement membrane in the epithelial- mesenchymal interface (i.. Ameloblasts with- draw from the cell cycle later that odontoblasts. Long-term subcultivation of A major aim for tissue engineers is to develop new keratinocytes in vitro permitted the formation of culture systems to change the way to conduct bio. indeed date skin can be regarded to as the first bioengi- detachment from the basement membrane seems to neered organ. Epidermal and dermal stem cells can be a prerequisite to undergo terminal differentiation be isolated from different sources. epi- Petri dishes in favour of organotypic three-dimen. 1990. The scheme summa- rizes corresponding stages of cell differentiation and extra- cellular matrix maturation. 1993. Review Figure 3.

270: 415-23. of proliferating cell populations during tooth development. Aurora Farina (Department Stark et al.. Socializing with the neighbors: stem and differentiation. Parenteau et al. et al.R. Gay tional and time profiles. 264: 261-72.A. 134: 293- regulate the mechanisms of epithelial histogenesis 300. Further experiments permitted the introduc. Casasco M. The application of our knowledge dine. and the existence of two cell populations within an epithelial stem niche. 1996. Cell Tissue Res 1992. 35: 421-8. Butler WT.b. multipotency. regulate epithelial histogenesis has been used to Cobourne MT. 1993). Tooth and jaw: molecular mechanisms of induce tissue regeneration in surgical procedures. and hairs in advanced models of artificial skin. Langerhans cells. where the need is to induce or enhance cell growth Fuchs E. Anat Rec. Margolis D. Tissue engineering experiments suggest that skin histogenesis is controlled by epithelial-mesenchy- References mal interactions (Smola et al. 245: 267-92. 1993. Cell 2004. J Molecular Histol 2004. Farina A. such as complete morphologic differ. thus ensuring the regula. Proliferative and functional stages of rat ameloblast differentiation as revealed by combined immuno- op. Icaro Cornaglia A. hypoxia and physical stimuli. Fuchs E. 272: 237-47. 1984. genetic Cell proliferation and differentiation in a model of human skin equiv- alent. 245: 162-73. patterning in the first branchial arch. discussed in Turksen.. This research was sup- dermal cells have been shown to have many in vivo. Dentin sialoprotein: biosynthesis and devolopmetal appear- ance in rat tooth germs in comparison with amelogenin. J Histochem Cytochem 1998. cytochemistry against enamel matrix proteins and bromodeoxyuri- cial equivalents. Sharpe PT. 48: 1-14. Epidermal differentiation: the bare essentials. Early determination and permissive expression of amelogenin transcription during mouse mandibular from the nature for the design of innovative thera. Falanga V. Casasco A. Changes in keratinocyte adhesion during terminal al extracellular matrices of the dermo-epidermal differentiation: reduction in fibronectin binding precedes α5β1 inte- grin loss from the cell surface. Guasch G. we might understand how engineer their artifi. Indeed. topical issue “The biology of dental tissues” 1996. Living tissue formed in vitro been implicated in the regulation of keratinocyte and accepted as skin-equivalent tissue of full thickness. junction and diffusible factors acting locally have Bell E. If we understand how tissues devel. 2001a. cine. 2005). Calligaro A. Casasco A. Acknowledgements ture (Bell et al. Auletta M. 98 . osteocalcin tion of cell proliferation. Cementogenesis reviewed: a comparison Future perspectives between human premolars and rat molars. physical factors play their role with specific posi. Lyaruu DM. Banca del Monte di Lombardia Foundation entiation. 1998) as well as high-fidelity mod. used in clinical practise (Gallico et al. 46: 135-42. Anat Rec 2001. The application of immunocytochemistry for the detection in epithelial histogenesis has permitted the genera. D'Sousa R. 118: 635-48. first molar development. Casasco A.. pres. Riccardo Rizzoli. els for quantitative research in biology and medi. Immunolocalization of epithelial and mes- of multiple signalling cascades. Maggiacomo F. This paper is dedicated to our master Prof. blood vessels all pertinent works. Cell kinetics in a model of artificial skin. Casasco et al. Science 1981. Emilio Casasco and our friend Prof. and molecular and enchymal matrix constituents in association with inner enamel epithelial cells. Icaro Cornaglia A. 1999. ported by grants from the University of Pavia like features. et al. cells and their niche. Moreover. Polak L.. Bronckers ALJJ. 45: 125-30. Calligaro A. Nakatsuji T. including tissue responses to drugs. Dev Biol 1994.).. Casasco M. 1991. Cell 2004. AC 2004-2006) and COFIN (AC. University and mesenchymal interactions (Casasco et al.A. 164: 290-9. We are grateful to Mrs. Tumbar T. Calligaro A. and collagen type-I. 63: 425-35. University of Pavia) for organotypic co-culture made of keratinocytes and valuable technical assistance. Alvarez O. The Authors apologizes to all contributors 2004). sever. of Experimental Medicine. Epithelial histogenesis involves dynamic patterns Bosshardt DD. information on the mechanisms that Differential distribution of elastic tissue in human natural skin and tissue-engineered skin. 1981. 211: 1052-4. Zerbinati N. Arch oral Biol 2003. according to biomimetics which derives principles Couwenhoven RI. Although the precise mechanisms are largely hypothetical. Nanci A. Casasco M. growth and differentiation and skin homeostasis Blancpain C. will find biotechnological and clinical application Fuchs E. tional assembly. and epithelial. Rapid heal- It is reasonable to believe that other factors which ing of venous ulcers and lack of clinical rejection with an allogenic cultured human skin equivalent. 116: 769-78.. Anat Rec . Casasco M. equivalents to reconstruct the entire skin architec. Research. Zerbinati N. peutical strategies and tissue engineering systems. Eur J Histochem 2001. Bosshardt DD. van Dijk S. Lowry WE. S. Mazzini G. alterations. (F. Cell 1990.. assembly of a basement membrane. Icaro Cornaglia A. 1998). AC 2003) ence of cells with stem-like features. 111: 2807-14. Casasco A. Buttle DJ. Adams JC. from the Italian Ministry of Education. Self-renew- (Smola et al.. Watt FM..The human skin equivalent investigators group. Altrman M. Snead ML. Geoghegan A. in skin and tooth research for inability to acknowledge tion of melanocytes. (AC. al. Calligaro A. Cell Tissue Res 1993. Arch Dermatol 1998. An immuno- Falanga et al. Schroeder HE. differentiation and func. tion in vitro of tissue equivalents that are currently topical issue “The biology of dental tissues”. Ehrlich HP. Zacchi et al. J Cell Biol 1990. Casasco A. histochemical and flow cytometric analysis.

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