Tung-Tien Sun

Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: Implications on epithelial stem cells

Despite the obvious importance of epithelial stem cells in tissue homeostasis and tumorigenesis, little is known about their specific location or biological characteristics. Using 3H-thymidine labeling, we have identified a subpopulation of corneal epithelial basal cells, located in the peripheral cornea in a region called limbus, that are normally slow cycling, but can be stimulated to proliferate in response to wounding and to a tumor promotor, TPA. No such cells can be detected in the central corneal epithelium, suggesting that corneal epithelial stem cells are located in the limbus. A comparison of various types of epithelial stem cells revealed a common set of features, including their preferred location, pigment protection, and growth properties, which presumably play a crucial role in epithelial stem cell function.

Involvement of Follicular Stem Cells in Forming Not Only the Follicle but Also the Epidermis

The location of follicular and epidermal stem cells in mammalian skin is a crucial issue in cutaneous biology. We demonstrate that hair follicular stem cells, located in the bulge region, can give rise to several cell types of the hair follicle as well as upper follicular cells. Moreover, we devised a double-label technique to show that upper follicular keratinocytes emigrate into the epidermis in normal newborn mouse skin, and in adult mouse skin in response to a penetrating wound. These findings indicate that the hair follicle represents a major repository of keratinocyte stem cells in mouse skin, and that follicular bulge stem cells are potentially bipotent as they can give rise to not only the hair follicle, but also the epidermis.

Correlation of specific keratins with different types of epithelial differentiation: Monoclonal antibody studies

We have prepared three monoclonal antibodies against human epidermal keratins. These antibodies were highly specific for keratins and, in combination, recognized all major epidermal keratins of several mammalian species. We have used these antibodies to study the tissue distribution of epidermis-related keratins. In various mammalian epithelia, the antibodies recognized seven classes of keratins defined by their immunological reactivity and size. The 40, 46 and 52 kilodalton (kd) keratin classes were present in almost all epithelia; the 50 kd and 58 kd keratin classes were detected in all stratified squamous epithelia, but not in any simple epithelia; and the 56 kd and 65-67 kd keratin classes were unique to keratinized epidermis. Thus the expression of specific keratin classes appeared to correlate with different types of epithelial differentiation (simple versus stratified; keratinized versus nonkeratinized).

Differentiation of the epidermal keratinocyte in cell culture: Formation of the cornified envelope

Human epidermal keratinocytes grow from single cells into stratified colonies. Cells in the upper layers of the colonies lose their ability to divide and begin terminal differentiation. In this process, there develops a cornified cell envelope that remains insoluble after heating in solutions of sodium dodecylsulfate and beta-mercaptoethanol. The insolubility of the cornified envelope depends upon proteins, since after treatment with proteolytic enzymes, the envelope becomes soluble in the detergent. Cells with cornified envelopes can be identified under the light microscope either in living colonies or following fixation and silver nitrate staining. Keratinocytes of the basal layer move in a characteristic way, but cornified cells do not move at all and form an immobile upper layer in the colonies. Keratinocytes disaggregated from growing colonies are of differing size and density, and can be separated on isopycnic gradients of Ficoll. The DNA-synthesizing cells are small (mean diameter 14 mum). The nonmultiplying cells are large and have a protein content proportionate to their size. Their final diameter may exceed 30 microns (volume increase greater than 10 fold). Cornified envelopes are found in some of the large cells but in none of the small. In growing colonies, usually 5-10% of the cells have cornified envelopes. The fraction is reduced in colonies growing in the presence of epidermal growth factor. Strain XB, an established keratinocyte line of mouse teratomal origin, also forms cornified envelopes, but the kinetics of the process are different, indicating that the program of terminal differentiation is not initiated at corresponding times in the two cell types.

Immunofluorescent staining of keratin fibers in cultured cells

Antibody prepared against a group of keratins purified from human stratum corneum was used to identify cells containing keratins by immunofluorescence. In sectioned tissue and in culture, keratinocytes of skin and other stratified squamous epithelia-whether human, rabbit of mouse-stained strongly, indicating homologous amino acid sequences in the keratins of these species. In all cases, the antibody revealed a dense cytoplasmic network of discrete fibers probably consisting of aggregated (tono-) filaments. The pattern of staining was not affected by cytochalasin B or colcemid. No keratins were detected in cultured cells of mesenchymal origin (3T3, NIL, BHK, human diploid fibroblasts) or in connective tissues, indicating that the 100 A filaments of fibroblasts are not related to the keratins. Keratinocytes at all stages of differentiation, including basal cells, stained brightly and therefore contained abundant keratins.

Intrinsic and extrinsic regulation of the differentiation of skin, corneal and esophageal epithelial cells

Abstract Basal cells of the stratified squamous epithelia of rabbit skin, cornea and esophagus appear morphologically similar. However, the histological features of their subsequent differentiation are different, and the three epithelia are characterized by distinctive keratin proteins. To analyze the relative importance of intrinsic versus extrinsic factors in regulating the differentiation of these epithelia, we compared their behavior under identical in vitro and in vivo conditions. When cultured in the presence of 3T3 feeder cells, keratinocytes from all three epithelia formed differentiating colonies. Although in culture the three cell types approached a common phenotype, they remained distinguishable morphologically and, in some cases, biochemically. When these cultured epithelial cells were trypsinized, suspended in medium and injected subcutaneously into athymic (nude) mice, each of the three cell types generated a characteristic cyst consisting of stratified squamous epithelium. Cultured skin, corneal and esophageal keratinocytes formed epithelia which were keratinized, nonkeratinized and parakeratinized, respectively. In addition, the injected skin and esophageal epithelial cells reacquired their distinctive in vivo keratin patterns. These data suggest that the three epithelia are not equipotential. Furthermore, since the distinctive in vivo phenotype of each epithelium was expressed when the cells were transplanted to the same subcutaneous site, the expression of these differences does not depend on specific mesenchymal instruction but on permissive factors not present in the culture system. Thus under in vivo conditions intrinsic divergence must play a predominant role in determining the characteristic phenotypes of the three epithelia. On the other hand, the finding that the morphological and biochemical differentiation of a given epithelium can be reversibly modulated by the external environment demonstrates that extrinsic factors may, under certain conditions, also play a role in regulating epithelial differentiation.

Proximal location of mouse prostate epithelial stem cells: a model of prostatic homeostasis

Stem cells are believed to regulate normal prostatic homeostasis and to play a role in the etiology of prostate cancer and benign prostatic hyperplasia. We show here that the proximal region of mouse prostatic ducts is enriched in a subpopulation of epithelial cells that exhibit three important attributes of epithelial stem cells: they are slow cycling, possess a high in vitro proliferative potential, and can reconstitute highly branched glandular ductal structures in collagen gels. We propose a model of prostatic homeostasis in which mouse prostatic epithelial stem cells are concentrated in the proximal region of prostatic ducts while the transit-amplifying cells occupy the distal region of the ducts. This model can account for many biological differences between cells of the proximal and distal regions, and has implications for prostatic disease formation.

Cultured epithelial cells of cornea, conjunctiva and skin: absence of marked intrinsic divergence of their differentiated states.

Keratinocytes of three different epithelia grown in cell culture express a large number of differentiation markers with either no differences or relatively small differences, depending on the species. Much of the distinctive phenotype of these epithelia in vivo must be due to external modulation and relatively little, at least in the case of the human, to permanent intrinsic divergence during development.

Comparison of Limbal and Conjunctival Autograft Transplantation in Corneal Surface Reconstruction in Rabbits

Destruction of corneal surface was created in one eye of 24 rabbits by n-heptanol corneal epithelial debridement and surgical removal of limbal zone. One month later, the animals were equally subdivided into three groups of eight for limbal transplantation, conjunctival transplantation, and control without transplantation. During a 6-month postoperative follow-up, all corneas in the control group showed progressive vascularization and conjunctivalization. All corneas with limbal transplantation showed progressive decrease of vascularity, verified by fluorescein angiography. In contrast, all but one of the eight corneas of conjunctival transplantation showed progressive vascularization (P = 0.01). More important, the resultant epithelia showed corneal phenotype in limbal transplantation, but remained conjunctival in conjunctival transplantation, as verified by monoclonal antibodies AM-3, APSM-1, and AE-5. These results support the concept of the limbal location of corneal epithelial stem cells, and indicate that complete destruction of the limbal zone resulted in corneal vascularization and conjunctivalization, and that limbal transplantation has a better efficiency than conjunctival transplantation in restoring such destroyed corneal surface.

Possible Mechanisms for the Loss of Goblet Cells in Mucin-deficient Disorders

By studying the pathological changes in human conjunctival flaps and the conjunctival transdifferentiation in rabbits, we have identified and correlated two pathological processes with the loss of goblet cells: (1) loss of vascularization, and (2) intense inflammation. Loss of vascularization may be correlated with the loss of goblet cells in the chronic cicatricial stage of various mucin-deficient disorders, whereas inflammation may be responsible for their absence in the acute inflammatory stage. The exact interrelationship between these two processes remains unknown. The loss of goblet cells appears to be an early sign of a spectrum of squamous metaplasia, an abnormality of epithelial differentiation. We therefore speculate that loss of vascularization and intense inflammation may have an important effect on epithelial differentiation.