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Objectives: Epithelial stem cells of the eye surface, of the cornea and of the conjunctiva, have the ability to give rise to self renewal and progeny production of differentiated cells with no apparent limit. The two epithelia are separated from each other by the transition zone of the limbus. The mechanisms adopted by stem cells of the two epithelia to accomplish their different characteristics, and how their survival, replacement and unequal division that generates differentiated progeny formation are controlled, are complex and still poorly understood. They can be learned only by understanding how stem cells/progenitors are regulated by their neighbouring cells, that may themselves be differently unspecialised, forming particular microenvironments, known as 'niches'. Stem cells operate by signals and a variety of intercellular interactions and extracellular substrates with adjacent cells in the niche. Technical advances are now making it possible to identify zones in the corneal limbus and conjunctiva that can house stem cells, to isolate and expand them ex vivo and to control their behaviour creating optimal niche conditions. With improvements in biotechnology, regenerative cornea and conjunctiva transplantation using adult epithelial stem cells becomes now a reality., Results and Conclusions: Here we review our current understanding of stem cell niches and illustrate recent significant progress for identification and characterization of adult epithelial stem cells/progenitors at cellular, molecular and mechanistic levels, improvement in cell culture techniques for their selective expansion ex vivo and prospects for a variety of therapeutic applications.

A 3D culture system was used to investigate the behaviour of mesothelial cells present in the wall of human processus vaginalis peritonei. Small tissue fragments placed on collagen sponges were cultured for 7, 14 and 21 days in medium supplemented with 10% FBS, and analysed for the expression and distribution of cytokeratins (CKAE1-AE3, CK19), p63, Ki-67, vimentin, CD34, and HBME-1. Before culture, flat mesothelial cells displayed immunoreactivity for cytokeratins, vimentin and HBME-1, while p63 and CD34 were negative. Mesenchymal cells within the stroma were vimentin-positive and endothelial cells of small vessels displayed positive staining for CD34. Cytokeratins, p63 and HBME-1 were negative in all stromal cells. In cultured fragments, flat mesothelial cells positive for vimentin, cytokeratins and HBME-1 proliferated, lining the fragment surface and migrating into the sponge. Capillaries showed morphological alterations; however, their immunoreactivity was comparable with the stroma prior to culture. Cells that had migrated into the sponge and displayed characteristics of mesothelial progenitors, predominantly spindleshaped and stellate, showed heterogeneous expression of markers especially in late phases of cultivation. These cells were constantly positive for vimentin, a small fraction was cytokeratin-positive and a few displayed HBME-1 immunoreactivity. CD34 was found in cells forming small cavities into the matrix, resembling newly formed blood vessels. Cells that had migrated into the sponge could be isolated and expanded in coculture with feeder NIH.3T3 fibroblasts. This system is suitable for studying growth and behaviour of mesothelial cells within their natural environment, providing a good method for isolation and expansion of their progenitor cells.

Rhesus monkey embryonic stem cells (ESCs) (R366.4), cultured on a three-dimensional (3D) collagen matrix with or without human neonatal foreskin fibroblasts (HPI.1) as feeder cells, or embedded in the collagen matrix, formed complex tubular or spherical gland-like structures and differentiated into phenotypes characteristic of neural, epithelial and endothelial lineages. Here, we analysed the production of endogenous extracellular matrix (ECM) proteins, cell-cell adhesion molecules, cell-surface receptors, lectins and their glycoligands, by differentiating ESCs, forming a micro-environment, a niche, able to positively influence cell behaviour. The expression of some of these molecules was modulated by HPI.1 cells while others were unaffected. We hypothesized that both soluble factors and the niche itself were critical in directing growth and/or differentiation of ESCs in this 3D environment. Creating such an appropriate experimental 3D micro-environment, further modified by ESCs and modulated by exogenous soluble factors, may constitute a template for adequate culture systems in developmental biology studies concerning differentiation of stem cells.

CM-S is an autonomous cell line of human hemopoietic precursor cells inducible to monocyte-macrophage differentiation in response to appropriate inducing agents. CM-S cells produce factors that stimulate their own growth and proliferation, and are also capable of stimulating clonal proliferation of human, but not mouse, monocytic and granulocytic bone marrow progenitor cells in viscous medium. Preliminary purification steps have demonstrated at least two species, one of which (MW 30,000-50,000) retains both these activities, while the other (MW less than or equal to 10,000) apparently retains only the autostimulatory activity. CM-S cells could thus be a useful source for the purification of human colony stimulating factors (CSFs). CM-S cells also respond to factors present in human placenta conditioned medium, known to contain human CSF. This suggests that CM-S cells could provide a homogeneous target cell population for testing CSFs from other human sources.

Epstein-Barr virus (EBV) DNA sequences were detected in four established monoblast or early monocytic cell lines (CM-S, ROV-S, CV-S and AD-S) obtained from bone marrow of children suffering from maturation defects of haematopoiesis. EBV is present in these cells in a latent state. The viral DNA in these cell lines was analysed by Southern blot hybridization, using a set of cloned EBV DNA fragments from the EBV strain B95-8 as probes. A common spectrum of highly related but distinguishable EBV DNA restriction enzyme sequences was found, suggesting some genomic diversity. Propagation of the cells in long-term culture revealed a gradual decrease of EBV copies per cell in all lines with some minor changes in the restriction pattern of the EBV DNA. These findings demonstrate that human precursor monocyte cells may be susceptible to infection by EBV.