The stem cells involved in renewal of the corneal epithelium are located in the basal region of the limbus, a narrow transition zone surrounding the cornea. In many ocular surface disorders loss of these stem cells results in partial or complete vision loss. Conventional corneal transplant in these patients is associated with dismal results. Stem cell transplantation offers new hope to such patients. The umbilical cord is emerging as an important source of stem cells that may have potential clinical applications. There are advantages to the use of umbilical cord stem cells as these cells are less immunogenic, non-tumorigenic, highly proliferative and ethically acceptable. In this study, we have confirmed the expression of several putative limbal stem cell markers such as HES1, ABCG2, BMI1, CK15 as well as cell adhesion-associated molecules INTEGRIN-α6, -α9, -β1, COLLAGEN-IV and LAMININ in our recently characterized CLEC-muc population derived from human umbilical cord. Ex vivo expansion of these cells on a human amniotic membrane substrate formed a stratified cell sheet that similarly expresses some of these molecules as well as cornea-specific cytokeratins, CK3 and CK12. Transplantation of a bioengineered CLEC-muc sheet in limbal stem cell-deficient rabbit eyes resulted in regeneration of a smooth, clear corneal surface with phenotypic expression of the normal corneal-specific epithelial markers CK3, CK12 but not CK4 or CK1/10. Our results suggest that CLEC-muc is a novel stem cell that can be ex vivo expanded for corneal epithelial regeneration in the treatment of various eye diseases.

Umbilical cord tissue is gaining attention as a novel source of multipotent stem cells because it is easily obtainable, ethically acceptable and the cells are immunologically naïve. In this study, we have isolated and characterized a new cell type expressing MUCIN1 (CD227) from human umbilical cord lining which we termed MUCIN-expressing Cord Lining Epithelial Cell (CLEC-muc). We found that CLEC-muc is highly proliferative and had significant clonogenic ability. These cells express embryonic stem cell markers OCT-4, NANOG, SSEA-4, REX1 and SOX2. Despite the abundant expression of epithelial cell marker MUCIN1 and cytokeratins, this population is also positive to the mesenchymal stem cell (MSC) marker CD166. CLEC-muc is unique in p63 expression that shuttles from the cytoplasm to the nucleus over time in culture. To understand p63 regulation and function in CLEC-muc, cells were treated with BMP4, a potent morphogen that plays a role in epidermal differentiation via p63 upregulation in ES cell and subsequent analyses were done. We found that BMP4 does not alter cytoplasmic expression of p63 that promotes cell proliferation. However, it increases nuclear p63 expression together with several other epithelial-associated genes such as GATA3, JAGGED1, NOTCH1, HES1 and IKKα. BMP4 has also been found to weakly induce deltaNp63 expression in CLEC-muc. Our results suggest that CLEC-muc is a novel stem cell-like population that can be further differentiated by BMP4 to generate specific cell-types probably destined to form non-keratinized epithelia.