Identification of progenitor cell-rich sites of the conjunctiva

The conjunctiva is a mucous membrane which forms the majority of the ocular surface, and plays a key role in ocular surface defence and maintenance of the tear film. Ex vivo expansion of conjunctival epithelial cells offers potential to reconstruct the ocular surface in cases of severe cicatrising disease; but in order to ensure long term success, conjunctival stem cells which produce both keratinocytes and goblet cells must be present. An initial biopsy rich in stem cells would aid this, however the distribution of human conjunctival stem cells has not been clearly elucidated. I hypothesised that human conjunctival progenitor cells reside in specific areas of the tissue. A surgical method to retrieve whole human conjunctival tissue for research purposes is described. Expression of the stem cell marker ABCG2 and the transit amplifying cell marker p63 was assessed across 22 different regions of such fixed paraffin-embedded tissue, with significantly higher expression of ABCG2 demonstrated basally in the medial canthal and inferior medial/central forniceal areas. Tissue was also cultured ex vivo, and clonogenic ability assessed across 8 different regions. Significantly higher colony forming efficiency was demonstrated in the medial canthal and inferior forniceal areas. Similar significant patterns were demonstrated for the expression of the stem cell markers ABCG2, ΔNp63 and Hsp70 in these cultures, with highest expression of each in these same areas, and significant associations between each marker. Increasing donor age and longer post mortem retrieval times were associated with significantly lower ABCG2 expression in fixed tissue, colony forming efficiency, and stem cell marker expression in cell cultures. Preliminary propagation studies demonstrated that conjunctival epithelial cell growth is supported by fibronectin, collagen IV and laminin 1. This is the first study to comprehensively assess the distribution of human conjunctival progenitor cells. Substantial evidence is here presented that progenitor cells are distributed basally throughout the human conjunctiva, but with highest levels in the medial canthal and inferior forniceal areas. This region may offer physical protection and niches which are rich in goblet cells, vasculature, melanocytes and immune cells. Biopsies from this area, from younger donors, and with short post mortem retrieval times offer the greatest potential to developing stem cell-rich epithelial constructs for transplantation