Multipotent Stem Cells in Human Corneal Stroma

Abstract

Keratocytes of the corneal stroma secrete a specialized extracellular matrix essential for vision. These quiescent cells exhibit limited capacity for self-renewal and after cell division become fibroblastic, secreting nontransparent tissue. This study sought to identify progenitor cells for human keratocytes. Near the corneal limbus, stromal cells expressed ABCG2, a protein present in many adult stem cells. The ABCG2-expressing cell population was isolated as a side population (SP) by cell sorting after exposure to Hoechst 33342 dye. The SP cells exhibited clonal growth and continued to express ABCG2 and also PAX6, product of a homeobox gene not expressed in adult keratocytes. Cloned SP cells cultured in medium with fibroblast growth factor-2 lost ABCG2 and PAX6 expression and upregulated several molecular markers of keratocytes, including keratocan, aldehyde dehydrogenase 3A1, and keratan sulfate. Cloned corneal SP cells under chondrogenic conditions produced matrix staining with toluidine blue and expressed cartilage-specific markers: collagen II, cartilage oligomatrix protein, and aggrecan. Exposure of cloned SP cells to neurogenic culture medium upregulated mRNA and protein for glial fibrillary acidic protein, neurofilament protein, and beta-tubulin II. These results demonstrate the presence of a population of cells in the human corneal stroma expressing stem cell markers and exhibiting multipotent differentiation potential. These appear to be the first human cells identified with keratocyte progenitor potential. Further analysis of these cells will aid elucidation of molecular mechanisms of corneal development, differentiation, and wound healing. These cells may be a resource for bioengineering of corneal stroma and for cell-based therapeutics. Stem Cells 2005;23:1266-1275

Stem cells of the adult cornea: From cytometric markers to therapeutic applications

“…In the same year, isolation of stromal stem cells from human cornea was also reported (139). In this latter study, some stromal cells have shown ABCG2 positivity (see also Fig.…”

“…Based on this observation, the side population was selected from the cells released from the corneal stroma by digestion with collagenase and hyaluronidase. In culture, these side population cells showed clonal growth and could be differentiated to express keratocyte, chondrogenic and neurogenic markers (139). The same group has concomitantly showed that while these undifferentiated corneal stromal stem cells predominantly express generic stem cell related genes (Bmi-1, Kit, Notch-1, Six2, Pax6, ABCG2, Spag10, p62/OSIL) in adherent cultures, when passaged in suspension in serum free medium with FGF2 and insulin, they form spheroid pellets, in which keratocyte-like cells secrete an ordered ECM and express mRNAs of known (keratocan, PTGDS, ALDH3A1) and potential (FLJ30046/SLAIN, CxAdR, PDK4, MTAC2D1, F13A1) keratocyte markers (140).…”

In Vivo Confocal Microscopic Analysis of Normal Human Anterior Limbal Stroma

“…Other researchers similarly described high cellularity in the anterior limbal stroma in phalloidin green and propidium iodide stained confocal sections. 19 The cellular components in this region include (i) ABCG2 and PAX6 positive multipotent stromal stem cells, 20 (ii) vimentin positive mesenchymal cells, expressing ESC markers 9 , and (iii) CD34, CD31, Flk-1,VWF positive stromal cells. 12 In another study, we demonstrated the presence of clusters of CD90 and CD105 positive MSCs in the native anterior limbal stroma, along with vimentin positive mature keratocytes distributed singly (unpublished data).…”

Multidrug Resistance Transporters – Roles in maintaining Cancer Stem-Like Cells

“…Since its initial application in bone marrow HSCs, the side population technique based on Hoechst 33342 efflux has been adapted to identify putative stem cells and progenitors in many normal tissues (Zhou et al, 2001;Asakura & Rudnucki, 2002;Leckner et al, 2002;Alvi et al, www.intechopen.com 2003; Summer et al, 2003;Budak et al, 2005;Du et al, 2005, Hussain, et al, 2005. SP cells have also been found in a number of established cancer cell lines as well as tumor samples and have been shown to have stem cell-like properties, overexpress ABCG2, and possess inherent drugresistance (Kondo et al, 2004;Hirschmann-Jax et al, 2004;Haraguchi et al, 2005;Seigel et al, 2005, Chiba et al, 2006.…”