Human limbal biopsy–derived stromal stem cells prevent corneal scarring

Abstract

Conventional allograft therapy for corneal scarring is widespread and successful, but donor tissue is not universally available, and some grafts fail owing to rejection and complications such as endothelial failure. We investigated direct treatment of corneal scarring using autologous stem cells, a therapy that, if successful, could reduce the need for corneal grafts. Mesenchymal cells were expanded from small superficial, clinically replicable limbal biopsies of human cadaveric corneo-scleral rims. Limbal biopsy–derived stromal cells (LBSCs) expanded rapidly in media containing human serum, were highly clonogenic, and could generate spheres expressing stem cell genes (ABCG2, Nestin, NGFR, Oct4, PAX6, and Sox2). Human LBSCs differentiated into keratocytes expressing characteristic marker genes (ALDH3A1, AQP1, KERA, and PTGDS) and organized a thick lamellar stroma-like tissue containing aligned collagen and keratan sulfate proteoglycans when cultured on aligned nanofiber substrata. When engrafted into mouse corneal wounds, LBSCs prevented formation of light-scattering scar tissue containing fibrotic matrix components. The presence of LBSCs induced regeneration of ablated stroma with tissue exhibiting lamellar structure and collagen organization indistinguishable from that of native tissue. Because the limbus can be easily biopsied from either eye of an affected individual and LBSCs capable of corneal stromal remodeling can be expanded under xeno-free autologous conditions, these cells present a potential for autologous stem cell–based treatment of corneal stromal blindness.

Alternatives to eye bank native tissue for corneal stromal replacement

“…Since the fibroblast phenotype is reversible in vivo, it is hypothesized that the fibroblasts in the stromal substitutes engineered using serum would revert into keratocyte-like cells once implanted. In accordance with this hypothesis, stem cells injected into the corneal stroma show evidence of differentiation into keratocytes (Basu et al, 2014).…”

Effect of culture medium on propagation and phenotype of corneal stroma–derived stem cells

“…Moreover, the addition of serum has been used to deliberately generate corneal fibroblasts rather than keratocytes [32], however, most researchers would prefer an undifferentiated/inactive phenotype. Cultures that contain a lower percentage of serum, such as 2% (v/v), retain a keratocyte phenotype more effectively, but have much lower proliferation rates [33].…”

“…The use of M199 with the addition of 20% FBS to culture keratocytes, generates MSC that adhere to ISCT criteria [18]. Some researchers have cultured cells in media containing recombinant growth factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and leukaemia inhibitory factor (LIF), in lieu of serum [32,36,37], in order to get a cell phenotype more indicative of a pluripotent stem cell. While this may prove more easily translatable to the clinic compared to serum, the addition of recombinant growth factors, both in research and clinically, can be costly.…”

Regenerative Therapies for the Ocular Surface

“…Recent studies have also shown that mesenchymal stem cells derived from various sources such as bone marrow, dental pulp, adipose tissues and limbal stroma are effective in controlling infl ammation and regeneration of corneal surface (Gu et al 2009 ;Gomes et al 2010 ;Nieto-Miguel et al 2013 ;Rohaina et al 2014 ;Katikireddy et al 2014 ;Acar et al 2015 ;Syed-Picard et al 2015 ). Another group has reported the presence of stromal stem cells at the limbal niche and demonstrated the usefulness of these autologous, mesenchymal-like stem cells in the treatment of corneal stromal scars (Li et al 2012 ;Basu et al 2014 ). Apart from various adult stem cell sources, pluripotent stem cell (PSCs) sources such as the human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been tested for their ability of to generate corneal epithelial, endothelial and stromal cells (Ahmad et al 2007 ;Yoshida et al 2011 ;Hayashi et al 2012 ;Chan et al 2013 ;Sareen et al 2014 ;Mikhailova et al 2014 ;Zhang et al 2014 ).…”