Abstract
Purpose :
Current ex-vivo protocols applied to LSCs primary culture lead to loss of stemness and proliferative capacity within 4-7 passages. An inhibitor of ROCK pathway (Y27632) has been shown to contribute to long term maintenance of these cells. The aim of this study is to assess the effect of this Rock Inhibitor (Ri) in ex-vivo cultured LSCs with a scope on their therapeutic (transplantation) potential and their usage as in vitro cell model. The question that we address is whether LSCs maintain their “identity” throughout passages while treated with Rock Inhibitor.
Methods :
LSCs were isolated from four human subjects (age: 52, 72, 78 and 80) and cultured on mitotically inactivated 3T3 feeder cells with (+) or without (-) Ri. Throughout the passages LSCs were assessed for their colony forming efficiency, population doubling, genomic stability and differentiation ability using air liquid interface (ALI) organotypic cultures. Samples were collected on each passage of Ri+ and Ri- LSCs to perform Bulk RNA-Seq. (fig.1)
Results :
Cultures without Ri ceased proliferating at passage 7, became senescent and changed their morphology while Ri(+) LESCs maintained their proliferative ability and morphology throughout the passages (passage 51 and ongoing), moreover Ri(+) condition had a higher proliferative rate. RNA-Seq analyses demonstrated a decrease of differentiation markers (KRT3, KRT12) and an increase in the expression of putative stem/progenitor cell marker TP63 throughout the passages in Ri(+) LSCs, indicating enrichment of LSCs through the passages and loss of the differentiated cells. Unlike Ri(-) condition, Ri(+) LSCs maintained a constant expression of mitogenic marker MKi67 and putative limbal stem/progenitor markers KRT14 and ABCG2.
Conclusions :
Our data indicates that LSCs can be maintained and differentiated ex-vivo with Ri for extended periods of time without losing their identity or proliferative capacity. This extended proliferative ability opens the window for in vitro genetic manipulation which has not been achieved before, making a significant advance in LSC biology and transplantation. Further work is needed to assess the potential of Ri cultured LSCs to maintain the corneal epithelium in vivo.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.