Static and RCCS dynamic spheroid expansion of Muse cells and the therapeutic potential for corneal scarring wound in mouse and tree shrew

Abstract

Purpose : Stem cell therapy for corneal scarring is useful and promising. Here, we use multilineage-differentiating stress-enduring (Muse) cells to study their differentiation and therapeutic potential for corneal injury.

Methods : Muse cells were isolated from lipoaspirate by severe stress conditions. Spheroid culture by static and dynamic rotary cell culture system (RCCS) were used to expand and activate Muse cells. These activated Muse spheroids were differentiated into corneal stromal cells (CSCs) in keratocyte differentiation medium. Tree shrews were used to establish an injury cornea debridement model. Muse-CSCs laden and orthogonally stacked two stretched compressed collagen (SCC) disk (3 mm diameter) was affixed to the wounded corneas (n=6). The microscopic and ultrastructural morphology of wounded eyes were evaluated.

Results : Muse cells in primary clusters had bi-phenotypic properties with both pluripotency and mesenchymal markers. They enabled to expand approximately 100-fold after 33 day static spheroid culture. We uncovered that Muse spheroids were activated by dynamic rotary cell culture system (RCCS), which is increased significantly by 26.5% and 20.3% in the percentage of live cells and EdU positive cells compared to static group (p=0.011, p=0.004). The gene and protein expressions of pluripotent markers of OCT3/4, SOX2 and Nanog as well as proliferation marker of Ki67 in Muse spheroids cultured under RCCS were significantly higher than those in static group. The activated Muse spheroids underwent maturation following CSC differentiation in vitro (Figure 2A). Furthermore, Muse-CSCs embedded in SCC membrane of engrafted group survived well in tree shrew debridement model. The Muse-CSC grafted group presented significantly higher nerve density and thicker epithelium as well as lesser neovascularization, scars area and CD45⁺ cells than those in control (Figure 2B).

Conclusions : This study provides opportunities for Muse cell-targeted strategies to rapidly and effectively restore vision from corneal diseases.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Graphical abstract showing the strategy for Muse cell isolation, amplification, activation, differentiation, and corneal therapeutic applications

 

(A) Static and dynamic culture and differentiation of Muse cells into CSCs which expressed positive keratocan and ALDH3A1. (B) Enhanced corneal regeneration by Muse-CSC implantation in tree shrew model.