Characterizing the impact of 2D and 3D culture conditions on the therapeutic effects of human mesenchymal stem cell secretome on corneal wound healing in vitro and ex vivo.

The therapeutic effects of secreted factors (secretome) produced by bone marrow-derived human mesenchymal stem cells (MSCs) were evaluated as a function of their growth in 2D culture conditions and on 3D electrospun fiber scaffolds. Electrospun fiber scaffolds composed of polycaprolactone and gelatin were fabricated to provide a 3D microenvironment for MSCs, and their mechanical properties were optimized to be similar to corneal tissue. The secretome produced by the MSCs cultured on 3D fiber matrices versus 2D culture dishes were analyzed using a Luminex immunoassay, and the secretome of MSCs cultured on the 3D versus 2D substrates showed substantial compositional differences. Concentrations of factors such as HGF and ICAM-1 were increased over 5 times in 3D cultures compared to 2D cultures. In vitro proliferation and scratch-based wound healing assays were performed to compare the effects of the secretome on corneal fibroblast cells (CFCs) when delivered synchronously from co-cultured MSCs through a trans-well co-culture system versus asynchronously after harvesting the factors separately and adding them to the media. Cell viability of CFCs was sustained for 6 days when co-cultured with MSCs seeded on the fibers but decreased with time under other conditions. Scratch assays showed 95% closure at 48 h when CFCs were co-cultured with MSCs seeded on fibers, while the control group only exhibited 50% closure at 48 h. Electrospun fibers seeded with MSCs were then applied to a rabbit corneal organ culture system, and MSCs seeded on fibers promoted faster epithelialization and less scarring. Corneas were fixed and stained for alpha smooth muscle actin (α-SMA), and then analyzed by confocal microscopy. Immunostaining showed that expression of α-SMA was lower in corneas treated with MSCs seeded on fibers, suggesting suppression of myofibroblastic transformation. MSCs cultured on electrospun fibers facilitate wound healing in CFCs and on explanted corneas through differential secretome profiles compared to MSCs cultured on 2D substrates. Future work is merited to further understand the nature and basis of these differences and their effects in animal models. STATEMENT OF SIGNIFICANCE: Previous studies have shown that the secretome of bone marrow-derived mesenchymal stem cells (MSC) is promotes corneal wound healing by facilitating improved wound closure rates and reduction of scarring and neovascularization. The present research is significant because it provides evidence for the modulation of the secretome as a function of the MSC culture environment. This leads to differential expression of therapeutic factors secreted, which can impact corneal epithelial and stromal healing after severe injury. In addition, this article shows that co-continuous delivery of the MSC secretome improves cell migration and proliferation over aliquoted delivery, and that MSCs grown on three-dimensional electrospun fiber constructs may provide a favorable microenvironment for cultured MSCs and as a carrier to deliver their secreted factors to the ocular surface.

Corneal Wound Healing Effects of Mesenchymal Stem Cell Secretome Delivered Within a Viscoelastic Gel Carrier.

Severe corneal injuries often result in permanent vision loss and remain a clinical challenge. Human bone marrow-derived mesenchymal stem cells (MSCs) and their secreted factors (secretome) have been studied for their antiscarring, anti-inflammatory, and antiangiogeneic properties. We aimed to deliver lyophilized MSC secretome (MSC-S) within a viscoelastic gel composed of hyaluronic acid (HA) and chondroitin sulfate (CS) as a way to enhance corneal re-epithelialization and reduce complications after mechanical and chemical injuries of the cornea. We hypothesized that delivering MSC-S within HA/CS would have improved wound healing effects compared the with either MSC-S or HA/CS alone. The results showed that a once-daily application of MSC-S in HA/CS enhances epithelial cell proliferation and wound healing after injury to the cornea. It also reduced scar formation, neovascularization, and hemorrhage after alkaline corneal burns. We found that combining MSC-S and HA/CS increased the expression of CD44 receptors colocalized with HA, suggesting that the observed therapeutic effects between the MSC-S and HA/CS are in part mediated by CD44 receptor upregulation and activation by HA. The results from this study demonstrate a reproducible and efficient approach for delivering the MSC-S to the ocular surface for treatment of severe corneal injuries. Stem Cells Translational Medicine 2019;8:478-489.