Engraftment and Survival of Human Umbilical Mesenchymal Stem Cells in the Mouse Cornea: An Immunofluorescent Computed Tomography Study

Abstract

Purpose: While the field of ocular regenerative medicine has advanced dramatically, little is known about the engraftment and migration characteristics of intra-stromal stem cell transplantation in the cornea. To better understand intra-stromal stem cell engraftment and migration, the purpose of this study was to use a novel imaging modality (immunofluorescent computed tomography or ICT) to localize and quantify DiO-labeled human umbilical mesenchymal stem cells (hUMSCs) transplanted into mouse corneas.

Methods: hUMSCs were DiO-labeled and injected (20,000 cells/cornea) using a 33-gauge needle and Hamilton syringe into the corneal stroma of twelve C57BL/6 mice. Cell survival following injection through the Hamilton needle and syringe was assessed by trypan blue exclusion. At two, four and twelve weeks post-injection, mice were sacrificed and corneas were excised and fixed in 2% PFA. The corneas were then dehydrated and embedded in butyl methyl methacrylate and polymerized under UV light at 4C. Once polymerized, corneas were serially sectioned (2µm thick), stained with DAPI and imaged using a Leica DMI6000B. Corneal volumes were then reconstructed using Amira and fiji software to quantify and characterize the distribution of DiO-labeled hUMSCs.

Results: At 2 weeks, 594 DiO labeled hUMSCs were identified in the stroma and migrated to occupy a volume of 1.23*107 μm3. At 4 weeks, 45 DiO labeled hUMSCs were detected in the stroma that occupied a volume of 4.28*107 μm3. At 12 weeks, only 19 DiO labeled hUMSCs in a stromal volume of 4.68*107 μm3 were identified. This suggests that the number of engrafted DiO-labeled hUMSC decreased over a 12-week period, although greater migration was observed over time. Cells injected through the Hamilton syringe showed cell viability averaging 11.66%.

Conclusions: ICT is a novel imaging modality that can track and quantify stem cell engraftment and migration over time. In this preliminary study, hUMSCs appear to migrate and occupy a larger volume over time, however, total cell engraftment appeared to markedly decline based on DiO labeling. This suggests that technique modifications may be required to optimize intra-stromal stem cell viability. Antibody staining for markers of human stem cell transplantation and keratocyte differentiation may also aid our understanding of corneal stem cell fate and verify loss of cells or labeling.

Keywords: 721 stem cells • 551 imaging/image analysis: non-clinical • 480 cornea: basic science