Corneal-Limbal Mesenchymal Stromal Cell Secretome is Antiangiogenic in Vitro

Abstract

Purpose : Corneal neovascularization is a visually devastating consequence of severe injuries or infections that result in inflammation and loss of the corneal anti-angiogenic milieu. We evaluated the angiogenic properties of corneal-limbal derived mesenchymal stromal/stem cell (CL-MSCs) secretome and compared it to bone-marrow derived MSC (BM-MSC) secretome in vitro.

Methods : BM-MSC were isolated from human healthy donors in a GMP facility. CL-MSCs were extracted from cadaver human corneas. MSCs at passage 4 to 6 from at least 5 donors were used for all experiments. MSCs were grown to confluency using MEM-alpha + 10% fetal bovine serum. Serum free secretome was collected after 48 hours. The effect of MSC secretome was evaluated on the endothelial cell tube formation using human umbilical vein endothelial cells (HUVECs). Fibrin gel bead (FIBA) assay was used to assess endothelial cell sprouting after the exposure to the MSCs secretome. Complete HUVEC media (medium 200 + 100% low serum growth supplement (LSGS)) was used as the positive control while 10% LSGS was used the base and mixed 1:1 with either MSC secretome or unconditioned media to evaluate pro/anti-angiogenic effect.

Results : Mean tubule length formation was 6810 ± 1060 µm/field with CL-MSC secretome which was significantly lower than negative control (8971 ± 1111 µm/field) in HUVEC assay (P<0.001). In contrast, BM-MSCs secretome induced angiogenesis (10534 ± 1200 µm/field) which was similar to positive control (10929 ± 1012 µm/field) (P=0.24). Likewise, in FIBA assay, the mean sprout count was 1.15 ± 1.22, 2.6 ± 1.66, and 5.15 ± 2.25 per bead 48 hours after incubation with CL-MSC secretome, negative control, and BM-MSC secretome, respectively (P<0.001 for all comparison). Mean length per sprout was 69.3 ± 74.89, 158.1 ± 92.54, and 259.6 ± 102.5 µm, after incubation with CL-MSC secretome, negative control, BM-MSC secretome, respectively (P<0.001 for all comparison).

Conclusions : These results demonstrate that CL-MSC secretome has distinct antiangiogenic effects whereas BM-MSCs secretome is proangiogenic. Further studies are necessary to determine if CL-MSC has superior anti-angiogenic effects in vivo.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.