Abstract
Purpose :
Mesenchymal stem cells (MSC) are a promising source of cells for cell based therapies in part based on their regenerative and immunomodulatory properties. MSCs have been isolated from many tissues such as bone marrow, fat, dental pulp, cornea, etc. In this study, we compared the rate of proliferation between Corneal Limbal derived MSCs (CL-MSC) and bone marrow derived MSCs (BM-MSC).
Methods :
MSCs were isolated from healthy human bone marrow donors and from human corneal limbus from research eyebank corneas (generously provided by Eversight). They were plated in 175 cm2 flasks at a seeding density of 2000 cells/cm2 in MEM-alpha with 10% fetal bovine serum. The cells were serially passaged at 80% confluency and doubling time was calculated. Cell proliferation at mid log proliferation phase were determined using the Click-iT EdU kit (Thermo-fisher) by fluorescent-activated cell sorting (BD LSR Fortessa). All experiments were repeated with at least five different donors.
Results :
The average doubling time of CL-MSC was 14.6 ± 1.8, 56.7 ± 16.2, 44.0 ± 1.4, 38.6 ± 10.8 and 36.9 ± 3.1 hours in passage 2, 3, 4, 5 and 6, respectively. For BM-MSC the doubling time was 64.6 ± 15.5, 83.7 ±12.3, 76.2 ± 17.0, 72.8 ± 11.0 and 75.3 ± 13.5 hours in passage 2, 3, 4, 5, and 6, respectively, which was significantly longer than CL-MSCs (p value for each comparison < 0.01). Incubation with 10 mM EdU for 24 hours resulted in 100.0 ± 5.1, 62.2 ± 18.1, 76.5 ± 14.4, 83.8 ± 12.7 and 76.2 ± 17.4 percent of EdU positive cells in passage 2 to 6 of CL-MSC, respectively. On the other hand, in BM-MSC 38.4 ± 9.7, 31.8 ± 12.1, 34.8 ± 11.5, 27.4 ± 16.3 and 38.5 ± 14.9 percent were EdU positive after 24 hours incubation (p value for each comparison < 0.01).
Conclusions :
These results demonstrate CL-MSCs have a faster proliferation rate compared to BM-MSCs and can be expanded in culture in greater numbers. Given the availability of eyebank corneal tissue and ease of isolation and rate of proliferation, CL-MSCs can be a suitable alternative for cell based therapy especially for ocular surface diseases.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.